Modern Industrial
Microbiology and Biotechnology
Modern Industrial
Microbiology and Biotechnology
Modern Industrial
Microbiology and Biotechnology
Nduka Okafor
Department of Biological Sciences
Clemson University, Clemson
South Carolina
USA
Science Publishers
Enfield (NH)
Jersey
Plymouth
SCIENCE PUBLISHERS
An imprint of Edenbridge Ltd., British Isles.
Post Office Box 699
Enfield, New Hampshire 03748
United States of America
Website: http://www.scipub.net
sales @scipub.net (marketing department)
editor@scipub.net (editorial department)
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Library of Congress Cataloging-in-Publication Data
Okafor, Nduka.
Modern industrial microbiology and bitechnology/Nduka Okafor.
p. cm.
Includes bibliographical references and index.
ISBN 978-1-57808-434-0(HC)
ISBN 978-1-57808-513-2(PB)
1. Industrial microbiology. 2.Biotechnology. I. Title
QR53.O355 2007
660.6’2--dc22
2006051256
ISBN
ISBN
978-1-57808-434-0 (HC)
978-1-57808-513-2 (PB)
© 2007, Nduka Okafor
All rights reserved. No part of this publication may be reproduced, stored in
a retrieval system, or transmitted in any form or by any means, electronic,
mechanical, photocopying, recording or otherwise, without the prior
permission.
This book is sold subject to the condition that it shall not, by way of trade or
otherwise, be lent, re-sold, hired out, or otherwise circulated without the
publisher’s prior consent in any form of binding or cover other than that in
which it is published and without a similar condition including this
condition being imposed on the subsequent purchaser.
Published by Science Publishers, Enfield, NH, USA
An imprint of Edenbridge Ltd.
Printed in India.
Dedication
This book is dedicated to the Okafor-Ozowalu family of Nri,
Anambra State, Nigeria, and their inlaws.
Modern Industrial
Microbiology and Biotechnology
Preface
The field of industrial microbiology has been undergoing rapid change in recent years.
First, what has been described as the ‘cook book’ approach has been largely abandoned
for the rational manipulation of microorganisms on account of our increased knowledge
of their physiology. Second, powerful new tools and technologies especially genetic
engineering, genomics, proteomics, bioinformatics and such like new areas promise
exciting horizons for man’s continued exploitation of microorganisms. Third, new
approaches have become available for the utilization of some traditional microbial
products such as immobilized enzymes and cells, site-directed mutation and metabolic
engineering. Simultaneously, microbiology has addressed itself to some current
problems such as the fight against cancer by the production of anti-tumor antibiotics; it
has changed the traditional practice in a number of areas: for example the deep sea has
now joined the soil as the medium for the search for new bioactive chemicals such as
antibiotics. Even the search for organisms producing new products has now been
broadened to include unculturable organisms which are isolated mainly on genes
isolated from the environment. Finally, greater consciousness of the effect of fossil fuels
on the environment has increased the call in some quarters for the use of more
environmentally friendly and renewable sources of energy, has led to a search for
alternate fermentation substrates, exemplified in cellulose, and a return to fermentation
production of ethanol and other bulk chemicals. Due to our increased knowledge and
changed approach, even our definitions of familiar words, such as antibiotic and species
seem to be changing. This book was written to reflect these changes within the context of
current practice.
This book is directed towards undergraduates and beginning graduate students in
microbiology, food science and chemical engineering. Those studying pharmacy,
biochemistry and general biology will find it of interest. The section on waste disposal
will be of interest to civil engineering and public health students and practitioners. For
the benefit of those students who may be unfamiliar with the basic biological
assumptions underlying industrial microbiology, such as students of chemical and civil
engineering, elements of biology and microbiology are introduced. The new elements
which have necessitated the shift in paradigm in industrial microbiology such as
bioinformatics, genomics, proteomics, site-directed mutation, metabolic engineering, the
human genome project and others are also introduced and their relevance to industrial
LEEE
Modern Industrial Microbiology and Biotechnology
microbiology and biotechnology indicated. As many references as space will permit are
included.
The various applications of industrial microbiology are covered broadly, and the
chapters are grouped to reflect these applications. The emphasis throughout, however, is
on the physiological and genomic principles behind these applications.
I would like to express my gratitude to Professors Tom Hughes and Hap Wheeler
(Chairman) of the Department of Biological Sciences at Clemson University for their help
and encouragement during the writing of the book. Prof Ben Okeke of Auburn University,
Alabama, and Prof Jeremy Tzeng of Clemson University read portions of the script and I
am deeply grateful to them.
My wife, Chinyelu was a source of constant and great support, without which the
project might never have been completed. I cannot thank her enough.
Clemson, South Carolina
Nduka Okafor
Contents EN
Contents
Preface
vii
SECTION A INTRODUCTION
1. Introduction: Scope of Biotechnology and
Industrial Microbiology
1.1 Nature of Biotechnology and Industrial Microbiology
1.2 Characteristics of Industrial Microbiology
1.2.1 Industrial vs medical microbiology 4
1.2.2 Multi-disciplinary or Team-work nature of
industrial microbiology 4
1.2.3 Obsolescence in industrial microbiology 5
1.2.4 Free communication of procedures in industrial microbiology 5
1.3 Patents and Intellectual Property Rights in
Industrial Microbiology and Biotechnology
1.4 The Use of the Word ‘Fermentation’ in Industrial Microbiology
1.5 Organizational Set-up in an Industrial Microbiology Establishment
Suggested Readings
3
3
4
5
9
10
13
SECTION B BIOLOGICAL BASIS OF PRODUCTIVITY IN
INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY
2. Some Microorganisms Commonly Used in
Industrial Microbiology and Biotechnology
2.1 Basic Nature of Cells of Living Things
2.2 Classification of Living Things: Three Domains of Living Things
2.3 Taxonomic Grouping of Micro-organisms Important in
Industrial Microbiology and Biotechnology
2.3.1 Bacteria 21
2.3.2 Eucarya: Fungi 29
2.4 Characteristics Important in Microbes Used in
Industrial Microbiology and Biotechnolgy
Suggested Readings
33
17
17
18
19
31
N Modern Industrial Microbiology and Biotechnology
3. Aspects of Molecular Biology and Bioinformatics of
Relevance in Industrial Microbiology and Biotechnology
3.1 Protein Synthesis
3.2 The Polymerase Chain Reaction
3.2.1 Some applications of PCR in industrial microbiology and
biotechnology 41
3.3 Microarrays
3.3.1 Applications of microarray technology 43
3.4 Sequencing of DNA
3.4.1 Sequencing of short DNA fragments 44
3.4.2 Sequencing of genomes or large DNA fragments 46
3.5 The Open Reading Frame and the Identification of Genes
3.6 Metagenomics
3.7 Nature of Bioinformatics
3.7.1 Some contributions of bioinformatics to biotechnology 51
Suggested Readings
34
39
42
44
46
48
50
52
4. Industrial Media and the Nutrition of Industrial Organisms
4.1 The Basic Nutrient Requirements of Industrial Media
4.2 Criteria for the Choice of Raw Materials Used in Industrial Media
4.3 Some Raw Materials Used in Compounding Industrial Media
4.4 Growth Factors
4.5 Water
4.6 Some Potential Sources of Components of Industrial Media
4.6.1 Carbohydrate sources 63
4.6.2 Protein sources 65
4.7 The Use of Plant Waste Materials in Industrial Microbiology Media:
Saccharification of Polysaccharides
4.7.1 Starch 67
4.7.2 Cellulose, hemi-celluloses and lignin in plant materials 73
Suggested Readings
34
54
54
56
58
62
62
63
66
76
5. Metabolic Pathways for the Biosynthesis of
Industrial Microbiology Products
5.1 The Nature of Metabolic Pathways
5.2 Industrial Microbiological Products as Primary and Secondary Metabolites
5.2.1 Products of primary metabolism 78
5.2.2 Products of secondary metabolism 79
5.3 Trophophase-idiophase Relationships in the Production of
Secondary Products
5.4 Role of Secondary Metabolites in the Physiology of
Organisms Producing Them
5.5 Pathways for the Synthesis of Primary and Secondary Metabolites of
Industrial Importance
5.5.1 Catabolism of carbohydrates 84
5.5.2 The Catabolism of hydrocarbons 88
77
77
78
81
82
83
Contents NE
5.6 Carbon Pathways for the Formation of Some
Industrial Products Derived from Primary Metabolism
5.6.1 Catabolic products 89
5.6.2 Anabolic products 89
5.7 Carbon Pathways for the Formation of Some Products of
Microbial Secondary Metabolism of Industrial Importance
Suggested Readings
99
100
109
115
120
120
7. Screening for Productive Strains and Strain
Improvement in Biotechnological Organisms
7.1 Sources of Microorganisms Used in Biotechnology
7.1.1 Literature search and culture collection supply 122
7.1.2 Isolation de novo of organisms producing
metabolites of economic importance 123
7.2 Strain Improvement
7.2.1 Selection from naturally occurring variants 126
7.2.2 Manipulation of the genome of
industrial organisms in strain improvement 126
Suggested Readings
89
98
6. Overproduction of Metabolites of Industrial Microorganisms
6.1 Mechanisms Enabling Microorganisms to Avoid Overproduction of
Primary Metabolic Products Through Enzyme Regulation
6.1.1 Substrate induction 101
6.1.2 Catabolite regulation 103
6.1.3 Feedback regulation 105
6.1.4 Amino acid regulation of RNA synthesis 107
6.1.5 Energy charge regulation 107
6.1.6 Permeability control 108
6.2 Derangement or Bypassing of Regulatory Mechanisms for
the Over-production of Primary Metabolites
6.2.1 Metabolic control 109
6.2.2 Permeability 114
6.3 Regulation of Overproduction in Secondary Metabolites
6.3.1 Induction 115
6.3.2 Catabolite regulation 115
6.3.3 Feedback regulation 117
6.3.4 ATP or energy charge regulation of secondary metabolites 117
6.4 Empirical Methods Employed to Disorganize Regulatory
Mechanisms in Secondary Metabolite Production
Suggested Readings
89
122
122
125
170
8. The Preservation of the Gene Pool in
Industrial Organisms: Culture Collections
171
8.1 The Place of Culture Collections in
Industrial Microbiology and Biotechnology
8.2 Types of Culture Collections
171
172
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Modern Industrial Microbiology and Biotechnology
8.3 Handling Culture Collections
8.4 Methods of Preserving Microorganisms
8.4.1 Microbial preservation methods based on the
reduction of the temperature of growth 174
8.4.2 Microbial preservation methods based on dehydration 176
8.4.3 Microbial preservation methods based on the
reduction of nutrients 178
8.4.4 The need for experimentation to determine the
most appropriate method of preserving an organism 178
Suggested Redings
SECTION C
173
173
178
BASIC OPERATIONS IN INDUSTRIAL FERMENTATIONS
9. Fermentors and Fermentor Operation
9.1 Definition of a Fermentor
9.2 The Aerated Stirred Tank Batch Fermentor
9.2.1 Construction materials for fermentors 185
9.2.2 Aeration and agitation in a fermentor 185
9.2.3 Temperature control in a fermentor 186
9.2.4 Foam production and control 188
9.2.5 Process control in a fermentor 192
9.3 Anerobic Batch Fermentors
9.4 Fermentor Configurations
9.4.1 Continuous fermentations 196
9.5 Fed-batch Cultivation
9.6 Design of New Fermentors on the
Basis of Physiology of the Organisms: Air Lift Fermentors
9.7 Microbial Experimentation in the Fermentation Industry:
The Place of the Pilot Plant
9.8 Inoculum Preparation
9.9 Surface or Solid State Fermentors
Suggested Readings
183
183
184
195
196
202
202
205
205
206
206
10. Extraction of Fermentation Products
10.1 Solids (Insolubles) Removal
10.1.1 Filtration 209
10.1.2 Centrifugation 210
10.1.3 Coagulation and flocculation 210
10.1.4 Foam fractionation 211
10.1.5 Whole-broth treatment 212
10.2 Primary Product Isolation
10.2.1 Cell disruption 212
10.2.2 Liquid extraction 213
10.2.3 Dissociation extraction 214
10.2.4 Ion-exchange adsorption 214
10.2.5 Precipitation 216
208
209
212
Contents
10.3 Purification
10.3.1 Chromatography 217
10.3.2 Carbon decolorization 217
10.3.3 Crystallization 218
10.4 Product Isolation
10.4.1 Crystalline processing 218
10.4.2 Drying 218
Suggested Readings
217
218
220
11. Sterility in Industrial Microbiology
221
11.1 The Basis of Loss by Contaminants
11.2 Methods of Achieving Sterility
11.2.1 Physical methods 222
11.2.2 Chemical methods 227
11.3 Aspects of Sterilization in Industry
11.3.1 The sterilization of the fermentor and its accessories 229
11.3.2 Media sterilization 229
11.4 Viruses (Phages) in Industrial Microbiology
11.4.1 Morphological grouping of bacteriophages 232
11.4.2 Lysis of hosts by phages 232
11.4.3 Prevention of phage contamination 232
11.4.4 Use of phage resistant mutants 234
11.4.5 Inhibition of phage multiplication with chemicals 234
11.4.6 Use of adequate media conditions and other practices 234
Suggested Readings
SECTION D
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221
222
229
230
234
ALCOHOL-BASED FERMENTATION INDUSTRIES
12. Production of Beer
237
12.1 Barley Beers
12.1.1 Types of barley beers 237
12.1.2 Raw materials for brewing 238
12.1.3 Brewery processes 242
12.1.4 Beer defects 253
12.1.5 Some developments in beer brewing 255
12.2 Sorghum Beers
12.2.1 Kaffir beer and other traditional sorghum beers
Suggested Readings
237
258
258
260
13. Production of Wines and Spirits
13.1 Grape Wines
13.1.1 Processes in wine making 262
13.1.2 Fermentation 263
13.1.3 Ageing and storage 263
13.1.4 Clarification 264
13.1.5 Packaging 265
262
262
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Modern Industrial Microbiology and Biotechnology
13.1.6 Wine defects 265
13.1.7 Wine preservation 265
13.1.8 Classification of wines 265
13.2 Palm Wine
13.3 The Distilled Alcoholic (or Spirit) Beverages
13.3.1 Measurement of the alcoholic strength of distilled beverages 274
13.3.2 General principles in the production of spirit beverages 275
13.3.3 The spirit beverages 276
Suggested Readings 278
14. Production of Vinegar
14.1 Uses
14.2 Measurement of Acetic Acid in Vinegar
14.3 Types of Vinegar
14.4 Organisms Involved
14.5 Manufacture of Vinegar
14.5.1 The Orleans (or slow) method 283
14.5.2 The trickling generators (quick) method
14.5.3 Submerged generators 286
14.6 Processing of Vinegar
Suggested Readings
SECTION E
270
274
280
280
281
281
282
283
284
288
289
USE OF WHOLE CELLS FOR FOOD RELATED PURPOSES
15. Single Cell Protein (SCP)
15.1 Substrates for Single Cell Protein Production
15.1.1 Hydrocarbons 294
15.1.2 Alcohols 297
15.1.3 Waste products 298
15.2 Microorganisms Used in SCP Production
15.3 Use of Autotrophic Microorganisms in SCP Production
15.4 Safety of Single Cell Protein
15.4.1 Nucleic acids and their removal from SCP 304
15.5 Nutritional Value of Single Cell Protein
Suggested Readings
293
294
300
300
303
305
305
16. Yeast Production
16.1 Production of Baker’s Yeast
16.1.1 Yeast strain used 308
16.1.2 Culture maintenance 309
16.1.3 Factory production 309
16.2 Food Yeasts
16.2.1 Production of food yeast 312
16.3 Feed Yeasts
16.4 Alcohol Yeasts
306
306
311
313
314
Contents NL
16.5 Yeast Products
Suggested Readings
314
314
17. Production of Microbial Insecticides
17.1 Alternatives to Chemical Insecticides
17.2 Biological Control of Insects
17.2.1 Desirable properties in organisms to be used for
biological control 317
17.2.2 Candidates which have been considered as
biological control agents 318
17.2.3 Bacillus thuringiensis Insecticidal toxin 321
17.3 Production of Biological Insecticides
17.3.1 Submerged fermentations 322
17.3.2 Surface culture 323
17.3.3 In vivo culture 323
17.4 Bioassay of Biological Insecticides
17.5 Formulation and Use of Bioinsecticides
17.5.1 Dusts 324
17.5.2 Liquid formulation 324
17.6 Safety Testing of Bioinsecticides
17.7 Search and Development of New Bioinsecticides
Suggested Readings
322
323
324
325
325
326
18. The Manufacture of Rhizobium Inoculants
18.1 Biology of Rhizobium
18.1.1 General properties 328
18.1.2 Cross-inoculation groups of rhizobium 328
18.1.3 Properties desirable in strains to be selected for
use as rhizobium inoculants 328
18.1.4 Selection of strains for use as rhizobial inoculants 329
18.2 Fermentation of Rhizobia
18.3 Inoculant Packaging for Use
18.3.1 Seed inoculants 331
18.3.2 Soil inoculants 332
18.4 Quality Control
Suggested Readings
315
315
316
327
328
330
331
333
333
19. Production of Fermented Foods
19.1 Introduction
19.2 Fermented Food from Wheat: Bread
19.2.1 Ingredients for modern bread-making 335
19.2.2 Systems of bread-making 339
19.2.3 Role of yeasts in bread-making 340
19.3 Fermented Foods Made from Milk
19.3.1 Composition of milk 343
334
334
335
343
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Modern Industrial Microbiology and Biotechnology
19.4
19.5
19.6
19.7
19.8
19.3.2 Cheese 344
19.3.3 Yoghurt and fermented milk foods 347
Fermented Foods from Corn
19.4.1 Ogi, koko, mahewu 349
Fermented Foods from Cassava: Garri, Foo-Foo, Chikwuange,
Kokonte, Bikedi, and Cinguada
19.5.1 Garri 351
19.5.2 Foo-foo, chikwuangue, lafun, kokonte,
bikedi, and cinguada 352
Fermented Vegetables
19.6.1 Sauerkraut 353
19.6.2 Cucumbers (pickling) 353
Fermentations for the Production of the
Stimulant Beverages: Tea, Coffee, and Cocoa
19.7.1 Tea production 354
19.7.2 Coffee fermentation 354
19.7.3 Cocoa fermentation 355
Fermented Foods Derived from Legumes and Oil Seeds
19.8.1 Fermented foods from Soybeans 355
19.8.2 Fermented foods from beans: Idli 359
19.8.3 Fermented foods from Protein-rich Oil-seeds 360
19.8.4 Food condiments made from fish 360
Suggested Readings
SECTION F
348
350
353
354
355
360
PRODUCTION OF METABOLITES AS BULK CHEMICALS OR
AS I NPUTS IN OTHER PROCESSES
20. Production of Organic Acids and Industrial Alcohol
20.1 Organic Acids
20.1.1 Production of citric acid 365
20.1.2 Uses of citric acid 365
20.1.3 Biochemical basis of the production of citric acid 366
20.1.4 Fermentation for citric acid production 368
20.1.5 Extraction 368
20.1.6 Lactic acid 369
20.2 Industrial Alcohol Production
20.2.1 Properties of ethanol 373
20.2.2 Uses of ethanol 374
20.2.3 Denatured alcohol 374
20.2.4 Manufacture of ethanol 374
20.2.5 Some developments in alcohol production 377
Suggested Readings
365
373
379
21. Production of Amino Acids by Fermentation
21.1 Uses of Amino Acids
365
380
380
Contents
21.2 Methods for the Manufacture of Amino Acids 384
21.2.1 Semi-fermentation 386
21.2.2 Enzymatic process 386
21.2.3 Production of amino acids by the direct fermentation 388
21.3 Production of Glutamic Acid by Wild Type Bacteria
21.4 Production of Amino Acids by Mutants
21.4.1 Production of amino acids by auxotrophic mutants 390
21.4.2 Production of amino acids by regulatory mutants 390
21.5 Improvements in the Production of Amino Acids Using
Metabolically Engineered Organisms
21.5.1 Strategies to modify the terminal pathways 392
21.5.2 Strategies for increasing precursor availability 393
21.5.3 Metabolic engineering to improve transport of
amino acids outside the cell 394
21.6 Fermentor Production of Amino Acid
21.6.1 Fermentor procedure 394
21.6.2 Raw materials 395
21.6.3 Production strains 395
21.6.4 Down stream processing 396
Suggested Readings
22.5
22.6
22.7
22.8
388
389
391
394
396
22. Biocatalysts: Immobilized Enzymes and Immobilized Cells
22.1
22.2
22.3
22.4
NLEE
398
Rationale for Use of Enzymes from Microorganisms
398
Classification of Enzymes
399
Uses of Enzymes in Industry
400
Production of Enzymes
406
22.4.1 Fermentation for enzyme production 406
22.4.2 Enzyme extraction 408
22.4.3 Packaging and finishing 408
22.4.4 Toxicity testing and standardization 408
Immobilized Biocatalysts: Enzymes and Cells
408
22.5.1 Advantages of immobilized biocatalysts in general 409
22.5.2 Methods of immobilizing enzymes 409
22.5.3 Methods for the immobilization of cells 412
Bioreactors Designs for Usage in Biocatalysis
414
Practical Application of Immobilized Biological Catalyst Systems
416
Manipulation of Microorganisms for Higher Yield of Enzymes
416
22.8.1 Some aspects of the biology of extracellular enzyme production 417
Suggested Readings
419
23. Mining Microbiology: Ore Leaching (Bioleaching) by
Microorganisms
23.1 Bioleaching
23.2 Commercial Leaching Methods
23.2.1 Irrigation-type processes 422
23.2.2 Stirred tank processes 423
421
421
422
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Modern Industrial Microbiology and Biotechnology
23.3 Microbiology of the Leaching Process
23.4 Leaching of Some Metal Sulfides
23.5 Environmental Conditions Affecting Bacterial Leaching
Suggested Readings
SECTION G
423
424
425
426
PRODUCTION OF COMMODITIES OF MEDICAL IMPORTANCE
24. Production of Antibiotics and Anti-Tumor Agents
429
24.1 Classification and Nomenclature of Antibiotics
24.2 Beta-Lactam Antibiotics
24.2.1 Penicillins 432
24.2.2 Cephalosporins 435
24.2.3 Other beta-lactam antibiotics 438
24.3 The Search for New Antibiotics
24.3.1 The need for new antibiotics 439
24.3.2 The classical method for searching for antibiotics:
random search in the soil 440
24.4 Combating Resistance and Expanding the Effectiveness of
Existing Antibiotics
24.4.1 Refinements in the procedures for
the random search for new antibiotics in the soil 444
24.4.2 Newer approaches to searching for antibiotics 445
24.5 Anti-Tumor Antibiotics
24.5.1 Nature of tumors 448
24.5.2 Mode of action of anti-tumor antibiotics 449
24.5.3 Search for new anti-tumor antibiotics 449
24.6 Newer Methods for Searching for Antibiotic and Anti-tumor Drugs
Suggested Readings
429
430
439
444
448
453
453
25. Production of Ergot Alkaloids
25.1
25.2
25.3
25.4
455
Nature of Ergot Alkaloids
Uses of Ergot Alkaloids and their Derivates
Production of Ergot Alkaloids
Physiology of Alkaloid Production
Suggested Readings
455
457
459
461
463
26. Microbial Transformation and Steroids and Sterols
26.1 Nature and Use of Steroids and Sterols
26.2 Uses of Steroids and Sterols
26.2.1 Sex hormones 466
26.2.2 Corticosteroids 467
26.2.3 Saponins 467
26.2.4 Heterocyclic steroids 467
26.3 Manufacture of Steroids
26.3.1 Types of microbial transformations in steroids and sterols
464
464
466
467
469
Contents NEN
26.3.2 Fermentation conditions used in steroid transformation 470
26.4 Screening for Microorganisms
Suggested Readings
471
27. Vaccines
27.1 Nature and Importance of Vaccines
27.2 Body Defenses against Communicable Diseases
27.2.1 Innate or non-specific immunity 475
27.3 Traditional and Modern Methods of Vaccine Production
27.3.1 Traditional vaccines 479
27.3.2 Newer approaches in vaccinology 480
27.4 Production of Vaccines
27.4.1 Production of virus vaccines 482
27.4.2 Production of bacterial toxoids 485
27.4.3 Production of killed bacterial vaccines 485
27.5 Control of Vaccines
27.6 Vaccine Production versus Other Aspects of Industrial Microbiology
Suggested Readings
471
472
472
472
479
482
486
487
487
28. Drug Discovery in Microbial Metabolites: The Search for
Microbial Products with Bioactive Properties
28.1 Conventional Processes of Drug Discovery
28.1.1 Cell-based assays 489
28.1.2 Receptor binding assays 491
28.1.3 Enzyme assays 491
28.2 Newer Methods of Drug Discovery
28.2.1 Computer aided drug design 492
28.2.2 Combinatorial chemistry 493
28.2.3 Genomic methods in the search for new drugs,
including antibiotics 494
28.2.4 Search for drugs among unculturable microorganisms 496
28.4 Approval of New Antibiotic and other Drugs by the Regulating Agency
28.4.1 Pre-submission work by the pharmaceutical firm 497
28.4.2 Submission of the new drug to the FDA 499
28.4.3 Approval 500
28.4.4 Post approval research 501
Suggested Readings
SECTION H
488
489
492
497
501
WASTE DISPOSAL
29. Treatment of Wastes in Industry
29.1 Methods for the Determination of Organic Matter Content in Waste Waters
29.1.1 Dissolved oxygen 506
29.1.2 The biological or biochemical oxygen demand (BOD) tests 506
29.1.3 Permanganate value (PV) test 506
505
505
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Modern Industrial Microbiology and Biotechnology
29.2
29.3
29.4
29.5
29.1.4 Chemical oxygen demand (COD) 507
29.1.5 Total organic carbon (TOC) 507
29.1.6 Total suspended solids (TSS) 507
29.1.7 Volatile suspended solids (VSS) 507
Wastes from Major Industries
Systems for the Treatment of Wastes
29.3.1 Aerobic breakdown of raw waste waters 509
Treatment of the Sludge: Anaerobic Breakdown of Sludge
Waste Water Disposal in the Pharmaceutical Industry
Suggested Readings
508
509
516
517
519
Glossary
520
Index
523
Section
Introduction
)
Modern Industrial
Microbiology and Biotechnology
+0)26-4
1
Introduction:
Scope of Biotechnology and
Industrial Microbiology
1.1
NATURE OF BIOTECHNOLOGY AND
INDUSTRIAL MICROBIOLOGY
There are many definitions of biotechnology. One of the broadest is the one given at the
United Nations Conference on Biological Diversity (also called the Earth Summit) at the
meeting held in Rio de Janeiro, Brazil in 1992. That conference defined biotechnology as
“any technological application that uses biological systems, living organisms, or
derivatives thereof, to make or modify products or processes for specific use.” Many
examples readily come to mind of living things being used to make or modify processes
for specfic use. Some of these include the use of microorganisms to make the antibiotic,
penicillin or the dairy product, yoghurt; the use of microorganisms to produce amino
acids or enzymes are also examples of biotechnology.
Developments in molecular biology in the last two decades or so, have vastly
increased our understanding of the nucleic acids in the genetic processes. This has led to
applications of biological manipulation at the molecular level in such technologies as
genetic engineering. All aspects of biological manipulations now have molecular biology
dimensions and it appears convenient to divide biotechnology into traditional
biotechnology which does not directly involve nucleic acid or molecular manipulations
and nucleic acid biotechnology, which does.
Industrial microbiology may be defined as the study of the large-scale and profitmotivated production of microorganisms or their products for direct use, or as inputs in
the manufacture of other goods. Thus yeasts may be produced for direct consumption as
food for humans or as animal feed, or for use in bread-making; their product, ethanol,
may also be consumed in the form of alcoholic beverages, or used in the manufacture of
perfumes, pharmaceuticals, etc. Industrial microbiology is clearly a branch of
biotechnology and includes the traditional and nucleic acid aspects.
"
Modern Industrial Microbiology and Biotechnology
1.2
CHARACTERISTICS OF INDUSTRIAL MICROBIOLOGY
The discipline of microbiology is often divided into sub-disciplines such as medical
microbiology, environmental microbiology, food microbiology and industrial
microbiology. The boundaries between these sub-divisions are often blurred and are
made only for convenience.
Bearing this qualification in mind, the characteristics of industrial microbiology can
be highlighted by comparing its features with those of another sub-division of
microbiology, medical microbiology.
1.2.1
Industrial vs Medical Microbiology
The sub-disciplines of industrial microbiology and medical microbiology differ in at least
three different ways.
First is the immediate motivation: in industrial microbiology the immediate motivation is profit and the generation of wealth. In medical microbiology, the immediate
concern of the microbiologist or laboratory worker is to offer expert opinion to the doctor
about, for example the spectrum of antibiotic susceptibility of the microorganisms
isolated from a diseased condition so as to restore the patient back to good health. The
generation of wealth is of course at the back of the mind of the medical microbiologist but
restoration of the patient to good health is the immediate concern.
The second difference is that the microorganisms per se used in routine medical
microbiology have little or no direct economic value, outside the contribution which they
make to ensuring the return to good health of the patient who may then pay for the
services. In industrial microbiology the microorganisms involved or their products are
very valuable and the raison d’etre for the existence of the industrial microbiology
establishment.
The third difference between the two sub-disciplines is the scale at which the
microorganisms are handled. In industrial microbiology, the scale is large and the
organisms may be cultivated in fermentors as large as 50,000 liters or larger. In routine
medical microbiology the scale at which the pathogen is handled is limited to a loopful or
a few milliliters. If a pathogen which normally would have no economic value were to be
handled on the large scale used in industrial microbiology, it would most probably be to
prepare a vaccine against the pathogen. Under that condition, the pathogen would then
acquire an economic value and a profit-making potential; the operation would properly
be termed industrial microbiology.
1.2.2
Multi-disciplinary or Team-work Nature of
Industrial Microbiology
Unlike many other areas of the discipline of microbiology, the microbiologist in an
industrial establishment does not function by himself. He is usually only one of a number
of different functionaries with whom he has to interact constantly. In a modern industrial
microbiology organization these others may include chemical or production engineers,
biochemists, economists, lawyers, marketing experts, and other high-level functionaries.
They all cooperate to achieve the purpose of the firm, which is not philanthropy, (at least
not immediately) but the generation of profit or wealth.
Introduction: Scope of Biotechnology and Industrial Microbiology
#
Despite the necessity for team work emphasized above, the microbiologist has a
central and key role in his organization. Some of his functions include:
a. the selection of the organism to be used in the processes;
b. the choice of the medium of growth of the organism;
c. the determination of the environmental conditions for the organism’s optimum
productivity i.e., pH, temperature, aeration, etc.
d. during the actual production the microbiologist must monitor the process for the
absence of contaminants, and participate in quality control to ensure uniformity of
quality in the products;
e. the proper custody of the organisms usually in a culture collection, so that their
desirable properties are retained;
f. the improvement of the performance of the microorganisms by genetic
manipulation or by medium reconstitution.
1.2.3
Obsolescence in Industrial Microbiology
As profit is the motivating factor in the pursuit of industrial microbiology, less efficient
methods are discarded as better ones are discovered. Indeed a microbiological method
may be discarded entirely in favor of a cheaper chemical method. This was the case with
ethanol for example which up till about 1930 was produced by fermentation. When
cheaper chemical methods using petroleum as the substrate became available in about
1930, fermentation ethanol was virtually abandoned. From the mid-1970s the price of
petroleum has climbed steeply. It has once again become profitable to produce ethanol by
fermentation. Several countries notably Brazil, India and the United States have officially
announced the production of ethanol by fermentation for blending into gasoline as
gasohol.
1.2.4
Free Communication of Procedures in
Industrial Microbiology
Many procedures employed in industrial microbiology do not become public property for
a long time because the companies which discover them either keep them secret, or else
patent them. The undisclosed methods are usually blandly described as ‘know-how’.
The reason for the secrecy is obvious and is designed to keep the owner of the secret one
step ahead of his/her competitors. For this reason, industrial microbiology textbooks
often lag behind in describing methods employed in industry. Patents, especially as they
relate to industrial microbiology, will be discussed below.
1.3
PATENTS AND INTELLECTUAL PROPERTY RIGHTS IN
INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY
All over the world, governments set up patent or intellectual property laws, which have
two aims. First, they are intended to induce an inventor to disclose something of his/her
invention. Second, patents ensure that an invention is not exploited without some
reward to the inventor for his/her innovation; anyone wishing to use a patented
invention would have to pay the patentee for its use.
$ Modern Industrial Microbiology and Biotechnology
The prerequisite for the patentability of inventions all over the world are that the
claimed invention must be new, useful and unobvious from what is already known
in ‘the prior art’ or in the ‘state of the art’. For most patent laws an invention is
patentable:
a. if it is new, results from inventive activity and is capable of industrial application,
or
b. if it constitutes an improvement upon a patented invention, and is capable of
industrial application.
For the purposes of the above:
a. an invention is new if it does not form part of the state of the art (i.e., it is not part of
the existing body of knowledge);
b. an invention results from inventive activity if it does not obviously follow from the
state of the art, either as to the method, the application, the combination of methods,
or the product which is concerns, or as to the industrial result it produces, and
c. an invention is capable of industrial application if it can be manufactured or used
in any kind of industry, including agriculture.
In the above, ‘the art’ means the art or field of knowledge to which an invention relates
and ‘the state of the art’ means everything concerning that art or field of knowledge
which has been made available to the public anywhere and at any time, by means of a
written or oral description, or in any other way, before the date of the filing of the patent
application.
Patents cannot be validly obtained in respect of:
a. plant or animal varieties, or essentially biological processes for the production of
plants or animals (other than microbiological processes and their products), or
b. inventions, the publication or exploitation of which would be contrary to public
order or morality (it being understood for the purposes of this paragraph that the
exploitation of an invention is not contrary to public order or morality merely
because its exploitation is prohibited by law).
Principles and discoveries of a scientific nature are not necessarily inventions for the
purposes of patent laws.
It is however not always as easy as it may seem to show that an invention is ‘new’,
‘useful’, and ‘unobvious’. In some cases it has been necessary to go to the law courts to
decide whether or not an invention is patentable. It is therefore advisable to obtain the
services of an attorney specializing in patent law before undertaking to seek a patent. The
laws are often so complicated that the layman, including the bench-bound microbiologist
may, without proper guidance, leave out essential details which may invalidate his claim
to his invention.
The exact wording may vary, but the general ideas regarding patentability are the
same around the world. The current Patent Law in the United States is the United States
Code Title 35 – Patents (Revised 3 August, 2005), and is administered by the Patents and
Trademarks Office while the equivalent UK Patent Law is the Patent Act 1977.
An examination of the patent laws of a number of countries will show that they often
differ only in minor details. For example patents are valid in the UK and some other
Introduction: Scope of Biotechnology and Industrial Microbiology
%
countries for a period of 20 years whereas they are valid in the United States for 17 years.
International laws have helped to bridge some of the differences among the patent
practices of various countries. The Paris Convention for the protection of Industrial
Property has been signed by several countries. This convention provides that each
country guarantees to the citizens of other countries the same rights in patent matters as
their own citizens. The treaty also provides for the right of priority in case of dispute.
Following from this, once an applicant has filed a patent in one of the member countries
on a particular invention, he may within a certain time period apply for protection in all
the other member countries. The latter application will then be regarded as having been
filed on the same day as in the country of the first application. Another international
treaty signed in Washington, DC came into effect on 1 June, 1968. This latter treaty, the
Patent Cooperation Treaty, facilitates the filing of patent applications in different
countries by providing standard formats among other things.
A wide range of microbiological inventions are generally recognized as patentable.
Such items include vaccines, bacterial insecticides, and mycoherbicides. As will be seen
below however, micro-organisms per se are not patentable, except when they are used as
part of a ‘useful’ process.
On 16 June, 1980 a case of immense importance to the course of industrial
microbiology was decided in the United States Court of Customs and Patent Appeals. In
brief, the court ruled that “a live human-made micro-organism is patentable”.
Dr. Ananda Chakrabarty then an employee of General Electric Company had introduced
into a bacterium of the genus Pseudomonas two plasmids (using techniques of genetic
engineering discussed in Chapter 7) which enabled the new bacterium to degrade
multiple components of crude oil. This single bacterium rather than a mixture of several
would then be used for cleaning up oil spills. Claims to the invention were on three
grounds.
a. Process claims for the method of producing the bacteria
b. Claims for an inoculum comprising an inert carrier and the bacterium
c. Claims to the bacteria themselves.
The first two were easily accepted by the lower court but the third was not accepted on
the grounds that (i) the organisms are products of nature and (ii) that as living things they
are not patentable. As had been said earlier the Appeals Court reversed the earlier
judgment of the lower court and established the patentability of organisms imbued with
new properties through genetic engineering.
A study of the transcript of the decision of the Appeals Court and other patents
highlights a number of points about the patentability of microorganisms.
First, microorganisms by themselves are not patentable, being ‘products of nature’ and
‘living things’. However they are patentable as part of a useful ‘process’ i.e. when they
are included along with a chemical or an inert material with which jointly they fulfill a
useful purpose. In other words it is the organism-inert material complex which is
patented, not the organism itself. An example is a US patent dealing with a bacterium
which kills mosquito larva granted to Dr L J Goldberg in 1979, and which reads thus in
part:
& Modern Industrial Microbiology and Biotechnology
What is claimed is:
A bacterial larvicide active against mosquito-like larvae comprising (this author’s
italics):
a. an effective larva-killing concentration of spores of the pure biological strain of
Bacillus thuringiensis var. WHO/CCBC 1897 as an active agent; and
b. a carrier….
It is the combination of the bacterial larvicide and the carrier which produced a unique
patentable material, not the larvicide by itself. In this regard, when for example, a new
antibiotic is patented, the organism producing it forms part of the useful process by
which the antibiotic is produced.
Second, a new organism produced by genetic engineering constitutes a ‘manufacture’
or ‘composition of matter’. The Appeals Court made it quite clear that such an organism
was different from a newly discovered mineral, and from Einstein’s law, or Newton’s law
which are not patentable since they already existed in nature. Today most countries
including those of the European Economic Community accept that the following are
patentable: the creation of new plasmid vectors, isolation of new DNA restriction
enzymes, isolation of new DNA-joining enzymes or ligases, creation of new recombinant
DNA, creation of new genetically modified cells, means of introducing recombinant
DNA into a host cell, creation of new transformed host cells containing recombinant
DNA, a process for preparing new or known useful products with the aid of transformed
cells, and novel cloning processes. Patents resulting from the above were in general
regarded as process, not substance, patents. (The above terms all relate to genetic
engineering and are discussed in Chapter 7.) The current US law specifically defines
biotechnological inventions and their patentability as follows:
“For purposes of (this) paragraph …. the term ‘biotechnological process’ means:
(A) a process of genetically altering or otherwise inducing a single- or multi-celled
organism to(i) express an exogenous nucleotide sequence,
(ii) inhibit, eliminate, augment, or alter expression of an endogenous nucleotide
sequence, or
(iii) express a specific physiological characteristic not naturally associated with
said organism;
(B) cell fusion procedures yielding a cell line that expresses a specific protein, such as
a monoclonal antibody; and
(C) a method of using a product produced by a process defined by subparagraph (A) or
(B), or a combination of subparagraphs (A) and (B).”
Third, the patenting of a microbiological process places on the patentee the obligation
of depositing the culture in a recognized culture collection. The larvicidal bacterium,
Bacillus thuringiensis, just mentioned, is deposited at the World Health Organization
(WHO) International Culture depository at the Ohio State University Columbus Ohio,
USA. The rationale for the deposition of culture in a recognized culture collection is to
provide permanence of the culture and ready availability to users of the patent. The
cultures must be pure and are usually deposited in lyophilized vials.
Introduction: Scope of Biotechnology and Industrial Microbiology
'
The deposition of culture solves the problems of satisfying patent laws created by the
nature of microbiology. In chemical patents the chemicals have to be described fully and
no need exists to provide the actual chemical. In microbiological patents, it is not very
helpful to describe on paper how to isolate an organism even assuming that the isolate
can be readily obtained, or indeed how the organism looks. More importantly, it is
difficult to readily and accurately recognize a particular organism based on patent
descriptions alone. Finally, since the organism is a part of the input of microbiological
processes it must be available to a user of the patent information.
Culture collections where patent-related cultures have been deposited include the
American Type Culture Collection, (ATCC), Maryland, USA, National Collection of
Industrial Bacteria (NCIB), Aberdeen, Scotland, UK, Agricultural Research Service
Culture Collection, Northern Regional Research Laboratory (NRRL), Peoria, Illinois,
USA. A fuller list is available in the World Directory of Cultures of Micro-organisms. Culture
collections and methods for preserving microorganisms are discussed in Chapter 8 of
this book.
Fourth, where a microbiologist-inventor is an employee, the patent is usually assigned
to the employer, unless some agreement is reached between them to the contrary. The
patent for the oil-consuming Pseudomonas discussed earlier went to General Electric
Company, not to its employee.
Fifth, in certain circumstances it may be prudent not to patent the invention at all, but
to maintain the discovery as a trade secret. In cases where the patent can be circumvented
by a minor change in the process without an obvious violation of the patent law it would
not be wise to patent, but to maintain the procedure as a trade secret. Even if the nature of
the compound produced by the microorganisms were not disclosed, it may be possible to
discover its composition during the processes of certification which it must undergo in
the hands of government analysts. The decision whether to patent or not must therefore
be considered seriously, consulting legal opinion as necessary. It is for this reason that
some patents sometimes leave out minor but vital details. As much further detail as the
patentee is willing to give must therefore be obtained when a patent is being considered
seriously for use.
In conclusion when all necessary considerations have been taken into account and it
is decided to patent an invention, the decision must be pursued with vigor and with
adequate degree of secrecy because as one patent law states:
…. The right to patent in respect of an invention is vested in the statutory inventor,
that is to say that person who whether or not he is the true inventor, is the first to
file…(the) patent application.
1.4
THE USE OF THE WORD ‘FERMENTATION’ IN
INDUSTRIAL MICROBIOLOGY
The word fermentation comes from the Latin verb fevere, which means to boil. It
originated from the fact that early at the start of wine fermentation gas bubbles are
released continuously to the surface giving the impression of boiling. It has three different
meanings which might be confusing.
Modern Industrial Microbiology and Biotechnology
The first meaning relates to microbial physiology. In strict physiological terms,
fermentation is defined in microbiology as the type of metabolism of a carbon source in
which energy is generated by substrate level phosphorylation and in which organic
molecules function as the final electron acceptor (or as acceptors of the reducing
equivalents) generated during the break-down of carbon-containing compounds or
catabolism. As is well-known, when the final acceptor is an inorganic compound the
process is called respiration. Respiration is referred to as aerobic if the final acceptor is
oxygen and anaerobic when it is some other inorganic compound outside oxygen e.g
sulphate or nitrate.
The second usage of the word is in industrial microbiology, where the term
‘fermentation’ is any process in which micro-organisms are grown on a large scale, even
if the final electron acceptor is not an organic compound (i.e. even if the growth is carried
out under aerobic conditions). Thus, the production of penicillin, and the growth of yeast
cells which are both highly aerobic, and the production of ethanol or alcoholic beverages
which are fermentations in the physiological sense, are all referred to as fermentations.
The third usage concerns food. A fermented food is one, the processing of which microorganisms play a major part. Microorganisms determine the nature of the food through
producing the flavor components as well deciding the general character of the food, but
microorganisms form only a small portion of the finished product by weight. Foods such
as cheese, bread, and yoghurt are fermented foods.
1.5
ORGANIZATIONAL SET-UP IN AN INDUSTRIAL
MICROBIOLOGY ESTABLISHMENT
The organization of a fermentation industrial establishment will vary from one firm to
another and will depend on what is being produced. Nevertheless the diagram in Fig. 1.1
represents in general terms the set-up in a fermentation industry.
The culture usually comes from the firm’s culture collection but may have been sourced
originally from a public culture collection and linked to a patent. On the other hand it
may have been isolated ab initio by the firm from soil, the air, the sea, or some other natural
body. The nutrients which go into the medium are compounded from various raw
materials, sometimes after appropriate preparation or modification including
saccharification as in the case of complex carbohydrates such as starch or cellulose. An
inoculum is first prepared usually from a lyophilized vial whose purity must be checked
on an agar plate. The organism is then grown in shake flasks of increasing volumes until
about 10% of the volume of the pilot fermentor is attained. It is then introduced into pilot
fermentor(s) before final transfer into the production fermentor(s) (Fig. 1.2).
The extraction of the material depends on what the end product is. The methods are
obviously different depending on whether the organism itself, or its metabolic product is
the desired commodity. If the product is the required material the procedure will be
dictated by its chemical nature. Quality control must be carried out regularly to ensure
that the right material is being produced. Sterility is important in industrial microbiology
processes and is maintained by various means, including the use of steam, filtration or by
chemicals. Air, water, and steam and other services must be supplied and appropriately
treated before use. The wastes generated in the industrial processes must also be disposed
Introduction: Scope of Biotechnology and Industrial Microbiology
Fig. 1.1 Set-up in an Industrial Microbiology Establishment
off. Packaging and sales are at the tail end, but are by no means the least important.
Indeed they are about the most important because they are the points of contact with the
consumer for whose satisfaction all the trouble was taken in the first instance. The items
in italics above are discussed in various succeeding chapters in this book.
Modern Industrial Microbiology and Biotechnology
Fig. 1.2 Flowchart of the Production Process in a Typical Industrial Microbiology Establishment
Introduction: Scope of Biotechnology and Industrial Microbiology
!
SUGGESTED READINGS
Anon. 1979. General Information Concerning Patents. United States Government Printing
Office. Washington, USA.
Anon. 1979. U.S. Patent 4,166,112 Goldberg, L.J., Mosquito larvae control using a bacterial
larvicide, Aug. 28, 1979.
Anon. 1980. Supreme Court of the United States. Diamond, (Commissioner of Patents and
Trademarks) v. Chakrabarty 447 US 303, 310, 206 USPQ 193, 197.
Anon. 1985. United States Patent Number 4,535,061 granted on August 13 1985 to Chakrabarty
et al.: Bacteria capable of dissimilation of environmentally persistent chemical compounds.
Washington, DC, USA.
Birch, R.G. 1997. Plant Transformation: Problems and Strategies for Practical Application Annual
Review of Plant Physiology and Plant Molecular Biology 48, 297-326.
Bull, A.T., Ward, A.C., Goodfellow, M. 2000. Search and Discovery Strategies For Biotechnology:.
The Paradigm Shift. Microbiology and Molecular Biology Reviews, 64, 573 - 548.
Dahod, S.K. 1999. Raw Materials Selection and Medium Development for Industrial
Fermentation Processes. In: Manual of Industrial Microbiology and Biotechnology. A.L.
Demain, J. E. Davies (eds) 2nd ed. American Society for Microbiology Press.
Doll, J.J. 1998. The patenting of DNA. Science 280, 689 -690.
Gordon, J. 1999. Intellectual Property. In: Manual of Industrial Microbiology and Biotechnology.
A.L. Demain, J.E. Davies (eds) 2nd ed. American Society for Microbiology Press.
Kimpel, J.A. 1999. Freedom to Operate: Intellectual Property Protection in Plant Biology And its
Implications for the Conduct of Research Annual Review of Phytopathology. 37, 29-51
Moran, K., King, S.R., Carlson, T.J. 2001. Biodiversity Prospecting: Lessons and Prospects. Annual
Review of Anthropology, 30, 505-526.
Neijseel, M.O., Tempest, D.W. 1979. In: Microbial Technology; Current State, Future Prospects.
A.T. Bull, D.C. Ellwood and C. Rattledge, (eds) Cambridge University Press, Cambridge, UK.
pp. 53-82.
Modern Industrial
Microbiology and Biotechnology
Section
*
Biological Basis of
Productivity in Industrial
Microbiology and Biotechnology
Modern Industrial
Microbiology and Biotechnology
+0)26-4
2
Some Microorganisms
Commonly Used in
Industrial Microbiology and
Biotechnology
2.1
BASIC NATURE OF CELLS OF LIVING THINGS
All living things are composed of cells, of which there are two basic types, the prokaryotic
cell and the eucaryotic cell. Figure 2.1 shows the main features of typical cells of the two
types. The parts of the cell are described briefly beginning from the outside.
Cell wall: Procaryotic cell walls contain glycopeptides; these are absent in eucaryotic
cells. Cell walls of eucaryotic cells contain chitin, cellulose and other sugar polymers.
These provide rigidity where cell walls are present.
Procaryotic cell (Bacillus sp)
Eucaryotic cell (Saccharomyces sp)
Fig. 2.1 Eucaryotic Cell (Yeast) and Procaryotic Cell (Bacillus)
&
Modern Industrial Microbiology and Biotechnology
Cell membrane: Composed of a double layer of phospholipids, the cell membrane
completely surrounds the cell. It is not a passive barrier, but enables the cell to actively
select the metabolites it wants to accumulate and to excrete waste products.
Ribosomes are the sites of protein synthesis. They consist of two sub-units. Procaryotic
ribosomes are 70S and have two sub-units: 30S (small) and a 50S (large) sub-units.
Eucaryotic ribosomes are 80S and have sub-units of 40S (small) and a 60S (large). (The
unit S means Svedberg units, a measure of the rate of sedimentation of a particle in an
ultracentrifuge, where the sedimentation rate is proportional to the size of the particle.
Svedberg units are not additive–two sub-units together can have Svedberg values that do
not add up to that of the entire ribosome). The prokaryotic 30S sub-unit is constructed from
a 16S RNA molecule and 21 polypeptide chains, while the 50S sub-unit is constructed
from two RNA molecules, 5S and 23S respectively and 34 polypeptide chains.
Mitochondria are membrane-enclosed structures where in aerobic eucaryotic cells the
processes of respiration and oxidative phosphorylation occur in energy release.
Procaryotic cells lack mitochondria and the processes of energy release take place in the
cell membrane.
Nuclear membrane surrounds the nucleus in eukaryotic cells, but is absent in procaryotic
cells. In procaryotic cells only one single circular macromolecule of DNA constitutes the
hereditary apparatus or genome. Eucaryotic cells have DNA spread in several
chromosomes.
Nucleolus is a structure within the eucaryotic nucleus for the synthesis of ribosomal RNA.
Ribosomal proteins synthesized in the cytoplasm are transported into the nucleolus and
combine with the ribosomal RNA to form the small and large sub-units of the eucaryotic
ribosome. They are then exported into the cytoplasm where they unite to form the intact
ribosome.
2.2
CLASSIFICATION OF LIVING THINGS: THREE
DOMAINS OF LIVING THINGS
The classification of living things has evolved over time. The earliest classification placed
living things into two simple categories, plants and animals. When the microscope was
discovered in about the middle of the 16th century it enabled the observation of
microorganisms for the first time. Living things were then divided into plants, animals
and protista (microorganisms) visible only with help of the microscope. This
classification subsisted from about 1866 to the 1960s. From the 1960s and the 1970s
Whittaker’s division of living things into five groups was the accepted grouping of living
things. The basis for the classification were cell-type: procaryotic or eucaryotic;
organizational level: single-celled or multi-cellular, and nutritional type: heterotrophy
and autotrophy. On the basis of these characteristics living things were divided by
Whitakker into five groups: Monera (bacteria), Protista (algae and protozoa), Plants,
Fungi, and Animals.
The current classification of living things is based on the work of Carl R Woese of the
University of Illinois. While earlier classifications were based to a large extent on
morphological characteristics and the cell type, with our greater knowledge of molecular
Some Microorganisms Commonly Used in Industrial Microbiology and Biotechnology
'
basis of cell function, today’s classification is based on the sequence of ribosomal RNA
(rRNA)in the 16S of the small sub-unit (SSU) of the procaryotic ribosome, and the 18S
ribosomal unit of eucaryotes. The logical question to ask is, why do we use the rRNA
sequence? It is used for the following reasons:
(i) 16S (or 18S) rRNA is essential to the ribosome, an important organelle found in all
living things (i.e. it is universally distributed);
(ii) its function is identical in all ribosomes;
(iii) its sequence changes very slowly with evolutionary time, and it contains variable
and stable sequences which enable the comparison of closely related as well as
distantly related species.
The classification is evolutionary and attempts to link all livings things with evolution
from a common ancestor. For this approach, an evolutionary time-keeper is necessary.
Such a time-keeper must be available to, or used by components of the system, and yet be
able to reflect differences and changes with time in other regions appropriate to the
assigned evolutionary distances. The 16S ribosomal RNAs meet these criteria as
ribosomes are involved in protein synthesis in all living things. They are also highly
conserved (remain the same) in many groups and some minor changes observed are
commensurate with expected evolutionary distances (Fig. 2.2).
Fig. 2.2
Diagram Illustrating Evolutionary Relationship between Organisms with Time
According to the currently accepted classification living things are placed into three
groups: Archae, Bacteria, and Eukarya. A diagram depicting the evolutionary
relationships among various groups of living things is giving in Fig. 2.3, while the
properties of the various groups are summarized in Table 2.1. Archae and Bacteria are
procaryotic while Eucarya are eucaryotic.
2.3
TAXONOMIC GROUPING OF MICRO-ORGANISMS
IMPORTANT IN INDUSTRIAL MICROBIOLOGY AND
BIOTECHNOLOGY
The microorganisms currently used in industrial microbiology and biotechnology are
found mainly among the bacteria and eukarya; the Archae are not used. However, as
discussed in Chapter 1, the processes used in industrial microbiology and biotechnology
are dynamic. Consequently, out-dated procedures are discarded as new and more efficient ones are discovered. At present organisms from Archae are not used for industrial
processes, but that may change in future. This idea need not be as far fetched as it may
Modern Industrial Microbiology and Biotechnology
Fig. 2.3
The Three Domains of Living Things Based on Woese’s Work
seem now. For as will be seen below, one of the criteria supporting the use of a microorganism for industrial purposes is the possession of properties which will enable the
organism to survive and be productive in the face of competition from contaminants.
Many organisms in Archae are able to grow under extreme conditions of temperature or
salinity and these conditions may be exploited in industrial processes where such physiological properties may put a member of the Archae at an advantage over contaminants.
Plants and animals as well as their cell cultures are also used in biotechnology, and
will be discussed in the appropriate sections below. Microorganisms have the following
advantages over plants or animals as inputs in biotechnology:
i. Microorganisms grow rapidly in comparison with plants and animals. The
generation time (the time for an organism to mature and reproduce) is about
12 years in man, about 24 months in cattle, 18 months in pigs, 6 months in chicken,
but only 15 minutes in the bacterium, E coli. The consequence is that
biotechnological products which can be obtained from microorganisms in a matter
of days may take many months in animals or plants.
ii. The space requirement for growth microorganisms is small. A 100,000 litre
fermentor can be housed in about 100 square yards of space, whereas the plants or
animals needed to generate the equivalent of products in the 100,000 fermentor
would require many acres of land.
iii. Microorganisms are not subject to the problems of the vicissitudes of weather
which may affect agricultural production especially among plants.
iv. Microorganisms are not affected by diseases of plants and animals, although they
do have their peculiar scourges in the form phages and contaminants, but there are
procedure to contain them.
Despite these advantages there are occasions when it is best to use either plants
or animals; in general however microorganisms are preferred for the reasons given
above.
Some Microorganisms Commonly Used in Industrial Microbiology and Biotechnology
Table 2.1
Summary of differences among the three domains of living things, (from Madigan
and Martimko, 2006)
S/No
Characteristic
Morphology and Genetics
1
Prokaryotic cell structure
2
DNA present in closed circular form
1
3
Histone proteins present
4
Nuclear membrane
5
Muramic acid in cell wall
6
Membrane lipids: Fatty acids or
Branched hydrocarbons
7
8
9
10
11
12
13
14
Ribosome size
Initiator tRNA
2
Introns in most genes
Operons
Plasmids
Ribosome sensitive to
diphtheria toxin
Sensitivity to streptomycin,
chloramphenicol, and kanamycin
4
Transcription factors required
3
Physiological/Special Structures
15
Methanogenesis
16
Nitrification
17
Denitrification
18
Nitrogen fixation
19
Chlorophyll based photosynthesis
20
Gas vesicles
21
Chemolithotrphy
22
Storage granules of poly-bhydroxyalkanoates
23
Growth above 80oC
24
Growth above 100oC
Bacteria
Archae
Eukarya
+
+
+
+
+
+
-
+
+
-
Fatty acids
Branched
hydrocarbons
70S
Methionine
Fatty acids
80S
Methionine
+
+
+
Rare
-
+
+
+
-
+
+
+
+
+
+
+
+
+
-?
+
+
+
+
+ (plants)
-
+
+
-
+
+
+
-
70S
Formylmethionine
+
+
1
Histone proteins are present in eucaryotic chromosomes; histones and DNA give structure to
chromosomes in eucaryotes. 2Non-coding sequences within genes; 3Operons: Typically present in
prokaryotes, these are clusters of genes controlled by a single operator; 4Transcription factor is a protein
that binds DNA at a specific promoter or enhancer region or site, where it regulates transcription.
2.3.1
Bacteria
Bacteria are described in two compendia, Bergey’s Manual of Determinative Bacteriology
and Bergey’s Manual of Systematic Bacteriology. The first manual (on Determinative
Bacteriology) is designed to facilitate the identification of a bacterium whose identity is
Modern Industrial Microbiology and Biotechnology
unknown. It was first published in 1923 and the current edition, published in 1994 is the
ninth. The companion volume (on Systematic Bacteriology) records the accepted published
descriptions of bacteria, and classifies them into taxonomic groups. The first edition was
produced in four volumes and published between 1984 and 1989. The bacterial
classification in the latest (second) edition of Bergey’s Manualof Sytematic Bacteriology is
based on 16S RNA sequences, following the work of Carl Woese, and organizes the
Domain Bacteria into 18 groups (or phyla; singular, phylum) It is to be published in five
volumes. Volume 1 which deals with the Archae and the deeply branching and
phototrophic bacteria was published in 2001; Volume 2 published in 2005, deals with the
Proteobacteria and has three parts while Volume 3 was published in 2006 and deals with
the low G+C Gram-positive bacteria. The last two volumes, Volume 4 (the high C + C
Gram-positive bacteria) and Volume 5 (The Plenctomyces, Spirochaetes, Fibrobacteres,
Bacteriodetes and Fusobacteria) will be published in 2007. The manuals are named after Dr
D H Bergey who was the first Chairman of the Board set up by the then Society of
American Bacteriologists (now American Society for Microbiology) to publish the books.
The publication of Bergey Manuals is now managed by the Bergey’s Manual Trust.
Of the 18 phyla in the bacteria, (see Fig. 2.4) the Aquiflex is evolutionarily the most
primitive, while the most advanced is the Proteobacteria. The bacterial phyla used in
industrial microbiology and biotechnology are found in the Proteobacteria, the
Firmicutes and the Actinobacteria.
Green sulfur
bacteria
Deinococci
Green non-sulfur
bacteria
Spirochetes
Deferribacter
Flavobacteria
Plectomyces/
Pirella
Cytophaga
Verucomicrobia
Thermotoga
Chlamydia
Cyanobacteria
Thermodesulfo
bacterium
Aquifex
Actinobacteria
Gram-positive bacteria
Nitrospira
e – Proteobacteria
d –Proteobacteria
a – Proteobacteria
b – Proteobacteria
g – Proteobacteria
Fig. 2.4 The 18 Phyla of Bacteria Based on 16S RNA Sequences (After Madigan and Matinko, 2006)
Some Microorganisms Commonly Used in Industrial Microbiology and Biotechnology
2.3.1.1
!
The Proteobacteria
The Proteobacteria are a major group of bacteria. Due to the diversity of types of bacteria
in the group, it is named after Proteus, the Greek god, who could change his shape.
Proteobacteria include a wide variety of pathogens, such as Escherichia, Salmonella, Vibrio
and Helicobacter, as well as free-living bacteria some of which can fix nitrogen. The group
also includes the purple bacteria, so-called because of their reddish pigmentation, and
which use energy from sun light in photosynthesis.
All Proteobacteria are Gram-negative, with an outer membrane mainly composed of
lipopolysaccharides. Many move about using flagella, but some are non-motile or rely on
bacterial gliding. There is also a wide variety in the types of metabolism. Most members
are facultatively or obligately anaerobic and heterotrophic, but there are numerous
exceptions.
Proteobacteria are divided into five groups: a (alpha), b (beta), g (gamma), d (delta), e
(epsilon). The only organisms of current industrial importance in the Proteobacteria are
Acetobacter and Gluconobacter, which are acetic acid bacteria and belong to the
Alphaproteobacteria. An organism also belonging to the Alphaproteobacteria, and
which has the potential to become important industrially is Zymomonas. It produces
copious amounts of alcohol, but its use industrially is not yet widespread.
2.3.1.1.1 The Acetic Acid Bacteria
The acetic acid bacteria are Acetobacter (peritrichously flagellated) and Gluconobacter
(polarly flagellated). They have the following properties:
i. They carry out incomplete oxidation of alcohol leading to the production of acetic
acid, and are used in the manufacture of vinegar (Chapter 14).
ii. Gluconobacter lacks the complete citric acid cycle and can not oxidize acetic acid;
Acetobacter on the on the other hand, has all the citric acid enzymes and can oxidize
acetic acid further to CO2.
iii. They stand acid conditions of pH 5.0 or lower.
iv. Their property of ‘under-oxidizing’ sugars is exploited in the following:
a. The production of glucoronic acid from glucose, galactonic aicd from
galactose and arabonic acid from arabinose;
b. The production of sorbose from sorbitol by acetic acid bacteria (Fig. 2.4),
an important stage in the manufacture of ascorbic acid (also known as
Vitamin C)
v. Acetic acid bacteria are able to produce pure cellulose when grown in an unshaken
culture. This is yet to be exploited industrially, but the need for cellulose of the
purity of the bacterial product may arise one day.
2.3.1.2 The Firmicutes
The Firmicutes are a division of bacteria, all of which are Gram-positive, in contrast to the
Proteobacteria which are all Gram-negative. A few, the mycoplasmas, lack cell walls
altogether and so do not respond to Gram staining, but still lack the second membrane
found in other Gram-negative forms; consequently they are regarded as Gram-positive.
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Modern Industrial Microbiology and Biotechnology
CH2OH
CH2OH
CO
Acetobacter
suboxydans
CH2OH
CH2OH
D-sorbitol
L-sorbose
Fig. 2.5 Conversion of Sorbitol to Sorbose
Originally the Firmicutes were taken to include all Gram-positive bacteria, but more
recently they tend to be restricted to a core group of related forms, called the low G+C
group in contrast to the Actinobacteria, which have high G+C ratios. The G+C ratio is an
important taxonomic characteristic used in classifying bacteria. It is the ratio of Guanine
and Cytosine to Guanine, Cytosine, Adenine, and Thymine in the cell. Thus the GC ratio
= G+C divided by G+C+A+T x 100. It is used to classify Gram-positive bacteria: low G+C
Gram-positive bacteria (ie those with G+C less than 50%) are placed in the Fermicutes,
while those with 50% or more are in Actinobacteria. Fermicutes contain many bacteria of
industrial importance and are divided into three major groups: i. spore-forming, ii. nonspore forming, and iii) wall-less (this group contains pathogens and no industrial
organisms.)
2.3.1.2.1 Spore forming firmicutes
Spore-forming Firmicutes form internal spores, unlike the Actinobacteria where the
spore-forming members produce external ones. The group is divided into two: Bacillus
spp, which are aerobic and Clostridium spp which are anaerobic. Bacillus spp are
sometimes used in enzyme production. Some species are well liked by mankind because
of their ability to kill insects. Bacillus papilliae infects and kills the larvae of the beetles in
the family Scarabaeidae while B. thuringiensis is used against mosquitoes (Chapter 17). The
genes for the toxin produced by B. thuringiensis are also being engineered into plants to
make them resistant to insect pests (Chapter 7). Clostridia on the other hand are mainly
pathogens of humans and animals.
2.3.1.2.2 Non-spore forming firmicutes
The Lactic Acid Bacteria: The non-spore forming low G+C members of the firmicutes
group are very important in industry as they contain the lactic acid bacteria.
The lactic acid bacteria are rods or cocci placed in the following genera: Enterococcus,
Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Streptococcus and are among some of
the most widely studied bacteria because of their important in the production of some
foods, and industrial and pharmaceutical products. They lack porphyrins and
cytochromes, do not carry out electron transport phosphorylation and hence obtain
Some Microorganisms Commonly Used in Industrial Microbiology and Biotechnology
#
energy by substrate level phosphorylation. They grow anaerobically but are not killed by
oxygen as is the case with many anaerobes: they will grow with or without oxygen. They
obtain their energy from sugars and are found in environments where sugar is present.
They have limited synthetic ability and hence are fastidious, requiring, when cultivated,
the addition of amino acids, vitamins and nucleotides.
Lactic acid bacteria are divided into two major groups: The homofermentative group,
which produce lactic acid as the sole product of the fermentation of sugars, and the
heterofermentative, which besides lactic acid also produce ethanol, as well as CO2. The
difference between the two is as a result of the absence of the enzyme aldolase in the
heterofermenters. Aldolase is a key enzyme in the E-M-P pathway and spits hexose
glucose into three-sugar moieties. Homofermentative lactic acid bacteria convert the Dglyceraldehyde 3-phosphate to lactic acid. Heterofermentative lactic acid bacteria receive
five-carbon xylulose 5 phosphate from the Pentose pathway. The five carbon xylulose is
split into glyceraldehyde 3-phosphate (3-carbon), which leads to lactic acid, and the twocarbon acetyl phosphate which leads to ethanol (Fig. 2.6).
Fig. 2.6
Splitting of 6-carbon Glucose into Three-carbon Compounds by the Enzyme
Fructose Diphposphate Aldolase
Use of Lactic Acid Bacteria for Industrial Purposes:
The desirable characteristics of lactic acid bacteria as industrial microorganisms include
a.
b.
c.
d.
e.
their ability to rapidly and completely ferment cheap raw materials,
their minimal requirement of nitrogenous substances,
they produce high yields of the much preferred stereo specific lactic acid
ability to grow under conditions of low pH and high temperature, and
ability to produce low amounts of cell mass as well as negligible amounts of other
byproducts.
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Modern Industrial Microbiology and Biotechnology
The choice of a particular lactic acid bacterium for production primarily depends on
the carbohydrate to be fermented. Lactobacillus delbreuckii subspecies delbreuckii is able to
ferment sucrose. Lactobacillus delbreuckii subspecies bulgaricus is able to use lactose while
Lactobacillus helveticus is able to use both lactose and galactose. Lactobacillus amylophylus
and Lactobacillus amylovirus are able to ferment starch. Lactobacillus lactis can ferment
glucose, sucrose and galactose and Lactobacillus pentosus has been used to ferment sulfite
waste liquor.
2.3.1.3
The Actinobacteria
The Acinobacteria are the Firmicutes with G+C content of 50% or higher. They derive
their name from the fact that many members of the group have the tendency to form
filaments or hyphae (actinis, Greek for ray or beam). The industrially important members
Table 2.2
Characteristics of the lactic acid bacteria
S/No Group
Description
Habit
Importance
Some in respiratory
tract, mouth, intestine;
others found in
fermenting
vegetable and silage
Found as commensals
in the human alimentary
canal; sometimes cause
urinary tract infections
Some cause sore
throat; nonpathogenic strains
used in yoghurt
manufacture
Can be used to
monitor water
quality, (like E. coli)
1
Streptococcus Cocci in pairs or
short chains
2
Enterococcus Cocco-bacilli
usually in pairs;
previously classified
Streptococcus
Lancefield Group D
Lactococcus Coccoid, usually occuring Plant material and
in pairs; hardly form
alimentary canals of
chains
animals
3
4
Pediococcus
Growth in tetrads
5
Leuconostoc
Cocco-bacili
Used as starter in
yoghurt manufacture;
Used as probiotic for
intestinal health;
Produces copious
amounts of lactic
acid.
Found on plant materials Spoils beer; but
required in special
beers such as lambic
beer drunk in parts
of Belgium
Associated with plant
Tolerates high conmaterials
centrations of salt
and sugar and
involved in the
pickling of
vegetables; produce
dextrans from
sucrose
Some Microorganisms Commonly Used in Industrial Microbiology and Biotechnology
%
Fig. 2.7 Formation of lacttic acid by homofermentative bacteria
Table 2.3 Distinguishing characteristics of lactic acid bacteria
Character
Lactobacillus Enterococcus Lactocococcus Leuconostoc Pediococcus Streptococcus
Tetrad formation
–
CO2 from glucose
±
Growth at 10°C
±
Growth at 45°C
±
Growth at 6.5% NaCl ±
Growth at pH 4.4
±
Growth at pH 9.6
–
Lactic acid (optical
orientation)
D, L, DL
–
–
+
+
+
+
+
–
–
+
–
–
±
–
–
+
+
–
±
±
–
+
–
±
±
±
+
–
–
–
–
±
–
–
–
L
L
D
L, DL
L
of the group are the Actinomycetes and Corynebacterium. Corynebacterium spp are
important industrially as secreters of amino acids (Chapter 21). The rest of this section
will be devoted to Actinomycetes.
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Modern Industrial Microbiology and Biotechnology
Enzymes involved: 1, Hexokinase; 2, Glucose-6-phosphate dehydrogenase; 3, 6-phosphogluconate
dehydrogenase; 4, Ribulose-5-phosphate 3-epimerase; Phosphoketolase; 6, Phosphotransacetylase; 7,
Acetaldehyde dehydrogenase; 8, Alcohol dehydrogense; 9, Enzymes of the homofermentative pathway
Fig. 2.8
Fermentation of Glucose by Heterofermentative Bacteria
Some Microorganisms Commonly Used in Industrial Microbiology and Biotechnology
Lactobacillus bulgaricus
'
Lactococcus lactis
Colour
Fig. 2.9 Photomicrographs of Lactic Acid Bacteria
2.3.1.3.1 The Actinomycetes
They have branching filamentous hyphae, which somewhat resemble the mycelia of the
fungi, among which they were originally classified. In fact they are unrelated to fungi, but
are regarded as bacteria for the following reasons. First they have petidoglycan in their
cell walls, and second they are about 1.0m in diameter (never more than 1.5m), whereas
fungi are at least twice that size in diameter.
As a group the actinomycetes are unsurpassed in their ability to produce secondary
metabolites which are of industrial importance, especially as pharmaceuticals. The best
known genus is Streptomyces, from which many antibiotics as well as non-anti-microbial
drugs have been obtained. The actinomycetes are primarily soil dwellers hence the
temptation to begin the search for any bioactive microbial metabolite from soil.
2.3.2
Eucarya: Fungi
Although plants and animals or their cell cultures are used in biotechnology,
microorganisms are used more often for reason which have been discussed. Fungi are
members of the Eucarya which are commonly used in industrial production.
The fungi are traditionally classified into the four groups given in Table 2.4, namely
Phycomycetes, Ascomycetes, Fungi Imprfecti, and Basidiomycetes. Among these the
following are those currently used in industrial microbiology
Phycomycetes (Zygomycetes)
Rhizopus and Mucor are used for producing various enzymes
Ascomycetes
Yeasts are used for the production of ethanol and alcoholic beverages
Claviceps purperea is used for the production of the ergot alkaloids
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Modern Industrial Microbiology and Biotechnology
Actinomyces
Actinoplanales
Micromonospora
Nocardia
Streptomyces
Saccharomonospora
Thermoactinomyces
Thermomonospora
Fig. 2.10 Different Actinomycetes
Fungi Imperfecti
Aspergillus is important because it produces the food toxin, aflatoxin, while Penicillium is
well-known for the antibiotic penicillin which it produces.
Some Microorganisms Commonly Used in Industrial Microbiology and Biotechnology
!
Basidiomycetes
Agaricus produces the edible fruiting body or mushroom
Numerous useful products are made through the activity of fungi, but the above are
only a selection.
Table 2.4
Description of the various groups of fungi
Group
Ordinary
Name
Septation
of hyphae
Sexual Spores
Representative
Zygomycetes
(Phycomycetes)
Ascomycetes
Bread molds
Non-septate
Zygospre
Rhizopus, Mucor
Sac fungi
Septate
Ascospore
(in Perithecia)
Basidiomycetes
Mushrooms
Septate
Deuteromycetes
Fungi imperfecti
Septate
Basidiomycetes
(Mushrooms)
None
Neurospora,
Saccharomyces
(Yeasts)
Agaricus
2.4
Penicillium,
Aspergillus
CHARACTERISTICS IMPORTANT IN MICROBES
USED IN INDUSTRIAL MICROBIOLOGY AND
BIOTECHNOLGY
Microorganisms which are used for industrial production must meet certain
requirements including those to be discussed below. It is important that these
characteristics be borne in mind when considering the candidacy of any microorganism
as an input in an industrial process.
i. The organism must be able to grow in a simple medium and should preferably not
require growth factors (i.e. pre-formed vitamins, nucleotides, and acids) outside
those which may be present in the industrial medium in which it is grown. It is
obvious that extraneous additional growth factors may increase the cost of the
fermentation and hence that of the finished product.
ii. The organism should be able to grow vigorously and rapidly in the medium in use.
A slow growing organism no matter how efficient it is, in terms of the production of
the target material, could be a liability. In the first place the slow rate of growth
exposes it, in comparison to other equally effective producers which are faster
growers, to a greater risk of contamination. Second, the rate of the turnover of the
production of the desired material is lower in a slower growing organism and
hence capital and personnel are tied up for longer periods, with consequent lower
profits.
iii. Not only should the organism grow rapidly, but it should also produce the desired
materials, whether they be cells or metabolic products, in as short a time as
possible, for reasons given above.
iv. Its end products should not include toxic and other undesirable materials,
especially if these end products are for internal consumption.
!
Modern Industrial Microbiology and Biotechnology
Rhizopus
Aspergillus
Perithecium with
Asci and ascospores
Penicillium
Yeast s with 8
Ascospores
Conidia
Sporangium
Sporophore
Mucor
Mucor
(Showing Zygospore)
Fig. 2.11
Basidiomycete Fruiting Bodies
(Mushrooms)
Representative Structures from Different Fungi
v. The organism should have a reasonable genetic, and hence physiological stability.
An organism which mutates easily is an expensive risk. It could produce
undesired products if a mutation occurred unobserved. The result could be
reduced yield of the expected material, production of an entirely different product
or indeed a toxic material. None of these situations is a help towards achieving the
goal of the industry, which is the maximization of profits through the production
of goods with predictable properties to which the consumer is accustomed.
vi. The organism should lend itself to a suitable method of product harvest at the end
of the fermentation. If for example a yeast and a bacterium were equally suitable for
manufacturing a certain product, it would be better to use the yeast if the most
appropriate recovery method was centrifugation. This is because while the
Some Microorganisms Commonly Used in Industrial Microbiology and Biotechnology
vii.
viii.
ix.
x.
!!
bacterial diameter is approximately 1m, yeasts are approximately 5m. Assuming
their densities are the same, yeasts would sediment 25 times more rapidly than
bacteria. The faster sedimentation would result in less expenditure in terms of
power, personnel supervision etc which could translate to higher profit.
Wherever possible, organisms which have physiological requirements which
protect them against competition from contaminants should be used. An organism
with optimum productivity at high temperatures, low pH values or which is able to
elaborate agents inhibitory to competitors has a decided advantage over others.
Thus a thermophilic efficient producer would be preferred to a mesophilic one.
The organism should be reasonably resistant to predators such as Bdellovibrio spp
or bacteriophages. It should therefore be part of the fundamental research of an
industrial establishment using a phage-susceptible organism to attempt to
produce phage-resistant but high yielding strains of the organism.
Where practicable the organism should not be too highly demanding of oxygen as
aeration (through greater power demand for agitation of the fermentor impellers,
forced air injection etc) contributes about 20% of the cost of the finished product.
Lastly, the organism should be fairly easily amenable to genetic manipulation to
enable the establishment of strains with more acceptable properties.
SUGGESTED READINGS
Asai, T. 1968. The Acetic Acid Bacteria. Tokyo: The University of Tokyo Press and Baltimore:
University Park Press.
Axelssson, L., Ahrne, S. 2000. Lactic Acid Bacteria. In: Applied Microbial Systematics, F.G. Priest,
M. Goodfellow, (eds) A.H. Dordrecht, the Netherlands, pp. 367-388.
Barnett, J.A. , Payne, R.W., Yarrow, D. 2000. Yeasts: Characterization and Identification. 3rd
Edition. Cambridge University Press. Cambridge, UK.
Garrity, G.M. 2001-2006. Bergey’s Manual of Systematic Bacteriology. 2nd Ed. Springer, New
York, USA.
Goodfellow, M., Mordaraski, M., Williams, S.T. 1984. The Biology of the Actinomycetes.
Academic Press, London, UK.
Madigan, M., Martimko, J.M. 2006. Brock Biology of Microorganisms. Upper Saddle River:
Pearson Prentice Hall. 11th Edition.
Major, A. 1975. Mushrooms Toadstools and Fungi: Arco New York, USA.
Narayanan, N., Pradip, K. Roychoudhury, P.K., Srivastava, A. 2004. L (+) lactic acid fermentation
and its product polymerization. Electronic Journal of Biotechnology 7, Electronic Journal of
Biotechnology [online]. 15 August 2004, 7, (3) [cited 23 March 2006]. Available from: http://
www.ejbiotechnology.info/content/vol2/issue3/full/3/index.html. ISSN 0717-3458.
Samson, R., Pitt, J.I. 1989. Modern Concepts in Penicillium and Aspergillus Classification. Plenum
Press New York and London.
Woese, C.R. 2002. On the evolution of cells Proceedings of the National Academy of Sciences of
the United States of America 99, 8742-8747.
!"
Modern Industrial Microbiology and Biotechnology
+0)26-4
3
Aspects of Molecular Biology
and Bioinformatics of Relevance
in Industrial Microbiology and
Biotechnology
In recent times giant strides have been taken in harnessing our knowledge of the
molecular basis of many biological phenomena. Many new techniques such as the
polymerase chain reaction (PCR) and DNA sequencing have arrived on the scene. In
addition major projects involving many countries such as the human genome project
have taken place. Coupled with all these exciting technological developments, new
vocabulary such as genomics has arisen. All this has transformed the approaches used
in industrial microbiology. New approaches anchored on developments in molecular
biology have been followed in many industrial microbiology processes and products
such as vaccines, the search for new antibiotics, and the physiology of microorganisms.
It therefore now appears imperative that any discussion of industrial microbiology and
biotechnology must take these developments into account. This chapter will discuss only
selected aspects of molecular biology in order to provide a background for understanding
some of the newer directions of industrial microbiology and biotechnology. The
discussion will be kept as simplified and as brief as possible, just enough in complexity
and length needed to achieve the purpose of the chapter. The student is encouraged to
look at many excellent texts in this field. In addition a glossary of some terms used in
molecular biology is included at the end of the book.
3.1
PROTEIN SYNTHESIS
Proteins are very important in the metabolism of living things. They are in hormones for
transporting messages around the body; they are used as storage such as in the whites of
eggs of birds and reptiles and in seeds; they transport oxygen in the form of hemoglobin;
they are involved in contractile arrangements which enable movement of various body
parts, in contractile proteins in muscles; they protect the animal body in the form of
antibodies; they are in membranes where they act as receptors, participate in membrane
transport and antigens and they form toxins such as diphtheria and botulism. The most
important function if it can be so termed is that form the basis of enzymes which catalyze
Aspects of Molecular Biology and Bioinformatics of Relevance in Industrial
!#
all the metabolic activities of living things; in short proteins and the enzymes formed from
them are the major engines of life.
In spite of the incredible diversity of living things, varying from bacteria to protozoa to
algae to maize to man, the same 20 amino acids are found in all living things. On account
of this, the principles affecting proteins and their structure and synthesis are same in all
living things.
The genetic macromolecules (i.e. the macromolecules intimately linked to heredity) are
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The genetic information
which determines the potential properties of a living thing is carried in the DNA present
in the nucleus, except in some viruses where it is carried in RNA. DNA is also present in
the organelles mitochondria and chloroplasts. (Just an interesting fact about mitochondrial
DNA. Individuals inherit the other kinds of genes and DNA from both parents jointly.
However, eggs destroy the mitochondria of the sperm that fertilize them. On account of
this, the mitochondrial DNA of an individual comes exclusively from the mother. Due to
the unique matrilineal transmission of mitochondrial DNA, data from mitochondrial
DNA sequences is used in the study of genelogy and sometimes for forensic purposes).
DNA consists of four nucleotides, adenine, cytosine, guanine and thymine. RNA is
very similar except that uracil replaces thymine (Fig. 3.1). RNA occurs in the nucleus and
in the cytoplasm as well as in the ribosomes.
Fig. 3.1 The Nucleic Acid Bases
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Modern Industrial Microbiology and Biotechnology
The processes of protein synthesis will be summarized briefly below. In protein
synthesis, information flow is from DNA to RNA via the process of transcription, and
thence to protein via translation. Transcription is the making of an RNA molecule from a
DNA template. Translation is the construction of a polypeptide from an amino acid
sequence from an RNA molecule (Fig. 3.2). The only exception to this is in retroviruses
where reverse transcription occurs and where a single-stranded DNA is transcribed from
a single-stranded RNA (the reverse of transcription); it is used by retroviruses, which
includes the HIV/AIDS virus, as well as in biotechnology.
Transcription
An enzyme, RNA polymerase, opens the part of the DNA to be transcribed. Only one
strand of DNA, the template or sense strand, is transcribed into RNA. The other strand,
Fig. 3.2
Transfer RNA Transferring Amino Acids to mRNA in Protein Synthesis
Aspects of Molecular Biology and Bioinformatics of Relevance in Industrial
!%
the anti-sense strand is not transcribed. The anti-sense strand is used in making ripe
tomatoes to remain hard. The RNA transcribed from the DNA is the messenger or mRNA
(Fig. 3.3). As some students appear to be confused by the various types of RNA, it is
important that we mention at this stage that there are two other types of RNA besides
mRNA. These are ribosomal or rRNA and transfer or tRNA; they will be discussed later
in this chapter. At this stage it is suffice to mention that in the analogy of a building,
messenger RNA, mRNA is the blueprint or plan for construction of a protein (building);
ribosomal RNA rRNA the construction site (plot of land) where the protein is made,
while transfer RNA, tRNA, is the vehicle delivering the proper amino acid (building
blocks) to the (building) site at the right time.
Fig. 3.3
Summary of Protein Synthesis Activities
When mRNA is formed, it leaves the nucleus in eukaryotes (there is no nucleus in
prokaryotes!) and moves to the ribosomes.
Translation
In all cells, ribosomes are the organelles where proteins are synthesized. They consist of
two-thirds of ribosomal RNA, rRNA, and one-third protein. Ribosomes consist of two
sub-units, a smaller sub-unit and a larger sub-unit. In prokaryotes, typified by E. coli , the
smaller unit is 30S and larger 50S. S is Svedberg units, the unit of weights determined
from ultra centrifuge readings. The 30S unit has 16S rRNA and 21 different proteins. The
50S sub-unit consists of 5S and 23S rRNA and 34 different proteins. The smaller sub-unit
has a binding site for the mRNA. The larger sub-unit has two binding sites for tRNA.
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Modern Industrial Microbiology and Biotechnology
The messenger RNA (mRNA) is the ‘blueprint’ for protein synthesis and is transcribed
from one strand of the DNA of the gene; it is translated at the ribosome into a polypeptide
sequence. Translation is the synthesis of protein from amino acids on a template of
messenger RNA in association with a ribosome. The bases on mRNA code for amino
acids in triplets or codons; that is three bases code for an amino acid. Sometimes different
triplet bases may code for the same amino acid. Thus the amino acid glycine is coded for
by four different codons: GGU, GGC, GGA, and GGG. However, a codon usually codes
for one amino acid. There are 64 different codons; three of these UAA, UAG, and UGA are
stop codons and stop the process of translation. The remaining 61 code for the amino
acids in proteins. (Table 3.1). Translation of the message generally begins at AUG, which
also codes for methionine. For AUG to act as a start codon it must be preceded by a
ribosome binding site. If that is not the case it simply codes for methionine.
Promoters are sequences of DNA that are the start signals for the transcription of
mRNA. Terminators are the stop signals. mRNA molecules are long (500-10,000
nucleotides).
Ribosomes are the sites of translation. The ribosomes move along the mRNA and bring
together the amino acids for joining into proteins by enzymes.
Table 3.1
The genetic code – codons
U
C
A
G
U
UUU = Phe
UUC = Phe
UUA = Leu
UUG = Leu
UCU = Ser
UCC = Ser
UCA = Ser
UCG = Ser
UAU = Tyr
UAC = Tyr
UAA = Stop
UGA = Stop
UGU = Cys
UGC = Cys
UAG = Stop
UGG = Trp
U
C
A
G
C
CUU = Leu
CUC = Leu
CUA = Leu
CUG = Leu
CCU = Pro
CCC = Pro
CCA = Pro
CCG = Pro
CAU = His
CAC = His
CAA = Gln
CAG = Gln
CGU = Arg
CGC = Arg
CGA = Arg
CGG = Arg
U
C
A
G
A
AUU = Ile
AUC = Ile
AUA = Ile
AUG = Met
ACU = Thr
ACC = Thr
ACA = Thr
ACG = Thr
AAU = Asn
AAC = Asn
AAA = Lys
AAG = Lys
AGU = Ser
AGC = Ser
AGA = Arg
AGG = Arg
U
C
A
G
G
GUU = Val
CUC = Val
GUA = Val
GUG = Val
GCU = Ala
GCC = Ala
GCA = Ala
GCG = Ala
GAU = Asp
GAC = Asp
GAA = Glu
GAG = Glu
GGU = Gly
GCG = Gly
GGA = Gly
GGG = Gly
U
C
A
G
AUG = start codon
UAA, UAG, and UGA = stop (nonsense) codons
Phe
Leu
Ile
Met
Val
= phenylalanine
= leucine
= isoleucine
= methionine
= valine
Ser
Pro
Thr
Ala
Tyr
Amino Acids
= serine
His = histidine
= proline
Gln = glutamine
= threonine
Asn = asparagine
= alanine
Lys = lysine
= tyrosine
Asp = aspartic acid
Glu
Cys
Trp
Arg
Gly
= glutamic acid
= cysteine
= tryptophan
= arginine
= glycine
Aspects of Molecular Biology and Bioinformatics of Relevance in Industrial
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Transfer RNAs (tRNAs) carry amino acids to mRNA for linking and elongation into
proteins. Transfer RNA is basically cloverleaf-shaped. (see Fig. 3.2) tRNA carries the
proper amino acid to the ribosome when the codons call for them. At the top of the large
loop are three bases, the anticodon, which is the complement of the codon. There are 61
different tRNAs, each having a different binding site for the amino acid and a different
anticodon. For the codon UUU, the complementary anticodon is AAA. Amino acid
linkage to the proper tRNA is controlled by the aminoacyl-tRNA synthetases. Energy for
binding the amino acid to tRNA comes from ATP conversion to adenosine
monophosphate (AMP).
Elongation terminates when the ribosome reaches a stop codon, which does not code
for an amino acid and hence not recognized by tRNA.
After protein has been synthesized, the primary protein chain undergoes folding:
secondary, tertiary and quadruple folding occurs. The folding exposes chemical groups
which confer their peculiar properties to the protein.
Protein folding (to give a three-dimensional structure) is the process by which a protein
assumes its functional shape or conformation. All protein molecules are simple
unbranched chains of amino acids, but it is by coiling into a specific three-dimensional
shape that they are able to perform their biological function. The three-dimensional
shape (3D) conformation of a protein is of utmost importance in determining the
properties and functions of the protein. Depending on how a protein is folded different
functional groups may be exposed and these exposed group influence its properties.
The reverse of the folding process is protein denaturation, whereby a native protein is
caused to lose its functional conformation, and become an amorphous, and nonfunctional amino acid chain. Denatured proteins may lose their solubility, and
precipitate, becoming insoluble solids. In some cases, denaturation is reversible, and
proteins may refold. In many other cases, however, denaturation is irreversible.
Denaturation occurs when a protein is subjected to unfavorable conditions, such as
unfavorable temperature or pH. Many proteins fold spontaneously during or after their
synthesis inside cells, but the folding depends on the characteristics of their surrounding
solution, including the identity of the primary solvent (either water or lipid inside the
cells), the concentration of salts, the temperature, and molecular chaperones. Incorrect
folding sometimes occurs and is responsible for prion related illness such as CreutzfeldtJakob disease and Bovine spongiform encephalopathy (mad cow disease), and amyloid
related illnesses such as Alzheimer’s Disease. When enzyme molecules are misfolded
they will not function.
3.2
THE POLYMERASE CHAIN REACTION
The Polymerase Chain Reaction (PCR) is a technology used to amplify small amounts of
DNA. The PCR technique was invented in 1985 by Kary B. Mullis while working as a
chemist at the Cetus Corporation, a biotechnology firm in Emeryville, California. So
useful is this technology that Muillis won the Nobel Prize for its discovery in 1993, eight
years later. It has found extensive use in a wide range of situations, from the medical
diagnosis to microbial systematics and from courts of law to the study of animal
behavior.
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The requirements for PCR are:
a.
b.
c.
d.
The DNA or RNA to be amplified
Two primers
The four nucleotides found in the nucleic acid,
A heat stable a thermostable DNA polymerase derived from the thermophilic
bacterium, Thermus aquaticus, Taq polymerase
The Primer: A primer is a short segment of nucleotides which is complementary to a
section of the DNA which is to be amplified in the PCR reaction.
Primers are anneal to the denatured DNA template to provide an initiation site for the
elongation of the new DNA molecule. For PCR, primers must be duplicates of nucleotide
sequences on either side of the piece of DNA of interest, which means that the exact order
of the primers’ nucleotides must already be known. These flanking sequences can be
constructed in the laboratory or purchased from commercial suppliers.
The Procedure: There are three major steps in a PCR, which are repeated for 30 or 40 cycles.
This is done on an automated cycler, which can heat and cool the tubes with the reaction
mixture at specific intervals.
a. Denaturation at 94°C
The unknown DNA is heated to about 94°C, which causes the DNA to denature and the
paired strands to separate.
b. Annealing at 54°C
A large excess of primers relative to the amount of DNA being amplified is added and the
reaction mixture cooled to allow double-strands to anneal; because of the large excess of
primers, the DNA single strands will bind more to the primers, instead of with each other.
Fig. 3.4
Primer-Template Annealing
c. Extension at 72°C
This is the ideal working temperature for the polymerase. Primers that are on positions
with no exact match, get loose again (because of the higher temperature) and donot give
an extension of the fragment. The bases (complementary to the template) are coupled to
the primer on the 3' side (the polymerase adds dNTP’s from 5' to 3', reading the template
from 3' to 5' side, bases are added complementary to the template).
d. The Amplification: The process of the amplification is shown in Fig. 3.3.
Aspects of Molecular Biology and Bioinformatics of Relevance in Industrial
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3.2.1 Some Applications of PCR in Industrial Microbiology
and Biotechnology
PCR is extremely efficient and simple to perform. It is useful in biotechnology in the
following areas:
(a) to generate large amounts of DNA for genetic engineering, or for sequencing, once
the flanking sequences of the gene or DNA sequence of interest is known;
(b) to determine with great certainty the identity of an organism to be used in a
biotechnological production, as may be the case when some members of a group of
organisms may include some which are undesirable. A good example would be
among the acetic acid bacteria where Acetobacter xylinum would produce slime
rather acetic acid which Acetobacter aceti produces.
Fig. 3.5
Diagrammatic Representation of PCR
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(c) PCR can be used to determine rapidly which organism is the cause of
contamination in a production process so as to eliminate its cause, provided the
primers appropriate to the contaminant is available.
3.3
MICROARRAYS
The availability of complete genomes from many organisms is a major achievement of
biology. Aside from the human genome, the complete genomes of many microorganisms
have been completed and are now available at the website of The Institue for Genomic
Research (TIGR), a nonprofit organization located in Rockville, MD with its website at
www.tigr.org. At the time of writing, TIGR had the complete genome of 294
microorganisms on its website (268 bacteria, 23 Archae, and 3 viruses). The major
challenge is now to decipher the biological function and regulation of the sequenced
genes. One technology important in studying functional microbial genomics is the use of
DNA Microarrays.
Microarrays are microscopic arrays of large sets of DNA sequences that have been
attached to a solid substrate using automated equipment. These arrays are also referred
to as microchips, biochips, DNA chips, and gene chips. It is best to refer to them as
microarrays so as to avoid confusing them with computer chips.
DNA microarrays are small, solid supports onto which the sequences from thousands
of different genes are immobilized at fixed locations. The supports themselves are
usually glass microscope slides; silicon chips or nylon membranes may also be used. The
DNA is printed, spotted or actually directly synthesized onto the support mechanically
at fixed locations or addresses. The spots themselves can be DNA, cDNA or
oligonucleotides.
The process is based on hybridization probing. Single-stranded sequences on the
microarray are labeled with a fluorescent tag or flourescein, and are in fixed locations on
the support. In microarray assays an unknown sample is hybridized to an ordered array
of immobilized DNA molecules of known sequence to produce a specific hybridization
pattern that can be analyzed and compared to a given standard. The labeled DNA strand
in solution is generally called the target, while the DNA immobilized on the microarray is
the probe, a terminology opposite that used in Southern blot. Microarrays have the
following advantages over other nucleic acid based approaches:
a. High through-put: thousands of array elements can be deposited on a very small
surface area enabling gene expression to be monitored at the genomic level. Also
many components of a microbial community can be monitored simultaneously in
a single experiment.
b. High sensitivity: small amounts of the target and probe are restricted to a small
area ensuring high concentrations and very rapid reactions.
c. Differential display: different target samples can be labeled with different
fluorescent tags and then hybridized to the same microarray, allowing the
simultaneous analysis of two or more biological samples.
d. Low background interference: non-specific binding to the solid surface is very low
resulting in easy removal of organic and fluorescent compounds that attach to
microarrays during fabrication.
Aspects of Molecular Biology and Bioinformatics of Relevance in Industrial
Condition
1
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Condition
2
Fig. 3.6 Representation of the Microarray Procedure, (after Madigan and Martinko 2006)
e. Automation: microarray technology is amenable to automation making it
ultimately cost-effective when compared with other nucleic acid technologies.
3.3.1
Applications of Microarray Technology
Microarray technology is still young but yet it has found use in a some areas which have
importance in microbiology in general as well as in industrial microbiology and
biotechnology, including disease diagnosis, drug discovery and toxicological research.
Microarrays are particularly useful in studying gene function. A microarray works by
exploiting the ability of a given mRNA molecule to bind specifically to, or hybridize to,
the DNA template from which it originated. By using an array containing many DNA
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samples, it is possible to determine, in a single experiment, the expression levels of
hundreds or thousands of genes within a cell by measuring the amount of mRNA bound
to each site on the array. With the aid of a computer, the amount of mRNA bound to the
spots on the microarray is precisely measured, generating a profile of gene expression in
the cell. It is thus possible to determine the bioactive potential of a particular microbial
metabolite as a beneficial material in the form of a drug or its deleterious effect.
When a diseased condition is identified through microarray studies, experiments can
be designed which may be able to identify compounds, from microbial metabolites or
other sources, which may improve or reverse the diseased condition.
3.4
3.4.1
SEQUENCING OF DNA
Sequencing of Short DNA Fragments
DNA sequencing is the determination of the precise sequence of nucleotides in a sample
of DNA.Two methods developed in the mid-1970s are available: the Maxim and Gilbert
method and the Sanger method. Both methods produce DNA fragments which are
studied with gel electrophoresis. The Sanger method is more commonly used and will be
discussed here. The Sanger method is also called the dideoxy method, or the enzymic
method. The dideoxy method gets its name from the critical role played by synthetic
analogues of nucleotides that lack the -OH at the 3' carbon atom (star position):
dideoxynucleotide triphosphates (ddNTP) (Fig. 3.7). When (normal) deoxynucleotide
Fig. 3.7
Normal and Dedeoxy Nucleotides
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triphosphates (dNTP) are used the DNA strand continues to grow, but when the dideoxy
analogue is incorporated, chain elongation stops because there is no 3' -OH for the next
nucleotide to be attached to. For this reason, the dideoxy method is also called the chain
termination method.
For Sanger sequencing, a single strand of the DNA to be sequenced is mixed with a
primer, DNA polymerase I, an excess of normal nucleotide triphosphates and a limiting
(about 5%) of the dideoxynucleotides labeled with a fluorescent dye, each ddNTP being
labeled with a different fluorescent dye color. This primer will determine the starting
point of the sequence being read, and the direction of the sequencing reaction. DNA
synthesis begins with the primer and terminates in a DNA chain when ddNTP is
incorporated in place of normal dNTP. As all four normal nucleotides are present, chain
elongation proceeds normally until, by chance, DNA polymerase inserts a dideoxy
nucleotide instead of the normal deoxynucleotide. The result is a series of fragments of
varying lengths. Each of the four nucleotides is run separately with the appropriate
ddNTP. The mix with the ddCTP produces fragments with C (cytosine); that with ddTTP
(thymine) produces fragments with T terminals etc. The fluorescent strands are separated
from the DNA template and electrophoresed on a polyacrilamide gel to separate them
according their lengths. If the gel is read manually, four lanes are prepared, one for each
of the four reaction mixes. The reading is from the bottom of the gel up, because the
smaller the DNA fragment the faster it is on the gel. A picture of the sequence of the
nucleotides can be read from the gel (Fig. 3.8). If the system is automated, all four are
(Arrow shows direction of the electrophoresis. By convention the autoradiograph is read from bottom to
the top).
Fig. 3.8
Diagram illustrating Autoradiograph of a Sequencing Gel of the Chain Terminating
DNA Sequencing Method
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mixed and electrphoresced together. As the ddNTPs are of different colors a scanner can
scan the gel and record each color (nucleotide) separately. The sanger method is used for
relatively short fragments of DNA, 700 -800 nucleotides. Methods for larger DNA
fragments are described below.
3.4.2
Sequencing of Genomes or Large DNA fragments
The best example of the sequencing of a genome is perhaps that of the human genome,
which was completed a few years ago. During the sequencing of the human genome, two
approaches were followed: the use of bacterial artificial chromosomes (BACs) and the
short gun approach.
3.4.2.1
Use of bacterial artificial chromosomes (BACs)
The publically-funded Human Genome Project, the National Institutes of Health and the
National Science Foundation have funded the creation of ‘libraries’ of BAC clones. Each
BAC carries a large piece of human genomic DNA of the order of 100-300 kb. All of these
BACs overlap randomly, so that any one gene is probably on several different
overlapping BACs. Those BACs can be replicated as many times as necessary, so there is
a virtually endless supply of the large human DNA fragment. In the publically-funded
project, the BACs are subjected to shotgun sequencing (see below) to figure out their
sequence. By sequencing all the BACs, we know enough of the sequence in overlapping
segments to reconstruct how the original chromosome sequence looks.
3.4.2.2
Use of the shot-gun approach
An innovative approach to sequencing the human genome was pioneered by a privatelyfunded sequencing project, Celera Genomics. The founders of this company realized that
it might be possible to skip the entire step of making libraries of BAC clones. Instead, they
blast apart the entire human genome into fragments of 2-10 kb and sequenced them. The
challenge was to assemble those fragments of sequence into the whole genome sequence.
It was like having hundreds of 500-piece puzzles, each being assembled by a team of
puzzle experts using puzzle-solving computers. Those puzzles were like BACs - smaller
puzzles that make a big genome manageable. Celera threw all those puzzles together into
one room and scrambled the pieces. They, however, had scanners that scan all the puzzle
pieces and used powerful computers to fit the pieces together.
3.5
THE OPEN READING FRAME AND THE
IDENTIFICATION OF GENES
Regions of DNA that encode proteins are first transcribed into messenger RNA and then
translated into protein. By examining the DNA sequence alone we can determine the
putative sequence of amino acids that will appear in the final protein. In translation
codons of three nucleotides determine which amino acid will be added next in the
growing protein chain. The start codon is usually AUG, while the stop codons are UAA,
UAG, and UGA. The open reading frame (ORF) is that portion of a DNA segment which
will putatively code for a protein; it begins with a start codon and ends with a stop codon.
Aspects of Molecular Biology and Bioinformatics of Relevance in Industrial
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Once a gene has been sequenced it is important to determine the correct open reading
frame. Every region of DNA has six possible reading frames, three in each direction
because a codon consists of three nucleotides. The reading frame that is used determines
which amino acids will be encoded by a gene. Typically only one reading frame is used in
translating a gene (in eukaryotes), and this is often the longest open reading frame. Once
the open reading frame is known the DNA sequence can be translated into its
corresponding amino acid sequence.
For example, the sequence of DNA in Fig. 3.9 can be read in six reading frames. Three
in the forward and three in the reverse direction. The three reading frames in the forward
direction are shown with the translated amino acids below each DNA sequence. Frame
1 starts with the ‘a’, Frame 2 with the ‘t’ and Frame 3 with the ‘g’. Stop codons are
indicated by an ‘*’ in the protein sequence. The longest ORF is in Frame 1.
5'
3'
atgcccaagctgaatagcgtagaggggttttcatcatttgaggacgatgtataa
1 atg ccc aag ctg aat agc gta gag ggg ttt tca tca ttt gag gac gat gta
taa
M
P
K
L
N
S
V
E
G
F
S
S
F
E
D
D
V
*
2 tgc cca agc tga ata gcg tag agg ggt ttt cat cat ttg agg acg atg tat
C
P
S
*
I
A
*
R
G
F
H
H
L
R
T
M
Y
gcc caa gct gaa tag cgt aga ggg gtt ttc atc att tga gga cga tgt
3
Fig. 3.9 Sequence from a Hypothetical DNA Fragment
Genes can be identified in a number of ways, which are discussed below.
i. Using computer programs
As was shown above, the open reading frame (ORF) is deduced from the start and stop
codons. In prokaryotic cells which do not have many extrons (intervening non-coding
regions of the chromosome), the ORF will in most cases indicate a gene. However it is
tedious to manually determine ORF and many computer programs now exist which will
scan the base sequences of a genome and identify putative genes. Some of the programs
are given in Table 3.2. In scanning a genome or DNA sequence for genes (that is, in
searching for functional ORFs), the following are taken into account in the computer
programs:
a. usually, functional ORFs are fairly long and are do not usually contain less than
100 amino acids (that is, 300 amino acids);
b. if the types of codons found in the ORF being studied are also found in known
functional ORFs, then the ORF being studied is likely to be functional;
c. the ORF is also likely to be functional if its sequences are similar to functional
sequences in genomes of other organisms;
d. in prokaryotes, the ribosomal translation does not start at the first possible (earliest
5’) codon. Instead it starts at the codon immediately down stream of the ShineDalgardo binding site sequences. The Shine-Dalgardo sequence is a short
sequence of nucleotides upstream of the translational start site that binds to
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Table 3.2
Some Internet tools for the gene discovery in DNA sequence bases (modified from
Fickett, (1996).
Category
Services
Database search
BLAST; search sequence bases
FASTA; search sequence bases
BLOCKS; search for functional
motifs
Profilescan
MotifFinder
FGENEH; integrated gene
identification
GeneID; integrated gene
identification
GRAIL; integrated gene
identification
EcoParse; integrated gene
identification
Gene
Identification
Organism(s)
Web address
Any
Any
Any
blast@ncbi.nlm.nih.gov
fasta@ebi.ac.uk
blocks@howard.flicr.org
Any
Any
Human
http://ulrec3.unil.ch.
motif@genome.ad.jp
service@theory.bchs.uh.
edu
geneid@bircebd.uwf.edu
Vetebrate
Human
grail@ornl.gov
Escherichia coli
ribosomal RNA and thereby brings the ribosome to the initiation codon on the
mRNA. The computer program searches for a Shine-Dalgardo sequence and
finding it helps to indicate not only which start codon is used, but also that the ORF
is likely to be functional.
e. if the ORF is preceded by a typical promoter (if consensus promoter sequences for
the given organism are known, check for the presence of a similar upstream region)
f. if the ORF has a typical GC content, codon frequency, or oligonucleotide
composition of known protein-coding genes from the same organism, then it is
likely to be a functional ORF.
ii. Comparison with Existing Genes
Sometimes it may be possible to deduce not only the functionality or not of a gene (i.e. a
functional ORF), but also the function of a gene. This can done by comparing an
unknown sequence with the sequence of a known gene available in databases such as
The Institute for Genomic Research (TIGR) in Maryland.
3.6
METAGENOMICS
Metagenomics is the genomic analysis of the collective genome of an assemblage of
organisms or ‘metagenome.’ Metagenomics describes the functional and sequence-based
analysis of the collective microbial genomes contained in an environmental sample
(Fig. 3.10). Other terms have been used to describe the same method, including
environmental DNA libraries, zoolibraries, soil DNA libraries, eDNA libraries,
recombinant environmental libraries, whole genome treasures, community genome,
whole genome shotgun sequencing. The definition applied here excludes studies that
use PCR to amplify gene cassettes or random PCR primers to access genes of interest since
these methods do not provide genomic information beyond the genes that are amplified.
Aspects of Molecular Biology and Bioinformatics of Relevance in Industrial
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Environmental Sample
Metagenomic Library Construction
Extract
DNA
Clone
Transform into
host bacterium
eg E coli
Metagenomic Library
Metagenomic
Analysis
Screen for
particular
sequences using
PCR or
Hybridization
Fig. 3.10
Random
Sequences
g
Screen for
expression of
particular
phenotypes
Schematic Procedure for Metagenomic Analysis (From: Riesenfeld, et al (2004))
Many environments have been the focus of metagenomics, including soil, the oral
cavity, feces, and aquatic habitats, as well as the hospital metagenome a term intended to
encompass the genetic potential of organisms in hospitals that contribute to public
health concerns such as antibiotic resistance and nosocomial infections.
Uncultured microorganisms comprise the majority of the planet earth’s biological
diversity. In many environments, as many as 99% of the microorganisms cannot be
cultured by standard techniques, and the uncultured fraction includes diverse
organisms that are only distantly related to the cultured ones. Therefore, cultureindependent methods are essential to understand the genetic diversity, population
structure, and ecological roles of the majority of microorganisms in a given
environmental situation. Metagenomics, or the culture-independent genomic analysis of
an assemblage of microorganisms, has potential to answer fundamental questions in
microbial ecology. It can also be applied to determining organisms which may be
important in a new industrial process still under study. Several markers have been used
in metagenomics, including 16S mRNA, and the genes encoding DNA polymerases,
because these are highly conserved (i.e., because they remain relatively unchanged in
many groups). The marker most commonly used however is the sequence of 16S mRNA.
The procedure in metagonomics is described in Fig. 3.10.
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Modern Industrial Microbiology and Biotechnology
Its potential application in biotechnology and industrial microbiology is that it can
facilitate the identification of uncultured organisms whose role in a multi-organism
environment such as sewage or the degradation of a recalcitrant chemical soil may be
hampered because of the inability to culture the organism. Indeed a method has been
patented for isolating organisms of pharmaceutical importance from uncultured
organisms in the environment. This is discussed in detail in Chapter 28 where
approaches to drug discovery are discussed.
3.7
NATURE OF BIOINFORMATICS
Bioinformatics is a new and evolving science and may be defined as the use of computers
to store, compare, retrieve, analyze, predict, or simulate the composition or the structure
of the genetic macromolecules, DNA and RNA and their major product, proteins.
Important research efforts in bioinformatics include sequence alignment, gene finding,
genome assembly, protein structure alignment, protein structure prediction, prediction of
gene expression and protein-protein interactions, and the modeling of evolution.
Bioinformatics uses mathematical tools to extract useful information from a variety of
data produced by high-throughput biological techniques. Examples of succesful
extraction of orderly information from a ‘forest’ of seemingly chaotic information include
the assembly of high-quality DNA sequences from fragmentary ‘shotgun’ DNA
sequencing, and the prediction of gene regulation with data from mRNA microarrays or
mass spectrometry.
The increased role in recent times of bioinformatics in biotechnology is due to a vast
increase in computation speed and memory storage capability, making it possible to
undertake problems unthinkable without the aid of computers. Such problems include
large-scale sequencing of genomes and management of large integrated databases over
the Internet. This improved computational capability integrated with large-scale
miniaturization of biochemical techniques such as PCR, BAC, gel electrophoresis, and
microarray chips has delivered enormous amount of genomic and proteomic data to the
researchers. The result is an explosion of data on the genome and proteome analysis
leading to many new discoveries and tools that are not possible in wet-laboratory
experiments. Thus, hundreds of microbial genomes and many eukaryotic genomes
including a cleaner draft of human genome have been sequenced raising the expectation
of better control of microorganisms. Bioinformatics has been used in the following four
areas:
a. genomics – sequencing and comparative study of genomes to identify gene and
genome functionality;
b. proteomics – identification and characterization of protein related properties and
reconstruction of metabolic and regulatory pathways;
c. cell visualization and simulation to study and model cell behavior; and
d. application to the development of drugs and anti-microbial agents.
The potential gains especially following from sequencing of the human genome and
many microorganisms are greater understanding of the genetics of microorganisms and
their subsequent improved control leading to better diagnosis of the diseases through the
use of protein biomarkers, protection against diseases using cost effective vaccines and
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rational drug design, and improvement in agricultural quality and quantity. Some of
these are discussed in Chapter 28 under the heading of drug discovery.
3.7.1
Some Contributions of Bioinformatics to
Biotechnology
Some contributions made by bioinformatics to biotechnology include automatic genome
sequencing, automatic identification of genes, identification of gene function, predicting
the 3D structure modeling and pair-wise comparison of genomes.
i. Automatic genome sequencing
The major contribution of the bioinformatics in genome sequencing has been in the: (i)
development of automated sequencing techniques that integrate the PCR or BAC based
amplification, 2D gel electrophoresis and automated reading of nucleotides, (ii) joining
the sequences of smaller fragments (contigs) together to form a complete genome
sequence, and (iii) the prediction of promoters and protein coding regions of the genome.
PCR (Polymerase Chain Reaction) or BAC (Bacterial Artificial Chromosome)-based
amplification techniques derive limited size fragments of a genome. The available
fragment sequences suffer from nucleotide reading errors, repeats – very small and very
similar fragments that fit in two or more parts of a genome, and chimera – two different
parts of the genome or artifacts caused by contamination that join end-to-end giving a
artifactual fragment. Generating multiple copies of the fragments, aligning the fragments,
and using the majority voting at the same nucleotide positions solve the nucleotide
reading error problem. Multiple experimental copies are needed to establish repeats and
chimeras. Chimeras and repeats are removed before the final assembly of the genomefragments. Using mathematical models, the fragments are joined. To join contigs, the
fragments with larger nucleotide sequence overlap are joined first.
ii. Automated Identification of Genes
After the contigs are joined, the next issue is to identify the protein coding regions or ORFs
(open reading frames) in the genomes. The identification of ORFs is based on the
principles described earlier. The two programs which are used are GLIMMER and
GenBank.
iii. Identifying gene function: searching and alignment
After identifying the ORFs, the next step is to annotate the genes with proper structure
and function. The function of the gene has been identified using popular sequence search
and pair-wise gene alignment techniques. The four most popular algorithms used for
functional annotation of the genes are BLAST, BLOSUM, ClustalX, and SMART
iv. Three-dimensional (3D) structure modeling
A protein may exist under one or more conformational states depending upon its
interaction with other proteins. Under a stable conformational state certain regions of the
protein are exposed for protein-protein or protein-DNA interactions. Since the function is
also dependent upon exposed active sites, protein function can be predicted by matching
the 3D structure of an unknown protein with the 3D structure of a known protein. With
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bioinformatics it is possible to predict the possible conformations of the protein coded for
by a gene and therefore the function of the protein.
v. Pair-wise genome comparison
After the identification of gene-functions, a natural step is to perform pair-wise genome
comparisons. Pair-wise genome comparison of a genome against itself provides the
details of paralogous genes – duplicated genes that have similar sequence with some
variation in function. Pair-wise genome comparisons of a genome against other genomes
have been used to identify a wealth of information such as ortholologous genes –
functionally equivalent genes diverged in two genomes due to speciation, different types
of gene-groups – adjacent genes that are constrained to occur in close proximity due to
their involvement in some common higher level function, lateral gene-transfer – gene
transfer from a microorganism that is evolutionary distant, gene-fusion/gene-fission,
gene-group duplication, gene-duplication, and difference analysis to identify genes
specific to a group of genomes such as pathogens, and conserved genes.
In conclusion, despite the recent emergence of bioinformatics it is already making big
impacts on biotechnology. Except for the availability of bioinformatics techniques, the
vast amount of data generated by genome sequencing projects would be unmanageable
and would not be interpreted due to the lack of expert manpower and due to the
prohibitive cost of sustaining such an effort. In the last decade bioinformatics has silently
filled in the role of cost effective data analysis. This has quickened the pace of discoveries,
the drug and vaccine design, and the design of anti-microbial agents. The major impact of
bioinformatics in microbiology and biotechnology has been in automating microbial
genome sequencing, the development of integrated databases over the Internet, and
analysis of genomes to understand gene and genome function. Programs exist for
comparing gene-pair alignments, which become the first steps to derive the gene-function
and the functionality of genomes. Using bioinformatics techniques it is now possible to
compare genomes so as to (i) identify conserved function within a genome family; (ii)
identify specific genes in a group of genomes; and (iii) model 3D structures of proteins
and docking of biochemical compounds and receptors. These have direct impact in the
development of antimicrobial agents, vaccines, and rational drug design.
SUGGESTED READINGS
Bansal, K.A. 2005 Bioinformatics in microbial biotechnology – a mini review, Microbial Cell
Factories 2005, 4, 19-30.
Dorrel, N., Champoin, O.L., Wren, B.W. 2002. Application of DNA Microarray for Comparative
and Evolutionary Genomics In: Methods in Microbiology. Vol 33, Academic Press
Amsterdam; the Netherlands pp. 83–99.
Handelsman, J., Liles, M., Mann, D., Riesenfeld, C., Goodman, R.M. 2002. In: Methods in
Microbiology. Vol 33, Academic Press Amsterdam; the Netherlands pp. 242–255.
Hinds, J., Liang, K.G., Mangan, J.A., Butecer, P.D. 2002. Glass Slide Microarrays for Bacterial
Genomes. In: Methods in Microbiology. Vol 33, Academic Press Amsterdam; the Netherlands
83–99.
Hinds, J., Witney, A.A., Vaas, J.K. 2002. Microarray Design for Bacterial Genomes. In: Methods in
Microbiology. Vol 33, Academic Press Amsterdam; the Netherlands, 67-82.
Aspects of Molecular Biology and Bioinformatics of Relevance in Industrial
#!
Madigan, M., Martinko, J.M. 2006. Brock Biology of Microorganisms 11th ed. Pearson Prentice
Hall, Upper Saddle River, USA.
Manyak, D.M., Carlson, P.S. 1999. Combinatorial GenomicsTM: New tools to access microbial
chemical diversity In: Microbial Biosystems: New Frontiers, C.R. Bell, M. Brylinsky, P.
Johnson-Green, (eds) Proceedings of the 8th International Symposium on Microbial Ecology
Atlantic Canada Society for Microbial Ecology, Halifax, Canada, 1999.
Priest, F., Austin, B. 1993. Modern Bacterial Taxonomy. Chapman and Hall. London, UK.
Riesenfeld, C.S., Schloss, P.D., Handelsman, 2004. Metagenomics: Genomic Analysis of Microbial
Communities. Annual Review of Genetics 38, 525-52.
Rogic, S., Mackworth, A.K., Ouellette, F.B.F. 2001. Evaluation of Gene-Finding Programs on
Mammalian Sequences Genome Research 11, 817-832.
Whitford, D. 2005. Proteins: Structure and Function. John Wiley and Sons Chichester, UK.
Zhou, J. 2002. Microarrays: Applications in Environmental Microbiology. In: Encyclopedia of
Environmental Microbiology Vol 4. Wiley Interscience, New York USA. pp. 1968-1979.
#"
Modern Industrial Microbiology and Biotechnology
+0)26-4
4
Industrial Media and the
Nutrition of Industrial
Organisms
The use of a good, adequate, and industrially usable medium is as important as the
deployment of a suitable microorganism in industrial microbiology. Unless the medium
is adequate, no matter how innately productive the organism is, it will not be possible to
harness the organism’s full industrial potentials. Indeed not only may the production of
the desired product be reduced but toxic materials may be produced. Liquid media are
generally employed in industry because they require less space, are more amenable to
engineering processes, and eliminate the cost of providing agar and other solid agents.
4.1
THE BASIC NUTRIENT REQUIREMENTS OF
INDUSTRIAL MEDIA
All microbiological media, whether for industrial or for laboratory purposes must satisfy
the needs of the organism in terms of carbon, nitrogen, minerals, growth factors,
and water. In addition they must not contain materials which are inhibitory to growth.
Ideally it would be essential to perform a complete analysis of the organism to be grown
in order to decide how much of the various elements should be added to the medium.
However, approximate figures for the three major groups of heterotrophic organisms
usually grown on an industrial scale are available and may be used in such calculations
(Table 4.1).
Carbon or energy requirements are usually met from carbohydrates, notably (in laboratory
experiments) from glucose. It must be borne in mind that more complex carbohydrates
such as starch or cellulose may be utilized by some organisms. Furthermore, energy
sources need not be limited to carbohydrates, but may include hydrocarbons, alcohols, or
even organic acids. The use of these latter substrates as energy sources is considered in
Chapters 15 and 16 where single cell protein and yeast productions are discussed.
In composing an industrial medium the carbon content must be adequate for the
production of cells. For most organisms the weight of organism produced from a given
weight of carbohydrates (known as the yield constant) under aerobic conditions is about
Industrial Media and the Nutrition of Industrial Organisms
Table 4.1
##
Average composition of microorganisms (% dry weight)
Component
Carbon
Nitrogen
Protein
Carbohydrates
Lipids
Nucleic Acids
Ash
Bacteria
48
12.5
55
9
7
23
6
(46-52)
(10-14)
(50 –60)
(6-15)
(5-10)
(15-25)
(4-10)
Yeast
48
7.5
40
38
8
8
6
Molds
(46-52)
(6-8.5)
(35-45)
(30-45)
(5-10)
(5-10)
(4-10)
48
6
32
49
8
5
4
(45-55)
(4-7)
(25-40)
(40-55)
(5-10)
(2-8)
(4-10)
Minerals (same for all three organisms)
Phosphorus
Sulfur, magnesium
Potassium, sodium
Iron
Zinc, copper, manganese
1.0 - 2.5
0.3 - 1.0
0.1 - 0.5
0.01 - 0.1
0.001 – 0.01
0.5 gm of dry cells per gram of glucose. This means that carbohydrates are at least twice
the expected weight of the cells and must be put as glucose or its equivalent compound.
Nitrogen is found in proteins including enzymes as well as in nucleic acids hence it is a
key element in the cell. Most cells would use ammonia or other nitrogen salts. The
quantity of nitrogen to be added in a fermentation can be calculated from the expected cell
mass and the average composition of the micro-organisms used. For bacteria the average
N content is 12.5%. Therefore to produce 5 gm of bacterial cells per liter would require
about 625 mg N (Table 4.1).
Any nitrogen compound which the organism cannot synthesize must be added.
Minerals form component portions of some enzymes in the cell and must be present in the
medium. The major mineral elements needed include P, S, Mg and Fe. Trace elements
required include manganese, boron, zinc, copper and molybdenum.
Growth factors include vitamins, amino acids and nucleotides and must be added to the
medium if the organism cannot manufacture them.
Under laboratory conditions, it is possible to meet the organism’s requirement by the
use of purified chemicals since microbial growth is generally usually limited to a few
liters. However, on an industrial scale, the volume of the fermentation could be in the
order of thousands of liters. Therefore, pure chemicals are not usually used because of
their high expense, unless the cost of the finished material justifies their use. Pure
chemicals are however used when industrial media are being developed at the laboratory
level. The results of such studies are used in composing the final industrial medium,
which is usually made with unpurified raw materials. The extraneous materials present
in these unpurified raw materials are not always a disadvantage and may indeed be
responsible for the final and distinctive property of the product. Thus, although alcohol
appears to be the desired material for most beer drinkers, the other materials extraneous
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Modern Industrial Microbiology and Biotechnology
to the maltose (from which yeasts ferment alcohol) help confer on beer its distinctive
flavor (Chapter 12).
4.2
CRITERIA FOR THE CHOICE OF RAW MATERIALS
USED IN INDUSTRIAL MEDIA
In deciding the raw materials to be used in the production of given products using
designated microorganism(s) the following factors should be taken into account.
(a) Cost of the material
The cheaper the raw materials the more competitive the selling price of the final product
will be. No matter, therefore, how suitable a nutrient raw materials is, it will not usually
be employed in an industrial process if its cost is so high that the selling price of the final
product is not economic. Thus, although lactose is more suitable than glucose in some
processes (e.g. penicillin production) because of the slow rate of its utilization, it is
usually replaced by the cheaper glucose. When used, glucose is added only in small
quantities intermittently in order to decelerate acid production. Due to these economic
considerations the raw materials used in many industrial media are usually waste
products from other processes. Corn steep liquor and molasses are, for example, waste
products from the starch and sugar industries, respectively. They will be discussed more
fully below.
(b) Ready availability of the raw material
The raw material must be readily available in order not to halt production. If it is seasonal
or imported, then it must be possible to store it for a reasonable period. Many industrial
establishments keep large stocks of their raw materials for this purpose. Large stocks help
beat the ever rising cost of raw materials; nevertheless large stocks mean that money
which could have found use elsewhere is spent in constructing large warehouses or
storage depots and in ensuring that the raw materials are not attacked during storage by
microorganisms, rodents, insects, etc. There is also the important implication, which is
not always easy to realize, that the material being used must be capable of long-term
storage without concomitant deterioration in quality.
(c) Transportation costs
Proximity of the user-industry to the site of production of the raw materials is a factor of
great importance, because the cost of the raw materials and of the finished material and
hence its competitiveness on the market can all be affected by the transportation costs.
The closer the source of the raw material to the point of use the more suitable it is for use,
if all other conditions are satisfactory.
(d) Ease of disposal of wastes resulting from the raw materials
The disposal of industrial waste is rigidly controlled in many countries. Waste materials
often find use as raw materials for other industries. Thus, spent grains from breweries
can be used as animal feed. But in some cases no further use may be found for the waste
from an industry. Its disposal especially where government regulatory intervention is
rigid could be expensive. When choosing a raw material therefore the cost, if any, of
treating its waste must be considered.
Industrial Media and the Nutrition of Industrial Organisms
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(e) Uniformity in the quality of the raw material and ease of standardization
The quality of the raw material in terms of its composition must be reasonably constant in
order to ensure uniformity of quality in the final product and the satisfaction of the
customer and his/her expectations. In cases where producers are plentiful, they usually
compete to ensure the maintenance of the constant quality requirement demanded by the
user. Thus, in the beer industry information is available on the quality of the barley malt
before it is purchased. This is because a large number of barley malt producers exist, and
the producers attempt to meet the special needs of the brewery industry, their main
customer. On the other hand molasses, which is a major source of nutrient for industrial
microorganisms, is a by product of the sugar industry, where it is regarded as a waste
product. The sugar industry is not as concerned with the constancy of the quality of
molasses, as it is with that of sugar. Each batch of molasses must therefore be chemically
analyzed before being used in a fermentation industry in order to ascertain how much of
the various nutrients must be added. A raw material with extremes of variability in
quality is clearly undesirable as extra costs are needed, not only for the analysis of the
raw material, but for the nutrients which may need to be added to attain the usual and
expected quality in the medium.
(f) Adequate chemical composition of medium
As has been discussed already, the medium must have adequate amounts of carbon,
nitrogen, minerals and vitamins in the appropriate quantities and proportions necessary
for the optimum production of the commodity in question. The demands of the
microorganisms must also be met in terms of the compounds they can utilize. Thus most
yeasts utilize hexose sugars, whereas only a few will utilize lactose; cellulose is not easily
attacked and is utilized only by a limited number of organisms. Some organisms grow
better in one or the other substrate. Fungi will for instance readily grow in corn steep
liquor while actinomycetes will grow more readily on soya bean cake.
(g) Presence of relevant precursors
The raw material must contain the precursors necessary for the synthesis of the finished
product. Precursors often stimulate production of secondary metabolites either by
increasing the amount of a limiting metabolite, by inducing a biosynthetic enzyme or
both. These are usually amino acids but other small molecules also function as inducers.
The nature of the finished product in many cases depends to some extent on the
components of the medium. Thus dark beers such as stout are produced by caramelized
(or over-roasted) barley malt which introduce the dark color into these beers. Similarly for
penicillin G to be produced the medium must contain a phenyl compound. Corn steep
liquor which is the standard component of the penicillin medium contains phenyl
precursors needed for penicillin G. Other precursors are cobalt in media for Vitamin B12
production and chlorine for the chlorine containing antibiotics, chlortetracycline, and
griseofulvin (Fig. 4.1).
(h) Satisfaction of growth and production requirements of the microorganisms
Many industrial organisms have two phases of growth in batch cultivation: the phase of
growth, or the trophophase, and the phase of production, or the idiophase. In the first
phase cell multiplication takes place rapidly, with little or no production of the desired
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Modern Industrial Microbiology and Biotechnology
Left: Vitamin B12. Please note that cobalt is highlighted. It must be present in the medium in which organisms
producing the vitamin are grown
Right: Top; The general structure of tetracyclines. Bottom; The structure of 7-Chlortetracycline; a chlorine
atom is present in position 7. Chlorine must be present in the medium for producing chlortetracyline; note that
chlorine is highlighted in position 7 in chlortetracycline.
Fig. 4.1
Vitamin B12 and Chlortetracycline Showing Location of Components Present as
Precursors in the Medium
material. It is in the second phase that production of the material takes place, usually
with no cell multiplication and following the elaboration of new enzymes. Often these
two phases require different nutrients or different proportions of the same nutrients. The
medium must be complete and be able to cater for these requirements. For example high
levels of glucose and phosphate inhibit the onset of the idiophase in the production of a
number of secondary metabolites of industrial importance. The levels of the components
added must be such that they do not adversely affect production. Trophophaseidiophase relationship and secondary metabolites are discussed in detail in Chapter 5.
4.3
SOME RAW MATERIALS USED IN COMPOUNDING
INDUSTRIAL MEDIA
The raw materials to be discussed are used because of the properties mentioned above:
cheapness, ready availability, constancy of chemical quality, etc. A raw material which is
Industrial Media and the Nutrition of Industrial Organisms
#'
cheap in one country or even in a different part of the same country may however not be
cheap in another, especially if it has already found use in some other production process.
In such cases suitable substitutes must be found if the goods must be produced in the new
location. The use of local substitutes where possible is advantageous in reducing the
transportation costs and even creating some employment in the local population. Prior
experimentation may however be necessary if such new local materials differ
substantially in composition from those already being used. Some well-known raw
materials will now be discussed. In addition, some of potential useability will also be
examined.
(a) Corn steep liquor
This is a by-product of starch manufacture from maize. Sulfur dioxide is added to the
water in which maize is steeped. The lowered pH inhibits most other organisms, but
encourages the development of naturally occurring lactic acid bacteria especially
homofermentative thermophilic Lactobacillus spp. which raise the temperature to
38-55°C. Under these conditions, much of the protein present in maize is converted to
peptides which along with sugars leach out of the maize and provide nourishment for
the lactic acid bacteria. Lactic fermentation stops when the SO2 concentration reaches
about 0.04% and the concentration of lactic acid between 1.0 and 1.5%. At this time the
pH is about 4. Acid conditions soften the kernels and the resulting maize grains mill
better while the gel-forming property of the starch is not hindered. The supernatant
drained from the maize steep is corn steep liquor. Before use, the liquor is usually filtered
and concentrated by heat to about 50% solid concentration. The heating process kills the
bacteria.
As a nutrient for most industrial organisms corn steep liquor is considered adequate,
being rich in carbohydrates, nitrogen, vitamins, and minerals. Its composition is highly
variable and would depend on the maize variety, conditions of steeping, extent of boiling
etc. The composition of a typical sample of corn steep liquor is given in Table 4.2. As corn
steep liquor is highly acidic, it must be neutralized (usually with CaCO3) before use.
(b) Pharmamedia
Also known as proflo, this is a yellow fine powder made from cotton-seed embryo. It is
used in the manufacture of tetracycline and some semi-synthetic penicillins. It is rich in
Table 4.2 Approximate composition of corn steep liquor (%)
Lactose
Glucose
Non-reducing carbohydrates (mainly starch)
Acetic acid
Glucose lactic acid
Phenylethylamine
Amino aids (peptides, mines)
Total solids
Total nitrogen
3.0-4.0
0.-0.5
1.5
0.05
0.5
0.05
0.5
80-90
0.15-0.2%
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Modern Industrial Microbiology and Biotechnology
protein, (56% w/v) and contains 24% carbohydrate, 5% oil, and 4% ash, the last of which
is rich in calcium, iron, chloride, phosphorous, and sulfate.
(c) Distillers solubles
This is a by-product of the distillation of alcohol from fermented grain. It is prepared by
filtering away the solids from the material left after distilling fermented cereals (maize or
barley) for whiskey or grain alcohol. The filtrate is then concentrated to about one-third
solid content to give a syrup which is then drum-dried to give distillers soluble. It is rich
in nitrogen, minerals, and growth factors (Table 4.3).
Table 4.3
Composition of maize distillers soluble
%
Moisture
Protein
Lipid
Fibre
Carbohydrate
Ash (mainly K, Na, Mg, CO3, and P)
5
27
9
5
43
11
(d) Soya bean meal
Soya beans (soja) (Glycine max), is an annual legume which is widely cultivated
throughout the world in tropical, sub-tropical and temperate regions between 50°N and
40°S. The seeds are heated before being extracted for oil that is used for food, as an antifoam in industrial fermentations, or used for the manufacture of margarine. The resulting
dried material, soya bean meal, has about 11% nitrogen, and 30% carbohydrate and may
be used as animal feed. Its nitrogen is more complex than that found in corn steep liquor
and is not readily available to most microorganisms, except actinomycetes. It is used
particularly in tetracycline and streptomycin fermentations.
(e) Molasses
Molasses is a source of sugar, and is used in many fermentation industries including the
production of potable and industrial alcohol, acetone, citric acid, glycerol, and yeasts. It
is a by-product of the sugar industry. There are two types of molasses depending on
whether the sugar is produced from the tropical crop, sugar cane (Saccharum officinarum)
or the temperate crop, beet, (Beta alba).
Four stages are involved in the manufacture of cane sugar. After crushing, a clear
greenish dilute sugar solution known as ‘mixed juice’ is expressed from the canes.
During the second stage known as clarification the mixed juice is heated with lime.
Addition of lime changes the pH of the juice to alkaline and thus stops further hydrolysis
(or inversion) or the cane sugar (sucrose), while heating coagulates proteins and other
undesirable soluble portions of the mixed juice to form ‘mud’. The supernatant juice is
then concentrated (in the third stage) by heating under high vacuum and increasing low
pressures in a series of evaporators. In the fourth and final stage of crystallization, sugar
crystals begin to form with increasing heat and under vacuum, yielding a thick brown
Industrial Media and the Nutrition of Industrial Organisms
$
syrup which contains the crystals, and which is known as ‘massecuite’. (In the beet
industry it is known as ‘fillmass’.) The massecuite is centrifuged to remove the sugar
crystals and the remaining liquid is known as molasses. The first sugar so collected is ‘A’
and the liquid is ‘A’ molasses. ‘A’ molasses is further boiled to extract sugar crystals to
yield ‘B’ sugar and ‘B’ molasses. Two or more boilings may be required before it is no
longer profitable to attempt further extractions. This final molasses is known as
‘blackstrap molasses’.
The sugar yielded with the production of black strap molasses is low-grade and
brown in color, and known as raw sugar, cargo sugar, or refining sugar. This raw sugar
is further refined, in a separate factory, to remove miscellaneous impurities including the
brown color (due to caramel) to yield the white sugar used at the table. The heavy liquid
discarded from the refining of sugar is known in the sugar refining industry as ‘syrup’
and corresponds to molasses in the raw sugar industry.
The above description has been of cane sugar molasses. In the beet sugar industry the
processes used in raw refined sugar manufacture are similar, but the names of the
different fractions recovered during purification differ. Cane and beet molasses differ
slightly in composition (Table 4.4). Beet molasses is alkaline while cane molasses is acid.
Table 4.4 Average composition of beet and cane molasses
Water
Sugars:
Sucrose
Fructose
Glucose
Raffinose
Non-sugar (nitrogeneous
Materials, acids, gums, etc.)
Ash
Beet Molasses
% (W/W)
Cane Molasses
% (W/W)
16.5
53.0
51.0
1.0
1.0
20.0
64.0
32.0
15.0
14.0
-
19.0
11.5
10.0
8.0
Even within same type of molasses – beet or cane – composition varies from year to
year and from one locality to another. The user industry selects the batch with a suitable
composition and usually buys up a year’s supply. For the production of cells the
variability in molasses quality is not critical, but for metabolites such as citric acid, it is
very important as minor components of the molasses may affect the production of these
metabolites.
‘High test’ molasses (also known as inverted molasses) is a brown thick syrup liquid
used in the distilling industry and containing about 75% total sugars (sucrose and
reducing sugars) and about 18% moisture. Strictly speaking, it is not molasses at all but
invert sugar, (i.e reducing sugars resulting from sucrose hydrolysis). It is produced by the
hydrolysis of the concentrated juice with acid. In the so-called Cuban method, invertase
is used for the hydrolysis. Sometimes ‘A’ sugar may be inverted and mixed with ‘A’
molasses.
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Modern Industrial Microbiology and Biotechnology
(f) Sulfite liquor
Sulfite liquor (also called waste sulfite liquor, sulfite waste liquor or spent sulfite liquor)
is the aqueous effluent resulting from the sulfite process for manufacturing cellulose or
pulp from wood. Depending on the type, most woods contain about 50% cellulose, about
25% lignins and about 25% of hemicelluloses. During the sulfite process, hemicelluloses
hydrolyze and dissolve to yield the hexose sugars, glucose, mannose, galactose, fructose
and the pentose sugars, xylose, and arabinsoe. The acid reagent breaks the chemical
bonds between lignin and cellulose; subsequently they dissolve the lignin. Depending on
the severity of the treatment some of the cellulose will continue to exist as fibres and can
be recovered as pulp. The presence of calcium ions provides a buffer and helps neutralize
the strong lignin sulfonic acid. The degradation of cellulose yields glucose. Portions of
the various sugars are converted to sugar sulfonic acids, which are not fermentable.
Variable but sometimes large amounts of acetic, formic and glactronic acids are also
produced.
Sulfite liquor of various compositions are produced, depending on the severity of the
treatment and the type of wood. The more intense the treatment the more likely it is that
the sugars produced by the more easily hydrolyzed hemicellulose will be converted to
sulfonic acids; at the same time the more intense the treatment the more will glucose be
released from the more stable cellulose. Hardwoods not only yield a higher amount of
sugar (up to 3% dry weight of liquor) but the sugars are largely pentose, in the form of
xylose. Hardwood hydrolyzates also contains a higher amount of acetic acid. Soft woods
yield a product with about 75% hexose, mainly mannose.
Sulfite liquor is used as a medium for the growth of microorganisms after being
suitably neutralized with CaCO3 and enriched with ammonium salts or urea, and other
nutrients. It has been used for the manufacture of yeasts and alcohol. Some samples do
not contain enough assaimilable carbonaceous materials for some modern
fermentations. They are therefore often enriched with malt extract, yeast autolysate, etc.
(g) Other Substrates
Other substrates used as raw materials in fermentations are alcohol, acetic acid,
methanol, methane, and fractions of crude petroleum. These will be discussed under
Single Cell Protein (Chapter 15). Barley will be discussed in the section dealing with the
brewing of beer (Chapter 12).
4.4
GROWTH FACTORS
Growth factors are materials which are not synthesized by the organism and therefore
must be added to the medium. They usually function as cofactors of enzymes and may be
vitamins, nucleotides etc. The pure forms are usually too expensive for use in industrial
media and materials containing the required growth factors are used to compound the
medium. Growth factors are required only in small amounts. Table 4.5 gives some
sources of growth factors.
4.5
WATER
Water is a raw material of vital importance in industrial microbiology, though this
importance is often overlooked. It is required as a major component of the fermentation
Industrial Media and the Nutrition of Industrial Organisms
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Table 4.5 Some sources of growth factors
Growth factor
Vitamin B
Vitamin B2
Vitamin B6
Nicotinamide
Panthothenic Acid
Vitamin B12
Source
Rice polishing, wheat germ, yeasts
Cereals, corn steep liquor
Corn steep liquor, yeasts
Liver, penicillin spent liquor
Corn steep liquor
Liver, silage, meat
medium, as well as for cooling, and for washing and cleaning. It is therefore used in
rather large quantities, and measured in thousands of liters a day depending on the
industry. In some industries such as the beer industry the quality of the product depends
to some extent on the water. In order to ensure constancy of product quality the water
must be regularly analyzed for minerals, color, pH, etc. and adjusted as may be necessary.
Due to the importance of water, in situations where municipal water supplies are likely to
be unreliable, industries set up their own supplies.
4.6
SOME POTENTIAL SOURCES OF COMPONENTS OF
INDUSTRIAL MEDIA
The materials to be discussed are mostly found in the tropical countries, including those
in Africa, the Caribbean, and elsewhere in the world. Any microbiological industries to
be sited in these countries must, if they are not to run into difficulties discussed above, use
the locally available substrates. It is in this context that the following are discussed.
4.6.1
Carbohydrate Sources
These are all polysaccharides and have to be hydrolyzed to sugar before being used.
(a) Cassava (manioc)
The roots of the cassava-plant Manihot esculenta Crantz serve mainly as a source of
carbohydrate for human (and sometimes animal) food in many parts of the tropical
world. Its great advantage is that it is high yielding, requires little attention when
cultivated, and the roots can keep in the ground for many months without deterioration
before harvest. The inner fleshy portion is a rich source of starch and has served, after
hydrolysis, as a carbon source for single cell protein, ethanol, and even beer. In Brazil it is
one of the sources of fermentation alcohol (Chapter 13) which is blended with petrol to
form gasohol for driving motor vehicles.
(b) Sweet potato
Sweet potatoes Ipomca batatas is a warm-climate crop although it can be grown also in sub
tropical regions. There are a large number of cultivars, which vary in the colors of the
tuber flesh and of the skin; they also differ in the tuber size, time of maturity, yield, and
sweetness. They are widely grown in the world and are found in South America, the USA,
Africa and Asia. They are regarded as minor sources of carbohydrates in comparison
with maize, wheat, or cassava, but they have the advantage that they do not require much
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Modern Industrial Microbiology and Biotechnology
agronomic attention. They have been used as sources of sugar on a semi-commercial
basis because the fleshy roots contain saccharolytic enzymes. The syrup made from
boiling the tubers has been used as a carbohydrate (sugar) source in compounding
industrial media. Butyl alcohol, acetone and ethanol have been produced from such a
syrup, and in quantities higher than the amounts produced from maize syrup of the same
concentration. Since sweet potatoes are not widely consumed as food, it is possible that it
may be profitable to grow them for use, after hydrolysis, in industrial microbiology media
as well as for the starch industry. It is reported that a variety has been developed which
yields up to 40 tonnes per hectare, a much higher yield than cassava or maize.
(c) Yams
Yams (Dioscorea spp) are widely consumed in the tropics. Compared to other tropical
roots however, their cultivation is tedious; in any case enough of this tuber is not
produced even for human food. It is therefore almost inconceivable to suggest that the
crop should be grown solely for use in compounding industrial media. Nevertheless
yams have been employed in producing various products such as yam flour and yam
flakes. If the production of these materials is carried out on a sufficiently large scale it is to
be expected that the waste materials resulting from peeling the yams could yield
substantial amounts of materials which on hydrolysis will be available as components of
industrial microbiological media.
(d) Cocoyam
Cocoyam is a blanket name for several edible members of the monocotyledonous (single
seed-leaf) plant of the family Araceae (the aroids), the best known two genera of which are
Colocasia (tano) and Xanthosoma (tannia). They are grown and eaten all over the tropical
world. As they are laborious to cultivate, require large quantities of moisture and do not
store well they are not the main source of carbohydrates in regions where they are grown.
However, this relative unimportance may well be of significance in regions where for
reasons of climate they can be suitably cultivated. Cocoyam starch has been found to be of
acceptable quality for pharmaceutical purposes. Should it find use in that area, starchy
by-products could be hydrolyzed to provide components of industrial microbiological
media.
(e) Millets
This is a collective name for several cereals whose seeds are small in comparison with
those of maize, sorghum, rice, etc. The plants are also generally smaller. They are
classified as the minor cereals not because of their smaller sizes but because they
generally do not form major components of human food. They are however hardy and
will tolerate great drought and heat, grow on poor soil and mature quickly. Attention is
being turned to them for this reason in some parts of the world. It is for this reason also
that millets could become potential sources of cereal for use in industrial microbiology
media. Millets are grown all over the world in the tropical and sub-tropical regions and
belong to various genera: Pennisetum americanum (pearl or bulrush millet), Setaria italica
(foxtail millet), Panicum miliaceum (yard millet), Echinochloa frumentacea (Japanese yard
millet) and Eleusine corcana (finger millet). Millet starch has been hydrolyzed by malting
for alcohol production on an experimental basis as far back as 50 years ago and the
Industrial Media and the Nutrition of Industrial Organisms
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available information should be helpful in exploiting these grains for use as industrial
media components.
(f) Rice
Rice, Oryza sativa is one of the leading food corps of the world being produced in all five
continents, but especially in the tropical areas. Although it is high-cost commodity, it has
the advantage of ease of mechanization, storability, and the availability of improved
seeds through the efforts of the International Rice Research Institute, Philippines and
other such bodies. The result is that this food crop is likely in the near future to displace,
as a carbohydrate source, such other starch sources as yams, and to a lesser extent
cassava in tropical countries. The increase in rice production is expected to become so
efficient in many countries that the crop would yield substrates cheap enough for
industrial microbiological use. Rice is used as brewing adjuncts and has been malted
experimentally for beer brewing.
(g) Sorghum
Sorghum, Sorghum bicolor, is the fourth in term of quantity of production of the world’s
cereals, after wheat, rice, and corn. It is used for the production of special beers in various
parts of the world. It has been mechanized and has one of the greatest potential among
cereals for use as a source of carbohydrate in industrial media in regions of the world
where it thrives. It has been successfully malted and used in an all-sorghum lager beer
which compared favorably with barley lager beer (Chapter 12)
(h) Jerusalem artichoke
Jerusalem artichoke, Helianthus tuberosus, is a member of the plant family compositae,
where the storage carbohydrate is not starch, but inulin (Fig. 4.2) a polymer of fructose
into which it can be hydrolyzed. It is a root-crop and grows in temperate, semi-tropical
and tropical regions.
4.6.2
Protein Sources
(a) Peanut (groundnut) meal
Various leguminous seeds may be used as a source for the supply of nitrogen in
industrial media. Only peanuts (groundnuts) Arachis hypogea will be discussed. The nuts
are rich in liquids and proteins. The groundnut cake left after the nuts have been freed of
oil is often used as animal feed. But just as is the case with soya bean, oil from peanuts
may be used as anti-foam while the press-cake could be used for a source of protein. The
nuts and the cake are rich in protein.
(b) Blood meal
Blood consists of about 82% water, 0.1% carbohydrate, 0.6% fat, 16.4% nitrogen, and 0.7%
ash. It is a waste product in abattoirs although it is sometimes used as animal feed.
Drying is achieved by passing live steam through the blood until the temperature reaches
about 100°C. This treatment sterilizes it and also causes it to clot. It is then drained,
pressed to remove serum, further dried and ground. The resulting blood-meal is
chocolate-colored and contains about 80% protein and small amounts of ash and lipids.
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Fig. 4.2 Structure of Inulin. This Polymer of Fructose Replaces Starch in some Plants
Where sufficient blood is available blood meal could form an important source of
proteins for industrial media.
(c) Fish Meal
Fish meal is used for feeding farm animals. It is rich in protein (about 65%) and, minerals
(about 21% calcium 8%, and phosphorous 3.5%) and may therefore be used for industrial
microbiological media production. Fish meal is made by drying fish with steam either
aided by vacuum or by simple drying. Alternatively hot air may be passed over the fish
placed in revolving drums. It is then ground into a fine powder.
4.7
THE USE OF PLANT WASTE MATERIALS IN
INDUSTRIAL MICROBIOLOGY MEDIA:
SACCHARIFICATION OF POLYSACCHARIDES
The great recommendation of plant agricultural wastes as sources of industrial
microbiological media is that they are not only plentiful but that in contrast with
petroleum, a major source of chemicals, they are also renewable. Serious consideration
has therefore been given, in some studies, to the possibility of deriving industrial
microbiological raw materials not just from wastes, but from crops grown deliberately for
the purpose. However, plant materials in general contain large amounts of
polysaccharides which are not immediately utilizable by industrial microorganisms and
which will therefore need to be hydrolyzed or saccharified to provide the more available
sugars. Thereafter the sugars may be fermented to ethyl alcohol for use as a chemical feed
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stock. The plant polysaccharides whose hydrolysis will be discussed in this section are
starch, cellulose and hemicelluloses.
4.7.1
Starch
Starch is a mixture of two polymers of glucose: amylose and amylopectin. Amylose is a
linear (1 ® 4) µ – D glucan usually having a degree of polymerization (D.P., i.e. number
of glucose molecules) of about 400 and having a few branched residues linked with (1 ®
6) bondings. Amylopectin is a branched D glucan with predominantly µ – D (1 ® 4)
linkages and with about 4% of the µ – D (1 ® 6) type (Fig. 4.3). Amylopectin consists of
amylose – like chains of D. P. 12 – 50 linked in a number of possible manners of which 3
in Fig. 4.4 seems most generally accepted. A comparison of the properties of amylose and
amylopectin is given in Table 4.6.
Table 4.6 Some properties of amylose and amylopectin
Property
Amylose
Amylopectin
Structure
Behavior in water
Linear
Precipitates
Irreversibly
103
103
Branched
Stable
104 -105
20-25
87
54
98
650
79
550
Degree of polymerization
Average chain length
Hydrolysis to maltose (%)
(a) b - amylase
(b) b - amylase and
debranching enzyme
Iodine Complex max (nm)
Starches from various sources differ in their proportion of amylopectin and amylose.
The more commonly grown type of maize, for example, has about 26% of amylose and
74% of amylopectin (Table 4.7). Others may have 100% amylopectin and still others may
have 80 – 85% of amylose.
4.7.1.1 Saccharification of starch
Starch occurs in discrete crystalline granules in plants, and in this form is highly
resistant to enzyme action. However when heated to about 55°C – 82°C depending on the
type, starch gelatinizes and dissolves in water and becomes subject to attack by various
enzymes.
Before saccharification, the starch or ground cereal is mixed with water and heated to
gelatinize the starch and expose it to attack by the saccharifying agents. The
gelatinization temperatures of starch from various cereals is given in Table 12.1. The
saccharifying agents used are dilute acids and enzymes from malt or microorganisms.
4.7.1.1.1
Saccharification of starch with acid
The starch-containing material to be hydrolyzed is ground and mixed with dilute
hydrochloric acid, sulfuric acid or even sulfurous acid. When sulfurous acid is used it
can be introduced merely by pumping sulfur dioxide into the mash.
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Top: a – D (1 ® 4) (left) and a – D (1 ® 6) (right)
Bottom: Part structure of amylose (a) and amylopectin (b)
0 = glucose units joined by a – D (I ® 4) linkages
® = a – D (1-6) linkages
Fig. 4.3 Linkages of D-glucose
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Table 4.7 Amylose contents of some starches
Source
Amylose Content %
Potato
Corn
Wheat
Oat
Waxymaize
Cassava
Sorghum
Rice
20-22
20-27
18-26
22-24
0-5
17-20
25-28
16-18
o = Terminal non-reducing end – groups
l = Reducing end group
® = a - D - (1 ® 6) linkage
— = Chain of 20 to 25 a - D - (1 ® 4) linked D-glucose residues
Fig. 4.4 Diagrams Representing Three Proposed Structures of Amylopectin
The concentrations of the mash and the acid, length of time and temperature of the
heating have to be worked out for each starch source. During the hydrolysis the starch is
broken down from starch (about 2,000 glucose molecules) through compounds of
decreasing numbers of glucose moieties to glucose. The actual composition of the
hydrolysate will depend on the factors mentioned above. Starch concentration is
particularly important: if it is too high, side reactions may occur leading to a reduction in
the yield of sugar.
At the end of the reaction the acid is neutralized. If it is desired to ferment the
hydrolysate for ethanol, yeast or single cell production, ammonium salts may be used as
they can be used by many microorganisms.
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4.7.1.1.2
Use of enzymes
Enzymes hydrolyzing starch used to be called collectively diastase. With increased
knowledge about them, they are now called amylases. Enzymatic hydrolysis has several
advantages over the use of acid: (a) since the pH for enzyme hydrolysis is about neutral,
there is no need for special vessels which must stand the high temperature, pressure, and
corrosion of acid hydrolysis; (b) enzymes are more specific and hence there are fewer side
reactions leading therefore to higher yields; (c) acid hydrolysis often yields salts which
may have to be removed constantly or periodically thereby increasing cost; (d) it is
possible to use higher concentrations of the substrates with enzymes than with acids
because of enzyme specificity, and reduced possibility of side reactions.
4.7.1.1.3
Enzymes involved in the hydrolysis of starch
Several enzymes are important in the hydrolysis of starch. They are divisible into six
groups.
(i) Enzymes that hydrolyse a – 1, 4 bonds and by-pass a – I, 6 bonding: The typical
example is a - amylase. This enzyme hydrolyses randomly the inner (1 ® 4) - a D - glucosidic bonds of amylose and amylopectin (Fig. 4.3). The cleavage can occur
anywhere as long as there are at least six glucose residues on one side and at least
three on the other side of the bond to be broken. The result is a mixture of branched
a - limit dextrins (i.e., fragments resistant to hydrolysis and contain the a - D (1 6)
linkage (Fig. 4.4) derived from amylopectin) and linear glucose residues especially
maltohexoses, maltoheptoses and maltotrioses. a - Amylases are found in virtually
every living cell and the property and substrate pattern of a - amylases vary
according to their source. Thus, animal a - amylases in saliva and pancreatic juice
completely hydrolyze starch to maltose and D-glucose. Among microbial a amylases some can withstand temperatures near 100°C.
(ii) Enzymes that hydrolyse the a – 1, 4 bonding, but cannot by-pass the a – 1,6 bonds: Beta
amylase: This was originally found only in plants but has now been isolated from
micro-organisms. Beta amylase hydrolyses alternate a – 1,4 bonds sequentially
from the non-reducing end (i.e., the end without a hydroxyl group at the C – 1
position) to yield maltose (Figs. 4.3 and 4.5). Beta amylase has different actions on
amylose and amylopectin, because it cannot by-pass the a – 1:6 – branch points in
amylopectin. Therefore, while amylose is completely hydrolyzed to maltose,
amylopectin is only hydrolyzed to within two or three glucose units of the a – 1.6 branch point to yield maltose and a ‘beta-limit’ dextrin which is the parent
amylopectin with the ends trimmed off. Debranching enzymes (see below) are able
to open up the a – 1:6 bonds and thus convert beta-limit dextrins to yield a mixture
of linear chains of varying lengths; beta amylase then hydrolyzes these linear
chains. Those chains with an odd number of glucose molecules are hydrolyzed to
maltose, and one glucose unit per chain. The even numbered residues are
completely hydrolyzed to maltose. In practice there is a very large population of
chains and hence one glucose residue is produced for every two chains present in
the original starch.
Industrial Media and the Nutrition of Industrial Organisms
o = glucose units joined by a-D-(1 ® 4) bonds
= non-reducing ends of chains
— = point of attack by enzyme
l
Fig. 4.5
Pattern of Attack of Alpha Amylase and Beta-amylase on Amylose and Amylopectin
Respectively
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(iii) Enzymes that hydrolyze (a —1, 4 and a — 1:6 bonds: The typical example of these
enzymes is amyloglucosidase or glucoamylase. This enzyme hydrolyzes a - D - (1
® 4) -D – glucosidic bonds from the non-reducing ends to yield D – glucose
molecules. When the sequential removal of glucose reaches the point of branching
in amylopectin, the hydrolysis continues on the (1 ® 6) bonding but more slowly
than on the (1 ® 4) bonding. Maltose is attacked only very slowly. The end product
is glucose.
(iv) De-branching enzymes: At least two de-branching enzymes are known: pullulanase
and iso-amylase.
Pullulanase: This is a de-branching enzyme which causes the hydrolysis of a — D
– (1 ® 6) linkages in amylopectin or in amylopectin previsouly attacked by alphaamylase. It does not attack a - D (1 ® 4) bonds. However, there must be at least two
glucose units in the group attached to the rest of the molecules through an a -D- (1
® 6) bonding.
Iso-amylase: This is also a de-branching enzyme but differs from pullulanase in that
three glucose units in the group must be attached to the rest of the molecules
through an a - D – (1 ® 6) bonding for it to function.
(v) Enzymes that preferentially attack a - 1, 4 linkages: Examples of this group are
glucosidases. The maltodextrins and maltose produced by other enzymes are
cleaved to glucose by a - glucosidases. They may however sometime attack
unaltered polysaccharides but only very slowly.
(vi) Enzymes which hydrolyze starch to non-reducing cyclic D-glucose polymers known as
cyclodextrins or Schardinger dextrins: Cyclic sugar residues are produced by Bacillus
macerans. They are not acted upon by most amylases although enzymes in
Takadiastase produced by Aspergillus oryzae can degrade the residues.
4.7.1.1.4 Industrial saccharification of starch by enzymes
In industry the extent of the conversion of starch to sugar is measured in terms of dextrose
equivalent (D.E.). This is a measure of the reducing sugar content, expressed in terms of
dextrose, determined under defined conditions involving Fehling’s solution. The D.E is
calculated as percentage of the total solids.
For the saccharification of starch in industry acid is being replaced more and more by
enzymes. Sometimes acid is used only initially and enzymes employed at a later stage.
Acid saccharification has a practical upper limit of 55 D.E. Beyond this, breakdown
products begin to accumulate. Furthermore, with acid hydrolysis reversion reactions
occur among the sugar produced. These two deficiencies are avoided when enzymes are
utilized. Besides, by selecting enzymes specific sugars can be produced.
Starch-splitting enzymes used in industry are produced in germinated seeds and by
micro-organisms. Barley malt is widely used for the saccharification of starch. It contains
large amounts of various enzymes notably b-amylase and a - glucosidase which further
split saccharides to glucose.
All the enzymes discussed above are produced by different micro-organisms and
many of these enzymes are available commercially. The most commonly encountered
organisms producing these enzymes are Bacillus spp, Streptomyeces spp, Aspergillus spp,
Penicillium spp, Mucor spp and Rhizopus spp.
Industrial Media and the Nutrition of Industrial Organisms
4.7.2
4.7.2.1
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Cellulose, Hemi-celluloses and Lignin in Plant Materials
Cellulose
Cellulose is the most abundant organic matter on earth. Unfortunately it does not exist
pure in nature and even the purest natural form (that found in cotton fibres) contains
about 6% of other materials. Three major components, cellulose, hemi-cellulose and
lignin occur roughly in the ratio of 4:3:3 in wood. Before looking more closely at cellulose,
the other two major components of plant materials will be briefly discussed.
4.7.2.2
Hemicelluloses
These are an ill-defined group of carbohydrates whose main and common characteristic
is that they are soluble in, and hence can be extracted with, dilute alkali. They can then be
precipitated with acid and ethanol. They are very easily hydrolyzed by chemical or
biological means. The nature of the hemicellulose varies from one plant to another. In
cotton the hemicelluloses are pectic substances, which are polymers of galactose. In
wood, they consist of short (DP less than 200) branched heteropolymers of glucose,
xylose, galactose, mannose and arabinose as well as uronic acids of glucose and
galactose linked by 1 – 3, 1 – 6 and 1 – 4 glycosidic bonding.
4.7.2.3
Lignin
Lignin is a complex three-dimensional polymer formed from cyclic alcohols. (Fig. 4.6). It
is important because it protects cellulose from hydrolysis.
Cellulose is found in plant cell-walls which are held together by a porous material
known as middle lamella. In wood the middle lamella is heavily impregnated with lignin
which is highly resistant and thus protects the cell from attack by enzymes or acid.
4.7.2.4
Pretreatment of cellulose-containing materials
before saccharification
In order to expose lignocellulosics to attack, a number of physical and chemical methods
are in use, or are being studied, for altering the fine structure of cellulose and/or breaking
the lignin-carbohydrate complex.
Table 4.8 Various pretreatment methods used in lignocellulose substrate preparation
Pretreatment type
Specific method
Mechanical
Irradiation
Thermal
Weathering and milling-ball, fitz, hammer, roller
Gamma, electron beam, photooxidation
Autohydrolysis, steam explosion, hydrothermolysis,
boiling, pyrolysis, moist or dry heat expansion
Sodium hydroxide, ammonium hydroxide
Sulfuric, hydrochloric, nitric, phosphoric, maleic
Peracetic acid, sodium hypochlorite, sodium chlorite,
hydrogen peroxide
butanol, phenol, ethylamine, acetone, ethylene glycol
Ammonia, chlorine, nitrous oxide, ozone, sulfur dioxide
Ligninolytic fungi
Alkali
Acids
Oxidizing agents
Solvents Ethanol,
Gases
Biological
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Modern Industrial Microbiology and Biotechnology
Fig. 4.6
Generalized Structure of Lignin
Chemical methods include the use of swelling agents such a NaOH, some amines,
concentrated H2SO4 or HCI or proprietary cellulose solvents such as ‘cadoxen’ (tris
thylene-diamine cadmium hydroxide). These agents introduce water between or within
the cellulose crystals making subsequent hydrolysis, easier. Steam has also been used as
a swelling agent. The lignin may be removed by treatment with dilute H2SO4 at high
temperature.
Physical methods of pretreatment include grinding, irradiation and simply heating
the wood.
4.7.2.5
Hydrolysis of cellulose
Following pretreatment, wood may be hydrolyzed with dilute HCI, H2SO4 or sulfites of
calcium, magnesium or sodium under high temperature and pressure as described for
sulfite liquor production in paper manufacture see section 4. above). When, however, the
aim is to hydrolyze wood to sugars, the treatment is continued for longer than is done for
paper manufacture.
A lot of experimental work has been done recently on the possible use of cellulolytic
enzymes for digesting cellulose. The advantage of the use of enzymes rather than harsh
chemicals methods have been discussed already. Fungi have been the main source of
cellulolytic enzymes. Trichoderma viride and T. koningii have been the most efficient
cellulase producers. Penicillicum funiculosum and Fusarium solani have also been shown
to possess equally potent cellulases. Cellulase has been resolved into at least three
components: C1, Cx, and b-glucosidases. The C1 component attacks crystalline cellulose
and loosens the cellulose chain, after which the other enzymes can attack cellulose. The
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Cx enzymes are b - (1 ® 4) glucanases and hydrolyse soluble derivatives of cellulose or
swoollen or partially degraded cellulose. Their attack on the cellulose molecule is
random and cellobiose (2-sugar) and cellotroise (3-sugar) are the major products of their
actions. There is evidence that the enzymes may also act by removing successive glucose
units from the end of a cellulose molecule. b-glucosidases hydrolyze cellobiose and shortchain oligo-saccharides derived from cellulose to glucose, but do not attack cellulose.
They are able to attack cellobiose and cellotriose rapidly. Many organisms described in
the literature as ‘cellulolytic’ produce only Cx and b-glucosidases because they were
isolated initially using partially degraded cellulose. The four organisms mentioned
above produce all three members of the complex.
5
4.7.2.4.1
Molecular structure of cellulose
Cellulose is a linear polymer of D-glucose linked in the Beta-1, 4 glucosidic bondage. The
bonding is theoretically as vulnerable to hydrolysis as the one in starch. However,
cellulose – containing materials such as wood are difficult to hydrolyze because of (a) the
secondary and tertiary arrangement of cellulose molecules which confers a high
crystallinity on them and (b) the presence of lignin.
The degree of polymerization (D. P.) of cellulose molecule is variable, but ranges from
about 500 in wood pulp to about 10,000 in native cellulose. When cellulose is hydrolyzed
with acid, a portion known as the amorphous portion which makes up 15% is easily and
quickly hydrolyzed leaving a highly crystalline residue (85%) whose DP is constant at
100-200. The crystalline portion occurs as small rod-like particles which can be
hydrolyzed only with strong acid. (Fig. 4.7)
A
B
C
D
= Original cellulose fibril
= Initial attack on amorphous region
= Residue crystalline region
= Attack on crystalline region
Fig. 4.7 Diagram Illustrating Breakdown of Crystalline Cellulose
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Modern Industrial Microbiology and Biotechnology
SUGGESTED READINGS
Barnes, A.C. 1974. The Sugar cane. Wiley, New York, USA.
Dahod, S.K. 1999. Raw Materials Selection and Medium Development for Industrial
Fermentation Processes. In: Manual of Industrial Microbiology and Biotechnology. A. L.
Demain and J. E. Davies (eds) American Society for Microbiology Press. 2nd Ed, Washington
DC.
Demain, A.L. 1998. Induction of microbial secondary Metabolism International Microbiology 1,
259–264.
Flickinger, M.C., Drew, S.W. (eds) 1999. Encyclopedia of Bioprocess Technology - Fermentation,
Biocatalysis, and Bioseparation, Vol 1-5. John Wiley, New York.
Ward, W.P., Singh, A. 2004. Bioethanol Technology: Developments and Perspectives Advance in
Applied Microbiology, 51, 53 – 80.
Metabolic Pathways for the Biosynthesis of Industrial Microbiology Products
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5
Metabolic Pathways for the
Biosynthesis of Industrial
Microbiology Products
5.1
THE NATURE OF METABOLIC PATHWAYS
In order to be able to manipulate microorganisms to produce maximally materials of
economic importance to humans, but at minimal costs, it is important that the physiology
of the organisms be understood as much as is possible. In this chapter relevant elements
of the physiology of industrial organisms will be discussed.
A yeast cell will divide and produce CO2 under aerobic conditions if offered a solution
of glucose and ammonium salts. The increase in cell number resulting from the growth
and the bubbling of CO2 are only external evidence of a vast number of chemical reactions
going on within the cell. The yeast cell on absorbing the glucose has to produce various
proteins which will form enzymes necessary to catalyze the various reactions concerned
with the manufacture of proteins, carbohydrates, lipids, and other components of the cell
as well as vitamins which will form coenzymes. A vast array of enzymes are produced as
the glucose and ammonium initially supplied are converted from one compound into
another or metabolized. The series of chemical reactions involved in converting a chemical (or a
metabolite) in the organism into a final product is known as a metabolic pathway. When the
reactions lead to the formation of a more complex substance, that particular form of
metabolism is known as anabolism and the pathway an anabolic pathway. When the
series of reactions lead to less complex compounds the metabolism is described as
catabolism. The compounds involved in a metabolic pathway are called intermediates and
the final product is known as the end-product (see Fig. 5.1).
Catabolic reactions have been mostly studied with glucose. Four pathways of glucose
breakdown to pyruvic acid (or glycolysis) are currently recognized. They will be
discussed later. Catabolic reactions often furnish energy in the form of ATP and
other high energy compounds, which are used for biosynthetic reactions. A second
function of catabolic reactions is to provide the carbon skeleton for biosynthesis.
Anabolic reactions lead to the formation of larger molecules some of which are
constituents of the cell.
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Modern Industrial Microbiology and Biotechnology
Fig. 5.1
Metabolism: Relationship between Anabolism and Catabolism in a Cell
Although anabolism and catabolism are distinct phenomena some pathways have
elements of both kinds Metabolic intermediates which are derived from catabolism and
which are also available for anabolism are known as amphibolic intermediates.
Methods for the study of metallic pathways are well reviewed in texts on microbial
physiology and will therefore not be discussed here.
5.2
INDUSTRIAL MICROBIOLOGICAL PRODUCTS AS
PRIMARY AND SECONDARY METABOLITES
Products of industrial microorganisms may be divided into two broad groups, those
which result from primary metabolism and others which derive from secondary
metabolism. The line between the two is not always clear cut, but the distinction is useful
in discussing industrial products.
5.2.1
Products of Primary Metabolism
Primary metabolism is the inter-related group of reactions within a microorganism
which are associated with growth and the maintenance of life. Primary metabolism is
essentially the same in all living things and is concerned with the release of energy, and
Metabolic Pathways for the Biosynthesis of Industrial Microbiology Products
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the synthesis of important macromolecules such as proteins, nucleic acids and other cell
constituents. When primary metabolism is stopped the organism dies.
Products of primary metabolism are associated with growth and their maximum
production occurs in the logarithmic phase of growth in a batch culture. Primary
catabolic products include ethanol, lactic acid, and butanol while anabolic products
include amino-acids, enzymes and nucleic acids. Single-cell proteins and yeasts would
also be regarded as primary products (Table 5.1)
Table 5.1
Some industrial products resulting from primary metabolism
Anabolic Products
1.
2.
3.
4.
5.
6.
7.
8.
Enzymes
Amino acids
Vitamins
Polysaccharides
Yeast cells
Single cell protein
Nucleic acids
Citric acid
5.2.2
Catabolic Products
1.
2.
3.
4.
5.
Ethanol and ethanol-containing products, e.g. wines
Butanol
Acetone
Lactic acid
Acetic acid (vinegar)
Products of Secondary Metabolism
In contrast to primary metabolism which is associated with the growth of the cell and the
continued existence of the organism, secondary metabolism, which was first observed in
higher plants, has the following characteristics (i) Secondary metabolism has no apparent
function in the organism. The organism continues to exist if secondary metabolism is
blocked by a suitable biochemical means. On the other hand it would die if primary
metabolism were stopped. (ii) Secondary metabolites are produced in response to a
restriction in nutrients. They are therefore produced after the growth phase, at the end of
the logarithmic phase of growth and in the stationary phase (in a batch culture). They can
be more precisely controlled in a continuous culture. (iii) Secondary metabolism appears
to be restricted to some species of plants and microorganisms (and in a few cases to
animals). The products of secondary metabolism also appear to be characteristic of the
species. Both of these observations could, however, be due to the inadequacy of current
methods of recognizing secondary metabolites. (iv) Secondary metabolites usually have
‘bizarre’ and unusual chemical structures and several closely related metabolites may be
produced by the same organism in wild-type strains. This latter observation indicates the
existence of a variety of alternate and closely-related pathways. (v) The ability to produce
a particular secondary metabolite, especially in industrially important strains is easily
lost. This phenomenon is known as strain degeneration. (vi) Owing to the ease of the loss
of the ability to synthesize secondary metabolites, particularly when treated with acridine dyes, exposure to high temperature or other treatments known to induce plasmid
loss (Chapter 5) secondary metabolite production is believed to be controlled by plasmids
(at least in some cases) rather than by the organism’s chromosomes. A confirmation of the
possible role of plasmids in the control of secondary metabolites is shown in the case of
leupetin, in which the loss of the metabolite following irradiation can be reversed by
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Modern Industrial Microbiology and Biotechnology
conjugation with a producing parent. (vii) The factors which trigger secondary metabolism, the inducers, also trigger morphological changes (morphogenesis) in the organism.
Inducers of Secondary Metabolites
Autoinducers include the g-butyrolactones (butanolides) of the actinomycetes, the Nacylhomoserine lactones (HSLs) of Gramnegative bacteria, the oligopeptides of Grampositive bacteria, and B-factor [3’-(1-butylphosphoryl)adenosine] of rifamycin
production in Amycolatopsis mediterrane. They function in development, sporulation,
light emission, virulence, production of antibiotics, pigments and cyanide, plasmiddriven conjugation and competence for genetic transformation. Of great importance in
actinomycete fermentations is the inducing effect of endogenous g-butyrolactones, e.g. Afactor (2-S-isocapryloyl-3R-hydroxymethyl-g-butyrolactone). A-factor induces both
morphological and chemical differentiation in Streptomyces griseus and Streptomyces
bikiniensis, bringing on formation of aerial mycelia, conidia, streptomycin synthases and
streptomycin. Conidia can actually form on agar without A-factor but aerial mycelia
cannot. The spores form on branches morphologically similar to aerial hyphae but they
do not emerge from the colony surface. In S. griseus, A-factor is produced just prior to
streptomycin production and disappears before streptomycin is at its maximum level. It
induces at least 10 proteins at the transcriptional level. One of these is streptomycin 6phosphotransferase, an enzyme which functions both in streptomycin biosynthesis and
in resistance. In an A-factor deficient mutant, there is a failure of transcription of the
entire streptomycin gene cluster. Many other actinomycetes produce A-factor, or related
a-butyrolactones, which differ in the length of the side-chain. In those strains which
produce antibiotics other than streptomycin, the g-butyrolactones induce formation of the
particular antibiotics that are produced, as well as morphological differentiation.
Secondary metabolic products of microorganism are of immense importance to
humans. Microbial secondary metabolites include antibiotics, pigments, toxins, effectors
of ecological competition and symbiosis, pheromones, enzyme inhibitors,
immunomodulating agents, receptor antagonists and agonists, pesticides, antitumor
agents and growth promoters of animals and plants, including gibbrellic acid, antitumor agents, alkaloids such as ergometrine, a wide variety of other drugs, toxins and
useful materials such as the plant growth substance, gibberellic acid (Table 5.2). They
have a major effect on the health, nutrition, and economics of our society. They often have
unusual structures and their formation is regulated by nutrients, growth rate, feedback
control, enzyme inactivation, and enzyme induction. Regulation is influenced by unique
low molecular mass compounds, transfer RNA, sigma factors, and gene products formed
during post-exponential development. The synthases of secondary metabolism are often
coded for by clustered genes on chromosomal DNA and infrequently on plasmid DNA.
Unlike primary metabolism, the pathways of secondary metabolism are still not
understood to a great degree. Secondary metabolism is brought on by exhausion of a
nutrient, biosynthesis or addition of an inducer, and/or by a growth rate decrease. These
events generate signals which effect a cascade of regulatory events resulting in chemical
differentiation (secondary metabolism) and morphological differentiation
(morphogenesis). The signal is often a low molecular weight inducer which acts by
negative control, i.e. by binding to and inactivating a regulatory protein (repressor
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Table 5.2 Some industrial products of microbial secondary metabolism
Product
Organism
Use/Importance
Antibiotics
Penicillin
Streptomycin
Penicillium chrysogenum
Streptomyces griseus
Clinical use
Clinical use
Anti-tumor Agents
Actinomyin
Bleomycin
Streptomyces antibioticus
Streptomyces verticulus
Clinical use
Clinical use
Toxins
Aflatoxin
Amanitine
Aspergiulus flavous
Amanita sp
Food toxin
Food toxin
Alkaloids
Ergot alkaloids
Claviceps purpurea
Pharmaceutical
Miscellaneous
Gibberellic acid
Kojic acid
Muscarine
Patulin
Gibberalla fujikuroi
Aspergillus flavus
Clitocybe rivalosa
Penicillium urticae
Plant growth hormone
Food flavor
Pharmaceutical
Anti-microbial agent
protein/receptor protein) which normally prevents secondary metabolism and
morphogenesis during rapid growth and nutrient sufficiency.
Thousands of secondary metabolites of widely different chemical groups and
physiological effects on humans have been found. Nevertheless a disproportionately
high interest is usually paid to antibiotics, although this appears to be changing. It would
appear that the vast potential utility of microbial secondary metabolites is yet to be
realized and that many may not even have been discovered. Part of this ‘lopsided’
interest may be due to the method of screening, which has largely sought antibiotics. The
general topic of screening, especially of secondary metabolites, will be discussed in
Chapters 7 and 28. In particular, an attempt will be made to discuss the screening of
drugs outside antibiotics.
5.3
TROPHOPHASE-IDIOPHASE RELATIONSHIPS IN THE
PRODUCTION OF SECONDARY PRODUCTS
From studies on Penicillium urticae the terms trophophase and idiophase were introduced
to distinguish the two phases in the growth of organisms producing secondary
metabolites. The trophophase (Greek, tropho = nutrient) is the feeding phase during
which primary metabolism occurs. In a batch culture this would be in the logarithmic
phase of the growth curve. Following the trophophase is the idio-phase (Greek, idio =
peculiar) during which secondary metabolites peculiar to, or characteristic of, a given
organism are synthesized. Secondary synthesis occurs in the late logarithmic, and in the
stationary, phase. It has been suggested that secondary metabolites be described as
‘idiolites’ to distinguish them from primary metabolites.
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5.4
ROLE OF SECONDARY METABOLITES IN THE
PHYSIOLOGY OF ORGANISMS PRODUCING THEM
Since many industrial microbiological products result from secondary metabolism,
workers have sought to explain the role of secondary metabolites in the survival of the
organism. Due to the importance of antibiotics as clinical tools, the focus of many workers
has been on antibiotics. This discussion while including antibiotics will attempt to
embrace the whole area of secondary metabolites.
Some earlier hypotheses for the existence of secondary metabolism are apparently no
longer considered acceptable by workers in the field. These include the hypotheses that
secondary metabolites are food-storage materials, that they are waste products of the
metabolism of the cell and that they are breakdown products from macro-molecules. The
theories in currency are discussed below; even then none of these can be said to be water
tight. The rationale for examining them is that a better understanding of the organism’s
physiology will help towards manipulating it more rationally for maximum productivity.
(i) The competition hypothesis: In this theory which refers to antibiotics specifically,
secondary metabolites (antibiotics) enable the producing organism to withstand
competition for food from other soil organisms. In support of this hypothesis is the
fact that antibiotic production can be demonstrated in sterile and non-sterile soil,
which may or may not have been supplemented with organic materials. As further
support for this theory, it is claimed that the wide distribution of b-lactamases
among microorganisms is to help these organisms detoxify the b-lactam
antibiotics. The obvious limitation of this theory is that it is restricted to antibiotics
and that many antibiotics exist outside Beta-lactams.
(ii) The maintenance hypothesis: Secondary metabolism usually occurs with the
exhaustion of a vital nutrient such as glucose. It is therefore claimed that the
selective advantage of secondary metabolism is that it serves to maintain
mechanisms essential to cell multiplication in operative order when that cell
multiplication is no longer possible. Thus by forming secondary enzymes, the
enzymes of primary metabolism which produce precursors for secondary
metabolism therefore, the enzymes of primary metabolism would be destroyed. In
this hypothesis therefore, the secondary metabolite itself is not important; what is
important is the pathway of producing it.
(iii) The unbalanced growth hypothesis: Similar to the maintenance theory, this
hypothesis states that control mechanisms in some organisms are too weak to
prevent the over synthesis of some primary metabolites. These primary metabolites
are converted into secondary metabolites that are excreted from the cell. If they are
not so converted they would lead to the death of the organism.
(iv) The detoxification hypothesis: This hypothesis states that molecules accumulated
in the cell are detoxified to yield antibiotics. This is consistent with the observation
that the penicillin precursor penicillanic acid is more toxic to Penicillium
chrysogenum than benzyl penicillin. Nevertheless not many toxic precursors of
antibiotics have been observed.
(v) The regulatory hypothesis: Secondary metabolite production is known to be
associated with morphological differentiation in producing organisms. In the
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fungus Neurospora crassa, carotenoids are produced during sporulation. In
Cephalospoium acremonium, cephalosporin C is produced during the idiophase
when arthrospores are produced. Numerous examples of the release of secondary
metabolites with some morphological differentiation have been observed in fungi.
One of the most intriguing relationships between differentiation and secondary
metabolite production, is that between the production of peptide antibiotics by
Bacillus spp. and spore formation. Both spore formation and antibiotic production
are suppressed by glucose; non-spore forming mutants of bacilli also do not
produce antibiotics, while reversion to spore formation is accompanied by
antibiotic formation has been observed in actinomycetes. Many roles have been
assigned to antibiotics in spore formers but the most clearly demonstrated has been
the essential nature of gramicidin in sporulation of Bacillus spp. The absence of the
antibiotic leads to partial deficiencies in the formation of enzymes involved in
spore formation, resulting in abnormally heat-sensitive spores. Peptide antibiotics
therefore suppress the vegetative genes allowing proper development of the
spores. In this theory therefore the production of secondary metabolites is
necessary to regulate some morphological changes in the organism. It could of
course be that some external mechanism triggers off secondary metabolite
production as well as the morphological change.
(vi) The hypothesis of secondary metabolism as the expression of evolutionary
reactions: Zahner has put forth a most exciting role for secondary metabolism. To
appreciate the hypothesis, it is important to bear in mind that both primary and
secondary metabolism are controlled by genes carried by the organism. Any genes
not required are lost. According to this hypothesis, secondary metabolism is a
clearing house or a mixed bag of biochemical reactions, undergoing tests for
possible incorporation into the cell’s armory of primary reactions. Any reaction in
the mixed bag which favorably affects any one of the primary processes, thereby
fitting the organism better to survive in its environment, becomes incorporated as
part of primary metabolism. According to this hypothesis, the antibiotic properties
of some secondary metabolites are incidental and not a design to protect the
microorganisms. This hypothesis is attractive because it implies that secondary
metabolism must occur in all microorganisms since evolution is a continuing
process. If that is the case, then the current range of secondary metabolites is
limited only by techniques sensitive enough to detect them. That this is a
possibility is shown by the increase in the number of antibiotics alone, since new
methods were recently introduced in the processes used in screening for them. If
therefore adequate methods of detection are devised it is possible that more
secondary metabolites of use for humans could be found.
5.5
PATHWAYS FOR THE SYNTHESIS OF
PRIMARY AND SECONDARY METABOLITES OF
INDUSTRIAL IMPORTANCE
The main source of carbon and energy in industrial media is carbohydrates. In recent
times hydrocarbons have been used. The catabolism of these compounds will be
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discussed briefly because they supply the carbon skeletons for the synthesis of primary
as well as for secondary metabolites. The inter-relationship between the pathways of
primary and the secondary metabolism will also be discussed briefly.
5.5.1
Catabolism of Carbohydrates
Four pathways for the catabolism of carbohydrates up to pyruvic acid are known. All
four pathways exist in bacteria, actinomycets and fungi, including yeasts. The four
pathways are the Embden-Meyerhof-Parmas, the Pentose Phosphate Pathways, the
Entner Duodoroff pathway and the Phosphoketolase. Although these pathways are for
the breakdown of glucose. Other carbohydrates easily fit into the cycles.
(i) The Embden-Meyerhof-Parnas (EMP Pathways): The net effect of this pathway is
to reduce glucose (C6) to pyruvate (C3) (Fig. 5.2). The system can operate under both
aerobic and anaerobic conditions. Under aerobic conditions it usually functions
with the tricarboxylic acid cycle which can oxidize pyruvate to CO2 and H2O.
Under anaerobic conditions, pyruvate is fermented to a wide range of fermentation
products, many of which are of industrial importance (Fig. 5.3).
(ii) The pentose Phosphate Pathway (PP): This is also known as the Hexose
Monophosphate Pathway (HMP) or the phosphogluconate pathway. While the
EMP pathway provides pyruvate, a C3 compound, as its end product, there is no
end product in the PP pathway. Instead it provides a pool of triose (C3) pentose
(C5), hexose (C6) and heptose (C7) phosphates. The primary purpose of the PP
pathway, however, appears to be to generate energy in the form of NADPA2 for
biosynthetic and other purposes and pentose phosphates for nucleotide synthesis
(Fig. 5.4)
(iii) The Entner-Duodoroff Pathway (ED): The pathway is restricted to a few bacteria
especially Pseudomonas, but it is also carried out by some fungi. It is used by some
organisms in the enaerobic breakdown of glucose and by others only in gluconate
metabolism (Fig. 5.5)
(iv) The Phosphoketolase Pathway: In some bacteria glucose fermentation yields lactic
acid, ethanol and CO2. Pentoses are also fermented to lactic acid and acetic acid.
An example is Leuconostoc mesenteroides (Fig. 5.6).
Pathways used by microorganisms
The two major pathways used by microorganisms for carbohydrate metabolism are the
EMP and the PP pathways. Microorganisms differ in respect of their use of the two
pathways. Thus Saccharomyces cerevisae under aeaobic conditions uses mainly the EMP
pathway; under anaerobic conditions only about 30% of glucose is catabolized by this
pathway. In Penicillium chrysogenum, however, about 66% of the glucose is utilized via the
PP pathway. The PP pathway is also used by Acetobacter, the acetic acid bacteria.
Homofermentative bacteria utilize the EMP pathway for glucose breakdown. The ED
pathway is especially used by Pseudomonas.
Metabolic Pathways for the Biosynthesis of Industrial Microbiology Products
Fig. 5.2 The Embden-Meyerhof – Parnas Pathway
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A
B
C
D
E
F
G
H
I
(End product, Lactate)
(End product, Acrylate)
(End product, Ethanol)
(Formic acid, H2, CO 2, Ethanol)
(H2, CO2, Ethanol)
(Acetoin, 2-3 Butanediol)
(Acetoin, 2-3 Butanediol)
(Acetone, Isoprpanol, Acetone)
(Propioninate)
Fig. 5.3
Lactic acid bacteria
Clostridium propinicum
Yeasts, Acetobacter, Zymomonas
Enterobacteriaceae
Clostridia
Aerobacter
Yeasts
Clostridia (butyric acid)
Propionic acid bacteria
Products of the Fermentation of Pyruvate by Different Microorganisms
Metabolic Pathways for the Biosynthesis of Industrial Microbiology Products
Fig. 5.4
The Pentose Phosphate Pathway
Fig. 5.5 The Enter-Doudoroff Pathway
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Fig. 5.6
5.5.2
The Phosphoketolase Pathway
The Catabolism of Hydrocarbons
Although the price of crude oil continues to rise, it is, along with other hydrocarbons still
used in some fermentations as energy and carbon skeleton sources. Compared with
carbohydrates, however, far fewer organism appear to utilize hydrocarbons.
Hydrocarbons have been used in single cell protein production and in amino-acid
production among other products. Their use by various organisms in industrial media is
discussed more fully in Chapter 15.
(i) Alkanes: Alkanes are saturated hydrocarbons that have the general formula
C2 Hn+2. When the alkanes are utilized, the terminal methyl group is usually
oxidized to the corresponding primary alcohol thus:
Metabolic Pathways for the Biosynthesis of Industrial Microbiology Products
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R.CH2CH2CH3 ® R.CH2 CH2 CH2 OH ® R CH2 CHO ® R.CH2 COOH
Alkane
Alcohol
Aldehyde
Fatty acid
The alcohol is then oxidized to a fatty acid, which then forms as ester with
coenzyme A. Thereafter, it is involved in a series of b-oxidations (Fig. 5.7) which
lead to the step-wise cleaving off of acetyl coenzyme A which is then further
metabolized in the Tricarboxylic Acid Cycle.
(ii) Alkenes: The alkenes are unsaturated hydrocarbons and contain many double
bonds. Alkenes may be oxidized at the terminal methyl group as shown earlier for
alkanes. They may also be oxidized at the double bond at the opposite end of the
molecule by molecular oxygen given rise to a diol (an alcohol with two –OH
groups). Thereafter, they are converted to fatty acid and utilized as indicated
above.
5.6
CARBON PATHWAYS FOR THE FORMATION OF
SOME INDUSTRIAL PRODUCTS DERIVED FROM
PRIMARY METABOLISM
The broad flow of carbon in the formation of industrial products resulting from primary
metabolism may be examined under two headings: (i) catabolic products resulting from
fermentation of pyruvic acid and (ii) anabolic products.
5.6.1
Catabolic Products
Industrial products which are catabolic products formed from carbohydrate
fermentation are derived from pyruvic acid produced via the EMP, PP, or ED pathway.
Those of importance are ethanol, acetic acid, 2, 3-butanediol, butanol, acetone and lactic
acid. The general outline for deriving these from pyruvic acid has already been shown in
Fig. 5.3. The nature of the products not only broadly depends on the species of organisms
used but also on the prevailing environmental conditions such as pH, temperature,
aeration, etc.
5.6.2
Anabolic Products
Anabolic primary metabolites of industrial interest include amino acids, enzymes, citric
acid, and nucleic acids. The carbon pathways for the production of anabolic primary
metabolites will be discussed as each product is examined.
5.7
CARBON PATHWAYS FOR THE FORMATION OF
SOME PRODUCTS OF MICROBIAL SECONDARY
METABOLISM OF INDUSTRIAL IMPORTANCE
The unifying features of the synthesis of secondary metabolic products by microorganisms can be summarized thus:
(i) conversion of a normal substrate into important intermediates of general
metabolism;
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Modern Industrial Microbiology and Biotechnology
Fig. 5.7 b-oxidation of Fatty Acids
(ii) the assembly of these intermediates in an unusual way, by means of a combination
of standard general mechanisms with a selection from a relatively small number of
special mechanism;
(iii) these special mechanisms while being peculiar to secondary metabolism are not
unrelated to general or primary mechanism;
(iv) the synthetic activity of secondary metabolism appears in response to conditions
favorable for cell multiplication.
From the above, it becomes clear that although secondary metabolites are diverse in
their intrinsic chemical nature as well as in the organism which produce them, they use
only a few biosynthetic pathways which are related to, and use the intermediates of, the
primary metabolic pathways. Based on the broad flow of carbon through primary
metabolites to secondary metabolites, (depicted in Fig. 5.8) the secondary metabolites
may then be classified according to the following six metabolic pathways.
(i) Secondary products derived from the intact glucose skeleton: The carbon skeleton of
glucose is incorporated unaltered in many antibiotics and other secondary
metabolites. The entire basic structure of the secondary product may be derived
from glucose as in streptomycin or it may form the glycoside molecule to be
combined with a non-sugar (aglycone portion) from another biosynthetic route.
Metabolic Pathways for the Biosynthesis of Industrial Microbiology Products
Main carbon routes
Primary metabolic routes
Secondary metabolic routes
Compounds in heavy type = secondary metabolites.
Biosynthetic Routes Between Primary and Secondary Metabolites
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Fig. 5.8
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Modern Industrial Microbiology and Biotechnology
The incorporation of the intact glucose molecule is more common among the
actinomycetes than among the fungi.
(ii) Secondary products related to nucleosides: The pentose phosphate pathway
provides ribose (5 carbon) for nucleoside biosynthesis. Many secondary
metabolites in this group are antibiotics and are produced mainly by
actinomycetes and fungi. Examples are nucleoside antibiotics such as bleomycin.
(Chapter 21).
(iii) Secondary products derived through the Shikimate-Chorismate Pathway:
Shikimic acid (C7) is formed by the condensation of erythrose-4- phosphate (C4)
obtained from the PP pathway with phosphoenolypyruvate (C3) from the EMP
pathway. It is converted to chorismic acid which is a key intermediate in the
formation of numerous products including aromatic aminoacids, such as
phynylalamine, tryrosine and tryptophan. Chorismic acid is also a precursor for a
number of secondary metabolites including chloramphenicol, p-amino benzoic
acid, phenazines and pyocyanin which all have anticrobial properties (Fig. 5.9).
The metabolic route leading to the formation of these compounds is therefore
Fig. 5.9 Metabolites in the Shikimic-Chorismate Pathway
Metabolic Pathways for the Biosynthesis of Industrial Microbiology Products
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referred to as the shikimate pathway. In view of this central role of chorismic acid,
however, the route is more widely known as the shikimate-chorismate route. The
shikimate-chorismate route is an important route for the formation of aromatic
secondary products in the bacteria and actinomycetes. Examples of such
secondary products include chloramphenicol and novobiocin. The route is less
used in fungi, where the polyketide pathway is more common for the synthesis of
aromatic secondary products.
(iv) The polyketide pathway: polyketide biosynthesis is highly characteristic of the
fungi, where more secondary metabolites are produced by it than by any other.
Indeed most of the known polyketide-derived natural products have been obtained
from the fungi, a much smaller number being obtained from bacteria and higher
plants. The triose (C3) derived from glucose in the EMP pathway is converted via
pyruvic acid to acetate, which occupies a central position in both primary and
secondary synthesis. The addition of CO2 to an acetate group gives a malonate
group. The synthesis of polyketides is very similar to that of fatty acids. In the
synthesis of both groups of compounds acetate reacts with malonate with the loss
of CO2. By successive further linear reactions between the resulting compound and
malonate, the chain of the final compound (fatty acid or polyketide) can be
successively lengthened.
However, in the case of fatty acid the addition of each malonate molecule is
followed by decarboxylation and reduction whereas in polyketides these latter
reactions do occur. Due to this a chain of ketones or a b-polyketomethylene (hence
the name polyketide) is formed (Fig. 5.10). The polyketide (b - poly-ketomethylene)
Fig. 5.10 Formation of Polyketides
chain made up of repeating C-CH2 or ‘C2 units’, is a reactive protein-bound
intermediate which can undergo a number of reactions, notably formation into
rings. Polyketides are classified as triketides, tetraketides, pentaketides, etc.,
depending on the number of ‘C2 units’. Thus, orsellenic acid which is derived from
the straight chain compound in Fig. 5.11 with four ‘C2-units’ is a tetraketide.
Although the polyketide route is not common in actinomycetes, a modified
polyketide route is used in the synthesis of tetracyclines by Streptomyces griseus.
(v) Terpenes and steroids: The second important biosynthetic route from acetate is that
leading via mevalonic acid to the terpenes and steroids. Microorganisms
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Modern Industrial Microbiology and Biotechnology
Fig. 5.11 Formation of the Triketide, Orsellenic Acid
especially fungi and bacteria synthesize a large number of terpenes, steroids,
carotenoids and other products following the ‘isoprene rule’. The central point of
this rule is that these compounds are all derivatives of isoprene, the five-carbon
compound.
Simply put the isoprene rules consist of the following (Fig. 5.12):
(i) Synthesis of mevalonate from acetate or leucine
(ii) Dehydratopm and decarboxylation to give isoprene followed by
condensation to give isoprenes of various lengths.
(iii) Cyclization (ring formation) e.g., to give steroids (Chapter 26)
Fig. 5.12 Isoprene Derivatives
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(iv) Further modification of the cyclised structure. The route leads to the
formation of essential steroid hormones of mammals and to a variety of
secondary metabolites in fungi and plants. it is not used to any extent in the
actinomycetes.
(vi) Compounds derived from amino acids: The amino acids are derived from various
products in the catabolism of glucose. Serine (C3N) and glycine (C2N) are derived
from the triose (C3) formed glucose; valine (C5N) is derived from acetate (C3);
aspartatic acid (C4N) is derived from oxeloacetic acid (C4) while glutamic acid
(C5N) is derived from oxoglutamic acid (C5). The biosynthetic pathways for the
formation of amino acids are shown in Fig. 5.13 from which it will be seen that
aromatic amino acids are derived via the shikimic pathway.
Secondary products may be formed from one, two or more amino acids. an example of
the first group (with one amino acid group) is hadacidin which inhibits plant tumors and
Fig. 5.13 Synthetic Routes of the Amino Acids
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Modern Industrial Microbiology and Biotechnology
is produced from glycine and produced by Penicillium frequentants according to the
formula shown below
H2NCH2CO2H ® HN(OH) CH2 CO2H ® OHCN(OH) CH2 CO2H
Glycine
Hadacidin
Other examples are the insecticidal compound, ibotenic acid (Amanita factor C)
produced by the mushroom Amanita muscaria and psilocybin, a drug which causes
hallucinations and produced by the fungus Psiolocybe (Fig. 5.14), the ergot alkaloids
(Chapter 25) produced by Clavicepts purpureae also belong in this group as does the
antibiotic cycloserine.
Among the secondary products derived from two amino acids are gliotoxin which is
produced by members of the Fungi Imperfecti, especially Trichoderma and which is a
highly active anti-fungal and antibacterial (Fig. 5.14) and Arantoin, an antiviral drug
also belongs to this group.
Top:
Middle:
Bottom:
Ibotenic acid (from one amino acid)
Indole (from one amino acid)
Gliotoxin (from two amino acids)
Fig. 5.14 Secondary Metabolites Formed from Amino Acids
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The secondary products derived from more than two amino acids include many
which are of immense importance to man. These include many toxins from mushrooms
e.g the Aminita toxins (Fig. 5.15) (phalloidin, amanitin) peptide antibiotics from Bacillus
app and a host of other compounds.
An example of a secondary metabolite produced from three amino acids is malformin
A (Fig. 5.15) which is formed by Aspergillus spp. It induces curvatures of beam shoots and
maize seedlings. It is formed from L-leucine, D-leucine, and cysteine.
Top: Malformin A, a secondary metabolite formed from three amino acids
Bottom: Amanitin, a secondary metabolite formed from two amino acids
Fig. 5.15 Secondary Metabolites from Amino Acids
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SUGGESTED READINGS
Bull, A.T., Ward, A.C., Goodfellow, M. 2000. Search and Discovery Strategies for Biotechnology:
The Paradigm Shift Microbiology and Molecular Biology Reviews 64, 573 – 606.
Demain, A.L. 1998. Induction of microbial secondary Metabolism International Microbiology 1,
259–264.
Herrmann, K.H., Weaver, L.M. 1999. The Shikimate Pathway. Annual Review of Plant
Physiology and Plant Molecular Biology. 50, 473–503.
Madigan, M.T., Martinko, J.M. 2006. Brock Biology of Micro-organisms. Pearson Prentice Hall
Upper Saddle River, USA.
Meurer, G., Hutchinson, C.R. 1999. Genes for the Synthesis of Microbial Secondary M etabolites.
In: Manual of Industrial Microbiology and Biotechnology. A.L. Demain and J.E. Davies, (eds).
ASM Press. 2nd Ed. Washington, DC, USA pp. 740-758.
Zahner, H. 1978. In: Antibiotics and other Secondary Metabolites. R. Hutter, T. Leisenger, J.
Nuesch, W. Wehrli (eds). Academic Press, New York, USA, pp. 1-17.
Overproduction of Metabolites of Industrial Microorganisms
+0)26-4
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6
Overproduction of
Metabolites of Industrial
Microorganisms
The complexity of the activities which go on within a cell was mentioned at the beginning
of Chapter 5 when we discussed the metabolism of a yeast cell introduced into an
aqueous solution of glucose and ammonium salts. The yeast cell must first permit the
entry into itself of the glucose and ammonium salts. Under suitable environmental
conditions such as pH and temperature it will grow by budding within about half an
hour. For these buds to occur, hundreds of activities will have gone on within the cell.
New proteins to be incorporated into enzymes and other structures will have been
synthesized; nucleic acids for the chromosomes and carbohydrates for the cell walls will
all have been synthesized. Hundreds of different enzymes will have participated in these
synthetic activities. The organism must synthesize each of the compounds at the right
time and in the appropriate quantities. If along side ammonium salts, amino acids were
supplied, the yeast cells would stop absorbing the ammonium salt and instead utilize the
supplied ‘readymade’ substrate.
A few yeasts can utilize starch. If our yeast belonged to this group and was supplied
nothing but starch and ammonium salts, it would secrete extracellular enzyme(s) to
breakdown the starch to sugars. These sugars would then be absorbed and would be
used with ammonium salts, for the synthetic activities we described earlier.
Clearly therefore, while the organism’s genetic apparatus determines in broad terms
the organism’s overall synthetic potentialities, what is actually synthesized depends on
what is available in the environment. Most importantly, the organism is not only able to
‘decide’ when to manufacture and secrete certain enzymes to enable it to utilize materials
in the environment, but it is able to decide to stop the synthesis of certain compounds if
they are supplied to it. These sensing mechanisms for the switching on and off of the
synthetic processes enable the organism to avoid the overproduction of any particular
compound. If it did not have these regulatory mechanisms it would waste energy and
resources (which are usually scarce in natural environments) in making materials it did
not require.
An efficient or ’stringent’ organism which does not waste its resources in producing
materials it does not require will survive well in natural environments where competition
Modern Industrial Microbiology and Biotechnology
is intense. Such an organism while surviving well in nature would not, however, be of
much use as an industrial organism. The industrial microbiologist or biotechnologist
prefers, and indeed, seeks, the wasteful, inefficient and ‘relaxed’ organism whose
regulatory mechanisms are so poor that it will overproduce the particular metabolite
sought. Knowledge of these regulatory mechanisms and biosynthetic pathways is
essential, therefore, to enable the industrial microbiologist to derange and disorganize
them so that the organism will overproduce desired materials.
In this chapter the processes by which the organism regulates itself and avoids overproduction using enzyme regulation and permeability control will first be discussed.
Then will follow a discussion of methods by which the microbiologist consciously
deranges these two mechanisms to enable overproduction. Genetic manipulation of
organisms will be discussed in the next chapter.
Regulatory methods and ways of disorganizing microorganisms for the overproduction of metabolites are far better understood in primary metabolites than they are
in secondary metabolites. Indeed for some time it was thought that secondary metabolites
did not need to be regulated since the microorganisms had no apparent need for them.
They are currently better understood and it is now known that they are also regulated.
In the discussions that follow, primary metabolites will first be considered. Only a
minimum of examples will be given in respect of regulatory mechanisms of primary
metabolites. Textbooks on microbial physiology may be consulted for the details.
6.1
MECHANISMS ENABLING MICROORGANISMS TO AVOID
OVERPRODUCTION OF PRIMARY METABOLIC
PRODUCTS THROUGH ENZYME REGULATION
Some of the regulatory mechanisms enabling organisms to avoid over-production are
given in Table 6.1. Each of these will be discussed briefly.
Table 6.1
Regulatory mechanisms in microorganisms
1. Substrate Induction
2. Catabolite Regulation
2.1 Repression
2.2 Inhibition
3. Feedback Regulation
3.1 Repression
3.2 Inhibition
3.3 Modifications used in branched pathways
3.3.1 Concerted (multivalent) feedback regulation
3.3.2 Cooperative feedback inhibition
3.3.3 Cumulative feedback regulation
3.3.4 Compensatory feedback regulation
3.3.5 Sequential feedback regulation
3.3.6 Isoenzyme feedback regulation
4. Amino acid Regulation of RNA synthesis
5. Energy Charge Regulation
6. Permeability Control
Overproduction of Metabolites of Industrial Microorganisms
6.1.1
Substrate Induction
Some enzymes are produced by microorganisms only when the substrate on which they
act is available in the medium. Such enzymes are known as inducible enzymes.
Analogues of the substrate may act as the inducer. When an inducer is present in the
medium a number of different inducible enzymes may sometimes be synthesized by the
organism. This happens when the pathway for the metabolism of the compound is based
on sequential induction. In this situation the organism is induced to produce an enzyme
by the presence of a substrate. The intermediate resulting from the action of this enzyme
on the substrate induces the production of another enzyme and so on until metabolism is
accomplished. The other group of enzymes is produced whether or not the substrate on
which they act, are present. These enzymes are known as constitutive.
Enzyme induction enables the organism to respond rapidly, sometimes within
seconds, to the presence of a suitable substrate, so that unwanted enzymes are not
manufactured.
Molecular basis for enzyme induction: The molecular mechanism for the rapid response
of an organism to the presence of an inducer in the medium relates to protein synthesis
since enzymes are protein in nature. Two models exist for explaining on a molecular
basis the expression of genes in protein synthesis: one is a negative control and the other
positive. The negative control of Jacob and Monod first published in 1961 is the better
known and more widely accepted of the two and will be described first.
6.1.1.1
The Jacob-Monod Model of the (negative) control of
protein synthesis
In this scheme (Fig. 6.1) the synthesis of polypeptides and hence enzymes protein is
regulated by a group of genes known as the operon and which occupies a section of the
chromosomal DNA. Each operon controls the synthesis of a particular protein. An
operon includes a regulator gene (R) which codes for a repressor protein. The repressor
can bind to the operator gene (O) which controls the activity of the neighboring structural
genes (S). The production of the enzymes which catalyze the transcription of the message
on the DNA into mRNA (namely, RNA polymerase) is controlled by the promoter gene
(P). If the repressor protein is combined with the operator gene (O) then the movement of
RNA polymerase is blocked and RNA complementary to the DNA in the structural genes
(S) cannot be made. Consequently no polypeptide and no enzyme will be made. In the
absence of the attachment of the repressor to the operator gene, RNA polymerase from the
promoter can move to, and transcribe the structural genes, S.
Inducible enzymes are made when an inducer is added. Inducers inactivate or remove
the repressor protein thus leaving the way clear for protein synthesis. Constitutive
enzymes occur where the regulator gene (R) does not function, produces an inactive
repressor, or produces a repressor to which the operator cannot bind. Often more than
one structural gene may be controlled by a given operator.
Mutations can occur in the regulator (R) and operator (O) genes thus altering the
nature of the repressor or making it impossible for an existing repressor to bind onto the
operator. Such a mutation is called constitutive and it eliminates the need for an inducer.
The structural genes of inducible enzymes are usually repressed because of the
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Fig. 6.1
Diagram Illustrating Negative Control of Protein Synthesis According to the Jacob
and Monod Model
attachment of the repressor to the operator. During induction the repressor is no longer a
hindrance, hence induction is also known as de-repression. In the model of Jacob and
Monod gene expression can only occur when the operator gene is free. (i.e., in the absence
of the attachment of the repressor protein the operator gene O. For this reason the control
is said to be negative.
6.1.1.2
Positive control of protein synthesis
Positive control of protein synthesis has been less well studied but has been established
in at least one system, namely the ara operon, which is responsible for L-arabinose
utilization in E. coli. In this system the product of one gene (ara C) is a protein which
combines with the inducer arabinose to form an activator molecule which in turn
initiates action at the operon. In the scheme as shown in Fig. 6.2, ‘C’ protein combines
with arabinose to produce an arabinose – ‘C’ protein complex which binds to the
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Fig. 6.2 Diagram Illustrating Positive Control of Protein Synthesis
Promoter P and initiates the synthesis of the various enzymes isomerase, kinase.
epimerase) which convert L-arabinose to D-xylulose-5-phosphate, a form in which it can
be utilized in the Pentose Phosphate pathway (Chapter 5). Positive control of protein
synthesis also operates during catabolite repression (see below).
6.1.2
Catabolite Regulation
The presence of carbon compounds other than inducers may also have important effects
on protein synthesis. If two carbon sources are available to an organism, the organism
will utilize the one which supports growth more rapidly, during which period enzymes
needed for the utilization of the less available carbon source are repressed and therefore
will not be synthesized. As this was first observed when glucose and lactose were
supplied to E. coli, it is often called the ‘glucose effect’, since glucose is the more available
of the two sugars and lactose utilization is suppressed as long as glucose is available. It
soon became known that the effect was not directly a glucose effect but was due to some
catabolite. The term catabolite repression was therefore adopted as more appropriate. It
must be borne in mind that other carbon sources can cause repression (see later) and that
sometimes it is glucose which is repressed.
The active catabolite involved in catabolite repression has been found to be a
nucleotide cyclic 3’5’-adenosine monophosphate (cAMP), (Fig. 6.3). In general, less
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Fig. 6.3 Action of Cyclic Amp on the Lac Operon
c-AMP accumulates in the cell during growth on carbon compounds supporting rapid
growth of the organism, vice versa.
During the rapid growth that occurs on glucose, the intracellular concentration of
cyclic AMP is low. C-AMP stimulates the synthesis of a large number of enzymes and in
necessary for the synthesis of the mRNA for all the inducible enzymes in E.coli. When it is
low as a result of growth on a favorable source the enzymes which need to be induced for
the utilization of the less available substrate are not synthesized.
Unlike the negative control of Jacob and Monod, c-Amp exerts a positive control.
Another model explains the specific action in catabolite repression of glucose. In this
model an increased concentration of c-AMP is a signal for energy starvation. When such
a signal is given, c-Amp binds to an intracellular protein, c-AMP-receptor protein (CRP)
for which it has high affinity. The binding of this complex to the promoter site of an
operon stimulates the initiation of operon transcription by RNA polymerase (Fig. 6.3).
The presence of glucose or a derivative of glucose inhibits adenylate cyclase the enzyme
which converts ATP to c-AMP. Transcription by susceptible operons is inhibited as a
result. In short, therefore, catabolite repression is reversed by c-AMP.
In recent times, for instance, it has been shown that c-AMP and CRP are not the only
mediators of catabolite repression. It has been suggested that while catabolite repression
in enterobacteria at least is exerted by the catabolite(s) of a rapidly utilized glucose source
it is regulated in a two-fold manner: positive control by c-AMP and a negative control by
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a catabolite modulation factor (CMF) which can interfere with the operation of operons
senstitive to catbolite repression. In Bacillus c-AMP has not been observed, but an
analogue of c-AMP is probably involved.
6.1.3
Feedback Regulation
Feedback or end-product regulations control exerted by the end-product of a metabolic
pathway, hence its name. Feedback regulations are important in the control over anabolic
or biosynthetic enzymes whereas enzymes involved in catabolism are usually controlled
by induction and catabolite regulation. Two main types of feedback regulation exist:
feedback inhibition and feedback repression. Both of them help adjust the rate of the
production of pathway end products to the rate at which macro-molecules are
synthesized (see Fig. 6.4).
6.1.3.1 Feedback inhibition
In feedback inhibition the final product of metabolic pathway inhibits the action of earlier
enzymes (usually the first) of that sequence. The inhibitor and the substrate need not
resemble each other, hence the inhibition is often called allosteric in contrast with the
isosteic inhibition where the inhibitor and substrate have the same molecular
conformation. Feedback inhibition can be explained on an enzymic level by the structure
of the enzyme molecule. Such enzymes have two type of protein sub-units. The binding
site on the sub-unit binds to the substrate while the site on the other sub-unit binds to the
feedback inhibitor. When the inhibitor binds to the enzyme the shape of the enzymes is
changed and for this reason, it is no longer able to bind on the substrate. The situation is
known as the allosteric effect.
6.1.3.2 Feedback Repression
Whereas feedback inhibition results in the reduction of the activity of an already
synthesized enzyme, feedback repression deals with a reduction in the rate of synthesis
of the enzymes. In enzymes that are affected by feedback repression the regulator gene (R)
is said to produce a protein aporepressor which is inactive until it is attached to
corepressor, which is the end-product of the biosynthetic pathway. The activated
repressor protein then interacts with the operator gene (O) and prevents transcription of
the structural genes (S) on to mRNA. A derivative of the end-product may also bring
about feedback repression. It is particularly active in stopping the over production of
vitamins, which are required only in small amounts (see Fig. 6.1).
While feedback inhibition acts rapidly, sometimes within seconds, in preventing the
wastage of carbon and energy in manufacturing an already available catabolite, feedback
repression acts more slowly both in its introduction and in its removal. About two
generations are required for the specific activity of the repressed enzymes to rise to its
maximum level when the repressing metabolite is removed; about the same number of
generations are also required for the enzyme to be repressed when a competitive
metabolite is introduced.
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6.1.3.3
Regulation in branched pathway
In a branched pathway leading to two or more end-products, difficulties would arise for
the organism if one of them inhibited the synthesis of the other. For this reason, several
patterns of feedback inhibition have been evolved for branched pathways of which only
six will be discussed. Each type of applicable to either feedback inhibition or feedback
repression The descriptions below refer to Fig. 6.4
(i) Concerted or multivalent feedback regulation: Individual end-products F and H
have little or no negative effect, on the first enzyme, E1, but together they are potent
inhibitors. It occurs in Salmonella in the branched sequence leading to valine,
leucine, isoleucine and pantothenic acid.
Fig. 6.4 Feedback Regulation (Inhibition and Repression) of Enzymes in Branched Pathways
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(ii) Cooperative feedback regulation: In this case the end-products F and H are
individually weakly inhibiting to the primary enzyme, E1, but together they act
synergistically, exerting an inhibition exceeding the sum of their individual
activities.
(iii) Cumulative feedback regulation: In this system an end-product for example (H),
inhibits the primary enzyme E1 to a degree which is not dependent on other
inhibitors. A second inhibitor further increases the total inhibition but not
synergistically. Complete inhibition occurs only when all the products (E, G, H in
Fig. 6.4) are present.
(iv) Compensatory antagonism of feedback regulation: This system operates where one
of the end-products, F, is an intermediate in another pathway J, K, F (Fig. 6.4). In
order to prevent the other end-product, H, of the original pathway from inhibiting
the primary Enzyme E1, and thus ultimately causing the accumulation of H, the
intermediate in the second pathway J, K is able to prevent its own accumulation by
decreasing the inhibitory effect of H on the primary enzyme E1.
(v) Sequential feedback regulation: Here the end-products inhibit the enzymes at the
beginning of the bifurcation of the pathways. This inhibition causes the
accumulation of the intermediate just before the bifurcation. It is the accumulation
of this intermediate which inhibits the primary enzyme of the pathway.
(vi) Multiple enzymes (isoenzymes) with specific regulatory effectors: Multiple
primary enzymes are produced each of which catabolyzes the same reaction from
A to B but is controlled by a different end-product. Thus if one end-product inhibits
one primary enzyme, the other end products can still be formed by the mediation of
one of the remaining primary enzymes.
6.1.4
Amino Acid Regulation of RNA Synthesis
Both protein synthesis and RNA synthesis stop when an amino acid requiring mutant
exhausts the amino acid supplied to it in the medium. In this way the cell avoids the
overproduction of unwanted RNA. Such economical strains are ’stringent’. Certain
mutant strains are however ‘relaxed’ and continue to produce RNA in the absence of the
required amino acid. The stoppage of RNA synthesis in stringent strains is due to the
production of the nucleotide guanosine tetraphosphate (PpGpp) and guanosine
pentaphosphate (ppGpp) when the supplied amino acid becomes limiting. The amount
of ppGpp in the cell is inversely proportional to the amount of RNA and the rate of
growth. Relaxed cells lack the enzymes necessary to produce ppGpp from guanosine
diphosphate and ppGpp from guanosine triphosphate.
6.1.5
Energy Charge Regulation
The cell can also regulate production by the amount of energy it makes available for any
particular reaction. The cell’s high energy compounds adenosine triphosphate, (ATP),
adenosine diphosphate (ADP), and adenosine monophosphate (AMP) are produced
during catabolism. The amount of high energy in a cell is given by the adenylate charge or
energy charge. This measures the extent to which ATP-ADP-AMP systems of the cell
contains high energy phosphate bonds, and is given by the formula.
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Energy charge =
( ATP) + 1/2 ( ADP)
( ATP) + ( ADP ) + AMP
Using this formula, the charge for a cell falls between 0 and 1.0 by a system resembling
feedback regulation, energy is denied reactions which are energy yielding and shunted to
those requiring it. Thus, at the branch point in carbohydrate metabolism
phosphoenolpyruvate is either dephosphorylated to give pyruvate or carboxylated to
give oxalocetate. A high adenylate charge inhibits dephosphorylation and so leads to
decreased synthesis of ATP. A high energy charge on the other hand does not affect
carboylation to oxoloacetate. It may indeed increase it because of the greater availability
of energy.
6.1.6
Permeability Control
While metabolic control prevents the overproduction of essential macromolecules,
permeability control enables the microorganisms to retain these molecules within the cell
and to selectively permit the entry of some molecules from the environment. This control
is exerted at the cell membrane.
A solute molecule passes across a lipid-protein membrane only if there is driving force
acting on it, and some means exists for the molecule to pass through the membrane.
Several means are available for the transportation of solutes through membranes, and
these can be divided into two: (a) passive diffusion, (b) active transport via carrier or
transport mechanism.
6.1.6.1
Passive transport
The driving force in this type of transportation is the concentration gradient in the case of
non-electrolytes or in the case of ions the difference in electrical charge across the
membrane between the internal of the cell and the outside. Yeasts take up sugar by this
method. However, few compounds outside water pass across the border by passive
transportation.
6.1.6.2
Transportation via specific carriers
Most solutes pass through the membrane via some specific carrier mechanism in which
macro-molecules situated in the cell membrane act as ferryboats, picking up solute
molecules and helping them across the membrane. Three of such mechanisms are
known:
(i) Facilitated diffusion: This is the simplest of the three, and the driving force is the
difference in concentration of the solute across the border. The carrier in the
membrane merely helps increase the rate of passage through the membrane, and
not the final concentration in the cell.
(ii) Active transport: This occurs when material is accumulated in the cell against a
concentration gradient. Energy is expended in the transportation through the aid
of enzymes known as permeases but the solute is not altered. The permeases act on
specific compounds and are controlled in many cases by induction or repression
so that waste is avoided.
Overproduction of Metabolites of Industrial Microorganisms
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(iii) Group translocation: In this system the solute is modified chemically during the
transport process, after which it accumulates in the cell. The carrier molecules act
like enzymes catalysing group-transfer reactions using the solute as substrate.
Group translocation can be envisaged as consisting of two separate activities: the
entrance process and the exit process. The exit process increases in rate with the
accumulation of cell solute and is carrier-mediated, but it is not certain whether the
same carriers mediate entrance and efflux.
Carrier-mediated transportation is important because it is selective, and also because
it is the rate-limiting step in the metabolism of available carbon and energy sources. As an
increased rate of accumulation of metabolisable carbon source can increase the extent of
catabolite repression of enzyme synthesis, the rate of metabolisable carbon transport may
have widespread effects on the metabolism of the entire organism.
6.2
DERANGEMENT OR BYPASSING OF REGULATORY
MECHANISMS FOR THE OVER-PRODUCTION OF
PRIMARY METABOLITES
The mechanisms already discussed by which microorganisms regulate their metabolism
ensure that they do not overproduce metabolites and hence avoid wastage of energy or
building blocks. From the point of view of the organism an efficient organism such as
Escherichia coli is one which does not permit any wastage: it switches on and off its
synthetic mechanisms only as they are required and makes no concessions to the need of
the industrial microbiologist to keep his job through obtaining excess metabolites from it!
The interest of the biotechnologist, the industrial microbiologist, and the biochemical
engineer and indeed the entire industrial establishment and even the consuming public,
is to see that the microorganism over produces desirable metabolites. If the
microorganism is highly efficient and economical about what it makes, then the adequate
approach is to disorganize its armamentarium for the establishment of order and thus
cause it to overproduce. In the previous section we discussed methods by which the
organism avoids overproduction. We will now discuss how these control methods are
disorganized. First the situation concerning primary metabolites will be discussed, and
later secondary metabolites will be looked at.
The methods used for the derangement of the metabolic control of primary metabolites
will be discussed under the following headings: (1) Metabolic control; (a) feedback
regulation, (b) restriction of enzyme activity; (2) Permeability control.
6.2.1
Metabolic Control
6.2.1.1 Feedback control
Feedback control is the major means by which the overproduction of amino acids and
nucleotides is avoided in microorganisms. The basic ingredients of this manipulation are
knowledge of the pathway of synthesis of the metabolic product and the manipulation of
the organism to produce the appropriate mutants (methods for producing mutants are
discussed in Chapter 7).
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(i) Overproduction of an intermediate in an unbranched pathway: The
accumulation of an intermediate in an unbranched pathway is the easiest of the
various manipulations to be considered. Consider the production of end-product E
following the series in Fig. 6.5.
E1
A
E2
B
A, B, C, D
E1 , E 2 , E 3 , E 4
ooooo
/
/
[C]
E3
C
=
=
=
=
=
=
=
oooooo o
E4
D
E
Intermediates
Enzymes
Biosynthetic routes
Feedback inhibition/repression
Interrupted biosynthetic route
Feedback interruption
Overproduced intermediate
Fig. 6.5 Scheme for the Overproduction of an Intermediate in an Unbranched Pathway
End-product E inhibits Enzyme 1 and represses Enzymes 2, 3, and 4. An
auxotrophic mutant is produced (Chapter 7) which lacks Enzyme 3. Such a mutant
therefore requires E for growth. If limiting (low levels) of E are now supplied to the
medium, the amount in the cell will not be enough to cause inhibition of Enzyme 1
or repression of Enzyme 2 and C will therefore be over produced, and excreted from
the cells. This principle is applied in the production of ornithine by a citrulline-less
mutant (citrulline auxotroph) of Corynebacterium glutamicum to which low level of
arginine are supplied (Fig. 6.6).
(ii) Overproduction of an intermediate of a branched pathway; Inosine –5monophosphate (IMP) fermentation: This is a little more complicated than the
previous case. Nucleotides are important as flavoring agents and the overproduction of some can be carried out as shown in Fig. 6.7. In the pathway shown
in Fig. 6.7 end-products adenosine 5- monophosphate (AMP) and guanosine –5monophsophate (GMP) both cumulatively feedback inhibit and repress the
primary enzyme [1].
Furthermore, AMP inhibits enzyme [11] which coverts IMP to xanthosine-5monophosphate (XMP). By feeding low levels of adenine to an auxotrophic mutant
of Corynebacterium glutamicum which lacks enzyme [11] (also known as
adenineless because it cannot make adenine) IMP is caused to accumulate. The
conversion of IMP to XMP is inhibited by GMP at [13]. When the enzyme [14] is
removed by mutation, a strain requiring both guanine and adenine is obtained.
Such a strain will excrete high amounts of XMP when fed limiting concentrations
of guanine and adenine.
Overproduction of Metabolites of Industrial Microorganisms
1, 2, 3 = Enzymes and enzymic steps
= Feedback
I = Inhibition
R = Repression
…… = Dotted lines denote absence of enzymic activity
– – –/
/– = Bypass of control mechanism
[ORNITHINE) = Overproduced metabolite
Fig. 6.6
Scheme for the Overproduction of Ornithine by a Citrulineless Mutant of
Corynebacterium glutamicum
(iii) Overproduction of end-products of a branched pathway: The overproduction of end
production of end-products is more complicated than obtaining intermediates.
Among end-products themselves the production of end-products of branched
pathways is easier than in unbranched pathways. Over-production of endproducts of branched pathways will be discussed in this section; unbranched
pathway will be dealt with later.
This is best illustrated (Fig. 6.8) using lysine, an important amino acid lacking in
cereals and therefore added as a supplement to cereal foods especially in animal
foods. It is produced using either Corynebacterium glutamicum or Brevibacterium
flavum. Lysine is produced in these bacteria by a branched pathway that also
produces methionine, isoleucine, and threonine. The initial enzyme in this
pathway aspartokinase is regulated by concerted feedback inhibition of threonine
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PHOSPHORIBOSYL PYROPHOSPHATE
(1)
PHOSPHORIBOSYLAMINE
(2)
GLYCINAMIDE RIBOTIDE
(3)
FORMYLGLYCINAMIDE RIBOTIDE
(4)
FORMYLGLYCINAMIDE RIBOTIDE
(5)
AMINOIMIDAZOLE RIBOTIDE
(6)
AMINOIMIDAZOLE CARBOXYLIC ACID RIBOTIDE
(7)
AMINOIMIDAZOLE-N-SOCCINO-CARBOXAMIDE RIBOTIDE
(8)
AMINOIMICAZOLE CARBOXAMIDE RIBOTIDE
(9)
FORMAMIDO – IMIDAZOLE
(10)
(12) ADENYLO-(11)
(13)
(14)
( I)
AMP
SUCCIATE
IMP
XMP
(R)
GMP
(I)
Key is as indicated in Fig. 6.6
Fig. 6.7
Scheme for the Overproduction of Inosinic Acid by an Adenine Auxotroph of
Corynebacterium glutamicum
and lysine. By mutational removal of the enzyme which converts aspartate semialdehyde to homoserine, namely homoserine dehydrogenase, the mutant cannot
grow unless methionine and threonine are added to the medium. As long as the
threonine is supplied in limiting quantities, the intracelluar concentration of the
amino acid is low and does not feed back inhibit the primary enzyme,
aspartokinase. The metabolic intermediates are thus moved to the lysine branch
and lysine accumulates in the medium (Fig. 6.8).
(iv) Overproduction of end-product of an unbranched pathway: Two methods are used
for the overproduction of the end-product of an unbranched pathway. The first is
the use of a toxic analogue of the desired compound and the second is to backmutate an auxotrophic mutant.
Overproduction of Metabolites of Industrial Microorganisms
!
ASPARTATE
aspartakinase
ASPARTYL PHOSPHATE
ASPARTATE SEMI -ALDEHYDE
dihydropicolinate
homoserine
synthetase
dehydrogenase
HOMOSERINE
THREONINE
METHIONINE
LYSINE
ISOLEUCINE
Key as in Fig. 6.6
Fig. 6.8 Lysine Overproduction Using a Mutant of Corynebacterium glutamicum Lacking
the Enzyme Homoserine Dehydrogenase
Use of toxic or feedback resistant analogues: In this method the organism (bacterial or
yeast cells, or fungal spores) are first exposed to a mutagen. They are then plated in a
medium containing the analogue of the desired compound, which is however also toxic
to the organism. Most of the mutagenized cells will be killed by the analogue. Those
which survive will be resistant to the analogue and some of them will be resistant to
feedback repression and inhibition by the material whose overproduction is desired.
This is because the mutagenized organism would have been ‘fooled’ into surviving on a
substrate similar to, but not the same as offered after mutagenesis. As a result it may
exhibit feedback inhibition in a medium containing the analogue but may be resistant to
feed back inhibition from the material to be produced, due to slight changes in the
configuration of the enzymes produced by the mutant. The net effect is to modify the
enzyme produced by the mutant so that it is less sensitive to feedback inhibition.
Alternatively the enzyme forming system may be so altered that it is insensitive to
feedback repression. Table 6.2 shows a list of compounds which have been used to
produce analogue-resistant mutants.
Use of reverse Mutation: A reverse mutation can be caused in the structural genes of an
auxotrophic mutant in a process known as reversion. Enzymes which differ in structure
from the original enzyme, but which are nevertheless still active, often result. It has been
reported that the reversion of auxotrophic mutants lacking the primary enzyme in a
metabolic pathway often results in revertants which excrete the end-product of the
pathway. The enzyme in the revertant is active but differs from the original enzyme in
being insensitive to feedback inhibition.
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Table 6.2
Excretion of end-products by analogue-resistant mutants
Analogue
Compound Excreted
Organism
p–Fluorophenylalanin
Phenylalanine
p–fluorophenylalanine
Thienylalanine
Thienylalanine
Ethionine
Tyrosine
Tyrosin +
phenylalanine
Phenylalanine
Methionine
Pseudomonas sp.
Mycobacterium sp.
Escherichia coli
E. coli
Norleucine
6-Methyltryptophan
5-Methyltryptophan
Methinonine
Tryptophan
Tryptophan
Canavanine
Trifluoroleucine
Valine
2–Thiazolealanine
3,4 – Dehydroproline
2, 6 – Diaminopurine
Arginine
Leucine
Isoleucine
Histidine
Proline
Adenine
6.2.1.2
E. coli
E. coli, candida utilis
Neurospora crassa
E. coli
Salmonella typhimurium
E. coli, escherichia
animdolica
E. coli
S. typhimurium
E. coli
E. coli, S. typhimurium
E. coli
S. typhimurium
Restriction of enzyme activity
In the tricarboxylic acid cycle the accumulation of citric acid can be encouraged in
Aspergillus niger by limiting the supply to the organism of phosphate and the metals
which form components of co-enzymes. These metals are iron, manganese, and zinc. In
citric acid production the quantity of these is limited, while that of copper which inhibits
the enzymes of the TCA cycle is increased (Chapter 20).
6.2.2
Permeability
Ease of permeability is important in industrial microorganisms not only because it
facilitates the isolation of the product but, more importantly, because of the removal of the
product from the site of feedback regulation. If the product did not diffuse out of the cell,
but remained cell-bound, then the cell would have to be disrupted to enable the isolation
of the product, thereby increasing costs. The importance of permeability is most easily
demonstrated in glutamic acid producing bacteria. In these bacteria, the permeability
barrier must be altered in order that a high level of amino acid is accumulated in the broth.
This increased permeability can be induced by several methods:
(i) Biotin deficiency: Biotin is a coenyme in carboxylation and transcarboxylation
reactions, including the fixation of CO2 to acetate to form malonate. The enzyme
which catalyses this is rich in biotin. The formation of malonyl COA by this
enzyme (acetyl-COA carboxylase) is the limiting factor in the synthesis of long
chain fatty acids. Biotin deficiency would therefore cause aberrations in the fatty
acid produced and hence in the lipid fraction of the cell membrane, resulting in
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#
leaks in the membrane. Biotin deficiency has been shown also to cause aberrant
forms in Bacillus polymax, B. megaterium, and in yeasts.
(ii) Use of fatty acid derivatives: Fatty acid derivatives which are surface-acting agents
e.g. polyoxylene-sorbitan monostearate (tween 60) and tween 40 (-monopalmitate) have actions similar to biotin and must be added to the medium before
or during the log phase of growth. These additives seem to cause changes in the
quantity and quality of the lipid components of the cell membrane. For example
they cause a relative increase in saturated fatty acids as compared to unsaturated
fatty acids.
(iii) Penicillin: Penicillin inhibits cell-wall formation in susceptible bacteria by
interfering with the crosslinking of acetylmuranmic-polypeptide units in the mucopeptide. The cell wall is thus deranged causing glutamate excretion, probably
due to damage to the membrance, which is the site of synthesis of the wall.
6.3 REGULATION OF OVERPRODUCTION IN
SECONDARY METABOLITES
The physiological basis of secondary metabolite production is much less studied and
understood than primary metabolism. Nevertheless there is increasing evidence that
controls similar to those discussed above for primary metabolism also occur in secondary
metabolites. Some examples will be given below:
6.3.1
Induction
The stimulatory effect of some compounds in secondary metabolite fermentation
resembles enzyme induction. A good example is the role of tryptophan in ergot alkaloid
fermentation by Claviceps sp. Although the amino acid is a precursor, its role appears to
be more important as an inducer of some of the enzymes needed for the biosynthesis of the
alkaloid. This is because analogues of tryptophan while not being incorporated into the
alkaloid, also induce the enzymes used for the biosynthesis of the alkaloid. Furthermore,
tryptophan must be added during the growth phase otherwise alkloid formation is
severely reduced. This would also indicate that some of the biosynthetic enzymes, or
some chemical reactions leading to alkaloid transformation take place in the
trophophase, thereby establishing a link between idiophase and the trophophase. A
similar induction appears to be exerted by methionine in the synthesis of cephalosporin
C by Cephalosporium ocremonium.
6.3.2
Catabolite Regulation
Catabolite regulation as seen earlier can be by repression or by inhibition. It is as yet not
possible to tell which of these is operating in secondary metabolism. Furthermore, it
should be noted that catabolite regulations not limited to carbon catabolites and that the
recently discovered nitrogen catabolite regulation noted in primary metabolism also
occurs in secondary metabolism
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Modern Industrial Microbiology and Biotechnology
6.3.2.1
Carbon catabolite regulation
The regulation of secondary metabolism by carbon has been known for a long time. In
penicillin production it had been known for a long time that penicillin is not produced in
a glucose-containing medium until after the exhaustion of the glucose, when the
idiophase sets in; the same effect has been observed with cephalosporin production.
Indeed the ‘glucose effect’ in which production is suppressed until the exhaustion of the
sugar is well known in a large number of secondary products. Although the
phenomenon where an easily utilizable source is exhausted before a less available is
used has been described as glucose effect, it is clearly a misleading term because other
carbon sources may be preferred in two-sugar systems when glucose is absent. Thus, bcarotene production by Mortierella sp. is best on fructose even though galoctose is a better
carbon-source for growth. Carbon sources which have been found suitable for secondary
metabolite production include sucrose (tetracycline and erythromycin), soyabena oil
(kasugamycin), glycerol (butirosin) and starch and dextrin (fortimicin). Table 6.3 shows
a list of secondary metabolites whose production is suppressed by glucose as well as
non- interfering carbon sources.
It is fairly easy to decide whether the catabolite is repressing or inhibiting the synthesis. In
catabolite repression the synthesis of the enzymes necessary for the synthesis of the
metabolite is repressed. It is tested by the addition of the test substrate just prior to the
initiation of secondary metabolite synthesis where upon synthesis is severely repressed.
To test for catabolite inhibition by glucose or other carbon source it is added to a culture
already producing the secondary metabolite and any inhibition in the synthesis noted.
Table 6.3
Secondary metabolites whose production is suppressed by glucose
Secondary
Metabolite
Organism
Non-interfering
Carbon Sources
Actinomycin
Indolmycin
Kanamycin
Mitomycin
Neomycin
Puromycin
Siomycin
Streptomycin
Bacitracin
Prodigiosin
Violacein
Cephalosporin C
Ergot alkaloids
Enniatin
Gibberellic acid
Penicillin
Streptomyces antibioticus
Streptomyces griseus
Streptomyces kanamyceticus
Streptomyces verticillatus
Streptomyces fradiae
Streptomyces alboniger
Streptomyces sioyaensis
Steptomyces griseus
Bacillus licheniformis
Seratia marcescens
Chromobacterium violaceum
Cephalosporium acremonium
Claviceps purperea
Fusarium sambucinum
Fusarium monoliforme
Penicillium chrysogenum
Galoactose
Fructose
Galactose
Low glucose
Maltose
Glycerol
Maltose
Mannan
Citrate
Galactose
Maltose
Sucrose
Lactose
Lactose
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6.3.2.2 Nitrogen catabolite regulation
Nitrogen catabolite regulation has also been observed in primary metabolism. It involves
the suppression of the synthesis of enzymes which act on nitrogen-containing
substances (proteases, ureases, etc.) until the easily utilizable nitrogen sources e.g.,
ammonia are exhausted. In streptomycin fermentation where soyabean meal is the
preferred substrate as a nitrogen source the advantage may well be similar to that of
lactose in penicillin, namely that of slow utilization. Secondary metabolites which are
affected by nitrogen catabolite regulation include trihyroxytoluene production by
Aspergillus fumigatus, bikaverin by Gibberella fujikuroi and cephamycins by Streptomyces
spp.
In all these cases nitrogen must be exhausted before production of the secondary
metabolite is initiated.
6.3.3
Feedback Regulation
That feedback regulation exists in secondary metabolism is shown in many examples in
which the product inhibits its further synthesis. An example is penicillin inhibition by
lysine. Penicillin biosynthesis by Penicillium chrysogenum is affected by feedback
inhibition by L-lysine because penicillin and lysine are end-products of a brack pathway
(Fig. 6.9). Feedback by lysine inhibits the primary enzyme in the chain, homocitrate
synthetase, and inhibits the production of a-aminoadipate. The addition of aaminoadipate eliminats the inhibitory effect of lysine.
Self-inhibition by secondary meabolites: Several secondary products or even their
analogues have been shown to inhibit their own production by a feedback mechanism.
Examples are audorox, an antibiotic active against Gram-positive bacteria, and used in
poultry feeds, chloramphenicol, penicillin, cycloheximids, and 6-methylsallicylic acid
(produced by Penicillium urticae). Chloramphenicol repression of its own production is
shown in Fig. 6.10, which also shows chorismic acid inhibition by tryptophan.
6.3.4
ATP or Energy Charge Regulation of
Secondary Metabolites
Secondary metabolism has a much narrower tolerance for concentrations of inorganic
phosphate than primary metabolism. A range of inorganic phosphate of 0.3-30 mM
permits excellent growth of procaryotic and eucaryotic organisms. On the other hand the
average highest level that favors secondary metabolism is 1.0 mM while the average
lower quantity that maximally suppresses secondary process is 10 mM High phosphate
levels inhibit antibiotic formation hence the antibiotic industry empirically selects media
of low phosphate content, or reduce the phosphate content by adding phosphatecomplexing agents to the medium. Several explanations have been given for this
phenomenon. One of them is that phosphate stimulates high respiration rate, DNA and
RNA synthesis and glucose utilization, thus shifting the growth phase from the
idiophase to the trophophase. This shift can occur no matter the stage of growth of the
organisms. Exhaustion of the phosphate therefore helps trigger off idiophase. Another
hypothesis is that a high phosphate level shifts carbohydrate catabolism ways from
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Modern Industrial Microbiology and Biotechnology
Peniciilin and lysine are synthesized by a branched pathway in a mutant of Penicillium chrysogenum, L2.
a-AAA is the branching intermediate. Mutant L2 is blocked before homoisocitrate and therefore
accumulates homocitrate. The first enzyme is repressed (R) and and inhibited (I) by L-lysine, but not by
penicillin G. 6-APA = 6-amino penicillanic acid.
Fig. 6.9 Penicillin Synthesis by a Mutant of Penicillium chrysogenum, L2
Fig. 6.10 Biosynthetic Pathway for Chloramphenicol
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Modern Industrial Microbiology and Biotechnology
HMP to the EMP pathway favoring glycolysis. If this is the case then NADPH would
become limiting of ridiolite synthesis.
6.4 EMPIRICAL METHODS EMPLOYED TO
DISORGANIZE REGULATORY MECHANISMS IN
SECONDARY METABOLITE PRODUCTION
Metabolic pathways for secondary metabolites are becoming better known and more
rational approaches to disrupting the pathways for overproduction are being employed.
More work seems to exist with regard to primary metabolites. Methods which are used to
induce the overproduction of secondary metabolites are in the main empirical. Such
methods include mutations and stimulation by the manipulation of media components
and conditions.
(i) Mutations: Naturally occurring variants of organisms which have shown
evidence of good productivity are subjected to mutations and the treated cells are
selected randomly and tested for metabolite overproduction. The nature of the
mutated gene is often not known.
(ii) Stimulatory effect of precursors: In many fermentations for secondary metabolites,
production is stimulated and yields increased by the addition of precursors. Thus
penicillin production was stimulated by the addition of phenylacetic acid present
in corn steep liquor in the early days of penicillin fermentation. For the
experimental synthesis of aflatoxin by Aspergillus parasiticus, methionine is
required. In mitomycin formation by Streptomyces verticillatus, L-citurulline is a
precursor.
(iii) Inorganic compounds: Two inorganic compounds which have profound effects of
fermentation for secondary metabolites are phosphate and maganese. The effect of
inorganic phosphate has been discussed earlier. In summary, while high levels of
phosphate encourage growth, they are detrimental to the production of secondary
metabolites. Manganese on the other hand specifically encourages idiophase
production particularly among bacilli, including the production of bacillin,
bacitracin, mycobacillin, subtilin, D-glutamine, protective antigens and
endospores. Surprisingly, the amount needed are from 20 to several times the
amount needed for growth.
(iv) Temperature: While the temperature range that permits good growth (in the
trophophase) spans about 25°C among microorganisms, the temperature range
within which secondary metabolites are produced is much lower, being in the
order of only 5-10°C. Temperatures used in the production of secondary
metabolites are therefore a compromise of these situations. Sometimes two
temperatures – a higher for the trophophase and a lower for the idiophase are used.
SUGGESTED READINGS
Betina, V. 1995. Differentiation and Secondary Metabolism in Some Prokaryotes and Fungi Folia
Microbiologica 40, 51–67.
Bull, A.T., Ward, A.C., Goodfellow, M. 2000. Search and Discovery Strategies for Biotechnology:
the Paradigm Shift. Microbiology and Molecular Biology Reviews, 64, 573–606.
Overproduction of Metabolites of Industrial Microorganisms
Demain, A.L. 1998. Induction of microbial secondary metabolism. International Microbiology, 1:
259–264.
Martin, J.F., Demain, A.L. 1980. Control of antibiotic biosynthesis. Microbiological Reviews 44,
230–251.
Krumphanzl, V., Sikyta, B., Vanck, Z. 1982. Overproduction of Microbial Products. Academic
Press, London and New York.
Spizek, J.J., Tichy, P. 1995. Some Aspects of Overproduction of Secondary Metabolites. Folia
Microbiologica 40, 43–50.
Vinci, A.V., Byng, G. 1999. Strain Improvement by Nono-recombinant Methods. In: Manual of
Industrial Microbiology and Biotechnology. A S M Press, 2nd Ed. Washington, DC, USA, pp.
103–113.
Watts, J.E.M., Huddleston-Anderson, A.S., Wellington, E.M.H. 1999. Bioprospecting. In: Manual
of Industrial Microbiology and Biotechnology. 2nd ed. A S M Press, Washington, DC, USA, pp.
631–641.
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7
Screening for
Productive Strains and
Strain Improvement in
Biotechnological Organisms
In the last several decades, a group of microbial secondary metabolites, the antibiotics,
has emerged as one of the most powerful tools for combating disease. So important are
antibiotics as chemotherapeutic agents that much of the effort in searching for useful
bioactive microbial products has been directed towards the search for them. Thousands
of secondary metabolites are, however, known and they include not only antibiotics, but
also pigments, toxins, pheromones, enzyme inhibitors, immunomodulating agents,
receptor antagonists and agonists, pesticides, antitumor agents and growth promoters of
animals and plants. When appropriate screening has been done on secondary
metabolites, numerous drugs outside antibiotics have been found. Some of such nonantibiotic drugs are shown in Table 7.1. It seems reasonable from this to conclude that the
exploitation of microbial secondary metabolites, useful to man outside antibiotics, has
barely been touched. A special effort is made in this book to discuss method for assaying
microbial metabolites for drugs outside of antibiotics (Chapter 28).
This section will therefore discuss in brief general terms the principles involved in
searching for microorganisms producing metabolites of economic importance. More
detailed procedures will be examined when various products are discussed. The genetic
improvement of strains of organisms used in biotechnology, including microorganisms,
plants and animals is also discussed.
7.1 SOURCES OF MICROORGANISMS USED IN
BIOTECHNOLOGY
7.1.1
Literature Search and Culture Collection Supply
If one was starting from scratch and had no idea which organism produced a desired
industrial material, then perhaps a search on the web and in the literature, including
patent literature, accompanied by contact with one or more of the established culture
collections (Chapter 8) and the regulatory offices dealing with patents (Chapter 1) may
Screening for Productive Strains and Strain Improvement
!
Table 7.1 Some microbial metabolites with non-antibiotic pharmacological activity
Compound
Activity
Producing Microorganism
Aspergillic acid
Astromentin
Siolipn
Azaserine
Ovalicin
Candicidin (and other
polyene Macrolides)
Streptozotocin
Zygosporin A
Fusaric acid
Leupeptin family
Pepstatin
Oosponol
Antihypertensive
Sommoth muscle relaxant
Acceleration of fibrin clot
Antidiuretic, antitumor
Immunosuppressive, antitumor
Cholesterol lowering
Aspergillus sp
Monascus sp
Streptomyces sioyaensis
Streptomyces fragilis
Pseudeurotum ovalis
Streptomyces noursei
Hyperglycemic, antitumor
Anti-inflammatory
Hypotensive
Plasmin inhibitor
Pepsin inhibitor
Dopamine b-hydroxlyase
inhibitor
Angiogenesis inhibitor
Streptomyces achromogenes
Cephalosporium acremonium
Fusarium oxysporum
Bacillus sp
Aspergiluus niger
Oospora adringens
Fumagallin
Aspergillus fumigatus
provide information on potentially useful microbial cultures. The cultures may, however,
be tied to patents, and fees may be involved before the organisms are supplied, along with
the right to use the patented process for producing the material. Generally, cultures are
supplied for a small fee from most culture collections irrespective of whether or not the
organism is part of a process patent.
7.1.2
Isolation de novo of Organisms Producing
Metabolites of Economic Importance
Although the well-known ubiquity of microorganism implies that almost any natural
ecological entity–water, air, leaves, tree trunks – may provide microorganisms, the soil is
the preferred source for isolating organisms, because it is a vast reservoir of diverse
organisms. Indeed microorganisms capable of utilizing virtually any carbon source will
be found in soil if adequate screening methods are used. In recent times, other ‘new’
habitats, especially the marine environment, have been included in habitats to be studied
in searches for bioactive microbial metabolites or ‘bio-mining’. Some general screening
methods are described below. Detailed methods for the discovery of new antibiotics and
other bioactive metabolites will be discussed in Chapter 21 and Chapter 28.
7.1.2.1 Enrichment with the substrate utilized by the
organism being sought
If the organism being sought is one which utilizes a particular substrate, then soil is
incubated with that substrate for a period of time. The conditions of the incubation can
also be used to select a specific organism. Thus, if a thermophilic organism attacking the
substrate is required, then the soil is incubated at an elevated temperature. After a period
of incubation, a dilution of the incubated soil is plated on a medium containing the
substrate and incubated at the previous temperature (i.e., elevated for thermopile search).
Organisms can then be picked out especially if some means has been devised to select
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Modern Industrial Microbiology and Biotechnology
them. Selection could, for instance, be based on the ability to cause clear zones in an agar
plate as a result of the dissolution of particles of the substrate in the agar. In the search for
a-amylase producers, the soil may be enriched with starch and subsequently suitable soil
dilutions are plated on agar containing starch as the sole carbon source. Clear halos form
around starch-splitting colonies against a blue background when iodine is introduced in
the plate.
Continuous culture (Chapter 9) methods are a particularly convenient means of
enriching for organisms from a natural source. The constant flow of nutrients over
material from a natural habitat such as soil will encourage, and after a time, select for
organisms able to utilize the substrate in the nutrient solution. Conditions such as pH,
temperature, etc., may also be adjusted to select the organisms which will utilize the
desired substrate under the given conditions. Agar platings of the outflow from the
continuous culture setup are made at regular intervals to determine when an optimum
population of the desired organism has developed.
7.1.2.2 Enrichment with toxic analogues of the substrate
utilized by the organism being sought
Toxic analogues of the material where utilization is being sought may be used for
enrichment, and incubated with soil. The toxic analogue will kill many organisms which
utilize it. The surviving organisms are then grown on the medium with the non-toxic
substrate. Under the new conditions of growth many organisms surviving from exposure
to toxic analogues over-produce the desired end-products. The physiological basis of this
phenomenon was discussed earlier in Chapter 6.
7.1.2.3
Testing microbial metabolites for bioactive activity
(i) Testing for anti-microbial activity
For the isolation of antibiotic producing organisms the metabolites of the test organism
are tested for anti-microbial activity against test organisms. One of the commonest
starting point is to place a soil suspension or soil particles on agar seeded with the test
organism(s). Colonies around which cleared zones occur are isolated, purified, and
further studied. This method is discussed more fully in Chapter 21 where discussion of
the search for antibiotics is included.
(ii) Testing for enzyme inhibition
Microorganisms whose broth cultures are able to inhibit enzymes associated with certain
disease may be isolated and tested for the ability to produce drugs for combating the
disease. Enzyme inhibition may be determined using one of the two methods among
those discussed by Umezawa in 1982. In the first method the product of the reaction
between an enzyme and its substrate is measured using spectroscopic methods. The
quantity of the inhibitor in the test sample is obtained by measuring (a) the product in the
reaction mixture without the inhibitor and (b) the product in the mixture with the
inhibitor (i.e., a broth or suitable fraction of the broth whose inhibitory potency is being
tested). The percentage inhibition (if any) is calculated by the formula
(a - b )
´ 100
a
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#
The second method determines the quantity of the unreacted substrate. For this
determination the following measurements of the substrate are made: (a) with the enzyme
and without the inhibitor (i.e., broth being tested); (b) with the enzyme and with the
inhibitor and; (c) without the enzyme and without the inhibitor. Percentage inhibition (if
any) is determined by (c-a) – (c-b) x 100. The results obtained above enable the assessment
of the existence of enzyme inhibitors and facilitate the comparison of the inhibitory
ability of broths from several sources.
(iii) Testing for morphological changes in fungal test organisms
The effect on spore germination or change in hyphal morphology may be used to detect
the presence of pharmacologically active products in the broth of a test organism. This
method does not rely on the death or inhibition of microbial growth, which has been so
widely used for detecting antibiotic presence in broths.
(iv) Conducting animal tests on the microbial metabolites
The effect of broth on various animal body activities such as blood pressure,
immunosuppressive action, anti-coagulant activity are carried out in animals to
determine the content of potentially useful drugs in the broth. This method is discussed
extensively in Chapter 21, which discusses details of the search for the production of
bioactive metabolites from microorganisms.
7.2
STRAIN IMPROVEMENT
Several options are open to an industrial microbiology organization seeking to maximize
its profits in the face of its competitors’ race for the same market. The organization may
undertake more aggressive marketing tactics, including more attractive packaging while
leaving its technical procedures unchanged. It may use its human resources more
efficiently and hence reduce costs, or it may adopt a more efficient extraction system for
obtaining the material from the fermentation broth. The operations in the fermentor may
also be improved by its use of a more productive medium, better environmental
conditions, better engineering control of the fermentor processes, or it may genetically
improve the productivity of the microbial strain it is using. Of all the above options, strain
improvement appears to be the one single factor with the greatest potential for
contributing to greater profitability.
While realizing the importance of strain improvement, it must be borne in mind that an
improved strain could bring with it previously non-existent problems. For example, a
more highly yielding strain may require greater aeration or need more intensive foam
control; the products may pose new extraction challenges, or may even require an entirely
new fermentation medium. The use of a more productive strain must therefore be
weighed against possible increased costs resulting from higher investments in
extraction, richer media, more expensive fermentor operations and other hitherto nonexistent problems. This possibility not withstanding, strain improvement is usually part
of the program of an industrial microbiology organization.
To appreciate the basis of strain improvement it is important to remember that the
ability of any organism to make any particular product is predicated on its capability for
the secretion of a particular set of enzymes. The production of the enzymes, themselves
depends ultimately on the genetic make-up of the organisms. Improvement of strains can
therefore be put down in simple term as follows:
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Modern Industrial Microbiology and Biotechnology
(i) regulating the activity of the enzymes secreted by the organisms;
(ii) in the case of metabolites secreted extracellularly, increasing the permeability of
the organism so that the microbial products can find these way more easily outside
the cell;
(iii) selecting suitable producing strains from a natural population;
(iv) manipulation of the existing genetic apparatus in a producing organism;
(v) introducing new genetic properties into the organism by recombinant DNA
technology or genetic engineering.
Items (i) and (ii) above have been discussed in Chapter 6. The other possible
procedures, namely selection from natural variants, modification of the genetic
apparatus without the introduction of foreign DNA and the use of foreign DNA will be
discussed below (Table 7.1).
7.2.1
Selection from Naturally Occurring Variants
In selection of this type, naturally occurring variants which over-produce the desired
product are sought. Strains which were encountered but not selected should not be
automatically discarded; the better ones are usually kept as stock cultures in the
organization’s culture collection for possible use in future genetic manipulations.
Selection from natural variants is a regular feature of industrial microbiology and
biotechnology. For example, in the early days of antibiotic production the initial increase
in yield was obtained in both penicillin and griseofulvin by natural variants producing
higher yields in submerged rather than in surface culture. Another example is lager beer
manufacture where the constant selection of yeasts that flocculate eventually gave rise to
strains which are now used for the production of the beverage. Similarly in wine
fermentation yeasts were repeatedly taken from the best vats until yeasts of suitable
properties were obtained.
Selection of this type is not only slow but its course is largely outside the control of the
biotechnologist, an intolerable condition in the highly competitive world of modern
industry. Strain improvement is therefore mostly achieved by other means described
below.
7.2.2
Manipulation of the Genome of Industrial Organisms
in Strain Improvement
The manipulation of the genome for increased productivity may be done in one of two
general procedures as shown in Table 7.2:
(a) manipulations not involving foreign DNA;
(b) manipulations involving foreign DNA .
7.2.2.1 Genome manipulations not involving Foreign DNA
or Bases: Conventional Mutation
Nature of conventional mutation
The properties of any microorganism depend on the sequence of the four nucleic acid
bases on its genome: adenine (A), thymine (T), cytosine (C), and guanine (G). The
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%
Table 7.2 Methods of manipulating the genetic apparatus of industrial organisms
A. Methods not involving foreign DNA
1. Conventional mutation
B. Methods involving DNA foreign to the organism (i.e. recombination)
2. Transduction
3. Conjugation
4. Transformation
5. Heterokaryosis
6. Protoplast fusion
7. Genetic engineering
8. Metabolic engineering
9. Site-directed mutation
arrangement of these DNA bases dictates the distribution of genes and hence the nature
of proteins synthesized. A mutation can therefore be described as a change in the
sequence of the bases in DNA (or RNA, in RNA viruses). It is clear that since it is the
sequence of these bases which is responsible for the type of proteins (and hence enzymes)
synthesized, any change in the sequence will lead ultimately to a change in the properties
of the organism.
Mutations occur spontaneously at a low rate in a population of microorganisms. It is
this low rate of mutations which is partly responsible for the variation found in natural
populations. An increased rate can however be induced by mutagens, (or mutagenic
agents) which can either be physical or chemical.
7.2.2.1.1 Physical agents
(i) ionizing radiations
(ii) ultraviolet light
(i) Ionizing radiations: X-rays, gamma rays, alpha-particles and fast neutrons are
ionizing radiations and have all been successfully used to induce mutation. X-rays are
produced by commercially available machines as well as van de Graaf generators.
Gamma rays are emitted by the decay of radioactive materials such as Cobalt60. Fast
neutrons are produced by a cyclotron or an atomic pile. Ionizing radiations are so called
because they knock off the outer electrons in the atoms of biological materials (including
DNA) thereby causing ionization in the molecules of DNA. As a result, highly reactive
radicals are produced and these cause changes in the DNA. Some authors do not advise
the use of ionizing radiations unless all other methods fail. This is party because the
equipment is expensive and hence not always readily available, but also because
ionizing radiations are apt to cause breakage in chromosomes.
(ii) Ultraviolet light: The mutagenic range of ultraviolet light lies between wave length
200 and 300 nm. ‘Low pressure’ UV lamps used for mutagenesis emit most of their rays in
the 254 nm region. The suspension of cells or spores to be mutagenized is placed in a Petri
dish 2-3 cm below a 15 watt lamp and stirred either by a rocking mechanism or by a
magnetic stirrer. The organisms are exposed for varying periods lasting from about
300 seconds to about 20 minutes depending on the sensitivity of the organisms. Since UV
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Modern Industrial Microbiology and Biotechnology
damage can be repaired by exposure to light in a process known as photo-reactivation all
manipulations should be conducted under a special light source such as 25 watt yellow
or red bulbs. A proportion of the organisms ranging from about 60–99.9% should be
killed by the radiation. The preference of workers as to the amount of kill varies, but the
higher the kill the more the likelihood of producing desirable mutants. Furthermore, the
higher the kill, the less likely it is that the killing is due to overheating consequent on
having the organism too close to the lamp. The initial concentration of the organisms
should also be in the order of 107 per ml.
The main effect of ultraviolet light on DNA is the formation of covalent bonds between
adjacent pyrimidine (thymine and cytosine) bases. Thymine is mainly affected, and
hence the major effect of UV light is thymine dimerization, although it can also cause
thymine-cytosin and cytosin-cytosin dimers. Dimerization causes a distortion of the
DNA double strand and the ultimate effect is to inhibit transcription and finally the
organism dies (Fig. 7.1).
Fig. 7.1
Schematic Representation of Thymine Dimerization by UV Light on DNA
7.2.2.1.2 Chemical mutagens
These may be divided into three groups:
(i) Those that act on DNA of resting or non-dividing organisms;
(ii) DNA analogues which may be incorporated into DNA during replication;
(iii) Those that cause frame-shift mutations.
(i) Chemicals acting on resting DNA
Some chemical mutagens, such as nitrous acid and nitrosoguanidine work by causing
chemical modifications of purine and pyrimidine bases that alter their hydrogenbonding properties. For example, nitrous acid converts cytosine to uracil which then
forms hydrogen bonds with adenine rather than guanine. These chemicals act on the
non-dividing cell and include nitrous acid, alkylating agents and nitrosoguanidine
(NTG) (also known as MNNG).
(a) Nitrous acid: This acid is rather harmless and the mutation can be easily performed
by adding 0.1 to 0.2 M of sodium nitrate to a suspension of the cells in an acid
Screening for Productive Strains and Strain Improvement
'
medium for various times. The acid is neutralized after suitable intervals by the
addition of appropriate amounts of sodium hydroxide. The cells are plated out
subsequently.
(b) Alkylating agents: These are compounds with one or more alkyl groups which can
be transferred to DNA or other molecules. Many of them are known but the
following have been routinely used as mutagens: EMS (ethyl methane sulphonate),
EES (ethyl ethane sulphonate) and DES (Diethyl sulphonate). They are liquids and
easy to handle. Cells are treated in solutions of about 1% concentration and
allowed to react from ¼ hour to ½ hour and thereafter are plated out.
Experimentation has to be done to decide the amount of kill that will provide a
suitable amount of mutation. While some are carcinostatic (i.e., stop cancers), some
are carcinogenic and must be handled carefully.
(c) NTG – nitrosoguanidine: also known as M-methyl-N-nitro-M-guanidine - MNNG:
it is one of the most potent mutagens known and must therefore should be handled
with care. Amounts ranging from 0.1 to 3.0 mg/ml have been used but for most
mutations the lower quantity is used. It is reported to induce mutation in closely
linked genes. It is widely used in industrial microbiology.
(d) Nitrogen mustards: The most commonly used of this group of compounds is
methyl-bis (Beta-chlorethyl) amine also referred to as ‘HN2’. Nitrogen mustards
were used for chemical warfare in World War I. Other members of the group are
‘HN,’ ‘HN1’, or ‘HN3’ from the wartime code name for mustard gas, H. The number
after the H denotes the number of 2-chloroethyl groups which have replaced the
methyl groups in trimethylamine. A spore or cell suspension is made in HN2
(methyl-bis [Beta-chloroethyl amine]) and after exposure to various concentrations
for about 30 minutes each, the reaction is ended by a decontaminating solution
containing 0.7% NaHCO3 and 0.6% glycine. The solution is then plated out for
survivors. Between 0.05 and 0.1% HN2 solutions in 2% sodium bicarbonate
solutions have been found satisfactory for Streptomyces. Sometimes the exposure
time may be extended
(ii) Base analogues
These are compounds which because they are similar to base nucleotides in composition
may be incorporated into a dividing DNA in place of the natural base. However, this
incorporation takes place only in special conditions. The best examples include 2-amino
purine, a compound that resembles adenine, and 5-bromouracil (5BU), a compound that
resembles thymine. The base analogs, however, do not have the hydrogen-bonding
properties of the natural base. Base analogues are not useful as routine mutagens because
suitable conditions for their use may be difficult to achieve. For example, with BU,
incorporation occurs only when the organisms is starved of thymine.
(iii) Frameshift mutagens (also known as intercalating agents)
Frameshift or intercalating agents are planar three-ringed molecules that are about the
same size as a nucleotide base pair. During DNA replication, these compounds can
insert or intercalate between adjacent base pairs thus pushing the nucleotides far enough
apart that an extra nucleotide is often added to the growing chain during DNA
replication. A mutation of this sort changes all the amino acids downstream and is very
likely to create a nonfunctional product since it may differ greatly from the normal
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Modern Industrial Microbiology and Biotechnology
i) Point Mutation or Substitution of a Nucleotide
ii) Deletion of a nucleotide
iii) Addition of a Nucleotide
iv) Substitution of a nucleotide: Results in one wrong
codon and one wrong amino acid
v) Substitution of a nucleotide: Results in a ‘stop’ codon
and premature termination of the protein
vi) Frameshift mutation: Results in a reading
frame shift. All codons.
Fig. 7.2
Different Types of Mutation
Screening for Productive Strains and Strain Improvement
!
protein. Furthermore, reading frames (i.e., the DNA base sequences) other than the correct
one often contain stop codons which will truncate the mutant protein prematurely.
Acridines are among the best known of these mutagens, which cause a displacement
or shift in the sequence of the bases. Although strongly mutagenic for some
bacteriophages, acridines have not been found useful for bacteria. However, certain
compounds, ICR (Institute for Cancer Research), (eg, ICR191) compounds in which an
acridine nucleus is linked to an alkylating side chain, induce mutations in bacteria.
Acridine, C13H9N, is an organic compound consisting of three fused benzene rings
(Fig. 7.3). Acridine is colorless and was first isolated from crude coal tar. It is a raw
material for the production of dyes. Acridines and their derivatives are DNA and RNA
binding compounds due to their intercalation abilities. Acridine Orange (3,6dimethylaminoacridine) is a nucleic acid selective metachromatic stain useful for cell
cycle determination. Another example is ethidium bromide, which is also used as a DNA
dye.
Fig. 7.3 Acridine
7.2.2.1.3 Choice of mutagen
Mutagenic agents are numerous but not necessarily equally effective in all organisms.
Should one agent fail to produce mutations then another should be tried. Other factors
besides effectiveness to be borne in mind are (a) the safety of the mutagen: many mutagens
are carcinogens, (b) simplicity of technique, and (c) ready availability of the necessary
equipment and chemicals.
Among physical agents, UV is to be preferred since it does not require much
equipment, and is relatively effective and has been widely used in industry. Chemical
methods other than NTG are probably best used in combination with UV. The
disadvantage of UV is that it is absorbed by glass; it is also not effective in opaque or
colored organisms.
7.2.2.1.4 The practical isolation of mutants
There are three stages before a mutant can come into use: the organisms must be exposed
to a suitable mutagen under suitable conditions; the treated cells must be exposed to
conditions which ideally select for the mutant; and finally, the mutant must then be tested
for productivity.
(i) Exposing organisms to the mutagen: The organism undergoing mutation should be
in the haploid stage during the exposure. Bacterial cells are haploid; in fungi and
actinomycetes the haploid stage is found in the spores. However, in non-sporing
strains of these organisms hyphae, preferable the tips, may be used. The use of
haploid is essential because many mutant genes are recessive in comparison to the
parent or wild-type gene.
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Modern Industrial Microbiology and Biotechnology
(ii) Selection for mutants: Following exposure to the mutagen the cells should be
suitably diluted and plated out to yield 50 – 100 colonies per plate. The selection of
mutants is greatly facilitated by relying on the morphology of the mutants or on
some selectivity in-built into the medium on which the treated cells or spores are
plated.
When morphological mutants are selected, it is in the hope that the desired
mutation is pleotropic (i.e., a mutation in which change in one property is linked
with a mutation in another character). The classic example of a pleotropic
mutation is to be seen in the development of penicillin-yielding strains of
Penicillium chrysogenum. It was found in the early days of the development work on
penicillin production that after irradiation, strains of Penicillium chrysogenum with
smaller colonies and which also sporulated poorly were better producers of
penicillin. Similar increases of metabolite production associated with a
morphological change have been observed in organisms producing other
antibiotics: cycloheximide, nystatin, and tetracyclines. In citric acid production it
was observed that mutants with color in the conidia produced more of the acid; in
some bacteria strains overproducing nucleic acid had a different morphological
characteristic from those which did not.
In-built selectivity of the medium for mutants over the parent cells may be
achieved by manipulating the medium. If, for example, it is desired to select for
mutants able to stand a higher concentration of alcohol, an antibiotic, or some
other chemical substance, then the desired level of the material is added to the
medium on which the organisms are plated. Only mutants able to survive the
higher concentration will develop. Toxic analogues may also be incorporated.
Mutants resisting the analogues develop and may, for reasons discussed in
Chapter 6, be higher yielding than the parent.
(iii) Screening: Screening must be carefully carried out with statistically organized
experimentation to enable one to accept with confidence any apparent
improvement in a producing organism. Shake cultures are preferred and about 6 of
these of 500 ml capacity should be used. Accurate methods of identifying the
desired product among a possible multitude of others should be worked out. It may
also be better in industrial practice where time is important to carry out as soon as
possible a series of mutations using ultraviolet, and a combination of ultraviolet
and chemicals and then to test all the mutants.
Isolation of auxotrophic mutants
Auxotrophic mutants are those which lack the enzymes to manufacture certain required
nutrients; consequently, such nutrients must therefore be added to the growth medium.
In contrast the wild-type or prototrophic organisms possess all the enzymes needed to
synthesize all growth requirements. As auxotrophic mutants are often used in industrial
microbiology, e.g., for the production of amino acids, nucleotides, etc., their production
will be described briefly below.
A procedure for producing auxotrophic mutants is illustrated in Fig. 7.4. The
organism (prototroph) is transferred from a slant to a broth of the minimal medium (mm)
which is the basic medium that will support the growth of the prototroph but not that of
the auxotroph. The auxotroph will only grow on the complete medium, i.e., the minimal
Screening for Productive Strains and Strain Improvement
GR1, GR2, GR3 = various growth factors added to the minimum medium (mm)
Fig. 7.4 Procedure for Isolating Auxotrophic Mutants
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medium plus the growth factor, amino-acid or vitamin which the auxotroph cannot
synthesize. The prototroph is shaken in the minimal broth for 22–24 hours, at the end of
which period it is subjected to mutagenic treatment. The mutagenized cells are now
grown on the complete medium for about 8 hours after which they are washed several
times. The washed cells are then shaken again in minimal medium to which penicillin is
added. The reason for the addition of penicillin is that the antibiotic kills only dividing
cells; as only prototrophs will grow in the minimal medium these are killed off leaving the
auxotrophs. The cells are washed and plated out on the complete agar medium.
In order to determine the growth factor or compound which the auxotroph cannot
manufacture, an agar culture is replica-plated on to each of several plates which contain
the minimal medium and various growth factors either single or mixed. The composition
of the medium on which the auxotroph will grow indicates the metabolite it cannot
synthesize; for example when the auxotroph requires lysine it is designated a ‘lysineless’ mutant (Table 7.3).
Table 7.3
Growth of various mutants, produced after treatment of a wild-type organism
S/N
Complete
medium
(cm)
Minimal
medium
(mm)
mm
+
lysine
Growth on
mm
+
biotin
mm
+
valine
1
2
3
4
5
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Remarks
Biotin-less mutant
Lysine-less mutant
Valine-less mutant
Biotin-and valine-less
Parent Prototype
Key: + = growth
– = no growth
7.2.2.2 Strain Improvement Methods Involving Foreign DNA
or Bases
7.2.2.2.1 Transduction
Transduction is the transfer of bacterial DNA from one bacterial cell to another by means
of a bacteriophage. In this process a phage attaches to, and lyses, the cell wall of its host.
It then injects its DNA (or RNA) into the host.
Once inside the cell the viral genome may become attached to the host DNA or remain
unattached forming a plasmid. Such a phage, which does not lyse the cell, is a temperate
phage and the situation is known as lysogeny. Sometimes the viral genome may direct the
host DNA to produce hundreds of copies of the phage. At the end of this manufacture the
host is lysed releasing the viral particles into the medium; the new phages carry portions
of the host DNA. If one of these viral particles now invades another bacterium, but is
lysogenic in the new host, the new host will acquire some nucleic acid, and hence, some
properties from the previous bacterial host. This process of the acquisition of new DNA
from another bacterium through a phage is transduction.
Transduction is two broad types: general transduction and specialized transduction. In
general transduction, host DNA from any part of the host’s genetic apparatus is
Screening for Productive Strains and Strain Improvement
!#
integrated into the virus DNA; in specialized transduction, which occurs only in some
temperate phages, DNA from a specific region of the host DNA is integrated into the viral
DNA and replaces some of the virus’ genes.
It is now possible by methods which will be discussed later under the section on
genetic engineering to excise genes responsible for producing certain enzymes and
attach them on the special mutant viral particles, which do not cause the lysis of their
hosts. Several hundreds of virus particles carrying the attached gene may therefore be
present in one single bacterial cell following viral replication in it. The result is that the
enzyme specified by the attached gene may be produced up to 1,000-fold. Gene
amplification by phage is much higher than that obtained by plasmids (see below). The
method is a well-established research tool in bacteria including actinomycetes but
prospects for its use in fungi appear limited.
7.2.2.2.2 Transformation
Transformation is a change in genetic property of a bacterium which is brought about
when foreign DNA is absorbed by, and integrates with the genome of, the donor cell. Cells
in which transformation can occur are ‘competent’ cells. In some cases competence is
artificially induced by treatment with a calcium salt. The transforming DNA must have a
certain minimum length before it can be transformed. It is cut by enzymes, endonucleases,
produced by the host before it is absorbed.
Reports of transformation in Streptomyces spp have been made. Transformation has
been used to introduce streptomycin production into Streptomyces olivaceus with DNA
from Streptomycin grisesus. Oxytetracycline producing ability was transformed into
irradiated wild-type S. rimosus, using DNA from a wild-type strain. The technique has
also been used to transform the production of the antifungal antibiotic thiolutin from S.
pimpirin to a chlortetracycline producing S. aureofaciens which subsequently produced
both antibiotics. An inactive strain of Bacillus was transformed to one producing the
antibiotic bacitracin with the same method. The method has also been used to increase
the level of protease and amylase production in Bacillus spp. The method therefore has
good industrial potential.
7.2.2.2.3 Conjugation
Conjugation involves cell to cell contact or through sex pili (singular, pilus) and the
transfer of plasmids. Conjugation involves a donor cell which contains a particular type
of conjugative plasmid, and a recipient cell which does not. The donor strain’s plasmid
must possess a sex factor as a prerequisite for conjugation; only donor cells produce pili.
The sex factor may on occasion transfer part of the hosts’ DNA. Mycelial ‘conjugation’
takes place among actinomycetes with DNA transfer as in the case of eubacteria. Among
sex plasmids of actinomycetes, perhaps the two best known are plasmids SCP1 and
SCP2. Plasmids play an important role in the formation of some industrial products,
including many antibiotics. Plasmids will be discussed in more detail later in this
chapter.
7.2.2.2.4 Parasexual recombination
Parasexuality is a rare form of sexual reproduction which occurs in some fungi. In
parasexual recombination of nuclei in hyphae from different strains fuse, resulting in the
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Modern Industrial Microbiology and Biotechnology
formation of new genes. Parasexuality is important in those fungi such as Penicillium
chrysogenum and Aspergiluss niger in which no sexual cycles have been observed. It has
been used to select organisms with higher yields of various industrial product such as
phenoxy methyl penicillin, citric acid, and gluconic acid. Parasexuality has not become
widely successful in industry because the diploid strains are unstable and tend to revert
to their lower-yielding wild-type parents. More importantly is that the diploids are not
always as high yielding as the parents.
7.2.2.2.5
Protoplast fusion
Protoplasts are formed from bacteria, fungi, yeasts and actinomycetes when dividing
cells are caused to lose their cell walls. Protoplasts may be produced in bacteria with the
enzyme lysozyme, an enzyme found in tears and saliva, and capable of breaking the
b-1-4 bonds linking the building blocks of the bacterial cell wall. Protoplast fusion
enables recombination in strains without efficient means of conjugation such as
actinomycetes. It has also been used previously to produce plant recombinants. The
technique involves the formation of stable protoplasts, fusion of protoplasts and
subsequent regeneration of viable cells from the protoplasts. Fusion from mixed
populations of protoplasts is greatly enhanced by the use of polyethylene glycol (PEG).
Protoplast fusion has been successfully done with Bacillus subtilis and B. megaterium and
among several species of Streptomyces (S. coeli-color, S. acrimycini, S. olividans, S. pravulies)
has been done between the fungi Geotrichum and Aspergillus. The method has great
industrial potential and experimentally has been used to achieve higher yields of
antibiotics through fusion with protoplasts from different fungi.
7.2.2.2.6 Site-directed mutation
The outcome of conventional mutation which we have discussed so far, is random, the
result being totally unpredictable. Recombinant DNA technology and the use of
synthetic DNA now make it possible to have mutations at specific sites on the genome of
the organism in a technique known as Site-Directed Mutagenesis. The mutation is
caused by in vitro change directed at a specific site in a DNA molecule. The most common
method involves use of a chemically synthesized oligonucleotide mutant which can
hybridize with the DNA target molecule; the resulting mismatch-carrying DNA duplex
may then be transfected into a bacterial cell line and the mutant strands recovered. The
DNA of the specific gene to be mutated is isolated, and the sequence of bases in the gene
determined (Chapter 3). Certain pre-determined bases are replaced and the ‘new’ gene is
reinserted into the organism. Site-directed mutagenesis creates specific, well-defined
mutations (i.e., specific changes in the protein product). It has helped to raise the
industrial production of enzymes, as well as to produce specific enzymes.
7.2.2.2.7 Metabolic engineering
Metabolic engineering is the science which enables the rational designing or redesigning
of metabolic pathways of an organism through the manipulation of the genes so as to
maximize the production of biotechnological goods. In metabolic engineering, existing
pathways are modified, or entirely new ones introduced through the manipulation of the
genes so as to improve the yields of the microbial product, eliminate or reduce
undesirable side products or shift to the production of an entirely new product. It is a
Screening for Productive Strains and Strain Improvement
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modern evolution of an existing procedure which as described earlier in Chapter 6, is
used to induce over production of products by blocking some pathways so as to shunt
productivity through another. In the older procedure the pathways are shut off by
producing mutants in which the pathways are lacking using the various mutation
methods described earlier. In metabolic engineering the desired genes are isolated,
modified and reintroduced into the organism. Metabolic engineering is the logical end of
site-directed mutagenesis. It has been used to overproduce the amino acid isoluecine in
Corynebacterium glutamicum, and ethanol by E. coli and has been employed to introduce
the gene for utilizing lactose into Corynebacterium glutamicum thus making it possible for
the organism to utilize whey which is plentiful and cheap. Through metabolic
engineering the gene for the utization of xylose was introduced into Klebsiella sp making
it possible for the bacterium to utilize the wood sugar.
It is equally applicable to primary and secondary metabolites alike. Among primary
metabolites the alcohol producing adhB gene from the high alcohol yielding bacterium,
Zymomonas mobilis was introduced into E. coli and Klebsiella oxytoca, enabling these
organisms to produce alcohol from a wide range of sugars, hexose and pentose. Other
primary metabolites which have been produced in other organisms by introducing genes
from extraneous sources are carotenoids, the intermediates in the manufacture of vitamin
A in the animal body, and 1,3 propanediol (1,3 PD) an intermediate in the synthesis of
polyesters. 1,3 PD is currently derived from petroleum and is expensive to produce.
1,3 PD has been produced by E. coli carrying genes from Klebsiella pneumoniae able to
anaerobically produce the diol.
Among secondary metabolites, increase in the production of existing antibiotics, and
the production of new antibiotics and anti-tumor agents have been enabled by metabolic
engineering. The transfer of genes from Streptomyces erythreus to Strep lividans facilitated
the production of erythromycin in the latter organism. In the field of anti-tumor drugs,
epirubicin has less cardiotoxicity than others such as the more frequently prescribed
doxorubicin. The chemical production of epirubicin is complicated and requires seven
steps. However using a metabolic engineering method in which the erythromycin
biosynthetic gene was introduced into Strep peucetius it has been possible to produce it
directly by fermentation.
7.2.2.2.8 Genetic engineering
Genetic engineering, also known as recombinant DNA technology, molecular cloning or
gene cloning. has been defined as the formation of new combinations of heritable
material by the insertion of nucleic acid molecules produced by whatever means outside
the cell, into any virus, bacterial plasmid or other vector system so as to allow their
incorporation into host organisms in which they do not naturally occur but in which they
are capable of continued propagation
The DNA to be inserted into the host bacterium may come from a eucaryotic cell, a
prokaryotic cell or may even be synthesized chemically. The vector-foreign DNA complex
which is introduced into the host DNA is sometimes known as a DNA chimera after the
Chimera of classical Greek mythology which had the head of lion, the body of a goat and
the tail of a snake.
A species has been described as a group of organisms which can mate and produce
fertile offspring. A dog cannot mate with a cat; even if they did the offspring would not be
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Modern Industrial Microbiology and Biotechnology
fertile. A horse and the donkey are not the same species. Although they can mate, the
offspring the mule, is not fertile. Genetic engineering has enabled the crossing of the
species barrier, in that DNA from one organism can now be introduced into another
where such exchange would not be possible under natural conditions. With this
technology engineered cells are now capable of producing metabolic products vastly
different from those of the unaltered natural recipient.
Procedure for the Transfer of the Gene in Recombinant DNA Technology
(Genetic Engineering)
In broad items the following are the steps involved in in vitro recombination or genetic
engineering. The bulk of the work done so far has been with E. coli as the recipient organism
1. Dissecting a specific portion from the DNA of the donor organism.
2. Attachment of the spliced DNA piece to a replicating piece of DNA (or vector),
which can be from either a bacteriophage or a plasmid.
3. Transfer of the vector along with the attached DNA (i.e., the DNA chimera) into the
host cell.
4. Isolation (or recognition) of cells successfully receiving and maintaining the vector
and its attached DNA.
7.2.2.2.8.1 Dissection of a portion of the DNA of the donor organism
The donor DNA may come from a plant, an animal, a microorganisms or may even be
synthesized in the laboratory.
The dissection of DNA at specific sites is done by enzymes obtained from various
bacteria and known as restriction endonucleases. They will be discussed briefly below.
(i) Nature and Types of restriction endonucleases
Restriction endonucleases are nucleic acid-splitting enzymes and are termed ‘restriction’
because they help a host cell destroy or restrict foreign DNA which enter the cell. The host
protects its DNA from its own restriction endonucleases by the introduction of methyl
groups at recognition sites where the cleavage of the DNA occurs. The host DNA so
protected is said to be ‘modified.’ For every restriction enzyme there is a modification one
hence the enzymes exist as restriction-modification complexes. Their
CH 3
5' G A A T T C 3'
3' C T T A A G 5'
5' G A A T T C 3'
3' C T T A A G 5'
CH 3
Restriction of DNA
Modification of DNA
discovery was an important landmark in molecular biology. Daniel Nathans and
Hamilton Smith received the 1978 Nobel Prize in Physiology and Medicine for their
isolation of restriction endonucleases, which are able to cut DNA at specific sites.
Conventionally restriction enzymes are denoted as the single stranded DNA; the
position of the restriction is written / while the position of the modification is written as
an asterisk *. Thus the representation for the above enzyme would 5’G/AA*TTC3’.
Screening for Productive Strains and Strain Improvement
!'
There are four different types of restriction endonucleases: Types I, II, III and IV (Type
IV is designated Type II S by some authors), but only Type II is used extensively in gene
manipulations. In Types I and III, one enzyme is involved for recognition of specific DNA
sequences for cleavage and methylation, but the cutting positions are at variable
distances from these sites (sometimes up to 1000 base pairs (bps)) away from these sites.
Type IV cuts only methylated DNA. As most molecular biology work is done with Type II
endonucleases and only they will be discussed.
Type II endonucleases have the following advantages over the others. Firstly in Type II
systems, restriction and modification are brought about by different enzymes and hence
it is possible to cut DNA in the absence of modification (note that in Types I and III a
single enzyme is involved); secondly, Type II enzymes are easier to use because they do
not require enzyme cofactors. Finally as will be seen below they recognize a defined
symmetrical sequence and cut within this sequence.
Type II restriction endonucleases recognize and cut DNA within particular sequences
of 4 to 8 nucleotides in an axis of symmetry in such a way that the sequences of the top
strand when read backwards are exactly like the bottom on the other side of the axis thus:
5’-A T G
C A T-3’
3’ -T A C
G T A-5’
Axis of symmetry
Such sequences are referred to as palindromes. Type II restriction endonucleases were
discovered in Haemophilus influenzae in 1970. About 3,000 of theses enzymes have now
been discovered and they cut in about 200 patterns; many of them are available
commercially.
(ii) Nomenclature of restriction endonucleases
The nomenclature of restriction endonucleases is based on the proposals of Smith and
Nathans and the currently adopted procedure is as follows:
(a) The species name of the host organisms is identified by the first letter of the genus
name and the first two letters of the species name to form a three-letter abbreviation
written in italics. For example, E. coli is Eco and Haemophilus inflenzae, Hin.
(b) Strain or type identification is supposed to be written as a subscript. Thus, E. coli
strain K, EcoK. In practice it is all written in one line Ecok.
(c) Where a particular host has several different restriction and modification systems,
these are identified by Roman numerals. Thus, those from H. influenzae strain Rd.
would be Hind I, Hind II, Hind III, in the order of their discovery.
(d) Restriction enzymes have the general name endonuclease R and in addition carry
the system name, thus endonuclease R. Hind I. Modification enzymes are named
methylase M; thus the modification enzyme from H. influenzae Rd. is named
methylase M. Hind I. Where the context makes it clear that restriction enzymes are
being discussed, ‘endonuclease R’ is left out leaving Hind I as in the example
quoted above.
(iii) Cutting DNA by Type II endonucleases
Type II endonucleases recognize and break DNA within particular sequences of four,
five, six, or seven nucleotides (Table 7.4A, B) which have a symmetry along a central axis.
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Modern Industrial Microbiology and Biotechnology
Table 7.4
Restriction Endonucleases
A: Patterns of Endonucleases Cutting);
B: Some Restriction Endoncleases and their Recognition Sequences
The same restriction endonuclease is used to cut the foreign DNA to be inserted into a
vector, as well as the vector itself, in order to open it up.
Restriction enzymes cut DNA between deoxyribose and phosphate groups, leaving a
phosphate at 5’ end and an OH group at the 3’. The restriction enzymes used in genetic
engineering cut within their recognition sites and generate one of three types of ends (see
Table 74.4A):
a) Single-stranded, “sticky” or cohesive ends as cut by Bam H1 (1, 5’ overhangs).
b) Single-stranded, “sticky” or cohesive ends as cut by Kpn 1 (2, 3’ overhangs).
c) Double-stranded, “blunt” ends as cut by Sma 1. (3, Blunts)
Screening for Productive Strains and Strain Improvement
"
The single-stranded sticky or cohesive ends of DNA ends (Table 7.4A and Fig. 7.5) will
join (anneal) with any DNA with sticky ends, having complimentary bases no matter the
origin of the DNA, provided that both DNA samples have been cut with the same
restriction enzyme.
Some restriction endonucleases and their recognition sequences are given in Table
7.4B.
7.2.2.2.8.2 The attachment of the spliced piece of DNA to a vector
(i) Joining DNA molecules: Three methods are used for the in vivo ‘tying’ of DNA
molecules. The first method uses an enzyme DNA ligase to tie sticky ends
produced by restriction endonucleases; the second is the use of another DNA
ligase produced by E. coli infected by T4 bacteriophages to link blunt ended DNA
fragments. The third method uses an enzyme terminal deoxynucleotidyl –
transferase isolated from calf thymus to introduce single-stranded complimentary
tails to two different DNA populations after which they anneal when mixed. Only
the first, method, i.e., the use of DNA ligase, will be discussed, because this has
been used extensively.
(ii) The use of DNA ligase to join foreign DNA to the vector: High concentrations of the
DNA of the previously circular vector (usually a plasmid) and of the foreign DNA
to be cloned onto the vector, are mixed. Both DNA types have sticky ends having
been treated with the same restriction endonuclease: in the case of the foreign DNA
to cut it from its source and in the case of the vector, to open it up. Complimentary
sticky ends from the foreign DNA and the vector anneal leaving however gaps
created by the absence of a few base pairs in opposite strands (Fig. 7.5).
The enzyme DNA ligase can repair these gaps to create an intact duplex. DNA
ligase is produced by E. coli and phage T4. The ligase from T4 can, however, join
blunt-ended DNA whereas that from E. coli cannot. The vector-foreign DNA
chimaera is then introduced into the bacterial cell by transformation.
To prevent recircularization of the linearized vector, it may be treated with
alkaline phosphotase. When it is so treated circularization can only occur when a
foreign DNA is introduced. A gap is left at each joint. These gaps are closed after
transformation by the hosts’ repair system.
(iii) Vectors used in recombinant DNA work: Two broad groups of cloning vehicles have
been used, namely plasmids and lamda phages. Both have replication systems
that are independent of that of the host cell.
7.2.2.2.8.3 Plasmids
Plasmids are circular DNA molecules with molecular weights ranging from a few million
to a few hundred million Daltons. Plasmids appear to be associated with virtually all
known bacterial genera. They replicate within the cell. Some of the larger plasmids,
known as conjugative plasmids, carry a set of genes which promote their own transfer in a
sexual process known as conjugation which has already been discussed. Smaller
plasmids are usually non-conjugative but their transfer can usually be promoted by the
presence of a conjugative plasmid in the same cell.
Besides genes for sexual transfer, plasmids usually carry genes for antibiotic or heavy
metal resistance. They often also carry genes for the production of toxins, bacteriocins,
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Modern Industrial Microbiology and Biotechnology
Fig. 7.5 Generalized Diagram of Procedure for Genetic Engineering
Screening for Productive Strains and Strain Improvement
"!
antibiotics, and unusual metabolites. In some cases they may carry genes for unusual
capabilities such as the breakdown of complex organic compounds. Plasmids are,
however, not essential for the cell’s survival.
Two important features of plasmids to be used in genetic experiments may be
compared by examining two plasmids. Plasmid psC101 has only two to five copies per
cell and replicates with its host DNA. It is said to be under ‘stringent’ control. However,
another plasmid pCol E 1 is found in about 25–30 copies per cell. It has a ‘relaxed control’
independent of the host and replicates without reference to the host DNA. When the host
cell is starved of amino-acids or its protein synthesis is inhibited in some other manner,
such as with the use of chloramphenicol, the Col E 1 plasmid continues to replicate for
several hours until there are 1,000 to 3,000 copies per cell. Due to this high level of gene
dosage (also referred to as gene amplification), products synthesized because of the
presence of these plasmids are produced in extremely high amounts, a property of
immense importance in biotechnology and industrial microbiology. Generally
conjugative plasmids are large, exhibit stringent control of DNA replication, and are
present in low copy numbers; on the other hand, non-conjugative plasmids are small,
show relaxed DNA replication, and are present in high numbers. Many other plasmid
vectors exist, some constructed in the laboratory (Table 7.5).
(i) Ideal properties in a plasmid used as a vector
A plasmid to be used in genetic engineering should ideally have the following properties:
(a) the plasmid should be as small as possible so the unwanted genes are not
transmitted, as well as to facilitate handling;
(b) it should have an origin of replication, the site where DNA replication initiates;
(c) it should have a relaxed mode of replication;
(d) it should have sites for several restriction enzymes;
(e) it should carry, preferably, two marker genes. Marker genes are those which
express characteristics by which the plasmid can be identified. Such
characteristics include resistance to one or more antibiotics. A marker of great
importance is the ability to satisfy auxotrophy, i.e., the ability to produce an amino
acid or other nutritional component which the host’s chromosome is incapable of
producing.
Table 7.5 Some commonly used plasmid cloning vehicles
Plasmid
Molecular weight
(x 10-6)
Marker*
pSC101
Col E1
pMB9
pBR313
5.8
4.2
3.6
5.8
Tc
Colimm
Tcr, Colimm
Tcr, Apr
Colimm
pBR322
2.6
r
Single restriction sites
BamHI, EcoRI, HindIII, Hpal, SalI, Smal
EcoRI
BamHI, EcoRI, HindIII, Hpal, SalI Smal
BamHI, EcorI, HindIII, Hpal, SalI, Smal
BamHI, EcoRI, HindIII, pstI, SalI
r
*Tc : tetracycline resistance. Ap : ampicillin resistance
colimm: colicin immunity
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Modern Industrial Microbiology and Biotechnology
(f) the nucleotide sequence of the plasmid should be known;
(g) for safety reasons the plasmid should not be able to replicate at mammalian body
temperatures so that should it enter the human body and be able to produce
deleterious substances, it should fail to replicate;
(h) for safety reasons also, it should not be highly transmissible by conjugation if it
controls the production of any material harmful to the mammalian body;
(i) the plasmid as a cloning vehicle should have a site for inducing transcription
across the inserted fragment. The plasmid-initiated transcription should be
controlled by the host (by induction or repression). Uncontrolled transcription
could be harmful to the host.
Table 7.5 shows some commonly used plasmid cloning vehicles. They carry various
markers based on tetracycline or ampicillin resistance or immunity against colicin attack.
The marker may be carried either on the plasmid or on the inserted DNA. If neither of
them carries a marker then DNA carrying a marker can be grafted on to either the vector
or the insert.
(ii) Plasmids currently in use for cloning
In the early years of genetic engineering, naturally occurring plasmids such as Col E1
and pSC 101 were used as cloning vectors. They were small and had single sites for the
common endonucleases. However they lacked markers which would help select
transformed organisms. New plasmids were therefore developed. The best and most
commonly used is pBR322 developed by Francisco Bolivar. (In naming plasmids p is
used to show it is a plasmid; p is followed by the initials of the worker who isolated or
developed the plasmid; numbers are used to denote the particular strain). Plasmid
pBR322 has all the properties expected in a plasmid vector: low molecular weight, two
markers, (resistance to ampicillin, ApR and tetracycline, TcR) an origin of replication, and
several single-cut replication sites. (see map of pBR322 in Fig 7.6). Modifications of the
original pBR322 have been made to suit special purposes, and consequently many
variants exist in the pBR322 family. A widely used variant of pBR322 is pAT153, which
some consider a better vector than its parent because it is present in more copies per cell
than pBR322 Another series of popular vectors is the pUC family of vectors (Fig. 7.7). It
has several unique restriction sites in a short stretch of DNA, which is an advantage in
some kinds of work.
7.2.2.2.8.4
Phages
Two types of phages have been developed for cloning, l lamda, and M13. Most of the
phages used for cloning are derivatives of the lamda phage of E. coli because so much is
already known about this phage. Derivatives are used because the wild-type phage is not
suitable as a vector as it has several targets of sites for most of the most commonly used
endonucleases. The chromosome of phage must be folded and encapsulated into the
head of the virus in order to provide a mature virion. The amount of DNA that can enter
the head is limited, and hence the available DNA in a phage is also limited. Therefore
unwanted phage DNA must be removed as well as all but one of restriction targets for the
chosen enzyme.
The DNA of phage lambda when it is isolated from the phage particle is linear and
double-stranded. At each end of the chain are single-stranded portions which are
Screening for Productive Strains and Strain Improvement
"#
Fig. 7.6 Genetic Map of Plasmid pBR322 Showing Unique Recognition Endonuclease Sites
and Genes for Tetracycline and Ampicillin Resistance
Fig. 7.7 Genetic Map of pUC18
complimentary to each other, much like the ‘sticky ends’ produced from DNA cutting by
restriction endonucleases (Fig. 7.9). These lamba DNA pieces are able to circularize and
replicate independently within the host.
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Modern Industrial Microbiology and Biotechnology
Fig. 7.8
Structure of l and M13 Bacteriophages
The middle portion of the linear double-stranded phage DNA is non-essential for
phage growth and it is here that the foreign DNA is introduced. The more distal positions
carry genes which code for essential components such as the head, tail of the
bacteriophage and the host lysis (Fig. 7.9)
(i) Transfection: The linear chimera can be introduced by transformation. (When
virus DNA is transformed the process is known as transfection.) However, much of
introduced chimeras are restricted in comparison to when pure phage DNA is
transfected.
(ii) Packaging the chimeras into virus heads: The recombinant DNA or chimera may be
packaged into a virus head and a tail attached by in vitro means. The procedure for
this packaging is outside the scope of this book but may be found elsewhere. Once
packaged, the synthetic virus can then inject its DNA into the host in the usual way.
7.2.2.2.8.5 Cosmids
Cosmids are plasmids constructed from phage DNA by circularization at the ‘sticky,’
single stranded ends or cos sites. Foreign DNA is attached to the cosmid which is then
packaged into a phage. When the cosmid is injected it circularizes like other virus DNA
Fig. 7.9 Map of Chromosome of Lamba (l) Phage
Screening for Productive Strains and Strain Improvement
"%
but it does not behave as a phage, rather it replicates like a plasmid. Drug resistance
markers carried on it help to identify it.
A number of commercially available vectors are based on phages and some are shown
in Table 7.6
Table 7.6 Phage-based vectors
Vector
Features
r
pBR322
pAT 153
pGEMTM-32
pClTM
pCMV-ScriptTM
r
Ap Tc
Single cloning sites
AprTcr
Single cloning sites
Apr
MCS
SP6/T7 promoters
lacZ a-peptide
Apr
MCS
T7 promoter
CMV enhance/promoter
Neor
Large MCS
CMV enhancer/promoter
Applications
General cloning and subcloning in E. coli
General cloning and subcloning in E. coli
General cloning and
in vitro transcription in E.
coli and mammalian cells
Expression of genes in
mammalian cells
Expression of genes in
mammalian cells
Cloning of PCR products
7.2.2.2.8.6 Transfer of the vector along with the attached DNA into the host all
The vector once spliced with the endonuclease cannot reform into a circular structure
unless a suitable fragment of the foreign DNA with a complimentary ‘sticky end’ fits in.
Foreign DNA digests produced by physical inactivation may also be used. If a large
enough amount of foreign DNA digest is used the probability is that a piece with the
appropriate complementary end will fit in. The new hybrid DNA is introduced into the
host cell by transformation. Transformation is facilitated by treating the host cells in
calcium salts, after washing them in magnesium salts.
7.2.2.2.8.7 Recognizing the transformed cell
The introduction of the new property into the host may be detected by growing the cells in
a medium containing antibiotics whose resistance is specified in the introduced foreign
DNA. Growth should occur if the resistance gene was transferred. If genes for the
synthesis of some products were introduced via the chimera, the transformed bacteria
should grow on the selective medium. The products are then examined for the synthesis
of the new compound. For the introduced gene to lead to protein synthesis a suitable
promoter must be present; it may be introduced from other organisms if an appropriate
one is not indigenous.
7.2.2.2.8.8
Gene transfer into organisms other than E. coli,
including plants and animals
The methods discussed above are those developed primarily for E. coli. The discussion
below will look at the introduction of DNA into bacteria other than E. coli as well into
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other organisms, including plants and animals. Some of the methods to be discussed are
also used on E. coli.
(i) Delivery into bacteria other than E. coli
(a) Electroporation
In the process of electroporation, cells into which DNA is to be introduced (i.e., cells to be
transfected) are exposed to high-voltage electric pulses. This creates temporary holes in
the cell membrane through which DNA can pass. Electroporation can be used for
transfecting cells of Bacilli spp and actinomycetes, especially when protoplasts are
produced from the cells. Electroporation is the short form for electric field-mediated
membrane permeabilization. It is still used for E. coli, especially when chimera longer
than about 100 kilobases (100 kb) are to be used. In general electroporation can be used for
transfecting bacteria and archae, after the appropriate electric voltage and other
parameters have been worked out. As will be seen below, it is also used for transfecting
plant cells.
(b) Conjugation
In some bacteria where other means of introducing DNA appear difficult or have failed,
the natural means of transferring DNA by plasmid mediated conjugation has been
exploited. A conjugative plasmid which is carrying the insert and which has the genes
for its own transfer is used. However where this is not possible, a conjugative plasmid
with its own transfer gene may first be introduced, followed by the non-conjugative (i.e.,
does not promote the transfer of DNA through pili) plasmid carrying the DNA insert.
This has been used in some strains of Pseudomonas.
(c) Use of Liposomes
When the DNA to be introduced is first entrapped in phospholipid droplets known as
liposomes, it enhances the entry of the DNA into protoplasts of Gram-positive Bacillus
and actinomycetes. Liposomes have also been used for delivering DNA into animal cells.
Liposomes are microscopic, fluid-filled vesicles whose walls are made of layers of
phospholipids identical to the phospholipids that make up cell membranes. The outer
layer of the vesicle is hydrophobic, while the inner layer is hydrophilic; this enables the
liposome to carry water soluble materials within it. They can be designed so that they
have cationic, anionic or neutral charges at the hydrophobic end depending on the
purpose for which they are meant. They are used for introducing DNA into animal, plant
or bacterial cells. When used for introducing DNA into plant cells, such cells must have
their cell walls removed, yielding protoplasts; with bacteria, the cell walls must also be
removed to yield sphaeroplasts. Liposomes have been used experimentally to carry
normal genes into a cell to replace defective, disease-causing genes in gene therapy.
Liposomes are used to deliver certain vaccines, enzymes, or drugs (e.g., insulin and some
cancer drugs) to the animal body. Liposomes are sometimes used in cosmetics because of
their moisturizing qualities.
(ii) Delivery of DNA into Plant cells
Plants are peculiar in that most single plant cells can be caused to develop into the entire
plants. Successfully transfecting (i.e., introducing foreign DNA into) a plant cell will
result in having the foreign DNA as part of the genetic apparatus of the transfected plant.
Screening for Productive Strains and Strain Improvement
"'
The introduction of foreign DNA into plants is done for the improvement of the
agricultural, ornamental, nutritional, or horticultural value of the plant. It is also done to
convert the plants into ‘living fermentors’ which with the appropriate genes can
manufacture cheaply, some industrially important materials, which it may not even be
possible to produce by chemical means. Several methods are available for the delivery of
DNA into plants
(a) Plant Transfection with Ti plasmid of Agrobacterium tumefaciens
The Gram-negative soil bacterium, Agrobacterium tumefaciens, is the causative agent of
crown gall, a disease which produces tumors in plants (mainly dicots) on entering
through a wounded plant cell.
The pathogenic properties of the bacterium are due to the Ti (tumor inducing) plasmid
which it carries. Part of the Ti plasmid, known as the T (transfer) DNA, is transferred to
the plant cell and is integrated into the genome of the host under the direction of the
virulence gene (Fig. 7.10). It is within the TDNA that foreign DNA can be introduced. A
section of the TDNA codes for the production of auxins and cytokins which lead to the
formation of galls or tumors. Another section codes for the production of conjugates of
amino acids and sugars known as opines and which are metabolized by Agrobacterium
tumefaciens residing in the tumor. The oncogenic (tumor-causing) and the
opine-producing portions of the TDNA of wild-type Agrobacterium tumefaciens are
removed, when it is to be used for cloning. Furthermore, marker genes (e.g., for kamycin
resistance) are introduced into the plasmids so that transformed plants can be identified.
Because the marker genes are of bacterical origin an origin of replication from E. coli is
also introduced. The TDNA is defined by the left and right borders. The sequences of the
right border are essential for the TDNA transfer and integration into the host plant. The
transfected plant cells therefore result in normal plants. Ti plasmids in which the
oncogenic section has been removed is said to be ‘disarmed’. Such disarmed plasmids
lack the sequences necessary to produce the phytohormones which give rise to diseased
conditions, gall or tumor. Other properties such as the transfer of DNA are still active and
the regeneration of healthy plants can still occur. The Agrobacterium tumefaciens Ti
plasmid has been successfully and widely used in cloning in plants. However it has been
more successful in dicots than in monocots.
Fig. 7.10 Map of Agrobacterium tumefaciens Ti Plasmid
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Modern Industrial Microbiology and Biotechnology
(b) Use of Viruses
The cauliflower mosaic virus (caMV) has been used as a powerful vector for introducing
DNA into plants. It is a double-stranded DNA virus with 8025 base pairs.
Certain portions of the virus are dispensable and foreign genes can be replace them.
However, it has a limited host range; furthermore foreign sequences are often unstable in
the caMV genome.
(c) Electroporation
Electroporation is widely used for transfecting plant cells. When plants are to be
transfected, protoplasts or whole plant cells placed in contact with exogenous DNA in
foil-lined cuvettes and exposed to high electrical current. The cells become permeable and
take up exogenous DNA, some of which integrate with the plant genome. It has been
successfully used in a wide variety of species using equipment which is relatively
inexpensive.
(d) Biollistic or Microprojectile methods
This is one of the commonest methods used for transfecting plants. In this method a socalled gene gun or particle gun is used to shoot tiny pellets of tungsten or gold coated
with the foreign DNA in question into the leaves or stem of the plant to be transformed. It
is a widely used and highly successful method of transfecting plants using plant
protoplasts, plant cell suspensions, callus cultures, even chloroplasts and mitochondria,
and indeed any form of plant preparation capable of regeneration in dicots, monocots,
The eight shaded boxes are the coding regions
Fig. 7.11
Genetic Map of the Cauliflower Mosaic Virus
Screening for Productive Strains and Strain Improvement
#
and conifers. Success occurs more with linear DNA than with circular; furthermore very
large DNA inserts tend to be broken during the projection. When inside the cell, some of
the introduced DNA get integrated with the plant DNA.
(e) Microinjection
This method involves immobilizing the cells and injecting DNA into protoplasts, walled
cells, or embryos. It is done with a fine needle under the microscope. The technique needs
a lot of skill. Some authors do not think it has much future because only one cell can be
injected at a time.
(iii) Delivery of DNA into Animal cells
Genetic engineering in plants differs in at least two respects from that in animals. Firstly
while plant cells are mostly totipotent (i.e., most plant cells are able to give rise to a new
plant), animal cells cannot give rise to whole animals once differentiated into specialized
cells. In animals the cells that become reproductive cells separate early from those that are
ordinary body (somatic) cells. Somatic cells do not give rise to new animals To create
transgenic animals the foreign DNA must be introduced into cells while they are still
totipotent and differentiation has not occurred. Generally this involves introducing the
DNA into stem cells (yet undifferentiated cells), an egg, the fertilized egg, (oocyte or
zygote) or early embryo.
Some of the methods discussed above for introducing foreign DNA into bacteria and
plants are also applicable to animal cells: electroporation, biollistic methods and
microinjection have all been successfully used in animals. In addition the liposome
(phospholipid) delivery seen in bacteria is also used in animal cells.
Genes are introduced into animal cells as well as in vivo by transduction via viruses in
gene therapy. Four groups of viral vectors are used for gene therapy in humans:
adenoviruses, baculoviruses, herpesvirus vectors, and retroviruses.
Changing the genetic make-up of animals, in large domesticated mammals such as
cows, pigs and sheep, allows a number of commercial applications. These applications
include the production of animals which express large quantities of exogenous proteins
in an easily harvested form (e.g., expression into the milk), the production of animals
which are resistant to infection by specific microorganisms and the production of
animals having enhanced growth rates or reproductive performance.
Most of the work on transgenic animals has been done with mice on account of their
small size and low cost of housing in comparison to that for larger vertebrates, their short
generation time, and their fairly well defined genetics. Foreign DNA is introduced in mice
in one of the following ways: DNA microinjection, embryonic stem cell-mediated gene
transfer and retrovirus-mediated gene transfer, sperm-mediated transfer, transfer into
unfertilized ova.
(a) DNA microinjection
This method involves the direct microinjection of a chosen gene construct (a single gene
or a combination of genes) from another member of the same species or from a different
species, into the pronucleus of a fertilized ovum. The introduced DNA may lead to the
over- or under-expression of certain genes or to the expression of genes entirely new to the
animal species. The insertion of DNA is, however, a random process, and there is a high
probability that the introduced gene will not insert itself into a site on the host DNA that
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Modern Industrial Microbiology and Biotechnology
will permit its expression. The manipulated fertilized ovum is transferred into the
oviduct of a recipient female, or foster mother that has been induced to act as a recipient
by mating with a vasectomized male. Such males cannot inject sperms into the female
because the tubes carrying the sperms, the vas deferens, have been cut. The major
advantage of this method is its applicability to a wide variety of species.
(b) Embryonic stem cell-mediated gene transfer
This method involves prior insertion of the DNA sequence by homologous recombination into an in vitro culture of embryonic stem (ES) cells. Stem cells are undifferentiated
cells that have the potential to differentiate into any type of cell (somatic and germ cells)
and therefore to give rise to a complete organism. These cells are then incorporated into
an embryo at the blastocyst stage of development. The result is a chimeric animal. ES cellmediated gene transfer is the method of choice for gene inactivation, the so-called
knock-out method. This technique is of particular importance for the study of the genetic
control of developmental processes. This technique works particularly well in mice. It
has the advantage of allowing precise targeting of defined mutations.
(c) Retrovirus-mediated gene transfer
To increase the probability of expression, gene transfer is mediated by means of a carrier
or vector, generally a virus or a plasmid. Retroviruses are commonly used as vectors to
transfer genetic material into the cell, taking advantage of their ability to infect host cells
in this way. Offspring derived from this method are chimeric, i.e., not all cells carry the
retrovirus. Transmission of the transgene is possible only if the retrovirus integrates into
some of the germ cells.
(d) Sperm-mediated Gene Transfer
Sperms may be coated with the target DNA or attached to the sperm through a linker
protein, and introduced through surgical oviduct insemination. It has been successfully
used in pigs.
With the above techniques the success rate in terms of live birth of animals containing
the transgene is extremely low. If there is birth, the result is a first generation (F1) of
animals that need to be tested for the expression of the transgene. The F1 generation may
result in chimeras. When the transgene has integrated into the germ cells, the germ line
chimeras are then inbred for 10 to 20 generations until homozygous transgenic animals
are obtained and the transgene is present in every cell. At this stage embryos carrying the
transgene can be frozen and stored for subsequent implantation.
7.2.2.2.8.9
Application of genetic engineering in
industrial microbiology and biotechnology in general
The unparalleled ability of DNA to replicate and reproduce itself is truly remarkable.
What this means is that, put crudely, DNA of a given sequence coding for the production
of a polypeptide or protein in organism A will lead to the production of the same
polypeptide or protein if the same sequence is put into organism B. This is the basic
assumption underlying the numerous advances in our manipulation of the biotic world
for the benefit of humans. This section looks only at some of the numerous positive
changes recombinant DNA technology has contributed to spreading a better quality of
life to millions of people around the world through improvements in agriculture, health
care delivery and industrial productivity.
Screening for Productive Strains and Strain Improvement
#!
(i) Production of Industrial Enzymes
Genetically engineered bulk enzymes are used mostly in the food industry (baking,
starch manufacture, fruit juices), the animal feed industry, in textile manufacture, and in
detergents. A leading manufacturer of these enzymes among world manufacturer is
Novo Enzymes of Denmark.
The advantages of using engineered enzymes are as follows:
(a) such enzymes have a higher specificity and purity;
(b) it is possible to obtain enzymes which would otherwise not be available due to
economical, occupational health or environmental reasons;
(c) on account of the higher production efficiency there is an additional environmental benefit through reducing energy consumption and waste from the production
plants;
(d) for enzymes used in the food industry particular benefits are for example a better
use of raw materials (juice industry), better shelf life of the final food and thereby
less wastage of food (baking industry) and a reduced use of chemicals in the
production process (starch industry);
(e) for enzymes used in the animal feed industry particular benefits include a
significant reduction in the amount of phosphorus released to the environment
from farming.
Two enzymes will be discussed briefly: chymosin (rennets) and bovine somatotropin
(BST).
Chymosin is also known as rennets or chymase and is used in the manufacture of cheese.
It used to be produced from rennets of farm animals, namely calves. Later it was produced
from fungi, Rhizomucor spp. Over 90% of the chymosin used today is produced by E. coli,
and the fungi, Kluyveromyces lactis and Aspergillus niger. Genetically engineered
Table 7.7
Some genetically engineered industrial enzymes (selected from brochure by Novo
Industries of Denmark)
Type of enzyme
Alpha-amylase/Bacillolysin/Xylanase
Amyloglucosidase
Cellulase
Decarboxylase
Glucoamylase
Glucose oxidase
Lipase
Lipase
Maltogenic amylase
Pectate lyase
Pectinesterase
Phytase
Protease
Pullulanase/Amyloglucosidase
Main application
Brewing industry, starch industry, baking industry
Alcohol industry, fruit processing
Detergent industry; textiles
Brewing industry
Alcohol industry, starch industry
Baking industry
Oils and fats industry; baking industry; dairy
industry; leather industry
Pasta/noodles
Starch industry, baking industry
Textile industry, fruit processing
Fruit processing
Animal feed industry
Meat industry; detergent industry
Starch industry, fruit processing
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Modern Industrial Microbiology and Biotechnology
chymosin is preferred by manufacturers because while it behaves in exactly the same
way as calf chymosin, it is purer than calf chymosin and is more predictable.
Furthermore, it is preferred by vegetarians and some religious organizations.
Bovine Somatotropin (BST) is a growth hormone produced by the pituitary glands of
cattle and it helps adult cows produce milk. It is produced by genetic engineering in E. coli
using a plasmid vector. Supplementing dairy cows with bovine somatotropin safely
enhances milk production and serves as an important tool to help dairy producers
improve the efficiency of their operations. The use of supplemental BST allows dairy
farmers to produce more milk with fewer cows, thereby providing them with additional
economic security. It is marketed by Monsanto as Posilac.
(ii) Enhancing the activities of Industrial Enzymes
Through protein engineering it has been possible to enhance the properties of proteins to
make them more stable to denaturation, more active in their biocatalytic ability and even
to design new properties in existing enzymes. The properties of proteins are due to their
conformation which is a result of their amino acid sequence. Certain amino acids in a
protein play important parts in determining the stability of the protein to high
temperatures, specificity and stability to acidity. In protein engineering changes are
caused to occur in the protein by changes in the nucleotide sequence; a change of even a
single nucleotide could lead to a drastic change in a protein. Many industrial processes
are carried out at elevated temperatures, which can unfold the proteins and cause them to
denature. The addition of disulphide bonds helps to stabilize them. Disulphide bonds
are usually added by engineering cysteine in positions where it is desired to have the
disulphide bonds. The addition of disulphide bonds not only increases stability towards
elevated temperatures, but in some instances also increases stability towards organic
solvents and extremes of pH. An example of the increase of stability to elevated
temperatures due to the addition of disulphide bonds is seen in xylanase.
Xylanase is produced from Bacillus circulans. During paper manufacture, wood pulp is
treated with chemicals to remove hemicelluloses. This treatment however leads to the
release of undesirable toxic effluents. It is possible to use xylanase to breakdown the
hemicellulose. However, at the time when bleaching is done, the pulp is highly acidic as
a result of the acid used to digest the wood chips to produce wood pulp. The acid is
neutralized with alkali, but the temperature is still high and would denature native
xylanase. In silico (i.e. computer) modeling showed the sites where disulphide bonds can
be added without affecting the enzyme’s activity. The introduction of the disulphide
bonds did increase the thermostability of the enzyme, making it possible to keep 85% of
its activity after 2 hours at 60°C whereas the native enzyme lost its activity after about 30
minutes at the same temperature.
Another way in which enzyme activity can be enhanced by protein engineering is to
actually increase the activity of the enzyme. This can be done only with an enzyme whose
conformation, including the active sites, is thoroughly understood. Using in silico
modeling, it is possible to predict the effect of changing amino acids at the active site of an
enzyme. This has been done with the enzyme tRNA synthase from Bacillus
stearothermophilus.
Various other properties have been engineered into proteins including a modification
of the metal co-factor and even a change in the specificity of enzymes.
Screening for Productive Strains and Strain Improvement
Native enzyme
##
Engineered enzyme
Disulfide bond
Fig. 7.12 Stabilizing Enzymes through the Introduction of Disulfide Bonds
(iii) Engineered Products or Activities Used for the Enhancement of Human Health
Engineered health care products and activities can be divided into: a) those used to
replace or supplement proteins produced by the human body in insufficient quantities or
not produced at all; b) those involving the replacement of a defective gene; c). those that
are used to treat disease, d) those that are used for prophylaxis or prevention of disease,
i.e., vaccines, or e) those that are used for the diagnosis of disease (Table 7.8). Only insulin
and edible vaccines will be discussed.
Table 7.8
Some genetically engineered health related products
Product
Hormones
Insulin
Human growth hormone (somatotropin)
Follicle stimulating hormone
Immune System Participants
Tumor necrosis factor
Interleukin 2
Lysozyme
A-Interferon
Blood components
Erythropoeitin
Tissue plasminogen activate
Factor VIII
Enzymes
Human DNase I
Application
Treatment of diabetes
Treatment of dwarfism
Treatment of some disorders of the
reproductive system
Anti-tumor agent
Treatment of some cancers
Anti-inflammatory agent
Antiviral
Treatment of anemia and cancers
Dissolves blood clots
Treating hemophilia
Treatment of cystic fibrosis
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Modern Industrial Microbiology and Biotechnology
(1) Insulin
Insulin is a hormone produced by the pancreas; hormones are small proteins. Insulin is
used to treat diabetes of which there are there three types, only two of which are relevant
to this discussion.
Type 1 diabetes (previously known as insulin-dependent diabetes) is an auto-immune disease
where the body’s immune system destroys the insulin-producing beta cells in the
pancreas. This type of diabetes, also known as juvenile-onset diabetes, accounts for 1015% of all people with the disease. It can appear at any age, although common under 40,
and is triggered by environmental factors such as viruses, diet or chemicals in people
genetically predisposed. To live, people with type 1 diabetes must inject themselves with
insulin several times a day and follow a careful diet and exercise plan.
Type 2 diabetes (previously known as non-insulin dependent diabetes) is the most common
form of diabetes, affecting 85-90% of all people with the disease. This type of diabetes,
also known as late-onset diabetes, is characterized by relative insulin deficiency. The
disease is strongly genetic in origin but lifestyle factors such as excess weight, inactivity,
high blood pressure and poor diet are major risk factors for its development. Symptoms
may not show for many years and, by the time they appear, significant problems may
have developed. People with type 2 diabetes are twice as likely to suffer cardiovascular
disease. Type 2 diabetes may be treated by dietary changes, exercise and/or medications.
Insulin injections may later be required.
The third type affects pregnant women, is less common, and will not be discussed.
Genetically engineered insulin was the first major product of biotechnology. As
insulin from some animals is similar to human insulin, beginning from the 1920s, insulin
isolated from the pancreas of farm animals, mainly pigs and cows, was used to treat
diabetes. There were several problems with this product. First it takes several months for
animals to mature and be ready to be slaughtered for their pancreas. This made animalbased insulin expensive since it was difficult to meet the demand. Furthermore such
animal insulin caused immune reactions in some patients and a few became intolerant or
resistant to animal insulin. For a more effective solution the then new technology of
recombinant DNA was resorted to. In 1978, in the laboratory of Herbert Boyer at the
University of California at San Francisco, a synthetic version of the human insulin gene
was constructed and inserted E. coli. In 1982 Eli Lilly Corporation was granted approval
for its genetically engineered insulin. Insulin is a small protein, and today’s insulin is
produced with a synthesized gene, which is expressed in a yeast.
Insulin consists of two amino acid chains: the A peptide chain which is acidic and
with 21 amino acids and the B peptide chain which is basic and has 30 amino acids.
When synthesized the A and B chains are further linked by a 30 amino acid C peptide
chain to produce a structure known as pro-insulin. Pro-insulin is cleaved enzymatically
to yield insulin. (Fig. 7.13).
(2) Edible vaccines
An innovative new approach to vaccine production is the surface expression of the
antigen of a bacterium in a plant. Most current immunization is done by injection
(parenteral delivery) and rarely results in specific protective immune responses at the
mucosal surfaces of the respiratory, gastrointestinal and genito-urinary tracts. Mucosal
Screening for Productive Strains and Strain Improvement
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Top: Structure of insulin. The A chain has 21 amino acids (represented by circles) and the B chain has 30
amino acids. The A and B chains are linked by disulfide bond between cysteine residues (filled circles)
Bottom: Synthesis of Insulin: Proinsulin, the Precursor of Insulin
When synthesized proinsulin consists of an 81-amino acid polypeptide. The C chain is then cleaved off by
a protease (P) to yield insulin.
Fig. 7.13
Structure and Synthesis of Insulin
immune responses represent a first line of defense against most pathogens. In contrast,
mucosally targeted vaccines achieve stimulation of both the systemic as well as the
mucosal immune networks. In addition, mucosal vaccines delivered orally increase
safety and compliance by eliminating the need for needles. In addition many vaccines
depend on the need of maintenance of a ‘cold chain’ (refrigeration) for delivery. Many
developing countries, lack the resources to maintain the chain giving rise to many cases
of vaccine failure, along with the fact they lack the resources and technology for
fermentation industries. Both of these factors create constraints in vaccine use in the
developing world, where these vaccines are needed the most. Combining a cost-effective
production system with a safe and efficacious delivery system, plant edible vaccines,
provide a compelling new opportunity.
Plant-based oral vaccines are cheap, safe and efficient. From the point of the little child
receiving the vaccine, a smile might be elicited when a ‘banana’ vaccine is eaten rather
the sharp cry of the pain of a needle! Vaccines have been produced in several plants,
including a vaccine against dental carries caused by Streptococcus mutans, which was
produced in the tobacco. Two other interesting examples are the expression of the rabies
external antigen in tomatoes and the hepatitis virus antigen in lettuce.
7.2.2.2.8.10 Genetically engineered plants
Plants have been engineered for the introduction of many new desirable properties.
Collectively these attainments represent a major triumph of biotechnology, enabling us to
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Modern Industrial Microbiology and Biotechnology
Table 7.9
Vaccines produced in plants
Vaccine against
Cholera
Foot and mouth disease
Herpes virus
Norwark (diarrhea) virus
Rabies
Plant
Potato
Arabidopsis
Tobacco
Potato
Tobacco
achieve in a few years what would take traditional plant breeding decades to attain, if at
all. Some genetic engineering achievements would be impossible with traditional plant
breeding methods since in the latter, the introduction of new genetic properties occurs
only through the exchange of sexual materials (in the pollen grains) of the same species.
In genetic engineering the natural species barrier is not recognized since the DNA
sequence introduced into a plant can come from another plant of a different species or
even from a non-plant source such as a bacterium, and indeed may even be synthesized.
The introduction of some genetically engineered foods has met with public resistance,
although many have been shown to be safe. What is required is continued public
education about their safety before their introduction, and constant sensitivity to public
opinion thereafter.
The ensuing discussion will be under two headings, a) improving field, production or
agronomic traits and b) modification of consumer products.
Improving Field or Production Characteristics
Numerous improvements have been made in agricultural crops by introducing into them
genes coding for the desired properties, but only plant engineering for herbicide
resistance, resistance to viral diseases, resistance to insect pests, and resistance to salt
stress will be discussed.
(i) Engineering Plants for Herbicide Resistance
An estimated US $10 billion is spent annually on weed killers. In spite of this about 10%
of world crop production is lost to weeds. Herbicides (weed killers) target processes that
are essential and unique to plants. These processes are however important to plants and
weeds alike, and getting methods that are selective for either is difficult. One method that
is used is to engineer crops so they become resistant to the weed killer. Plants can become
resistant to herbicides in one of the four following ways:
(a) overproduction of the herbicide sensitive target, so that some is still left for the
proper cell function despite presence of the herbicide in the cell;
(b) reduction of the ability of the herbicide-sensitive target protein to bind to the
herbicide;
(c) engineering into plants the ability to metabolically inhibit the herbicide;
(d) inhibition of the uptake of the herbicide.
It is important to have some idea of the modes of action of herbicides so as to
understand how the genetic engineering is to proceed. The most common modes of action
are given below and examples of herbicides in each group are given in parenthesis:
Screening for Productive Strains and Strain Improvement
#'
• Auxin mimics (2,4-D, clopyralid, picloram, and triclopyr), which mimic the plant
growth hormone auxin causing uncontrolled and disorganized growth in
susceptible plant species;
• Mitosis inhibitors (fosamine), which prevent re-budding in spring and new
growth in summer (also known as dormancy enforcers);
• Photosynthesis inhibitors (hexazinone, bromoxynil), which block specific
reactions in photosynthesis leading to cell breakdown;
• Amino acid synthesis inhibitors (glyphosate, imazapyr, and imazapic), which
prevent the synthesis of amino acids required for construction of proteins;
• Lipid biosynthesis inhibitors (fluazifop-p-butyl and sethoxydim), that prevent the
synthesis of lipids required for growth and maintenance of cell membranes.
Engineering plants for resistance to glyphosate will be discussed. Glyphosate (Nphosphonomethylglycine) is a non-selective, broad spectrum herbicide that is
systematically translocated to the meristems of growing plants. It causes shikimate
accumulation through inhibition of the chloroplast localized EPSP synthase (5enolpyruvylshikimate-3-phosphate synthase; EPSPs) [EC 2.5.1.19]. Resistance to the
herbicide glyphosate has been developed for soybean. Glyphosate is said to be
environmentally-friendly as it does not accumulate in the environment because it is
easily broken by soil bacteria. Glyphosate kills plants by preventing the synthesis of
certain amino acids produced by plants but not animals. It acts by inhibiting the enzyme
5-enolpyruvyshikimate-3-phosphate synthase (EPSPS), an enzyme in the shikimate
pathway (Fig 7.14) and plays an important part in the synthesis of aromatic amino acids
in plants and bacteria. An EPSPS gene isolated from a glyphosate-resistant E. coli was
linked to a plant promoter and termination transcription sequence and cloned into plant
cells. Tobacco, tomato, potato, petunia, and cotton have been transfected with glyphosate
Fig. 7.14
Mode of Action of Action Glyphosphate
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resistance in this way. They produce large amounts of EPSPS, enough to leave some for
the plants cells to utilize for their metabolism after neutralization of a portion by
glyphosate.
An example of a case where the plant is engineered to inactivate the herbicide before it
can act is bromoxynil, a photosynthesis inhibitor. Plants were made resistant to this
herbicide by engineering into them the gene for nitrilase obtained from the bacterium,
Klebsiella ozaenae. Nitrilase inactivates bromoxynil before it can act.
(ii) Engineering Plants for Pathogenic Microbe Resistance
The majority of microbes attacking plants are fungi, but some bacterial diseases of plants
do exist. Plants are conventionally sprayed with chemicals to eliminate fungal
pathogens. Such chemicals sometimes are not always easily biodegradable, and they
may also find their way into food. A genetic approach which bypasses this problem is to
engineer into plants anti-fungal proteins such as the gene coding for chitinase, an
enzyme which hydrolyzes chitin, a polymer of the amino sugar N-acetyl glucosamine.
Chitinase gene from bean has been cloned into tobacco where chitinase stopped the
attack by the fungus, Rhizoctonia solani. Chitinase is one of the ‘pathogen-related
proteins’ (PRs) synthesized by plants; they also synthesize ant-fungal peptides known
as defensins. Genes coding for these are sought from source of high productivity and
cloned into plant to protect them.
Plant resistance to bacterial disease has also been genetically engineered. For example,
the a-thionin gene from barley has been shown to confer resistance to a bacterial
pathogen, Pseudomonas syringae in transgenic tobacco.
With regard to engineering plants against viruses, when the viral coat of a plant virus
is engineered into a plant, that plant usually becomes resistant to the virus from which
the coat comes. Often the plant is also resistant against other unrelated viruses.
(iii) Engineering Plants for Insect Resistance
Insect pests are devastating to crops, about US $5 billion are currently being used to
control them annually with chemicals. The advantages of using biological means of
controlling insect pest have been highlighted in Chapter 17. The methods described
relate to the use of biological insecticides which are sprayed on plants. Such sprayed
insecticides have the disadvantage that thet are inactivated by ultraviolet rays from the
sun or may be washed away by the rain. Genetic engineering of crops for resistance
against insect pests has the advantage that the active constituents are protected from the
environment and remain within the plant.
The major strategy of producing plants resistant to insect pests is to engineer the gene
for producing the toxic crystals of Bacillus thuringiensis (Bt) into plants. These crystals are
produced in Bt but in no other Bacillus sp. They are small proteins and are highly specific
against given insects. In such susceptible insects they bind to receptors in the gut lining
of the insects, dissolve in the alkali milieu therein and create holes in the gut lining
through which gut contents leak out, leading to death. The gene for Bt toxin has been
engineered into cotton, tomatoes and numerous other plants (Fig. 7.15).
Alternative strategies which have been inspired by the fact that Bt toxins do not affect
some insects, is to engineer into plants two groups of enzymes which inhibit digestive
enzymes in the insect gut: amylase inhibitors and protease inhibitors. In effect the insect
starves to death.
Screening for Productive Strains and Strain Improvement
Fig. 7.15
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Cloning Vector Carrying a B. thuringiensis (Bt) Insecticidal Toxin Gene
Another strategy for developing insect resistance in plants is to engineer into the plant
the gene for cholesterol oxidase, which is present in many bacteria. Cholesterol oxidase
catalizes 3-hydroxysteroids to ketosteroids and hydrogen peroxide (Chapter 26). Small
amounts of this enzyme are very lethal to the larvae of boll weevil which attacks cotton. It
is possible that the cholesterol oxidase acts by disrupting the insect larva’s alimentary
canal epithelium leading to its death.
(iv) Genetically Engineering Plants to Survive Water and Salt Stress
Many parts of the world have desert or near desert conditions where water is in short
supply. Added to this is the fact that salt used for treating ice in the winter finds its way
into agricultural land. These factors create conditions which bring plants into conditions
of water (drought) and salt stress. To survive under
these conditions, many plants synthesize compounds known as osmoprotectants. They help the
plant increase its water uptake as well as retain the
water absorbed. Osmoprotectants include sugars,
alcohols and quartenary ammonium compounds.
The quartenary ammonium compound, betaine, is a
Fig 7.16 Betaine
powerful osmoprotectant and the gene encoding it
obtained from E. coli has enabled plants into which it was cloned survive drought better
than un-engineered plants.
Modification of Plant Consumer Products
This section looks at how genetic engineering has been used to modify the plant food
which comes to the consumer as opposed to the previous section which dealt with the
concerns of the farmer or the producer.
(i) Maintenance of Hardness and Delayed Ripeness in Fruits
During post-harvest transportation of fruits to supermarkets these fruits sometimes ripen
and become soft due to the natural processes which go on within the fruit. These natural
processes include the production of polygalacturonase (PG) (which hydrolyzes pectin)
and cellulases by the fruit. In tomatoes the softening of the fruit is inhibited by
engineering an anti-sense PG producing gene into the plant, enabling the fruit to ripen on
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Modern Industrial Microbiology and Biotechnology
the plant before harvesting instead of harvesting them while still green. Such tomatoes
have a longer shelf life while retaining the taste of regular tomatoes. The genetically
engineered tomato known as Flavr Savr was approved by the FDA in 1994 as safe for
human use. In anti-sense technology, a gene sequence is inserted in the opposite
direction, so that during transcription, mRNA complimentary to the normal RNA is
produced. The anti-sense mRNA therefore binds to the normal inhibiting translation.
The net result is that the gene is shut off and in the particular case of PG the fruit-softening
enzyme is reduced to about 1% of the normal, thereby inhibiting softening of the fruit and
possible microbial attack thereafter.
In climacteric fruits (i.e., fruits that are picked before they are ripe) such as tomatoes,
avocados, and bananas, the initiation of ripening is associated with a burst in ethylene
biosynthesis. After harvesting unripe fruits such as bananas may be treated with
ethylene to induce simultaneous ripening. Ethylene has been described as a gaseous
plant hormone: extraneous ethylene and ethylene generated by the plant equally induce
ripening. Ethylene is a gaseous effector with a very simple structure. In higher plants,
ethylene is produced from L-methionine (Fig 7.17). A major step is the production of the
non-protein amino acid 1-aminocyclopropane-1-carboxylic acid (ACC), catalysed by the
enzyme ACC synthase. It has numerous functions in higher plants. It stimulates the
following activities: the release of dormancy, leaf and shoot abscission, leaf and flower
senescence, flower opening and fruit ripeneing.
Two biotechnological strategies have been pursued to control ethylene action on fruit
ripening. One approach taken in tomato was designed to inhibit biosynthesis of ethylene
within the plant by the use of antisense expression of ACC synthase. In a second
approach, a mutated ethylene receptor from Arabidopsis was introduced into tomato and
petunia. This resulted in delayed fruit ripening.
(ii) Engineering Sweetness into Foods
The taste of fresh tomatoes and lettuce is well known in sandwiches. Some enjoy these
items with greater relish with the addition of sweet tasting tomato ketchup. Sweet taste
has been engineered into tomatoes and lettuce by cloning into them the synthesized gene
coding for monellin. Monellin is a protein which is 3,000 times sweeter than sucrose by
weight; it is naturally obtained from the red berries of the West African plant,
Dioscoreophyllum comminsii Diels, and has been expressed in yeast. A major attraction of
sweeting tomatoes and lettuce with this protein is that it is ‘weight-friendly’. Several
sweet proteins which might be similarly engineered into foods are shown in Table 7.11.
(iii) Modification of Starch for Industrial Purposes
Starch consists of amylose in which the glucose molecules are configured in a straight
chain in the a-1-4 linkage, and the branched chain amylopectin which has a-1,4 and a1,6 linkages (Chapter 4). Starches from different plants have different percentages of
amylase and amylopectin, but generally in the order of 30% amylase to 70 to 80%
amylopectin. Starch is used for making several industrial products such as glue, gelling
agent or thickener. For some purposes it may be desirable to have starch that has a
preponderance of amylase. When that is the case, antisense technology has been used to
block the formation of the amylopectin component of starch, giving rise to a product with
only about 20%.
Screening for Productive Strains and Strain Improvement
Fig. 7.17
$!
Synthesis of Ethylene
Table 7.10 Some sweet tasting proteins produced by plants
S/No
1
2
3
4
Name
Plant
Thaumatin
Monellin
Brazzein
Curculin
Thaumatococcus danielli Benth
Dioscoreophyllum cumminsii Diels
Pentadiplandra brazzeana
Curculingo latifolia
Sweetness ratio
over sucrose (w/w)
3,000
3,000
2,000
550
One further modification is the engineering into a starch source the enzymes needed to
convert starch to high fructose syrup. In the production of high fructose syrup, the starch
is first converted to glucose by a-amylase and thereafter the resulting glucose is converted
to high fructose syrup by glucose isomerase. Both operations are normally done
sequentially. However, both enzymes have been linked together and engineered into
potato and the potato starch converted into fructose in one operation with consequent
saving in costs.
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Modern Industrial Microbiology and Biotechnology
Table 7.11
Modification of Canola oil for different purposes
Seed product
Commercial use(s)
40% Stearic
40% Lauric
60% Lauric
80% Oleic
Petroselinic
“Jojoba” wax
40% Myristate
90% Erucic
Ricinoleic
Margarine, cocoa butter
Detergents
Detergents
Food, lubricants, inks
Polymers, detergents
Cosmetics, lubricants
Detergents, soaps, personal care items
Polymers, cosmetics, inks, pharmaceuticals
Lubricants, plasticizers, cosmetics, pharmaceuticals
(iv) Modifying Flower Pigmentation and Delaying Wilting and Abscision in Flowers
The flower business is of the order of many billions of dollars annually. Most of the
market centers around four flowers: roses, carnations, tulips, and chrysanthemums.
Hundreds of different flowers differring in shape, size, color, fragrance, and structure
have become available through tradional plant breeding. But the usual shortcomings
have also affected this industry: the slow pace of the plant breeding, the uncertainty of the
the results of the efforts and the limitation imposed by the paucity of the genes available
in traditional plant breeding.
Genetic engineering has now been introduced and has helped to extend the range of
the variety of flowers. A group of flavonoids, anthocyanins (Chapter 22) are commonest
pigments in flowers. Anthocyanins are glucosides of phenolic compounds produced in
plants, some being colorless, while many are responsible for the colors in plants. The
aglycone (non-sugar) protions of anthocyanins are derived from the amino acid
phenylalanine. The color which they bear is determined by the chemical nature of the
side chain substituent. By blocking some of the genes in the pathway of anthocyanin
synthesis using anti-sense technology or introducing toally new genes it is possible to
create flowers with new colors (Fig. 7.18).
It is also known that flower wilting and abscission are controlled by ethylene in the
same way as it does with fruits. When a mutated ethylene receptor from Arabidopsis was
introduced into petunia, it led to delayed petal fading, and in delayed flower abscission.
(v) Modification of Nutritional Capabilities of Crops
Genetic engineering has enabled the introduction of new nutritional capabilities in
crops, in a much shorter time and in a range of qualities impossible with traditional
breeding. Unlike genetic engineering which can cross the species barrier, plant breeding
deals with the collection of genes within the species. The amino acid content of foods, the
lipid composition, the amylose/amylopectin ratio of starch, the vitamin contents and
even the mineral contents of foods have all been modified by genetic engineering.
(a) Engineering Vitamin A into Rice
‘Golden rice’ has been prepared by engineering it beta-carotene, a substance which the
body can convert to Vitamin A to combat vitamin A deficiency (VAD), a condition which
afflicts millions of people in developing countries, especially children and pregnant
women. Severe Vitamin A deficiency (VAD) can cause partial or total blindness; less
severe deficiencies weaken the immune system, increasing the risk of infections such as
Screening for Productive Strains and Strain Improvement
$#
Most flowers derive their color from anthocyanins which are synthesized from the amino acid phenyl
alanine. The color of the flower depends on the possession by the plant of genes which can code for the
the enzymes whose reactions result in the various colors in flowers. In the figure above the plant must
possess the gene coding for the enzyme CHI (chalcone synthase) which produces 4,2’,4’,6’Tetrahydroxychalcone from the two intermediates indicated and gives rise to yellow flowers. Lower down
the chain the critical enzymes are DFR ( Dihydroflavonol 4-reductase) and 3GT (UDP-glucose: flavonoid 3O-glucosytransferase. These two enzymes DFR and 3GT will convert intermediates to compounds which
will give rise brick red, red, or yellow flowers. By manipulating the pathway through introducing various
genes, flowers of different colors can be produced at will (see text).
Fig. 7.18 Synthesis of Anthocyanins in Flowers
measles and malaria. Women with VAD are more likely to die during or after childbirth.
Each year, it is estimated that VAD causes blindness in 350,000 preschool age children,
and it is implicated in over one million deaths. Golden rice was created by transforming
rice with three beta-carotene biosynthesis genes: psy (phytoene synthase) and lyc
(lycopene cyclase) both from daffodil (Narcissus pseudonarcissus), and crt1 from the soil
bacterium Erwinia uredovora. The psy, lyc, and crt1 genes were transformed into the
nuclear genome and placed under the control of an endosperm specific promoter, so that
they are only expressed in the endosperm. The plant endogenous enzymes process the
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Modern Industrial Microbiology and Biotechnology
lycopene to beta-carotene in the endosperm, giving the rice the distinctive yellow color
which gave it the name ‘golden’.
(b) Engineering Amino Acids into Legumes and Cereals
The seed storage compounds in cereals such as corn usually contain proteins deficient in
the essential amino acids lysine and methionine. Storage proteins found in many
legumes are sometimes deficient in these two essential amino acids, or cysteine. Corn and
many grain legumes are used as animal feeds, and feeds made from them have to be
supplemented with the deficient amino acids. Legumes such as lupine have been
engineered to express sunflower seed albumin which is unusually rich in the sulfurcontaining amino acids methionine and cysteine. High-lysine corn is currently available,
but engineering lysine, methionine and or cysteine into corn is almost certainly a matter
of time
(c) Modifying Fats and Oils for Various Purposes
Plant oils are derived from soybean, oil palm, sunflower, and rapeseed (canola) and to a
lesser extent from the endosperm of corn. Most of the oils is used for margarine
manufacture, as fats for baking, for salads and for frying. The extent to which an oil is
liquid at room temperature depends on the degree of unsaturation, i.e., the number of
double bonds it has. For industrial purposes oils are also used in cosmetics, in detergents,
soaps, confectionaries, and as drying agents in paints and inks. Each use to which the
oils are put requires a different property. For example oils which contain conjugated
double bonds (in contrast to those which contain double bonds separated by methyl
groups– CH2 (Fig. 7.19) require less oxgene for polymerization and hence dry more
quickly in paints and inks. Genetic engineering has been used to modify oils for various
uses. Thus canola oil from rapeseed has been genetically modified for use in various
products.
CH2==CH—CH==CH—CH3
Fig. 7.19
CH2==CH—CH2—CH==CH2
Cojugated Double Bonds
Soyoil has been genetically modified by DuPont to make it more suitable as an edible
oil and also for certain industrial uses including the manufacture of inks, paints,
varnishes, resins, plastics, and biodiesel.
Soybean oil is a complex mixture of five fatty acids (palmitic, stearic, oleic, linoleic, and
linolenic acids) that have vastly differing melting points, oxidative stabilities, and
chemical functionalities. The most notable example, developed by researchers at DuPont,
is the transgenic production of soybean seeds with oleic acid content of approximately
80% of the total oil. Conventional soybean oil, by comparison, contains oleic acid at levels
of 25% of the total oil. The high oleic acid trait was obtained by down regulating the
expression of FAD2 genes that encode the enzyme, which converts the monounsaturated
oleic acid to the polyunsaturated linoleic acid. High-oleic oils with elevated oleic acid
content are generally considered to be healthier oils than conventional soybean oil,
which is an omega-6 or linoleic acid-rich oil. From an industrial perspective, the high
content of oleic acid and low content of polyunsaturated fatty acids result in an oil that
has high oxidative stability. In addition, soybean oil is naturally rich in the vitamin E
antioxidant gamma-tocopherol, which also contributes to the oxidative stability of high
oleic acid soybean oil. High oxidative stability is a critical property for lubricants.
Screening for Productive Strains and Strain Improvement
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Genetic engineering can also be used to produce soybean oil with high levels of
linolenic acid, a polyunsaturated fatty acid with low oxidative stability. Soybean seeds
with linolenic acid content in excess of 50% of the total oil have been generated by
increasing the expression of the FAD3 gene, which encodes the enzyme that converts
linoleic acid to linolenic acid The linolenic acid content of conventional soybean oil, in
contrast, is approximately 10% of the total oil. The low oxidative stability associated with
high linolenic acid oil is a desirable property for drying oils that are used in coating
applications, such as paints, inks, and varnishes.
Significant progress has been made in the development of chemical methods for
enhancing the functionality of soybean oil for the production of polyols from soybean oil
which may eventually lead to a number of industrial applications, including the
production of polyurethanes.
7.2.2.2.8.11 Transgenic animals and plants as biological fermentors (or Bioreactors)
Transgenic animals and plants have been used to produce high-quality pharmaceutical
substances or diagnostics. The procedure is known as ‘pharming’ from a parody of the
word pharmaceutical; it is also known as ‘molecular farming’ or ‘gene pharming’ and
the transgenic plants or animals used are sometimes referred to as animal or plant
‘bioreactors’ or ‘fermentors’. Therapeutically active proteins already on the market are
usually produced in bacteria, fungi, or animal cell cultures. However microorganisms
usually produce comparatively simple proteins; furthermore microorganisms are not
always able to correctly assemble and fold complex proteins. If the protein structure is
very complicated, such microorganisms may produce defective clumps.
In pharming, transgenic animals are mostly used to make human proteins that have
medicinal value. The protein encoded by the transgene is secreted into the animal’s milk,
eggs or blood or even urine, and then collected and purified. Livestock such as cattle,
sheep, goats, chickens, rabbits and pigs have already been modified in this way to
produce several useful proteins and drugs. Some human proteins that are used as drugs
require biological modifications that only the cells of mammals, such as cows, goats and
sheep, can provide. For these drugs, production in transgenic animals is a good option.
Using farm animals for drug production has many advantages: they are reproducible,
have flexible production, and are easily maintained. Since the mammary gland and milk
are not part of the main life support systems of the animal, there is not much risk of harm
to the animal making the transgenic protein. To ensure that the protein coded in the
transgene is secreted in the milk, the transgene is attached to a promoter which is only
active in the mammary gland. Although the transgene is present in every cell of the
animal, it is only active where the milk is made. Some examples of the drugs currently
being tested for production in animals are antithrombin III and tissue plasminogen
activator used to treat blood clots, erythropoietin for anemia, blood clotting factors VIII
and IX for hemophilia, and alpha-1-antitrypsin for emphysema and cystic fibrosis.
A good example of the need for processing a protein in an animal is seen in the silk of
the golden spider, Nephila clavipes. The dragline form of spider silk is regarded as the
strongest material known; it is five times stronger than steel. People have actually tried
starting ‘spider farms’ to harvest silk, but the spiders are too aggressive and territorial to
live close together. They also like to eat each other. Though the genes for dragline silk
were isolated several years ago, attempts to produce it in bacterial and mammalian cell
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culture have failed. When the genes were put into a goat and expressed in the mammary
glands, however, the animal produced silk proteins in its milk that could be spun into a
fine thread with all the properties of spider-made silk. This can be used to make lighter,
stronger bulletproof vests, thinner thread for surgery and stitches or indestructible clothes.
The advantage of using biological fermentors have been put as follows: lower drug
prices for consumers, production of drugs unavailable any other way, new value-added
products for farmers, and inexpensive vaccines for the developing world.
(a) Lower drug costs
Expected savings on infrastructure and production costs lead companies producing
‘pharm’ and industrial crops to predict drug prices 10 to 100 times lower than current
prices. The cost of treating a patient with Fabry’s disease, currently as much as US
$400,000 a year, for example, is predicted to drop to approximately US $40,000 annually.
Similarly, it is claimed that the leaves from only 26 tobacco plants could make enough
glucocerebrosidase, currently one of the most expensive drugs in the world, to treat a
patient with Gaucher’s disease for a whole year. Regarding plants, the biggest factor in
reducing costs is the high yields of recombinant proteins attainable in transgenic plants.
Production costs for corn systems are estimated at between US $10 and US $100 per gram
for proteins that currently cost as much as US $1,000 per gram. Dollar figures based on
large-scale tobacco production vary widely from less than US $10 per gram to US $1000
per gram. If realized, these projections would represent substantial savings over current
costs.
(b) Faster, more flexible manufacturing
Abundantly available commodity like corn and the environment as a production inputs
could cut not only production costs but also capital investment in, and the time it takes to
increase, manufacturing infrastructure. Very rapid scale-up (or scale-down) of a
production pipeline in response to the market or other factors and new drugs could,
theoretically, become available sooner.
(c) Drugs unavailable any other way
Cheap production also means that drugs that could not be produced cheaply enough at
high volume through conventional methods might become economically viable using
genetically engineered crops. Monoclonal antibodies (‘plantibodies’) fall into this
category. One company’s idea for such a product is a monoclonal antibody against
bacteria responsible for tooth decay, which could be used as a dental prophylactic. A
topical therapeutic for herpes, as well as antibodies for the treatment of many other
diseases, are also under development.
(d) New value-added agricultural products
These crops producing pharmaceutical products could be a boon to farmers, as they
could be economically viable alternatives to commodity production of corn or tobacco.
Special Advantages of Plants as Biological Fermentors
Apart from the advantages accruing in the use of plants and animals as bioreactors, a
sector of the biotechnology industry views plants (in comparison with animals) as
preferable for protein production for the following reasons.
Screening for Productive Strains and Strain Improvement
Table 7.12
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Human proteins synthesized in animals
Protein
Use
Animal(s)
a-1-anti-protease inhibitor
a-1-antirypsin
Antithrombin III
a-1-antirypsin deficiency
Anti-inflammatory
Sepsis and disseminated intravascular
coagulation (DIC)
Goat
Goat, sheep
Goat
Collagen
Factor VII and IX
Fibrinogen
Human fertility hormones
Human hemoglobin
Human serum albumin
Lactoferrin
LAtPA
Monoclonal antibodies
Tissue plasminogen activator
Burns, bone fracture, incontinence
Hemophilia
“Fibrin glue,” burns, surgery localized
chemotherapeutic drug deliver
Infertility, contraceptive vaccines
Blood replacement for transfusion
Burns, shock, trauma, surgery
Bacterial gastrointestinal infection
Venous stasis ulcers
Colon cancer
Heart attacks, deep vein thrombosis,
pulomary embolism
Cow
Sheep, pig
Pig, sheep
Goat, cow
Pig
Goat, cow
Cow
Goat
Goat
Goat
Table 7.13 Some therapeutic agents produced in transgenic plants
Protein
Plant(s)
Human protein C
Human hirudin variant 2
Tobacco
Anticoagulant
Tobacco, canola, Ethiopian Anticoagulant
mustard
Human gramulocyte-macrophage
colony-stimulating factor
Human erythropoientin
Human enkephalins
Tobacco
Tobacco
Thale cress, canola
Human epidermal growth factor
Tobacco
Human a-interferon
Human serum albumin
Human hemoglobin
Human homotrimetic collagen I
Human a-1-antitrypsin
Rice, turnip
Potato, tobacco
Tobacco
Tobacco
Rice
Human growth hormone
Human aprotinin
Tobacco
Corn
Angiotension-1-converting enzyme Tobacco, tomato
a-Tricosanthin
Tobacco
Glucocerebrosidase
Tobacco
Application
Neutropenia
Anemia
Antihyperanalgesic by
opiate activity
Wound repair/control of
cell proliferation
Hepatitis C and B
Liver cirrhosis
Blood substitute
Collagen synthesis
Cystic fibrosis, liver
disease, hemorrhage
Dwarfism, wound healing
Trypsin inhibitor for
transplantation surgery
Hypertension
HIV therapy
Gaucher disease
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Modern Industrial Microbiology and Biotechnology
(a) Plants are less controversial than animal production systems
Plants do not normally transmit animal pathogens (that can cause diseases like mad
cow). Products obtained by ‘pharming’ plants are promoted as safer than animalsourced proteins. It must, however, be borne in mind that plants as bioreactors may also
present their own risks of product contamination from mycotoxins, pesticides, herbicides
or endogenous plant secondary metabolites such as nicotine and glycoalkaloids.
Using plants as bioreactors also avoids any animal welfare and certain ethical
concerns associated with cloning animals and using them as bioreactors
(b) Inexpensive, easily delivered vaccines
Food plants engineered to contain pieces of disease agents can function as orally
administered vaccines, avoiding the need for injection and syringes. Currently, tomatoes
and other vegetables are under development for that purpose. Although the vaccines
would still have to be standardized for dose and delivered to patients one at a time, it is
hoped that the lower production costs and the convenience of avoiding refrigeration
would make the products attractive to the developing world.
SUGGESTED READINGS
Bleecker A.B., Kende, H. 2000. Ethylene: a gaseous signal molecule in plants. Annual Review of
Cell Devision and Biology 16, 1-18.
Deacon, J. 2006. Fungal Biology. 4th Blackwell. Malden USA.
Gelvin, S.B. 2003. Agrobacterium-Mediated Plant Transformation: the Biology behind the “GeneJockeying” Tool Microbiology and Molecular Biology Reviews, 67, 16-37.
Glick, B.R., Pasternak, J.J. 2003. Molecular Biotechnology: Principles and Applications of
Recombinant DNA. ASM Press Washington DC, USA.
Glover, S.W., Hopwood, D.A. 1981 eds. Genetics as a Tool in Microbiology Cambridge
University Press, Cambridge, UK.
Keiji, K.Y., Miura, Y., Sone, H., Kobayashi, K., Hiroshi lijima, H., Parek, S. 2004. Strain
Improvement: High-level expression of a sweet protein, monellin, in the food yeast Candida
utilis.
Koffas, M., Roberge, C., Lee, K., Stephanopoulos, G. 1999. Metabolic Engineering. Annual
Reviews of Biomedical Engineering. 1, 535- 557.
Kondo, K., Miura, Y., Sone, H., Kobayashi, K., lijima, H. 1997. High-level expression of a sweet
protein, monellin, in the food yeast Candida utilis. Nature Biotechnology, 15, 453-457.
Labeda, D.P., Shearer, M.C. 1990. Isolation of Actinomycetes for Biotechnological Aplications. In:
Isolation of Biotechnological Organisms from Nature. D.P. Labeda. (ed) McGraw-Hill, New
York, USA. pp. 1-20.
Murooka, Y., and Imanaka, T. (eds) 1994. Recombinant Microbes for Industrial and Agricultural
Applications. Marcel Dekker, New York, USA.
Parek, S. 2004. Strain Improvement. In: the motherland. The Desk Encyclopedia of Microbiology.
M Schaechter (ed.) Elsevier Amsterdam: pp. 960–973.
Steele, D.B., Stowers, M.D. 1991. Techniques for the Selection of Industrially Important
Microorganisms. Annual Review of Microbiology, 45, 89–106.
Velandez, W., Lubon, H., Drohan, W. 1997. Transgenic Livestock as Drug Factories. Scientific
American, 1: 55.
Wei, L.N. 1997. Transgenic Animals as New Approaches in Pharmacological Studies. Annual
Reviews of Pharmacology and Toxicology 37, 119–141.
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8
The Preservation of the
Gene Pool in Industrial
Organisms: Culture
Collections
8.1
THE PLACE OF CULTURE COLLECTIONS IN
INDUSTRIAL MICROBIOLOGY AND
BIOTECHNOLOGY
The central importance of a microorganism in an industrial microbiological
establishment may sometimes be taken for granted. While a raw material may be fairly
easily substituted, the use of an organism different from one already in existence may
involve extensive experimentation and modification of established processes if the usual
products are to be obtained. It is therefore important that organisms whose genetic
potentials remain unchanged be constantly available. In other words, the gene pool of
organisms with desirable properties must be preserved and be constantly available.
The gene pool is the group of genes which collectively define a species and create the
distinctions which exist between one species and another. Thus, while genes which give
rise to the variations among humans exist in the gene pool of humans, such that they are
short or tall or fat or thin humans, humans are clearly distinct from other animals such as
cats, which have a completely different gene pool. It should also be mentioned that even
within the gene pool, there are groups of genes which define strains within the species. In
industrial microbiology, the strain is often more valuable than the species as the ability to
produce the unique characteristics of a product resides in the strain.
Industrial microbiological establishments usually keep a collection of the
microorganisms which possess the gene pools for producing the goods manufactured by
the establishment. This stock of organisms is known as a culture collection and ensures a
regular supply of organisms to be used in the manufacturing process. Organisms in a
culture collection are maintained in a low metabolic state in which replication of the cells
is kept to a minimum or even entirely restricted. Industrially important microorganisms
are often mutants, and the condition of low metabolism in which they are kept, limits
their tendency to revert to their low-yielding ancestors.
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Modern Industrial Microbiology and Biotechnology
In some circumstances organisms are maintained for comparatively short periods of
days in an active state in which they are immediately ready for use in fermentations; such
organisms are called working stock. In many breweries, for example, the producing yeasts
are reused sometimes for up to eight runs or more before being discarded. In the interval
between inoculations such yeasts are regarded by some workers as working stocks. It
must be borne in mind that working stocks stand the chance of contamination and/or
mutation, two serious problems inherent in industrial fermentations.
8.2
TYPES OF CULTURE COLLECTIONS
Culture collections in general, are an important part of the science of microbiology but, as
will be shown below, they are specially important in industrial microbiology. Culture
collections maintained by industrial establishments are usually specialized and store
mainly those used in that particular organization.
There are various kinds of culture collections. Some national culture collections
handle a wide variety of organisms, of whatever kind. The best known in this category is
the American Type Culture Collection (ATCC). Other collections are specialized and may
handle only pathogenic microorganisms, such as the National Collection of Type
Cultures (NCTC) in Colindale, London, UK or industrial microorganisms, such as
National Collection of Industrial Bacteria (NCIB) in Aberdeen, Scotland. Still others
almost exclusively handle one type of organism such as Center vor Braunsveitzer (CBS)
in Holland, which handles fungi exclusively. Many universities all over the world have
culture collections which reflect their range of microbiological interests.
Culture Collections around the world are linked by the World Federation of Culture
Collections (WFCC). The WFCC is an affiliate of the International Union of
Microbiological Societies (IUMS) the organization which links national microbiological
societies world wide. The WFCC is concerned with the collection, authentication,
maintenance, and distribution of cultures of microorganisms and cultured cells. Its aim is
to promote and support the establishment of culture collections and related services, to
provide liaison and set up an information network between the collections and their
users, and work to ensure the long term perpetuation of important collections.The WFCC
pioneered the development of an international database on culture resources worldwide.
The result is the WFCC World Data Center for Microorganisms (WDCM).
Culture Collections are organized on regional and international basis for the
exchange of cultures and ideas and include the Asian Network on Microbial Research
(ANMR), BCCCM (Belgium Co-ordinated Collections of Microorganisms), ECCO
(European Culture Collection Organization), JFCC (Japanese Federation of Culture
Collections), MICRO-NET (Microbial Information Network of China), MSDN (Microbial
Strain Data Network, UK), UKNCC (United Kingdom National Culture Collection),
USFCC (United States Federation of Culture Collections, USA). The WFCC maintains a
World Data Center for Microorganisms (WDCM) at the National Institute of Genetics
(NIG) in Japan, and has records on about 500 culture collections from 60 countries. A list
of culture collections around the world will be found in the Kirsop and Doyle, 1991.
Culture collections may be specialized and in-house such as those in industrial
establishments. Others are public and have the function of acquiring, identifying,
The Preservation of the Gene Pool in Industrial Organisms: Culture Collections
%!
preserving and distributing microorganisms and for a fee will supply cultures for in
teaching, research or to industry. Such culture collections receive cultures from all over
the world and thus serve the overall purpose of maintaining worldwide microbiological
biodiversity.
In addition to making available organisms for industrial use, the major culture
collections serve the important function of acting as depositories for microorganisms
mentioned in the patenting of microbiological processes.
8.3
HANDLING CULTURE COLLECTIONS
Cultures are expensive to purchase. They are usually, however, supplied at a discount
when used for reaching. Universities can however build their own cultures collections by
preserving cultures arising from their research.
An industrial process may be initiated with organisms obtained through the Patent
Office in connection with a patent. Often only one vial of such an organism is usually
available. Once growth has been obtained from that vial the organism should be
multiplied and stored in one or more of the several manners described below for the
preservation of primary stock organisms in a Culture Collection. No matter what the
source of a valuable organism, it is important that several replicates are stored
immediately for fear of contamination while tests are carried out to ascertain its potential
for fulfilling the expected activity. If the tests show that the expected antibiotic or other
desired metabolite is being produced in the expected quantity then stored organisms are
retained. The stocks of those organisms which proved negative at first sampling should
not be discarded in a hurry because further examination may show that poor
productivity was due to factors extrinsic to the organism such as an inadequate medium.
In order to identify the organisms they must be properly labeled and accurate records
kept of the handling of the organism. Date of transfer, the medium and the temperature of
growth, etc., must all be carefully recorded to afford a means of assessing the effect of the
preservation method.
8.4
METHODS OF PRESERVING MICROORGANISMS
Several methods have been devised for preserving microbial cultures. None of them can
be said to apply exclusively to industrial microorganisms. Furthermore, no one method is
suitable for preserving all organisms. The method most suited to any particular organism
must therefore be determined by experimentation unless the information is already
available.
Methods employed in the preservation of microorganisms all involve some limitation
on the rate of metabolism of the organism. A low rate of spontaneous mutation exists
during the growth of microorganisms, about once in every 109 division. Lowering the
metabolic rate of the organism will further reduce the chances of occurrence of mutations.
Preservation methods will be discussed under the following three headings, although it
should be understood that in practice the methods combine one or more of the following
three principles. The principles involved in preserving microorganisms are:
(a) reduction in the temperature of growth of the organism;
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Modern Industrial Microbiology and Biotechnology
(b) dehydration or desiccation of the medium of growth;
(c) limitation of nutrients available to the organism.
All three principles lead to a reduction in the organisms metabolism.
8.4.1
8.4.1.1
Microbial Preservation Methods Based on the
Reduction of the Temperature of Growth
Preservation on agar with ordinary refrigeration (4 – 10°C)
Organisms growing on suitable agar at normal growth temperatures attain the stationary
phase and begin to die because of the release of toxic materials and the exhaustion of the
nutrients. Agar-grown organisms are therefore refrigerated as soon as adequate growth
is attained as to preserve them.
a) Aerobic organisms
Agar slants: Aerobic organisms may be grown on agar slants and refrigerated at 4 – 10°C
as soon as they have shown growth.
Petri dishes: Aerobic organisms may also be stored on Petri dishes. The plates may be
sealed with electrical tapes to prevent the plates from drying out on account of
evaporation. Electrical tapes of different colors may be used to identify special attributes
or groups among the cultures.
b) Anaerobic organisms: Anaerobic organisms may be stored on agar stabs which are then
sealed with sterile molten petroleum jelly.
Storage using the above agar methods has advantages and disadvantages.
The advantage is that agar storage methods are inexpensive because they do not
require any specialized equipment.
The disadvantages are
(a) The organisms must be sub-cultured at intervals which have to be worked for each
organism, medium used, laboratory practice, etc. This is because the temperature of
the refrigeration is not low enough to limit growth completely.
(b) Consequent on regular sub-culturing is the possibility that contaminations and or
mutations may occur.
(c) The third disadvantage is that Petri dishes occupies a lot of space in the refrigerator when compared with agar slants. But even agar slants are too bulky in
comparison with the small vials in which lyophilized (freeze-dried) cultures are
stored. Since plates occupy a lot of space, test tubes are usually preferred for storage
in refrigerators.
(d) The process of sub-culturing is tedious apart from the possibility of contamination
and mutation.
(e) When petroleum jelly is used as a seal, the arrangement can be messy.
Oil overlay
With the method of oil overlay whose function is to limit oxygen diffusion many bacteria,
especially anaerobes and facultatives, and fungi survive for up to three years, and most of
them for at least one year.
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Medium for storing organisms on agar
The nature of the agar medium on which organisms are stored is of importance. A
medium prepared from natural components rather than a chemically defined material is
preferable, since a defined medium may, because it lacks some components present in the
natural components, select for organisms specifically capable of growing on it. A stock
culture medium should also not be unduly rich in carbohydrates such as glucose which
will lead to early production of acid and hence possible early microbial death. Where
glucose is used, such as for lactic bacteria, the medium should be buffered with calcium
carbonate.
Popularity of agar storage methods
In spite of its shortcomings storage on agar is very popular and is the most widely used
after lyophilization.
8.4.1.2
Preservation in Deep Freezers at about -20°C, or
between -60°C and -80°C
The regular home freezer attains a temperature of about -20 °C.
Laboratory deep freezers used for molecular biology work range in temperature
between -60 °C and -80 °C. It is possible to store microorganisms in either type of deep
freezers in the form of agar plugs or on sterile glass beads coated with the organism to be
stored.
Preservation on glass beads
The bacteria to be preserved are placed in broth containing cryoprotective compounds
such as glycerol, raffinose, lactose, or trehalose. Sterile glass beads are placed in the glass
vials containing the bacterial cultures. The vials are gently shaken before being put in the
deep freezers.
To initiate a culture a glass bead is picked up with a pair of sterile forceps and dropped
into warm broth. Growth develops from the organisms coating the bead. The growth is
introduced onto an agar plate containing the appropriate medium and checked for purity
before use.
Storage of agar cores with microbial growth
Bacteria, but especially moulds, yeasts, and actinomycetes may be stored as agar plugs
made from plates of the confluent growths bacteria or of hyphe of filamentous organisms.
It consists of placing agar plugs of confluent growth of bacteria and yeasts and hyphe of
moulds or actinomycete in glass vials containing a suitable cryoprotectant and freezing
the vials in deep freezers as above. To initiate growth a plug is placed in warm broth and
plated out.
Freezing is rapidly gaining acceptance for preserving organisms because of its dual
use for working and primary stock maintenance as well as its storage effectiveness for up
to three years. It is useful for a wide range of organisms, and survival rates have been
shown to be as good as freeze-drying in many organisms.
Advantages of the above freezing methods
(a) the methods are simple to use and require a minimum of equipment;
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Modern Industrial Microbiology and Biotechnology
(b) they save space as many hundreds of cultures can be stored in a small space;
(c) beads thaw rapidly and hence the method saves time,
(d) differently bead colors can represent different bacteria and so recognizing them is
easy;
( e) the methods can be adapted for both aerobic and anaerobic organisms;
(f) the methods are suitable for situations or countries where power outages occur, as
the freezer can remain cold for some time during power failures.
8.4.1.3 Storage in low temperature liquid or vapor phase
nitrogen (-156°C to -196°C)
The liquid or vapor phase of nitrogen at -156°C to -196°C is widely used for preserving
microorganisms and cultured cells. Fungi, bacteriophages, viruses, algae, protozoa,
bacteria, yeasts, animal and plant cells, and tissue cultures have all been successfully
preserved in it. It is a major method for organisms which will not survive freeze-drying.
The period of survival and the number of surviving organisms are higher for most
organisms than when freeze drying is used. In many laboratories it is the choice method
for storing very valuable organisms. Some organisms are prone to losing numbers with
this method, but the loss is reduced with the use of cryoprotectants. Some of the most
commonly used cryoprotectants are (vol/vol) 10-20% glycerol and 5-10% dimethyl
sulfoxide (DMSO) in broth culture of the organism in vials which are then frozen in liquid
nitrogen. Vials for storing organisms in low temperature nitrogen may be made of glass or
fashioned from ordinary polypropylene (plastic) drinking straws. Straws (4 mm
diameter) are usually cut into pieces 40 mm long and made into ampoules by sealing the
ends with heat.
Freezing at –156°C to -196°C has the following disadvantages:
(a) As liquid nitrogen evaporates, it has to be replenished regularly; if not replenished
the cultures may be lost.
(b) A risk of explosion exists when cultures are frozen in liquid nitrogen in improperly
sealed glass vials which permit entry of liquid nitrogen into the vials. Such vials
may explode when warmed to thaw them. Discarding poorly sealed glass vials
removes such risks; vapor phase storage removes such dangers.
(c) Although it is not labor intensive the equipment is expensive.
(d) Finally it is not a convenient method for transporting organisms.
8.4.2
Microbial Preservation Methods Based on Dehydration
Just as reduction in temperature limits the metabolism of the organism, dehydration
removes water a necessity for the metabolism of the organism. Several methods may used
to achieve desiccation as a basis for preserving microorganisms.
8.4.2.1
Drying on sterile silica gel
Many organisms including actinomycetes and fungi are dried by this method. Screw-cap
tubes half-filled silica gel are sterilized in an oven. On cooling a skim-milk suspension of
spores and the cells of the fungus or actinomycetes is placed over the silica gel and
The Preservation of the Gene Pool in Industrial Organisms: Culture Collections
%%
cooled. They are dried at 25°C, cooled and stored in closed containers containing
desiccants.
8.4.2.2 Preservation on sterile filter paper
Spore-forming microorganisms such as fungi, actinomycetes, or Bacillus spp may be
preserved on sterile filter paper by placing drops of broth containing the spores on sterile
filter paper in a Petri dish and drying in a low temperature oven or in a dessicator.
Alternatively, sterile filter paper may be soaked in the broth culture of the organism to be
dried, placed in a tube, which is then evacuated and sealed. After drying the filter paper
may be placed in sterile screw caps bottles and stored either at room temperature or in the
refrigerator.
8.4.2.3 Preservation in sterile dry soil
The most commonly used form of storage in a dry state is the use of dry sterile soil. In this
method dry soil is sterilized by autoclaving. It is then inoculated with a broth or agar
culture of the organism. The soil is protected from contamination and allowed to dry over
a period of time. Subsequently it may be refrigerated. The method has been widely and
successfully used to store sporulating organisms especially clostridia and fungi; it has
also been used for bacilli and Azotobacter sp. Some non-sporulating bacteria which do not
survive well under Lyophilization, may be stored in soil.
8.4.2.4 Freeze-drying (drying with freezing), lyophilization
Freeze-drying or lyophilization is widely employed and a lot has been written about it. The
principle of the method is that the organism is first frozen. Subsequently, water is
removed by direct vaporization of the ice with the introduction of a vacuum. As the
suspension is not in the liquid state, distortion of shape and consequent cell damage is
minimized. At the end of the drying the ampoule containing the organism may be stored
under refrigeration although survival for many years has also been obtained by storage at
room temperature. The initial freezing (before the drying) may be achieved in a number of
ways including the use of freezing mixtures of CO2 and alcohol, salt and ice, or in a
chamber of a freeze-drying machine in which the evaporation of water vapor from the
material causes enough cooling to freeze the material. A desiccant, usually phosphorous
pentoxide, is used to absorb water vapor during the freezing.
The suspending medium must be carefully chosen, because of differences in the
cryoprotection properties of different substrates. Horse blood is usually used; others
which have been successfully used are inositol, various disaccharides, and
polyalcohols. Unless the information already exists the best suspending medium can
only be decided by experimentation. The ampoule is usually evacuated after freezedrying. It may however be filled with nitrogen; CO2 or argon but the survival of organisms
with them is lower than in vacuum, or with nitrogen.
Lyophilization is preferred for the preservation of most organisms because of its
success with a large number of organisms, the relatively inexpensive equipment, the
scant demand on space made by ampoules, but above all, the longevity (up to 10 years or
more in some organisms) of most organisms stored by lyophilization.
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Modern Industrial Microbiology and Biotechnology
8.4.2.5
L-drying (liquid drying, drying without refrigeration)
This is considered a modification of drying methods, since unlike freeze-drying, the
organisms are not frozen, but dried from the liquid state. It has been used to preserve nonspore formers sensitive to freeze-drying, such as Cytophaga, Spirillum and Vibrio. Liquid
drying has been effectively used to preserve organisms such as anaerobes that are
damaged by freezing.
Small vials made of glass are filled with a mixture of skim milk, medical grade
activated charcoal and myo-inositol , autoclaved and thereafter frozen at about -40°C for
a few hours. The vials are then freeze-dried and this leads to a disc of freeze-dried carrier
material in the vials. The broth of the organism to be dried is placed on the disc and the
material is subjected to a vacuum in the liquid unfrozen state at 20°C.
8.4.3
8.4.3.1
Microbial Preservation Methods Based on the
Reduction of Nutrients
Storage in distilled water
Many organisms die in distilled water because of water absorption by osmosis. However
some have been known to survive for long periods in sterile distilled water. Usually such
storage is accompanied by refrigeration; some organisms are however, harmed by
refrigeration. Among organisms which have been stored for long periods with this
method are Pseudomonas solanaceanum, Saccharomyces cerevisiae, and Sarcina lutea. The
attractiveness of this method is its simplicity and inexpensiveness; since so few
organisms seem to be storable in this manner, it should not, for fear of losing the
organism, be adopted as the sole method for storing a newly acquired or isolated
organism until it has been shown to be suitable.
8.4.4
The Need for Experimentation to Determine the Most
Appropriate Method of Preserving an Organism
No one method can be said to suitable for the preservation of all and every organism. The
appropriate method must be determined for each organism unless prior literature
information exists. Even then such information must be used with caution, because a
minor change in the medium composition may affect the outcome of the effort. The
criterion to be used for determining the success of a method may not always necessarily
be growth.
The preservation method must retain the characteristics which are desirable in the
organism and this is crucial for industrial microorganisms. For example, the
characteristic brick-red color of Sarcina lutea was lost in some preservation methods,
while the production of rennet by Rhizomucor sp and of antibiotics by some actinomycetes
were respectively affected by the method used for their preservation.
SUGGESTED READINGS
Anony. 1980. National Work Conference on Microbial Collections of Major Importance to
Agriculture. American Phytopathological Society St Paul, Minnesota, USA.
The Preservation of the Gene Pool in Industrial Organisms: Culture Collections
%'
Calam, C.T. 1980. The long-term storage of microbial cultures in industrial practice. The Stability
of Industrial Organisms. B.E. Kirsop, (ed) In: Commonwealth Mycological Institute, Kew,
England.
Demain, A.L., Solomon, N.A. (eds) 1985. Biology of Industrial Microorganisms. The Benjamin/
Cummings Publishing Co., California, USA.
Kirsop, B.E., Doyle, A. (eds) 1991. Maintenance of Microorganisms and Cultured Cells. Academic
Press London and San Diego.
Kurtzman, C.P. 1992. Culture Collections: Methods and Distribution In: Encyclopedia of
Microbiology J, Lederberg, (ed) Vol 1 Academic Press, San Diego, USA. pp. 621–625.
Lamana, C. 1976. The Role of Culture Collections in the Era of Molecular Biology. Rita Colwell,
(ed) In: The Role of Culture Collections in the Era of Molecular Biology. American Society for
Microbiology Washington, DC, USA.
Monaghan, R.L., Gagliardi, M.M., Streicher, S.L. 1999. Culture Collections and Inoculum
Development. In: Manual of Industrial Microbiology and Biotechnology. A.L. Demain, J.E.
Davies, ASM Press, Washington, DC, USA, pp. 29-48; 2nd Ed.
Newman, Y.M., Ring, S.G., Colago, C. 1993. In: Biotechnology and Genetic Engineering Reviews
M. P. Tombs, (ed). Vol 11. Intercept Press, Andover USA, pp. 263–294.
Stevenson, R.E., Hatt, H. 1992. Culture Collections, Functions In: Encyclopedia of Microbiology J,
Lederberg, (ed), Vol 1 Academic Press, San Diego, USA, pp. 615-625.
Modern Industrial
Microbiology and Biotechnology
Section
+
Basic Operations in Industrial
Fermentations
Modern Industrial
Microbiology and Biotechnology
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9
Fermentors and Fermentor
Operation
9.1
DEFINITION OF A FERMENTOR
A fermentor (or fermenter) is a vessel for the growth of microorganisms which, while not
permitting contamination, enables the provision of conditions necessary for the maximal
production of the desired products. In other words, the fermentor ideally should make it
possible to provide the organism growing within it with optimal pH, temperature,
oxygen, and other environmental conditions. In the chemical industry, vessels in which
reactions take place are called reactors. Fermentors are therefore also known as
bioreactors.
Fermentors may be liquid, also known as submerged or solid state, also known as
surface. Most fermentors used in industry are of the submerged type, because the
submerged fermentor saves space and is more amenable to engineering control and
design. The discussions in most of the chapter will be therefore be on submerged
fermentors; solid state fermentors will be discussed at the end of the chapter.
Depending on the purpose, a fermentor can be as small as 1 liter or up to about 20 liters
in laboratory-scale fermentors and range from 100,000 liters to 500,000 liters
(approximately 25,000 – 125,000 gallons) for factory or production fermentors. Between
these extremes are found pilot fermentors which will be discussed later in this chapter. It
should be noted that while fermentor size is measured by the total volume, only about
75% of the volume is usually utilized for actual fermentation, the rest being left for foam
and exhaust gases. Several types of fermentors are known and they may be grouped in
several ways: shape or configuration, whether aerated or anaerobic and whether they are
batch or continuous. The most commonly used type of fermentor is the Aerated Stirred
Tank Batch Fermentor. So widely used is this type that unless specifically qualified, the
word fermentor usually refers to the Aerated Stirred Tank Batch Fermentor. This type will
be discussed early in the chapter. Other types will be discussed later. Major differences
between this and other fermentor types in configuration and operation will also be
discussed.
The construction and design of a fermentor are the province of the engineer and only
enough as will help the biotechnologist or microbiologist understand and utilize it
efficiently will be discussed.
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Modern Industrial Microbiology and Biotechnology
9.2
THE AERATED STIRRED TANK BATCH FERMENTOR
A typical fermentor of this type (Fig. 9.1) is an upright closed cylindrical tank fitted with
four or more baffles attached to the side of the wall, a water jacket or coil for heating and/
or cooling, a device for forcible aeration (known as sparger), a mechanical agitator
usually carrying a pair or more impellers, means of introducing organisms and nutrients
and of taking samples, and outlets for exhaust gases. Modern fermentors are highly
automated and usually have means of continuously monitoring, controlling or recording
pH, oxidation-reduction potential, dissolved oxygen, effluent O2 and CO2, and chemical
components of the fermentation broth (or fermentation beer as the broth is called before it
is extracted). Nevertheless the fermentor need not have all these gadgets and many
automated activities can also be prosecuted manually.
It is important that the type of fermentation required be clearly understood when a
fermentor is being planned; a fermentor is expensive and once installed it may be
unnecessarily expensive to drastically remodify it. Furthermore, because of its expense, a
Fig. 9.1 Structure of a Typical Fermentor (Stirred Tank Batch Bioreactor)
Fermentors and Fermentor Operation
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fermentor will be expected to enable the organization to recover the outlay made on it by
being in use over a reasonably long period. It may therefore be wise for small
establishments to set up general-purpose fermentors such as has been described above,
with provision for, if not for actual installation of, as many components as are likely to be
needed in the future.
9.2.1
Construction Materials for Fermentors
A simple batch fermentor may consist of no more than an open tank made of wood,
concrete or carbon-steel if contamination is not a serious problem and provided that no
need exits for strict pH and temperature control, or that the temperature is controlled in
the building. Thus, many breweries, particularly those making top-fermented beers for
many years had open fermentors. Although it is not the practice, feed yeasts for the
consumption of farm animals may also be grown in open fermentors. Serious
contamination is restricted because of the acidity of the medium usually used. However,
for fermentations with strict sterility requirements and closely controlled environmental
needs, such as in the antibiotic industry, a material which can withstand regular steam
sterilization is necessary. Furthermore, the hydrostatic pressure of a large volume of
liquid can be enormous. Stainless steel is therefore normally used for pilot and
production fermentors. Laboratory scale fermentors are usually made of Pyrex glass to
enable autoclaving.
Where a highly corrosive material is fermented, e.g. citric acid, the fermentor should
definitely be made of stainless steel. It is inevitable that small quantities of the material of
which the fermentor is made will dissolve in the medium. Some materials, e.g., iron may
inhibit the productivity of organisms in certain fermentations. It is for this reason that
carbon-steel fermentors are often lined with glass, or ‘plastic’ materials e.g. a phenolicepoxy coating. The material used for lining depends on the expected abrasion on the wall
of the fermentor by medium constituents. Glass lining is employed only for small
fermentors because of the high cost and the possibility of breakage.
In order to avoid contamination, fermentor vessels of all types should be of welded
construction throughout. The welds should be free of pinpoints where organisms can
develop in small bits of old media, and shielded from sterilization. The joint inlets and
outlets of the fermentor should be designed so as to provide smooth surfaces and
eliminate pockets difficult to sterilize. If gaskets are used at joints these should be nonporous.
9.2.2
Aeration and Agitation in a Fermentor
Oxygen is essential for growth and yield of metabolites in aerobic organisms. In those
fermentations where aerobic organisms are used, the supply of oxygen is therefore
critical. For the oxygen to be absorbed by microorganisms it must be dissolved in aqueous
solution along with the nutrients. Unfortunately not only does air ordinarily contain
only 20% of oxygen, but oxygen is also highly insoluble in water. At 20°C for example,
water holds only about nine parts per million of oxygen. Furthermore, the higher the
temperature the less oxygen (and other gases) water can hold. For some highly aerobic
fermentations such as the growth of yeast or production of citric acid, oxygen is so critical
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that even if the broth were entirely saturated with oxygen it would contain only a 15
second supply for the organisms. In other fermentations, the aeration requirement need
not be as intense but must be presented to the organisms at a controlled level. The
foregoing would have shown that oxygen control in industrial fermentations is as
important as pH, temperature and other environmental controls.
The air used in most fermentation is sterile and produced as discussed in Chapter 11.
However, in some fermentations where sterility is not necessary such as in yeast
fermentation, the air is merely scrubbed by passing it through glycerol. The air used in
fermentation, whether, sterile or not, is forced under pressure into the bottom of the
fermentor just below the lowest impeller the air enters through a sparger which is a pipe
with fine holes. The smaller the holes the finer the bubbles and the more effective the
supply of oxygen to the microorganisms. However, if the holes are too small, then a
greater pressure will be required to force the air through, with consequent higher
consumption of energy and therefore of costs. A balance must be struck between wide
holes which may become plugged and holes small enough to release fine bubbles.
Plugging by hyphae of filamentous fungi or by other particles in the medium may occur.
Usually holes of about 0.25-3.0 cm in diameter meet this compromise. Since the size of the
holes is fixed, the amount of oxygen fed into the medium (usually measured in feet/sec)
can be controlled by altering the pressure of the incoming air.
For many fermentations especially where filamentous fungi and actinomycetes are
involved, or the broth is viscous, it is necessary to agitate the medium with the aid of
impellers. In large-scale operations, where aeration is maintained by agitator-created
swarms of tiny air bubbles floating through the medium, the cost is very high and for this
reason careful aeration is done based on mathematical calculations conducted by
chemical engineers.
Agitators with their attached impellers serve a number of ends. They help to distribute
the incoming air as fine bubbles, mix organisms uniformly, create local turbulence, as
well as ensure a uniform temperature. The optimal number and arrangement of impellers
have to be worked out by engineers using information from pilot plant experiments. The
viscosity of the broth affects the effectiveness of the impellers. Since the viscosity of the
broth may alter as fermentation proceeds, a satisfactory compromise of size, shape, and
number of impellers must be worked out. In unbaffled fermentors a vortex or inverted
pyramid of liquid forms and liquid is thrown up on the side of the fermentor. The result is
that heavier particles sediment and thorough agitation is not achieved. The insertion of
baffles helps eliminate the formation of a vortex and interferes with the upward throw of
liquid against the side of the fermentor. A similar effect can be observed by stirring a cup
of coffee or water rapidly with the handle of a spoon and inserting the handle of the
spoon thereafter along the side of the cup. If four spoon ends were stuck simultaneously
in the (storm in a ) tea cup (!) the effective mixing of the liquid can be easily visualized. The
use of baffles thus ensures not only a more thorough mixing of the nutrient and air but
also the breakup of the air bubbles. In order to understand the importance of fine bubbles,
it is important to appreciate the several barriers through which oxygen must theoretically
pass before reaching the organism in the two film gas model which is commonly used
(Fig. 9.2).
These barriers are indicated in Fig. 9.2 and include the following:
(i) Gas-film resistance between gas and interface;
Fermentors and Fermentor Operation
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3
1
5
&%
6
4
7
Air bubble
Microbial
cell
Fig. 9.2 The Various Barriers through which Gas Passes to Reach the Microorganism in a Liquid
(ii)
(iii)
(iv)
(v)
(vi)
(vii)
Gas-liquid interfacial resistance;
Liquid-film resistance between interface and the bulk of the liquid;
Liquid-path resistance characterized by oxygen gradient in bulk;
Liquid-film resistance around cell or cell-clump;
Inter-cellular or intra-clump resistance;
Resistance to reaction (‘absorption’) of oxygen with the cell respiratory enzymes.
In this model, in the transfer of oxygen from a gas bubble to a liquid, a stagnant gas film
and a stagnant liquid film exist on both sides of a gas-liquid inter-phase. The resistance
of these films to the transfer of solutes depends to a large extent on the degree of agitation.
In the case of oxygen the only significant resistance is that of the liquid film which is
broken by agitation. On the other hand, cell-liquid resistance, becomes important when
there is clumping of organisms.
In terms of the above theory, the function of agitation of the fermentation may be taken
as follows:
(i) Gas dispersion or the creation of a large air-liquid interfacial area;
(ii) Reduction in the thickness, and hence to resistance to oxygen diffusion of the
liquid film which surrounds each bubble;
(iii) Bulk mixture of the culture;
(iv) Control of clump size.
It is clear from the figure that the finer the bubbles, the greater will be the total surface
area of oxygen presented to the organism by a given volume of air. Provisions for
agitation and aeration are thus very important components of an Aerated Stirred Tank
Fermentor. In large-scale operations, where aeration is achieved by swarms of tiny air
bubbles floating through thousand of liters of medium, the cost of aeration and agitation
could be high, hence aeration and agitation have been, and are still, the subject of intense
study by chemical engineers. From such studies the size and shape of the impellers in
comparison to the rest of the fermentor (i.e., tank geometry), the airflow, the power
requirement, etc., are calculated.
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The exhaust air from the fermentor is passed through a filter which is sterilized with
steam from time to time. This is especially necessary if the organism being grown is
pathogenic (e.g., for vaccines). The exhaust pipe is positioned away from the incoming
sterile air to avoid any chance of contamination. Furthermore, the agitation shaft which is
impelled by a motor is fitted with a special seal at the point where it enters the fermentor
in order to avoid contamination.
Sterile air is needed in some aerobic fermentations and it is produced in several ways
including irradiation, electrostatic absorption of particles, the use of heat resulting from
the compression of the gas. But the most commonly used method is the passage of the air
through filters either made of materials such as cellulose nitrate, or more commonly of
cotton and sometimes other materials. Sterility will be discussed in more detail in
Chapter 11. Besides supplying oxygen to the organisms, the provision of air under
pressure helps remove inhibitory volatile metabolites, and contributes to the reduction of
contaminants by providing a positive air pressure.
9.2.3
Temperature Control in a Fermentor
Many fermentation processes release heat, which must be removed so as to maintain the
optimum temperature for the productivity of the organism. In small laboratory fermentors
temperature control may be achieved by immersing the tank in a water bath; in mediumsized ones control may be achieved by a jacket of cold water circulating outside the tank
or merely by bathing the unjacketed cylinder with water. In large fermentors temperature
is maintained by circulating refrigerated water in pipes within the fermentor and
sometimes outside it as well. A heating coil is also provided to raise the temperature
when necessary.
The area required for the transfer of heat may be determined theoretically on the basis
of the expected heat release from the fermentation, the energy input from the agitator, the
work done by the air stream, and the amount of heat involved if the broth were sterilized
in situ in the fermentor. Heat losses to be taken account of include that lost by the effluent
air and to the cooling water.
9.2.4
Foam Production and Control
Foams are dispersions of gas in liquid. In fermentation they usually occur as a result of
agitation and aeration. In a few industrial processes, e.g. in the beer industry (where foam
head retention is a desirable quality), or in the manufacture of foam latex, foam is a
welcome property. However, in most industrial fermentations, foam has undesirable
microbiological, economic and chemical engineering consequences, as follows:
(i) The need to accommodate foams means that a substantial head space is left in
industrial fermentations. By reducing foaming it has been possible to increase the
total fermentation by 30-45%.
(ii) If the fermentation medium is such that it encourages rapid foaming, then the
maximum aeration and agitation possible cannot be introduced because of
excessive foaming. The effect of this is that the oxygen transfer rate is reduced.
(iii) If the foam escapes, then contamination may be introduced when foam bubbles
coalesce and fall back into the medium after wetting the filters and other non-sterile
portions of the fermentor.
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(iv) Organic nutrients or inorganic ions with complex organic compounds may be
removed from the medium by foam floatation, a phenomenon well known in beer
fermentation, when proteins, hop-resins, dextrin’s, etc., concentrate in the foam
layer. A loss of nutrient from fermentations in this way could lead to reduced yield.
(v) It can be seen that the fermentation product may also be removed should it be
amenable to foam floatation. Such a loss has actually been observed in a laboratory
experiment with the antibiotic, monamycin.
(vi) Loss of microorganisms could also easily occur by floatation thereby leading also
to reduced yields.
The above has dealt with surface foams which occur on the top of the medium. The
more stable surface foams are the most troublesome. The unstable ones breakdown in
about 20 seconds and cause no further havoc. In contrast with surface foams are the socalled fluid foams which occur within the broth. These are common in highly viscous
mycelial fermentations and in unbaffled vortex fermentors.
9.2.4.1
Foaming patterns
In order to understand methods of dealing with foams, it is important to discuss some
factors leading to their formation and their behavior during the progress of fermentation.
Fermentation media are usually made up of complex materials whose compositions are
not always precisely known. Of the compounds which give rise to foams, proteins
produce the most stable foams. A medium consisting of only inorganic compounds will
not foam unless suitable metabolites are produced by the organisms.
It is sometimes possible to reduce foaming by altering the medium composition of the
fermentation. Thus, it was possible to use a larger broth volume by reducing foam from a
yeast fermentation following the absorption of caramel and organic acids with bentonite
from a sample of molasses. Furthermore, the concentration of nutrients, the pH, the
method of preparing the medium components e.g. sterilization time, etc., can all affect
foam formation and stability
The pattern of peak foam formation and disappearance during the course of
fermentation depends on the composition of the medium and the nature and the activity
of microorganisms taking part in the fermentation. Four or five foaming patterns have
been recognized (Fig. 9.3).
In the first type (designated 1 in Fig. 9.3) the foam remains constant throughout the
fermentation. This is not common in media made of complex materials and is more
frequent in defined media consisting mainly of inorganic components. In the second type
the foam falls from a fairly high level to a low but constant level, following the utilization
of foam stabilizers in the nutrients by micro-organisms. In this type the microorganisms
themselves produce neither foam stabilizers nor defoamers. In the third type foam
life-time falls at first, but then rises. Under this condition the foam stabilizers in the
original medium are metabolized but the organism also produces foam-stabilizing
metabolites. In the fourth type the medium initially contains only a low amount of foam
stabilizers. These increase as autolysis of the mycelium sets in. If these are later
metabolized the foaming may once more drop resulting in a fifth pattern. In practice
combinations of all or some of these may occur simultanously.
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Modern Industrial Microbiology and Biotechnology
Fig. 9.3 Foaming Patterns in Industrial Fermentations
9.2.4.2
Foam control
Foams in industrial fermentations are controlled either by chemical or mechanical
means. Chemicals controlling foams have been classified into antifoams, which are added
in the medium to prevent foam formation, and defoamers which are added to knock
down foams once these are formed. Some may not see much in the distinction and in this
discussion the term antifoam will refer to both.
Foams are formed via froths which are temporary dispersals of gas bubbles in a liquid
of no foam formation ability. Bubbles in a froth coalesce as they rise to the surface. In a
foam however, they do not coalesce. Rather, the liquid film between two bubbles thins to
a lamella. Materials which yield foam forming aqueous solutions such as proteins,
peptides, synthetic detergents, soaps, and natural products such as saponin, lower the
surface tension of the solution and permit foam formation. An analogy which seems to
explain this is to imagine a fermentation liquid as being covered by various sheets of
rubber (or other elastic materials) of varying thickness representing surface tensions. A
thick sheet in this analogy represents high surface tension and thin sheets, low surface
tension. The thinner the sheet the easier it is to blow balloons from it. Solutes which lower
the surface tension of water are surfactants (although this name applies to a particular
group of chemicals). In general, surfactants have a positive hydrophobic or water
repellent end and a negative, hydrophilic on water-absorbing end.
The foam-forming properties of a surfactant may be seen as resulting from the
repulsion of positive charges surrounding the bubbles. Some commercial surfactants can
lower the surface tension of water from 92 dyne cm-1 (7.2 ´ 10-2Nm-1) to about
27 dyne cm-1 (27 ´ 10-2Nm-1). The positively absorbed surfactant layer confers on the
liquid a phenomenon known as film elasticity which prevents local thinning during
bubble formation in the same manner as a rubber sheet stretches and holds together.
Basically, antifoams enter the lamella between the bubbles by spreading over or
mixing with the positively absorbed surfactants monolayer and thus destroying the film
elasticity. The result is that the film collapses. Ideally, therefore, the antifoam should be
miscible with the foaming liquid. Antifoams used in industrial fermentation should
ideally have the following properties. They should:
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(i) be non-toxic to microorganisms and higher animals, especially if the fermentation
product is for internal use.
(ii) have no effect on taste and odor; a change in the usual organoleptic properties of
the finished goods due to the antifoam or other components of the medium may
result in consumer rejection of the goods. It is significant that a silicone antifoam
has been used to limit foaming during wort boiling in beer manufacture. The
silicone was subsequently removed and had no effect on the quality, including the
foam head retention, of the beer.
(iii) be autoclavable.
(iv) not be metabolized by the microorganisms; sometimes as when natural oils are
used, the antifoam may be utilizable in which case they must be replaced regularly.
(v) not impair oxygen transfer.
(vi) be active in small concentrations, cheap, and persistent.
Some chemical antifoams are discussed in Table 9.1. Many antifoams work best if
dispersed in suitable carriers. Thus for Alkaterge C (Trade name) paraffin oil was found
to be the best carrier.
Table 9.1 Some antifoams which have been used in industry
Category
Example
Chemical nature
Remarks
Natrual oils
and fats
Peanut
oil, soybean oil
Esters of glycerol
and long chain
mono-basic acids
Alcohols
Sorbitan alcohol
Mainly alcohols with
8-12 carbon atoms
Sorbitan
derivatives
Sorbitan monolaureate
(Span 20-Atlas)
Polyethers
P400, P1200, P2000
(Dow Chemical Co.)
Antifoam A (Dow
Corning Ltd.)
Derivatives of sorbitol
produced by reacting
it with H2SO4 or ethylene
Polymers of ethylene
oxide & propylene oxide
Polymers of polydimethyl
-siloxane fluids
Not very efficient.
Used as carriers for
other antifoams; may
be metabolized.
Not very efficient;
may be toxic or
may be metabolized.
Span 20 active in
extremely small
amounts.
Active, but varies
with fermentation.
Very active; inert,
highly dispersable,
low toxity; expensive.
Silicones
Antifoams may be added manually when foam is observed. This entails a close watch
and may be expensive. Automatic antifoam additions are now very common and depend
on a probe which is activated when foams rise and make contact with the probe. One of
the earliest is the wick defoamer (Fig. 9.4) in which the foam drew some antifoam on
making contact with a wick. Modern methods are electrically activated systems. Other
systems which have been used include antifoam introduction via the sparging air, or
continous drip-feeding.
Mechnanical defoamers of various designs have been described. In general they act by
physically dispersing the foams by rapidly breaking them up.
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Fig. 9.4
9.2.5
Wick Type Anti-foam
Process Control in a Fermentor
The course of a fermentation may be followed by monitoring various operational
parameters within the fermentor e.g. pH, air input, effluent gases, temperature; factors
such as cell yield, or the output of metabolites may also be followed. The degree of
accuracy of the monitoring depends of course on the instruments being used for the
purpose. The purpose of this section is to discuss the principles involved in the operation
of some of the various instruments used. Lists of manufacturers will be found in various
publications, and on the Internet. It is not considered important to lay emphasis on
manufacturers and their equipment as these are subject to changes dictated by the
market.
9.2.5.1
pH measurement and control
The importance of the control of pH in microbial growth is well known. In some
industrial fermentations, good yield depends on accurate control (and hence accurate
measurement) of the pH of the fermentation broth. Sometimes the control of pH is
achieved by natural buffers present in the medium; phosphates and calcium carbonate
may also be used for this purpose. The buffering effect of these compounds is however
usually temporary. The broth must therefore be sampled and the pH adjusted as desired
with either acid or base. This method is laborious and may not accurately reflect the
continuous change taking place in the pH of the broth. Sterilizable pH probes have
become available and these are inserted in the fermentor or in a suitable projection
therefrom in which the broth bathes the electrode. With these electrodes it is now possible
to use an arrangement which will monitor pH changes and automatically induce the
introduction into the medium of either acid or alkali. In many fermentations acidity
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rather alkalinity is the situation to be combated. Such acidity usually arises from
microbial activity. It is therefore usual to arrange for the introduction of anhydrous
ammonia as acidity increases.
9.2.5.2 Carbon dioxide measurement
Water and carbon dioxide are two of the most common end-products of aerobic
fermentations. The measurement of CO2 therefore helps determine the course of the
fermentation as well as the carbon balance. At least three principles are employed in
current equipment for CO2 determination. The first method, which is the most widely
used, depends on the ability of CO2 to absorb infrared rays. A sensitive sensor translates
this absorption to a gauge or record, from which it can be read off. In another principle,
the effluent gas emerging from the broth is bubbled through a dilute solution of NaOH
containing phenol red. The change in color of the phenol red is reflected in a photocell
and the amount of CO2 may be calculated from a standard curve. The third method
depends on the thermal conductivities of the various gases in a mixture.
9.2.5.3 Oxygen determination and control
A number of methods are available for determining the oxygen concentration in a
fermentation broth.
Of the chemical methods, the best known is that of Winkler which is routinely used to
determine the biochemical oxygen demand (B.O.D) of water (Chapter 29). This method
relies on the back-titration, using iodine and starch, of unoxidized manganous salt
added to the liquid to be analyzed. Interfering substances are usually present in
fermentation broths. Furthermore, the method is cumbersome. Modern sensing methods
are not, however, based on this method. They rather sample the dissolved oxygen (DO) in
the medium. Modern dissolved oxygen probes are autoclavable and are based on one or
the other of two principles: the polarographic or the galvanic method.
In the polarographic method, a negative electric current 0.6-0.8 in voltage is passed
through an electrode immersed in an electrolyte made of neutral potassium chloride. This
negative electrode (cathode) is made of a noble metal such as platinum or gold. The anode
is calomel or Ag/Ag CI. Under this condition the dissolved oxygen is reduced at the
surface of the cathode according to the following reactions:
Cathode:
Anode:
Overall:
O2 + 2H2O + 2e ® H2O2 + 2OH–
H2O2 + 2e– ® 2OH–
Ag + Cl– ® Ag Cl + e–
4 Ag + O2 + 2H2 + 4 Cl ® 4 Ag Cl + 4OH–
The current which is measured after it has passed through the electrolyte is
proportional to the dissolved oxygen reacting at the cathode. A plastic membrane
permeable to gases but not ions separates the cathode, anode, and the electrolyte from the
liquid to be studied. The dissolved oxygen diffuses through the membrane and its
reaction at the cathode is measured at the current meter (Fig. 9.5). The electrolyte soon
becomes depleted by the constant replacement of Cl- by OH - (see equations above) and
the electrolyte has to be replaced.
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Modern Industrial Microbiology and Biotechnology
Fig. 9.5
Structure of Oxygen Electrodes: (A) Polarographic (B) Galvanic
In the galvanic method, no external source of electricity is applied. Instead the
electricity generated between a base metal anode (zinc, lead, or cadmium) and noble
metal cathode (silver or gold) is sufficient to cause the reduction of oxygen at the cathode.
The reactions are thus:
Cathode:
Anode:
Overall:
O2 + 2H2 + 4e ® 4 OH
Pb ® Pb2+ + 2e
O2 + 2Pb + 2H2O ® 2Pb(OH)2
The principle remains the same otherwise. The electric current generated in the system
is proportional to the quantity of oxygen reacting at the cathode. The electrolyte does not
however participate but the anode surface is gradually oxidized.
9.2.5.4
Pressure
It is important to know the pressure of gases in order to ensure that a positive pressure is
maintained. A positive pressure helps eliminate contamination and contributes to the
maintenance of proper aeration. Pressure may be determined with aid of a manometer.
9.2.5.5
Computer control
The fermentation industry, especially the antibiotic manufacturing aspect, usually
compares its operations with those in the chemical industry. Leaders in the fermentation
industry usually point to the fact that the fermentation industry in the early 1970s lagged
behind chemical industries in applying computers in regulating and managing
fermentations. The situation today is different and fermentation procedures are now
highly automated. Automation is an engineering problem and the expected advantages
of computerization have been given as follows:
(i) It should reduce labor by eliminating manual intervention.
Fermentors and Fermentor Operation
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(ii) The use of a computer should render an operator’s work easier and reduce human
error; it should, however, be possible to make changes while fermentation is on.
(iii) Automatic recording of all aspects of the fermentation is possible with a computer
and is useful in meeting any regulatory requirements as well as in improving
fermentation operations.
(iv) Experimentation should be easier as it should be much easier to study the effect of
altering any variables such as dissolved oxygen, temperature, pH, air flow,
nutrient addition, etc.
(v) Quality control should be easier to carry out.
(vi) In the event of power failure, and other emergencies, the system should be able to
shut up itself and restart and gradually build up to the original level of activity.
Commercial sensors are available for a wide variety of parameters in a fermentor. This
includes various ions, redox potential, cell mass measurement, carbohydrate
measurements to name a few. The computerization of these parameters makes it fairly
easy to monitor the operations in modern fermentation operations.
9.3
ANAEROBIC BATCH FERMENTORS
Some processes do not require the high levels of oxygen needed in aerobic fermentation;
indeed some, such as clostridial fermentations do not require oxygen at all. These are
collectively referred to as ‘anaerobic’ fermentations although in strict terms some may be
micro-aerophilic. Anaerobic fermentors, whether strict or micro-aerophilic (i.e., requiring
small amounts of oxygen) are not commonly used in industry. When they do (i.e., require
oxygen), they are essentially the same as the descriptions given above in the typical
(aerobic) fermentor. They, however, differ in the construction and operation as given
below.
(i) Vigorous aeration through air sparging is absent, as oxygen is not required.
(ii) Agitation when done is aimed only at achieving an even distribution of organisms,
nutrients and temperature, but not for aeration. In some cases agitation may be
essential only initially; the evolution of CO2 and H2 in anaerobic fermentors may
stir the medium.
(iii) The medium is introduced into the fermentor while hot to prevent the absorption of
gases; and usually it is also introduced at the bottom of the fermentor.
(iv) The fermentor itself is filled as much as possible, in order to avoid an airspace
which would introduce oxygen.
(v) If strict anaerobiosis is desirable, then an inert gas such as nitrogen may be blown
through the fermentation, at least initially, to remove oxygen.
(vi) Some low redox compounds, such as cysteine, may be introduced into the medium.
The same typical fermentor already described may be used for both aerobic and
anaerobic fermentations. It is especially important that it be possible for aerobic or
anaerobic fermentations to be carried in the same vessel as some fermentatons such as
alcohol manufacture require an earlier aerobic stage in which cells are produced in large
numbers and a later stage in which alcohol is produced anaerobically. But even the
strictly anaerobic fermentations can be carried out in the stirred tank batch fermentor
already described above.
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Modern Industrial Microbiology and Biotechnology
Two strictly anaerobic fermentaiton processes include acetone-butanol fermentation
(Clostridium acetobutylicum) and anti-tetanus toxoid production (Clostridium tetani). An
example of a micro-aerophilic fermentation which requires only a small amount of
oxygen is lactic acid production, while one which has a primary aerobic and a secondary
anaerobic system is alcohol production. Other examples are dextran production and the
production of 2-3 butylene-glycol.
9.4
FERMENTOR CONFIGURATIONS
Based on the nomenclature of the chemical engineering industry fermentors have been
grouped into four:
(i) Batch fermentors (Stirred Tank Batch Fermentors): (designated BF in Fig. 9.6) The
major features of this type of fermentor have been described already in Section 9.2
and Fig. 9.1. The other three are continuous fermentors and these are described
below:
(ii) Continuous stirred tank fermentors: (CSTF in Fig. 9.6) The tank used in this
system is essentially similar to that of the batch fermentor. It differs only in so far as
there is provision for the inlet of medium and the outlet of broth. The system has
been described under continuous cultivation.
(iii) Tubular fermentors: (TF in Fig. 9.6) The tubular fermentor was originally so
named because it resembled a tube. In general tubular fermentors are continuous
unstirred fermentors in which the reactants move in a general direction. Reactants
enter at one end and leave from the other and no attempt is made to mix them. Due
to the absence of mixing, there is a gradual fall in the substrate concentration
between the entry point and the outlet while there is an increase in the product in
the same direction.
(iv) The fluidized bed fermentor: This is essentially similar to the tubular fermentor.
In both the continuous stirred fermentor and the tubular fermentor there is a real
danger of the organisms being washed out (Fig. 9.10). The fluidized bed reactor is
an answer to this problem because it is intermediate in nature between the stirred
tank and the tubular fermentor. The microorganisms which are in a fluidized bed
fermentor are kept in suspension by a medium flow rate whose force just balances
the gravitational force. If the flow were lower, the bed would remain ‘fixed’ and if
the flow rate was at a force higher than the weight of the cells then ‘elutriation’
would occur with the particles being washed away from the tube. The tower
fermentor for the brewing of beer and production of vinegar (Chapter 14) is an
example of a fluidized bed fermentor.
9.4.1
Continuous Fermentations
Continuous fermentations are those in which nutrients are continuously added, and
products are also continuously removed. Continuous fermentations contrast with batch
fermentations in which the products are harvested, the fermentor cleaned up and
recharged for another round of fermentation. In the chemical industry continuous
processing has replaced many batch processes. This is because for products for which
there is a high and constant demand continuous processing offers several advantages.
Fermentors and Fermentor Operation
'%
Fluidized Bed Fermentor (FB)
Fig. 9.6
Different Fermentor Configurations (Left) and Graphs Depicting Substrate Usage in the Various
Configurations (Left) (see also Fig. 9.7)
'&
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These advantages when adapted to the microbiological industries, potentially include
the following:
(i) More intensive use of the equipment, especially the fermentor, and therefore greater
return on the initial capital outlay made in installing them. A great deal of time
involved in the cycle of batch production is not employed in direct production of
the final goods. Part of such ‘dead’ time is used in emptying the batch fermentor
during harvest, for cleaning, sterilizing, cooling and recharging with fresh
medium in between each batch. Furthermore, much of the period of a batch
fermentation is required for a lag period when the organisms are merely growing
and not yet producing (where the product is a metabolite), or the maximum
population has not been attained (where the product is the cell itself). In a
continuous fermentation, as soon as the steady state has been attained and
provided no contaminations occur, and other production activities permit the
plant to run for a reasonably long time, the ‘dead’ time required for all the above is
eliminated.
(ii) Allied to the above are savings in labor which do not have to repeatedly perform
the various operations linked with the ‘unproductive’ portions of batch
fermentation.
(iii) Continuous processes are more easily automated. This helps eliminate human
error and thus ensures greater uniformity in the quality of the products.
Automation also further saves labor costs additional to those mentioned in (ii).
Despite the possible advantages of continuous fermentation, the fermentation
industry has not in general adopted it. The areas where it has been employed include beer
brewing, food and feed yeast production, vinegar manufacture, and sewage treatment.
The reasons for the slow adoption of continuous fermentation since interest developed
in it several decades ago, are to be found in technical and economic factors. One of the
early deterrents was the fact that many early continuous fermentations became easily
contaminated. It is easy to see that while slow growing contaminants might not have
developed to the point where they can be noticed in the 4, 5, or 10 days of a batch
fermentation they can pose a serious threat to production, in a continuous culture which
goes on for up to three, six, or nine months. If the contaminant is fast growing then the
danger while serious in a batch fermentation is infinitely more so in a continuous
fermentation. Another problem was that mutants better adapted to the environment of the
continuous fermentor are easily selected. Where they perform better than the parent type
the difference was hardly noticed, except perhaps that a particular continuous
fermentation was inbued with an apparently inexplicable efficiency. On the other hand,
where the mutants were less productive, the reputation of continuous fermentation was
not helped.
9.4.1.1
Theory of continuous fermentation
In a batch culture four or five phases of growth are well recognized: the lag phase, the
phase of exponential or logarithmic growth, the stationary phase, the death or decline
phase. Some others add the survival phase. In the lag phase individual cells increase
somewhat in size but there is no substantial increase in the size of the population. In the
Fermentors and Fermentor Operation
''
exponential phase, the population doubles at a constant rate, in an environment in
which the various nutritional requirements are present in excess. As the population
increases, various nutrients are used up and inhibitory materials, including acids, are
produced; in other words the environment changes. The change in the environment soon
leads to the death of some organisms. In the stationary phase the rate of growth of the
organisms is the same as the rate of death. The net result is a constant population. In the
death phase, the rate of death exceeds the growth rate and the population declines at an
exponential rate.
If however during the exponential phase of growth, a constant volume is maintained
by ensuring an arrangement for a rate of broth outflow which equals the rate of inflow of
fresh medium, then the microbial density (i.e., cells per unit volume) remains constant.
This is the principle of one method of the continuous culture in the laboratory, namely,
the turbidostat.
As discussed above, the stationary phase sets in partly because of the exhaustion of
various nutrients and partly because of the introduction of an unfavorable environment
produced by metabolites such as acid. Either of these two groups of factors can be used to
maintain the culture at a constant density. Usually nutrients are used and their use for
this purpose will be discussed.
In a batch culture the various nutrients required by an organism are usually initially
present in excess. If all but one of the nutrients are present in adequate amount, then the
rate of growth of the organisms will depend on the proportion of the limiting nutrient that
is added. Thus if 100 grams per liter of the limiting nutrients are required for maximum
growth but only 90 grams per liter are added, then the rate of growth will be 90% of the
maximum. It is then possible to control the growth at any given rate but which rate is less
than the maximum possible, by letting in fresh nutrient at the same rate as broth is
released and also supplying one of the nutrients at a level slightly less than the
maximum. This principle is employed in the chemostat method of continuous growth.
In both the chemostat and the turbidostat the rate of nutrient inflow and broth outflow
must relate to the generation time or growth rate of the organism. If the rate of nutrient
addition is too high, then sufficient time is denied to the organism to develop an adequate
population. The organisms are then washed out in the outflow. If on the other hand the
rate of nutrient addition is too low, a stationary phase may set in and the population may
begin to decline.
The above is a simple non-mathematical description of the two basic procedures
which have been employed in the laboratory study and industrial application of
continuous individual cultivation. More detailed studies are widely available in texts on
microbial physiology.
To summarize, in the turbidostat a device exists for ensuring that a constant volume of
a microbial culture is maintained at constant density or turbidity. All the nutrients are
present in excess and the density or turbidity is monitored by a photo-cell which
translates any change to a mechanism which automatically reduces or increases the rate
of medium inlet and broth output, as necessary.
In the chemostat method a constant population is maintained in a constant volume by
the use of sub-maximal amounts of nutrient(s).
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In the laboratory and in practice the chemostat is far more widely used than the
turbidostat, probably because of the slightly more complex set up of the turbidostat which
follows from the need for constant density monitoring of the broth.
9.4.1.2
Classification of continuous microbial cultivation
It is important to understand the physiology of the production of the fermentation
product in order to enable the designing of an efficient continuous fermentation set-up.
The classification given below enables such a selection (Fig. 9.7).
Fig. 9.7
Various Types of Continuous Culture Arrangements (S Denotes Substrate Addition) A = Stirred
Batch Fermentor. B-E, Continuous Fermentors
Fermentors and Fermentor Operation
9.4.1.2.1
Single-state continuous fermentations
There are fermentations in which the entire operation is carried out in one vessel, the
nutrient being added simultaneously with broth outflow. This system is suited for
growth related fermentations such as yeast, alcohol, or organic acid production.
9.4.1.2.2
Multiple-stage continuous fermentation
This consists of a battery of fermentation tanks. The medium is led into the first and the
outflow into the second, third, or fourth as the case may be. This is most frequently used
for the fermentation involving metabolites. The first tank may be used for the growth
phase and subsequent tanks for production, depending on the various requirements
identified for maximal productivity.
9.4.1.2.3
Recycled single or multiple stage continuous fermentation
The out flowing broth may be freed of the organisms by centrifugation and the
supernatant returned to the system. This system is particularly useful where the
substance is difficult to degrade or not easily miscible with water such as in
hydrocarbons. Recycling can be applied in a single stage fermentor. In a multiple stage
fermentor, recycling may involve all or some of the fermentation vessels in the series
depending on the need.
9.4.1.2.4
Semi-continuous fermentations
In semi-continuous fermentations, simultaneous nutrient addition and outflow
withdrawal are carried out intermittently, rather than continuously. There are two types
of semi-continuous fermentation, namely;
(i) ‘cyclic-continuous’; (ii) ‘cell reuse’.
In Cyclic-continuous, a single vessel is usually employed, although a series of vessels
may be used. Fermentation proceeds to completion or near completion and a volume of
the fermentation broth is removed. Fresh medium of a volume equivalent to that
withdrawn is introduced into the vessel. As the size of the fresh medium is reduced, the
time taken to complete the fermentation cycle is reduced until eventually the intermittent
feeding becomes continuous. This system has been said to ensure a compromise, between
the desirable and undesirable features of batch and continuous fermentation;
productivity has however been shown theoretically and experimentally to be lower than
in continuous fermentation.
In cell reuse, cells are centrifuged from the fermentation broth and used to reinoculate
fresh medium. It is continuous only in the sense that cells are reused; in essence it is a
batch fermentation.
9.4.1.3 Applications of continuous cultivation
The literature is full of various areas of potential application of continuous fermentation,
experimented upon either in the laboratory or in pilot plants. These include single cell
protein production, organic solvents such as ethanol, acetone, butanol, isopropanol,
acetic acid from traditional raw materials such as sugar, starch, and molasses. Cellulose
is also being considered as a substance for these and the continuous culture of cellulose
digesting enzymes from Trichoderma is an important step. In agriculture, continuous
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cheese making, continuous yoghurt starter production and continuous use of lactore in
whey are being vigorously pursued. Medical and veterinary applications include the
continuous production of vaccines, and cell cultivation.
Continuous waste digestion for sewage chemical wastes outside the activated sludge
exist as also do the continuous brewing of beer, the continuous production of wine and
the continuous manufacture of yeasts, vinegar and alcohol.
9.5
FED-BATCH CULTIVATION
Fed-batch cultivation is a modification of batch cultivation in which the nutrient is added
intermittently to a batch culture. It was developed out of cultivation of yeasts on malt,
where it was noticed that too high a malt concentration lead to excessively high yeast
growth leading to anaerobic conditions and the production of ethanol instead of yeast cells.
After its successful introduction in yeast cultivation, the original method or
modifications of it have been used to achieve higher yields or more efficient media
utilization in the production of various antibiotics, amino acids, vitamins, glycerol,
acetone, butanol, and lactic acid. Some of the modifications include continuous (rather
than intermittent) addition of single or multiple media components, withdrawal of a
portion of the broth from the growth vessel and immediate dilution of the residue with
fresh medium and the use of diffusion capsules. The latter are cylindrical capsules to one
end of which a semi-permeable membrane is fixed. The nutrient diffuses slowly out
through the membrane into the medium.
9.6
DESIGN OF NEW FERMENTORS ON THE BASIS OF
PHYSIOLOGY OF THE ORGANISMS: AIR LIFT
FERMENTORS
The Stirred Tank Batch Fermentor already described is the most widely used type of
fermentor. Increasing knowledge of the physiology of industrial microorganisms and
better instrumentation have provided the bases for more efficient manipulation of the
organisms in the existing batch fermentors:
(i) More sophisticated instrumentation is now used to monitor such fermentor
parameters as dissolved oxygen and carbon dioxide, redox potential, and control
of the fermentation leading to higher yields.
(ii) Different levels of pH, temperature, and phosphate concentration are sometimes
needed during the trophophase and the idiophase for the production of secondary
metabolites. These differences have been exploited in some fermentations for
higher yields.
(iii) By careful monitoring using automated sensing devices, it is now possible to add
just enough of the nutrients required by a growing culture so that feedback
inhibition is avoided.
The above are a few examples to show that the existing fermentors can be better
utilized when greater knowledge of microbial physiology is harnessed for that purpose.
Despite these improvements, needs have arisen for drastic change from the typical stirred
tank batch fermentor, and these needs would appear not to be fully met by automation of
batch fermentors. Some of the needs call for the design of new fermentors based on the
following:
Fermentors and Fermentor Operation
!
(i) The diversification of fermentation products and new attendant problems.
Examples are the production of single cell protein by continuous fermentation;
production of microbial polysaccharides; fermentor cultivation of animal and
plant cells; the growing re-emergence of anaerobic fermentations such as for
ethanol.
(ii) The unusual properties of the substrates or products involved in this diversification such as insolubility in water (for example, of petroleum fractions, agricultural
wastes, or hydrogen gas) or high viscosity (for example, microbial capsules).
(iii) Greater knowledge or awareness of the physiology of the organisms during their
growth in a fermentor especially:
(a) The need for high amounts of dissolved oxygen.
(b) Adequate mixing of fermentation broths
(c) The problem of clumping or aggregation especially in filamentous organisms
such as actinomycetes.
(d) The need to avoid feedback inhibition by the removal of inhibitory products.
To solve some of these problems most of the newly designed fermentors have moved
away from the structure of the commonly used Stirred Tank Batch Fermentor. They in fact
lack stirrers; instead they are of the recycle, loop, or airlift type in which stirring is
replaced by pumping of air. Some of the problems these fermentors or arrangements are
designed to solve are given below.
(i) Need for high amounts of dissolved oxygen: Many industrial fermentations require
large amounts of oxygen, and yields are severely limited when the gas is in short supply.
To solve this problem especially in regard to the utilization of novel carbon sources, from
hydrocarbons, the airlift fermentor was designed (Fig. 9.8). In this fermentor high levels of
dissolved oxygen are achieved by using the air pressure to lift the broth. According to
(A)
(B)
In the airlift type (A), air is forced through a sparger; in the plunging jet type (B) air is forced into the broth
in a jet. There are no moving parts in loop fermenters.
Fig. 9.8 Loop Fermentors
"
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some authors the airlift fermentor is a modification of the batch fermentor differing in the
absence of stirring. It is in fact one of several types of loop fermentors.
(ii) Mixing of the broth: Poor mixing reduces yields in yeasts grown on alkanes.
Aggregates consisting of alkane droplets and yeast cells float to the top of the broth in
poorly mixed fermentations. The nutrients cannot therefore get to the yeasts which
become starved as a consequence. The problem was solved using a completely filled
circulating fermentor which operates on the same principle as a shake flask (Fig. 9.9).
Fig. 9.9
Circulating Fermentor: 1, Vessel; 2&3, Draught Type; 4, Baffle; 5, Stirrer; 6 & 7, Foam
Breaker; 8 & 9, Air Sparger; 10, Outer Section; 11, Inner Section.
(iii) Aggregate of cells: In filamentous cells, e.g., actinomycetes and fungi, the cells tend to
aggregate and only those at the periphery of the clump grow. A steep gradient
concentration of the product therefore exists from the outside to the inside. The avoidance
of clumps and the production of loosely organized cells are achieved in the airlift
fermentor.
(iv) Removal of inhibitory products: In the high concentration of components of a
fermentation broth, feedback inhibition easily limits production. One manner of dealing
with the problem is to subject the broth to dialysis. This can be achieved by constantly
circulating the broth in an external membrane in contact with water. Volatile end
products may be removed as they are formed by applying reduced pressure.
Fermentors and Fermentor Operation
9.7
#
MICROBIAL EXPERIMENTATION IN THE FERMENTATION
INDUSTRY: THE PLACE OF THE PILOT PLANT
When the microorganism used in a fermentation is new, experimentation must be carried
out to determine conditions for its maximum productivity. It is usual to initiate the
studies in a series of conical flasks of increasing size and to progress through a 10-20 liter
fermentor to a pilot plant (100-500 liter) and finally to a production plant (10,000200,000 liters). The processes involved in the increasing scale of operation culminating in
the production plant are known as scaling up.
On the other hand, in a well-established fermentation procedure, any change to be
introduced must be experimented on and tested out in a pilot plant whose function is to
simulate the conditions and structures of the production plant. This procedure is often
referred to as scaling down. The processes of scaling up and scaling down are essentially
in the domain of the chemical engineer who depends on data supplied by the
microbiologist.
Information gathered at the shake flask stage is used to predict requirements in the
pilot plant which itself serves a similar purpose for the production plant. The optimum
requirements of medium composition, aeration, temperature, redox potential, pH,
foaming, etc., are determined and extrapolated for the next higher scale. The pilot
fermentor is also used for training new recruits in the fermentation industry; it may also
be used for continuous fermentation where a large enough number of them exist.
One approach which helps facilitate translation of information from the pilot plant to
production is to reproduce the production plant as a geometrical replica of the pilot plant.
Baffles, agitators, etc., are increased exactly according to a predetermined scale. This,
however, does not entirely solve the problem because the mere increase in volume
immediately poses its own problems. If the same level of productivity as encountered in
the pilot study is to be maintained, then agitation and aeration may be applied at a level
higher than that expected in a proportional increase in the production fermentor.
9.8
INOCULUM PREPARATION
The conditions needed for the development of industrial fermentations often differ from
those in the production plant. This is because except in a few examples where the cells
themselves are the required product, e.g., in single cell protein, or in yeast manufacture,
most fermentation products are metabolites. Cells to be used must be actively growing,
young and vigorous and must therefore be in the phase of logarithmic growth. Since
organisms used in most fermentations are aerobes, the inocula will usually be vigorously
aerated in order to encourage maximum cell development, although they may need less
aeration in subsequent incubation. The chemical composition of the medium may differ
in the inoculum and production stages. The inoculum usually forms 5-20% of the final
size of the fermentation. By having an inoculum of this size the actual production time is
considerably shortened.
The initial source of the inoculum is usually a single lyophilized tube. If the content of
such a tube were introduced directly into a 100,000 liter pilot fermentor, the likelihood is
that it would take an intolerably long time to achieve a production population, during
$
Modern Industrial Microbiology and Biotechnology
which period the chance of contamination is created. For these reasons inocula are
prepared in several stages of increasing volume. At each step, the growth is checked for
the absence of contamination by plating. When the lyophilized vial is initially plated out
and shown to be pure, the entire plate instead of a single colony is scraped off and
transferred to the shake flask so as to avoid picking mutants (Fig. 1.2, Chapter 1)
9.9
SURFACE OR SOLID STATE FERMENTORS
In solid state fermentors rice bran or some such solid is used. Molasses may be added and
a nutrient solution of ammonium and phosphate may be introduced. It is used mainly in
Japan for enzyme production, and has been used for citric acid production. Fungal
bioinsecticides are also cultivated as surface cultures. Certain mushrooms are also
grown in tray fermentors.
In the surface fermentor shown in Fig. 9.10, a series of shallow trays no more than
about 7 cm in depth is used, the solid medium not being more than about 5 cm so that air
can penetrate into the solid medium. Humid air is blown into the chamber containing the
trays. The incoming air and the out going air may be filtered especially when fungi are
used to save the dissemination of the spores in the atmosphere. In some fermentations
some form of temperature control is imposed through blowing cold air into the fermentor
and also by cooling the room where the fermentor is located.
Fig. 9.10
Diagram of a Solid-state (Surface)Tray Fermentor Humid Cooled, Sometimes Filtered, Air is let
into the Fermentor; the Exhaust Air is also Filtered (see text)
SUGGESTED READINGS
Ahuja, S. 2000. Handbook of Bioseparations. Vol 2 Academic Press. San Diego, USA.
Dobie, M., Kruthiventi, A.K., Gaikar, V.G. 2004. Biotransformations and Bioprocesses. Marcel
Dekker, New York, USA.
Endo, I., Nagamune, T., Katoh, S., Yonemoto (eds) 1999. Bioseparation Engineering. Elsevier
Amsterdam the Netherlands.
Fermentors and Fermentor Operation
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Flickinger, M.C., Drew, S.W. (eds) 1999. Encyclopedia of Bioprocess Technology - Fermentation,
Biocatalysis, and Bioseparation, Vol 1-5. John Wiley, New York, USA.
Garcia, A.A., Bonem, M.R., Ramirez-Vick, J., Saddaka, M., Vuppu, A. 1999. Bioseparation Process
Science. Blackwell Science Massachussets USA.
Harrison, R.G., Todd, P., Rudge, S.R., Petrides, D.P. 2003. Bioseparation Science and Engineering.
Oxford University Press, New York, USA.
Kalyanpur, M. 2000. Downstream Processing in Biotechnology ln: Downstream Processing of
Proteins: Methods and Protocols. M Desai, (ed) Humana. Totowa, NJ, USA pp. 1–10.
Naglak, T.J., Hettwer, D.J., Wang, H.Y. 1990. Chemical permeabilization of cells for intracellular
product release In: Separation Processes In Biotechnology, Marcel Dekker, New York, USA.
&
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+0)26-4
10
Extraction of
Fermentation Products
Judging from the extent of discussion on the fermentor and its accessories one might be
led to feel that they consume nearly all of the capital investment in a fermentation
industry. This is however not so: not only is the investment in recovery equipment high,
but isolation costs represent a good proportion (sometimes up to 60%) of the cost of the
final product. In one antibiotic factory, recovery equipment cost four times more than the
fermentor. The necessity of having a well-planned and reliable recovery process and an
efficient recovery plant is therefore of utmost importance. In this discussion only broad
outlines of the principles of extraction will be given, more detailed consideration being
given when each product is discussed.
The central problem in the extraction of fermentation products from the fermentation
‘beer’ or broth is that the required product usually (but not always) forms a small
proportion of a complex heterogeneous mixture of cell debris, other metabolic product,
and unused portions of the medium. The following are the factors borne in mind in
deciding the extraction method to be used:
(i) the value of the final product;
(ii) the degree of purity required to make the final product acceptable, bearing in mind
its revenue-yielding potential;
(iii) the chemical and physical properties of the product;
(iv) the location of the product in the mixture i.e. whether it is free within the medium or
is cell-bound;
(v) the location and properties of the impurities; and finally;
(vi) the cost-effectiveness or the economic attractiveness of the available alternate
isolation procedures.
The various steps followed in the extraction of fermentation products together with the
approximate level of purification obtained in each stage are given in Table 10.1.
The procedure followed within each stage depends of course on the material being
extracted, and are discussed hereunder. The product sought could be the cells themselves
such as in yeast manufacture, or lodged in the cells (such as in streptomycin or some
enzymes) or free in the medium as with penicillin.
Extraction of Fermentation Products
10.1
'
SOLIDS (INSOLUBLES) REMOVAL
In general the initial step separates solids from the liquid fraction thereby facilitating
further extractive steps, such as sorption, solvent extraction which would be wasteful or
near impossible if the cells were not separated. When the required product is solid or is
lodged in the insoluble portion liquid removal helps concentrate the solids.
Table 10.1 Conventional steps* followed in the purification of products in the soluble portion
of ‘beer’
Step Process
Hypothetical degree of purity (%)
1a. Removal of insolubles
Filtration
Centrifugation
Decantation
0.1-1.0 (if product solubles)
90-99 if product is cell
such as yeasts
1b. Disruption of cells
2 Primary foam isolation of the product
Sorption physical and/or ion exchange
Solvent extraction
Precipitation
Ultracentrifugation
3 Purification
Fractional precipitation
Chromatography (adsorption,
partition, ion exchange, affinity)
Chemical derivatization
Decolorization
4. Final product isolation
Crystallization
Drying
Solvent removal
1-10
50-80
90-100
*Some modern extraction methods combine steps 1 and 2
In a few cases no separation takes place such as in the acetone butanol fermentation,
where the entire beer is used. In most cases, however, the separation methods used are
filtration, centrifugation, decantation, and foam fractionation. Where the required
fraction is in the cells then much of the impurities are removed with the filtrate after the
cells have been isolated. The various methods used in solids removal are discussed
below.
10.1.1
Filtration
The rotary vacuum filter: One of the most commonly used filters in industry is the
rotary vacuum filter which is available in several forms. Essentially the filter consists of a
hollow rotating cylinder divided into four partitions and covered with a metal or cloth
gauze. A vacuum is applied in the cylinder and as it rotates the vacuum sucks liquid
materials from the shallow trough in which the rotating cylinder is immersed. For thick
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slurries which are difficult to filter (e.g. aminoglycoside broths) a thin layer of filter aid
(e.g. Kiesselghur) is first allowed to be absorbed on the cylinder. Later the filter cylinder
with its thin coating of the filter aid is allowed to rotate in the trough in which the broth
is now placed. The rotating cylinder, the vacuum still on, is washed with a sprinkle of
water; a knife whose edge is positioned just short of the layer of filter aid scrapes off the
solids picked up from the broth.
When it is used for easily filtered broth such as in penicillin broth no filter aid is used.
Instead an arrangement of strings coupled with a release of the vacuum in the segment of
the cylinder helps release the material picked up from the broth.
Fig. 10.1
Transverse Section of the Rotary Vacuum Filter Illustrating its Operation
Ring and wire type filters: These filters consist of a coating of diatomaceous earth on a
wire-mesh supported by a frame of metal rods. The liquid to be filtered is introduced
under a pressure of 75 p.s.i rather than under a vacuum as in the rotary vacuum filter.
They are used when the load is light such as for polishing beer or fruit juices. They can be
cleaned by back flushing with water.
10.1.2
Centrifugation
Centrifugation is not widely used for the primary separation of solids from broth in
fermentation beer because of the thickness of these slurries and the fact that many
industries have operated successfully with filters. Only in a few cases will a centrifuge
de-water a broth to anywhere near the extend a filter would. In the enzyme isolation
industry, however, centrifugation is preferred to filtration, probably because unwanted
cell debris are quite efficiently removed by this method. A large number of centrifuges are
available in the market and a new fermentation industry or a change in the production
method of old processes may require the use of centrifuges for primary separation.
10.1.3
Coagulation and Flocculation
Coagulation is the cohesion of dispersed colloids into small flocs; in flocculation these
flocs aggregate to form larger masses. The first is induced by electrolytes and the latter by
polyelectrolytes, high molecular weight, water soluble compounds that can be obtained
in ionic, anionic, or cationic forms. Bacteria and proteins being negatively charged
colloids are easily flocculated by electrolytes or polyelectrolytes. Sometimes clay, or
Extraction of Fermentation Products
activated charcoal may be used. The net effect of the flocculation is that colloid removal
facilitates filtration. It may even be possible to merely decant the supernatant once large
enough flocs remove the solid portion of the ‘beer’ of them which to use and low much to
use among the various flocculants must be worked out by experimentation. Since
flocculation depends on cell wall characteristics, the agents must meet the following
requirements especially if the cells, and not the liquid, are the required products. The
flocculants should have the following properties.
(i)
(ii)
(iii)
(iv)
They must react rapidly with the cells.
They must be non-toxic.
They should not alter the chemical constituents of the cell.
They should have a minimum cohesive power in order to allow for effective
subsequent water removal by filtration.
(v) Neither high acidity nor high alkalinity should result from their addition.
(vi) They should be effective in small amounts and be low in cost.
(vii) They should preferably be washable for reuse.
10.1.4
Foam Fractionation
Foam formation has been described in Chapter 9. The principle of foam fractionation is
that in a liquid foam system the chemical composition of a given substance in the bulk
liquid is usually different from the chemical composition of some substance in the foam.
Foam is formed by sparging the bulk liquid containing the substance to be fractionated
with an inert gas. The gas is fed at the bottom (Fig. 10.2) of a tower and the foam created
overflows at the top carrying with it the solutes to be fractionated. Surfactants or (surface
Overflow
Foam
Breaker
Foam
Collapsed
Foam
Liquid
Gas
Fig. 10.2 Foam Fractionation
Modern Industrial Microbiology and Biotechnology
active substances that reduce surface tension e.g. teepol) may be added in liquids that do
not foam. This method has been used to collect a wide range of microorganisms and
although mainly experimental it may be used on a large scale in industry.
10.1.5
Whole-broth Treatment
As had been indicated earlier, in some fermentations such as the acetonebutanol
fermentation, the whole unseparated broth is stripped of its content of the required
product. In the antibiotic industry a similar situation was achieved before it became
possible to directly absorb the antibiotics streptomycin (using cationic-exchange resin)
and novobiocin (on an anionic resin.) The antibiotics are eluted from the resins and then
crystallized. This process saves the capital and recurrent expense of the initial separation
of solids from the broth.
10.2
PRIMARY PRODUCT ISOLATION
After separation of the broth into soluble and insoluble fractions, the next process
depends on the location of desired product as follows: the cells themselves as in yeasts
may be desired product; they are dried or refrigerated and the liquid discarded. Further
treatment such as drying is discussed later in the chapter.
The required product may be bound to the mycelia or to bacterial cells as in the case of
bound enzymes or antibiotics. The cells then have to be disrupted with any of the several
ways available – heat, mechanical disruption, etc. The cell debris are now removed by
centrifugation, filtration or any of the other methods for removing solids, described
above.
Where the material is extracellularly available or if it has been obtained by leaching
with or without cell disruption then it is treated by one of the following methods: liquid
extraction, dissociation extraction, sorption, or precipitation.
10.2.1
Cell Disruption
A lot of biological molecules are inside the cell, and they must be released from it. This is
achieved by cell disruption (lysis). Cell disruption is a sensitive process because of the
cell wall’s resistance to the high osmotic pressure inside them. Furthermore, difficulties
arise from a non-controlled cell disruption, that results from an unhindered release of all
intracellular products (proteins nucleic acids, cell debris) as well as the requirements for
cell disruption without the desired product’s denaturation. There are mechanical and
non-mechanical cell disruption methods.
10.2.1.1
Mechanical methods
When the target material is intracellular, the means microorganisms are disrupted
mainly by mechanical disruption of the cells. Equipment for cell disruption includes:
i) Homogenizers. These pump slurries through restricted orifice or valves at very high
pressure (up to 1500 bar) followed by an instant expansion through a special
exiting nozzle. The sudden pressure drop upon discharge, causes an explosion of
the cell. The method is applied mainly for the release of intracellular molecules.
Extraction of Fermentation Products
!
ii) Ball Mills. In a ball mill, cells are agitated in suspension with small abrasive
particles. Cells break because of shear forces, grinding between beads, and
collisions with beads. The beads disrupt the cells to release biomolecules.
iii) Ultrasonic disruption. This method of cell lysis is achieved with high frequency
sound that is produced electronically and transported through a metallic tip to an
appropriately concentrated cellular suspension. It is expensive and is used mainly
in laboratories.
10.2.1.2
Non-mechanical methods
Cells can be caused to disrupt by permeabilization thorough a number of ways:
(i) Chemical Permeabilization. Many chemical methods have been employed in order to
extract intra cellular components from microorganisms by permeabilizing (i.e., making
them permeable) the outer-wall barriers. It can be achieved with organic solvents that act
by the creation of canals through the cell membrane: toluene, ether, phenylethyl alcohol
DMSO, benzene, methanol, chloroform. Chemical permeabilization can also be achieved
with antibiotics, thionins, surfactants (Triton, Brij, Duponal), chaotropic agents, and
chelates. A very important chemical is EDTA (chelating agent) which is widely used for
permeabilization of Gram negative microorganisms. Its effectiveness is a result of its
ability to bond the divalent cations of Ca++, Mg++. These cation stabilize the structure of
outer membranes by bonding the lipopolysaccharides to each other. The removal of these
cations EDTA, increases the permeability areas of the outer walls.
(ii) Mechanical Permeabilization. One method of mechanical permeabilization is osmotic
shock. While cells exposed to slowly varying extracellular osmotic pressure are usually
able to adapt to such changes, cells exposed to rapid changes in external osmolarity, can
be mechanically injured. This procedure is typically conducted by first allowing the cells
to equilibrate internal and external osmotic pressure in a high sucrose medium, and then
rapidly diluting away the sucrose. The resulting immediate overpressure of the cytosol is
assumed to damage the cell membrane. Enzymes released by this method are believed to
be periplasmic, or at least located near the surface of the cell.
(iii) Enzymatic Permeabilization. Enzymes can also be employed to permeabilize cells, but
this method is often limited to releasing periplasmic or surface enzymes. In these
procedures, they often use EDTA in order to destabilize the outer membrane of Gram
negative cells, making the peptidoclycan layer accessible to the enzyme used. Enzymes
used for enzymatic permeabilization are: beta(1-6) and beta(1-3) glycanases, proteases,
and mannase.
10.2.2
Liquid Extraction
Also known as solvent extraction, or liquid-liquid extraction this procedure is widely
used in industry. It is used to transfer a solute from one solvent into another in which it is
more soluble. It also can be used to separate soluble solids from the mixture with
insoluble material by treatment with a solvent.
The law on which liquid-liquid extraction is based states that when an organic solute
is exposed to a two-phase immiscible liquid system the ratio of the solute concentration in
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the two phases is constant for a given temperature. This ratio, K, is the partition or
distribution coefficient, given as:
K = C1/C2
where C1 and C2 are the concentrations in phase 1 and phase 2 respectively. The equation
is effective, however, for dilute solutions and breaks down for very concentrated
solutions. The selectivity of a solvent solution is indicated by the ratio of the distribution
coefficients of the components in question. Which solvents are actually used will depend
on a number of factors including the distribution properties of the solute in them,
volatility, ease of recovery and cost.
In this method the broth to be extracted is shaken with a hydrophobic solvent (i.e., one
that will not mix with water), allowed to settle and the solvent which should contain
more of the material to be extracted is removed. This may be done in a small laboratory
scale in separating funnels or in a stirred tank in industry.
The separation may be done in a stirred tank in one of several ways (Fig. 10.3): (1) batch
wise in a single tank and the solvent with its solute drained; or (2) continuous with a
mixing and a setting tank. More efficient extractions are achieved with continuous
addition of solvent in (3) a cross-current arrangement in which successive solvent
extracts will be progressively more dilute or in a (4) counter-current fashion in which
efficient extraction is achieved with less solvent usage.
The counter-current multi-stage system is most commonly used and a wide variety of
equipment incorporating this system exist. In many applications a vertical column may
be used with the heavier liquid introduced from the top and the lighter from the bottom.
Mixing of the liquid may occur via a stirring shaft or by the turbulence created by a series
of plates placed in the column. A series of such columns may be set up with provision for
automatic transfer of liquid from one column to the next. A set-up similar to this has been
used to separate radio-active materials. This principle has also found use in penicillin
separation, but because penicillin would be destroyed in the acidified broth by prolonged
contact and also because such prolonged contact enable protein present in the medium to
stabilize, separation is done quickly.
10.2.3
Dissociation Extraction
Dissociation extraction is a special case of liquid-liquid extraction. Many fermentation
products are either weak bases or acids. When solvent extraction is employed the pH is so
selected that the material to be isolated is unionized since the ionized form is soluble in
the aqueous phase and the unionized form is soluble in the solvent phase. Weak bases
are therefore extracted under high pH conditions and weak acids under low pH
conditions. The result is a rapid and complete extraction of the solute and materials
similar to it.
10.2.4
Ion-exchange Adsorption
Ion exchange adsorption is one of several adoption methods which include
chromatography, and charcoal adsorption. These will be discussed later.
Ionic filtrates of fermentation broths can be purified and concentrated using ion
exchange resins packed in columns. An ion exchange resin is a polymer (normally
Extraction of Fermentation Products
Fig. 10.3
Schematic Representations of Various Methods of Solvent Extraction
#
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Modern Industrial Microbiology and Biotechnology
polystyrene) with electrically charged sites at which one ion may replace another.
Synthetic ion exchange resins are usually cast as porous beads with considerable
external and pore surface where ions can attach. Whenever there is a great surface area,
adsorption plays a role. If a substance is adsorbed to an ion exchange resin, no ion is
liberated. Testing for ions in the effluent will distinguish between removal by adsorption
and removal by ion exchange. While there are numerous functional groups that have
charges, only a few are commonly used for manmade ion exchange resins. These are:
•
•
•
•
•
-COOH which is weakly ionized to -COO¯
-SO3H which is strongly ionized to -SO3¯
-NH2 that weakly attracts protons to form NH3+
-secondary and tertiary amines that also attract protons weakly
-NR3+ that has a strong, permanent charge (R stands for some organic group)
These groups are sufficient to allow selection of a resin with either weak or strong
positive or negative charge. The resins are usually branched polymers of high molecular
weight-containing easily exchanged ions which are in equilibrium with ions in the
surrounding solution. The resins are however usually used in neutral salt forms: cation
exchangers in the sodium form and anion exchangers in the chloride form. The resins lose
the labile ions and in exchange bind suitable materials in the liquid percolating down the
column. The efficiency of the exchange depends on the following factors:
(i) The capacity of the resin for the ion to be adsorbed, usually expressed in milliequivalents.
(ii) The size of the resin spheres: the smaller, the more the exchange.
(iii) The flow rate; the slower, the greater the adsorption.
(iv) Temperature: the higher, the more rapid the exchange.
The choice of the resin depends on the chemical and physical properties of both resin
and product as well as on the contaminating materials. CaCO3, for example, is often left
out of media for streptomycin fermentation, because Ca++ ions are preferentially adsorbed
onto the resin in place of streptomycin cations.
As indicated earlier, streptomycin is extracted over a resin ( a carboxylic acid resin)
with prior separation of the soluble from the insolubles. The broth is passed successively
through two resin columns which have previously been flushed with NaOH to convert
them to the sodium phase. The resin absorbs a large amount of the streptomycin which is
eluted with HCI converting the streptomycin to chloride and the resin to the hydrogen
form. In this way the streptomycin is both purified and concentrated.
10.2.5
Precipitation
The insolubility of many salts is used in the selective isolation of some industrial
products. It is particularly useful in the elimination of proteinaceous impurities or in the
isolation of enzymes. Salts are precipitated by one of several methods: adding inorganic
salts and (or) reducing the solubility with the addition of organic solvents such as
alcohol in the case of enzymes. Lactate and oxalate salts of erythromycin have been so
isolated and citric acid has been isolated with its calcium salt.
Extraction of Fermentation Products
10.3
%
PURIFICATION
The methods described earlier isolate mixtures of materials similar in chemical and
physical properties to the required product. The methods used in this section are finer
and further eliminate the impurities thus leaving the desired product purer.
10.3.1
Chromatography
In chromatography, the components of a mixture of solutes migrate at different rates on a
solid because of varying solubilities of the solutes in a particular solvent. The mixture of
solutes is introduced (usually as a solution) at one end of the solid phase and the solvent
(i.e., the solution which separates the mixture) flows through this initial point of the
mixture application. Fermentation products are separated by any of the following
chromatographic methods, where the separation of the solids occur for the reasons given
in each of the following.
(i) Adsorption chromatography: (e.g., paper chromatography) variations in the weak
(Van der Wall) forces binding solutes to the solid phase;
(ii) Partition chromatography: A mobile solvent is passed through a column containing
an immobilized liquid phase; the solvent and immobilized liquid phase are
immiscible. Separation occurs by the different distribution or partition coefficients
of the solutes between the mobile and immobilized liquid phases.
(iii) Ion exchange chromatography: The difference in the strength of the chemical bonding
between the various solutes and the resin constitutes the basis for this method.
(iv) Gel Filtration: This depends on the ability of molecules of different sizes and shapes
to permeate the matrix of a gel swollen in the desired solvent. The gel can be
considered as containing two types of solvent; that within the gel particle and that
outside it. Large particles which cannot penetrate the gel appear in the column
effluent after a volume equivalent to the solvent outside the gel has emerged from
the column. Small molecules which permeate the matrix appear in the effluent after
a volume equivalent to the total liquid volume within the matrix has emerged.
10.3.2
Carbon Decolorization
Some solids are able to adsorb and concentrate certain substances on their surfaces when
in contact with a liquid solution (or gaseous mixture). These include activated charcoal,
oxides of silicon, aluminum, and titanium and various types of absorbent clays.
Absorbents have been used for the adsorption of antibiotics from broths, removal of
colored impurities from a solution of an antibiotic, sugar or even from gasoline. In the
fermentation industry activated charcoal has been most widely used because of its
extensive pores which confer on it a large surface. Furthermore, the pores are large
enough to allow the passage of the solvent.
Activated carbon, powdered or granular, is used to remove color. Thus penicillin
solution is usually treated with activated carbon before the crystallization of the amino
salt. A single-stage batch-wide system of mixing the solution with carbon followed by
filtration may be used. Multi-stage counter-current decolorization is far more efficient per
unit of carbon than batch. Before using an adsorbent it is important to determine
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Modern Industrial Microbiology and Biotechnology
experimentally the most efficient depth of the absorption zone which will thoroughly
remove all color.
10.3.3
Crystallization
Crystallization is the final purification method for those materials which can stand heat.
The solution is concentrated by heating and evaporation at atmospheric pressure to
produce a super saturated solution. Many fermentation products will not however stand
heat and the initial water removal is made by heating at reduced pressure or by lowering
the temperature to form crystals which can be centrifuged off leaving a concentrated
liquor. It yields compounds which are highly potent more stable and free from colored
impurities. To obtain crystals, first a super saturated solution is produced; secondly,
minute nuclei or seeds are formed and thirdly, the molecules of the solute build on the
nuclei. Crystalline particles from a previous preparation may be deliberately introduced
to produce the nuclei. In procaine penicillin production, fine crystals are used to induce
crystallization whereas in dehydrostreptomycin sulfate, addition of methanol brings
about crystallization.
10.4
PRODUCT ISOLATION
The final isolation of the product is done in one of the two following ways:
(i) processing of crystalline products.
(ii) drying of products direct from solution.
10.4.1
Crystalline Processing
Crystalline products are free-filtering and non-compressible and therefore may be filtered
on thick beds under high pressure. This is usually done on a centrifugal machine capable
of developing very high (about 1,000 fold) gravitational force. The crystals are washed to
remove adhering mother liquor. After washing they are dried by spinning for further
drying or solvent removal.
10.4.2
Drying
Drying consists of liquid removal (either organic solvent or water) from wet crystals such
as was described above from a solution, or from solids or cells isolated from the very
earliest operation. Several methods of drying exist and the one adopted will depend on
such factors as the physical nature of the finished product, its heat sensitivity, the form
acceptable to the consumer, and the competitiveness of the various methods in relation to
the cost of the finished product. Drying can be considered under two heads: (i) liquidphase moisture removal, and (ii) solid-phase moisture removal.
10.4.2.1
Liquid-phase moisture removal
Liquid-phase moisture removal involves drying by heat. When drying is done by heating,
the processes may be broken down to the supply of heat to the material and the removal
of the resulting water vapor. The simplest method is by direct heating in which heated
Extraction of Fermentation Products
'
atmospheric air both heats the material and removes the water vapor. In others, the
heating is done at reduced pressure to facilitate evaluation of the water vapor. Under
such conditions, indirect heating from a heated surface, radiation (e.g., infra-red) or both
is used to supplement the heat introduced by reduced vapor pressure.
The actual method of heating is done in a number of different mechanical contraptions
which will be mentioned briefly below.
(i) Tray Driers: The most commonly used in
some fermentation industries is the
vacuum tray drier. It is versatile and
consists simply of heated shelves in a
single cabinet which can be vacuum
evacuated. In some, the trays may have
provision for vibration or shaking to
hasten evaporation. As it can be
evacuated, heating at fairly low
temperature is possible and hence it is
useful for heat-labile materials.
(ii) Drum dryers: In this method the broth or
slurry is applied to the periphery of a
revolving heated drum. The drum may
Fig. 10.4 Drum Drier
be single or in pairs. High temperature is
applied though for a short time on the
material to be dried and some
destruction may occur. One arrangement
of drum driers is illustrated in Fig. 10.4.
(iii) Spray drying: This method is used
extensively in the food and fermentation
industries for drying heat-sensitive
materials such as drugs, plasma and
milk. The conventional spray consists of
an arrangement for introducing a fine
spray of the liquid to be dried against a
counter-current of hot air. As the
material is exposed to high temperature
for only a short while, a matter of a few
seconds, very little damage usually
occurs. Furthermore, it is convenient
Fig. 10.5 Conventional Spray Drying
because of its continuous nature.
Sometimes the material is introduced simultaneously with air (Fig. 10.5).
10.4.2.2
Solid-phase moisture removal (freeze-drying)
The equipment used in freeze-drying is essentially the same as in the vacuum drier
described earlier. The main difference is that the material is first frozen. In this frozen
state, the water evaporates straight from the material. It is useful for heat-labile materials
such as enzymes, bacteria, and antibiotics.
Modern Industrial Microbiology and Biotechnology
SUGGESTED READINGS
Ahuja, S. 2000. Handbook of Bioseparations. Vol 2 Academic Press. San Diego, USA.
Dobie, M., Kruthiventi, A.K., Gaikar, V.G. 2004. Biotransformations and Bioprocesses. Marcel
Dekker, New York, USA.
Endo, I., Nagamune, T., Katoh, S., Yonemoto (eds) 1999. Bioseparation Engineering. Elsevier
Amsterdam, the Netherlands.
Garcia, A.A., Bonem, M.R., Ramirez-Vick, J., Saddaka, M., Vuppu, A. 1999. Bioseparation Process
Science. Blackwell Science, Massachussets, USA.
Harrison, R.G., Todd, P., Rudge, S.R., Petrides, D.P. 2003. Bioseparation Science and Engineering.
Oxford University Press, New York, USA.
Kalyanpur, M. 2000. Downstream Processing in Biotechnology In: Downstream Processing of
Proteins: Methods and Protocols. M. Desai, (ed) Humana. Totowa, NJ: USA. pp. 1–10.
Naglak, T.J., Hettwer, D.J., Wang, H.Y, 1990. Chemical Permeabilization of cells for intracellular
product release. In: Separation Processes In Biotechnology, Marcel Dekker, New York, USA.
+0)26-4
11
Sterility in Industrial
Microbiology
In the microbiology laboratory, sterility is a most important consideration and ways of
achieving it form the earliest portions of the training of a microbiologist. In the
fermentation industry contamination by unwanted organisms could pose serious
problems because of the vastly increased scale of the operation in comparison with
laboratory work. If Pediococus streptococcus damnosus which causes sourness in beer were
to contaminate the fermentation tanks of a brewery then hundreds of thousand of liters of
beer may have to be discarded, with consequent loss in revenue to the brewery. The
situation would be similar if a penicillianase-producing Bacillus sp were to contaminate
a penicillin fermentation, or lytic phages an acetone-butanol mash.
11.1
THE BASIS OF LOSS BY CONTAMINANTS
Contaminations in industrial microbiology as seen above could lead to huge financial
losses to a fermentation firm. Losses due to contaminations may be explained in one or
more of the following ways:
(i) The contaminant may utilize the components of the fermentation broth to produce
unwanted end-products and therefore reduce yield. When slime-forming
Leuconostoc mesenteroides invades a sugar factory, it utilizes sucrose to form the
polysaccharide in its capsule which forms the slime. Similarly, in the beer industry
when lactic acid bacteria contaminate the fermentating wort, they utilize sugars
present therein to produce unwanted lactic acid which renders the beer sour.
(ii) The contaminant may alter the environmental conditions such as the pH or
oxidation-reduction potential of the fermentation and render it unsuitable for
maximum production of the required product. Thus, if E. coli which grows much
more rapidly than the highly aerobic Streptomyces griseus should contaminate a
streptomycin fermentation it may use up a large proportion of the oxygen thereby
reducing the yield of the antibiotic, because less than optimal amounts of oxygen
are available to the actinomycete.
(iii) Contamination by lytic organisms such as bacteriophages or Bdellovibrio could
lead to the entire destruction of the producing organism.
Modern Industrial Microbiology and Biotechnology
(iv) Finally, it is conceivable that contaminants could even, if they did not reduce yield
in a product, produce by-products not removable in the extraction process already
established in the factory. The result could be losses in manpower time needed to
devise means of dealing with the product.
Although contaminants are generally undesirable, not all fermentation need to be
carried out under strict asepsis, depending on the selling price of the end-product. Thus
while the high cost of antibiotics justifies strict sterility during production, such sterility
is not called for in such bulk products as yeasts or industrial alcohol.
11.2
METHODS OF ACHIEVING STERILITY
The various methods for achieving sterility are well-known and include physical and
chemical methods.
11.2.1
11.2.1.1
Physical Methods
Asepsis
Asepsis involves general cleanliness and is a procedure routinely observed in many
microbiological, pharmaceutical and food industries. In such organizations, laboratory
coats, face masks, gloves, and other protective clothing are often worn to prevent the
transfer of organisms from the individual to the product. Hands are regularly washed;
pipes, utensils, fermentation vats, and floors are washed with water and disinfectants. In
some industries such as those concerned with parenteral (injection) material, or with
vaccines, even the incoming air must be sterile. The maintenance of asepsis does not
sterilize but it helps reduce the load of microorganisms and hence lessens the stringency
of the sterility measures employed. It also helps to remove foci of microbial growth such
as particles of food, or media which could be sources of future contaminations.
11.2.1.2
Filtration
Filtration is used in industry and in the laboratory to free fluids (i.e., gases and liquids) of
dust and other particles and microorganisms. If properly used, it is highly effective and
also relatively inexpensive. Large volumes of sterile air and other gases are sometimes
required for ‘sterile’ areas where in the pharmaceutical industries, injections and
vaccines are handled, and for aeration in most fermentations.
Two types of air filters are available, the so-called absolute filters which are usually
made of ceramic and are so called because their pores are not large enough to admit a
microorganism and hence, they should theoretically be highly efficient. Their
disadvantage is that they are suitable for only small volumes of the gas being sterilized.
The second group, fibrous filters, is made of fibers of wool, cotton, glass or mineral slag,
whose diameters are in the order of 0.5-15 m. Fibrous filters are not absolute; nevertheless
they are quite effective and hold back organisms of the diameter of about 1.0m or even
viruses. The factors which contribute to their removal of microorganisms include direct
interception by the fibers, settlement by gravity electrostatic attraction between fiber and
particles, Brownian movement and convection (Fig. 11.1).
Sterility in Industrial Microbiology
!
Note the fibers placed in the central portion of a steel casing.
Fig. 11.1 Fibrous Filter
Prefilters usually consist of discs of mats of asbestos of the type used in Seitz filters. They,
however, let in fine fibers, which are undesirable in injectable materials. The fine fibers
are removed in the final filter. Prefilters also absorb large amounts of the liquid, although
such absorbed liquid can be re-extracted by flushing the filter at the end of filtration with
sterile nitrogen. The filters may also be made of compressed paper pulp; filter paper
coated with Kieselghur may be placed between the filter pads.
Final filters which may be of unglazed porcelain are usually made in the form of
cylimerial candles over which the liquid to be filtered flows. The filtrate is drained from
the inside of the cylinder. This type of arrangement increases the surface area available
for filtration. The candles may be sterilized by autoclaving. Sintered glass is usually
made in form of discs and, like porcelain, they are fragile. Membrane (‘Millipore’) filters of
cellulose acetate may also be used as final filters. They can be autoclaved.
Sterilizing filters should have pores with maximum diameters of 0.2 m. They should be
themselves sterilized before being used. Membrane filters can be sterilized by chemical
sterilants (such as ethylene oxide, hydrogen peroxide in vapor form, propylene oxide,
formaldehyde, and glutaraldehyde), radiant energy sterilization (such as c-irradiation)
or steam sterilization. The most common method of sterilization is steam sterilization.
Steam sterilization of a membrane filter can be accomplished either by an autoclave or
by in situ steam sterilization.
11.2.1.3
Heat
Heat may be applied dry or moist:
Dry heat: Not only is dry heat used to sterilize glassware on a small scale in industry
associated laboratories, more importantly it is used on a large scale in industry for
sterilizing some types of air filters. Principally, however, it is used for sterilizing air by
compression. When air is compression the temperature rises in accordance with the gas
law, PV = RT where P is the pressure, V the volume, R the gas constant and T temperature.
If P and V are increased, T, the temperature would rise as shown in Table 11.1. However,
compression is expensive. Furthermore, heated air must be at a high temperature (at a
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Modern Industrial Microbiology and Biotechnology
Table 11.1
Temperature of air after compression
Final pressure (p.s.i.g.)
Temp. (oC)
20
40
60
80
100
150
200
78
117
140
169
189
229
261
much higher pressure than that at which it will be used) and for fairly long holding
periods. Although not a very practical method, compression could reduce the microbial
population of air.
Other methods which have been explored include direct or indirect heating with the
gases and also with electrical heating. In each case while the procedure was effective, it
was too expensive.
Moist heat: Moist heat can be employed in industry to kill microorganisms during boiling,
tyndallization, and autoclaving.
Tyndallization consists of boiling the material for one half hour on three consecutive days.
Vegetative cells are killed on the first day’s boiling. Spores are not but they germinate.
During the second day’s boiling, the vegetative spores resulting from the spores not killed
on the first day, are killed. Any spore still surviving after the second day will be killed
during boiling on the third day as the spores would have germinated. After the third
day’s boiling the medium is expected to be sterile. It is a method which can be used for
sterilizing heat-labile media where filtration is not possible for whatever reason,
including that the medium is too viscous for filtration.
Pasteurization is very widely used in the food industry. It is used for treating beer and
wine. It consists of exposing the food or material to a temperature for a sufficiently long
period to destroy pathogenic or spoilage organisms. Pasteurization can either be batch or
continuous. The low temperature long time (LTLT) technique usually involves heating at
about 60°C for one half hour and is used in batch pasteurization whereas the high
temperature short time (HTST) of flash method involves heating at about 70°C for about
15 seconds. The flash method is employed in continuous pasteurizing.
When batch pasteurization is used on a large scale the final temperature of
pasteurization is attained by gradual increases. Similarly, the temperature is lowered
gradually to cool it; for 600 ml bottles in many breweries batch pasteurization time is a
total of about 90 minutes divided equally between raising the temperature, holding at the
pasteurization temperature, and cooling. This prolonged time during which the material
is exposed to high temperature and which may give rise to a ‘burnt’ odor is the major
deficiency of batch in comparison with continuous pasteurization.
Steam under pressure: Steam is useful as a sterilizer for the following reasons:
(i) It has a high heat content and hence a high sterilizing ability per unit weight or
volume; this heat is rapidly released.
Sterility in Industrial Microbiology
#
(ii) Steam releases its heat at a readily controlled and constant temperature.
(iii) It can be fairly easily produced and distributed.
(iv) No obnoxious waste products result from its use and it is clean, odorless and
tasteless.
Its disadvantages are that it is not suitable for sterilizing anhydrous soils, greases,
powders, and its effectiveness, as will be seen later, may be limited in the presence of air.
Steam is widely used for the sterilization of equipment in the laboratory as well as in
industry. Pipes, fermentors and media are all sterilized with the steam. Steam used for
this purpose is under pressure because the higher the pressure the higher the
temperature. The relationship between steam temperature and pressure will be
discussed further later in this section.
There are three ‘types’ of steam.
Wet steam is steam in which sufficient heat is lacking to keep all the steam in the
gaseous vapor phase. The effect of this is that some liquid water is present in the steam.
In ‘saturated’ (or sometimes wrongly called dry saturated) steam, all the steam is in the
vapor phase; its heat content is such that there is an equilibrium between it and water at
any temperature and pressure. Saturated steam is water vapor in the condition in which
it is generated from the water with which it is in contact. Saturated steam cannot undergo
a reduction in temperature without a lowering of its pressure, nor can the temperature be
increased without expanding the pressure. When steam is saturated therefore, it can be
described either by its pressure or its temperature, with which the two characteristics are
linked. Wet steam has far less heat than saturated steam per unit weight of steam.
Furthermore, wet steam introduces a lot more water than necessary in the material being
sterilized; for example media in fermentors may become diluted. One major reason for the
occurrence of wet steam is the use of long poorly insulated pipes.
In superheated steam no liquid water is present, and the temperature is higher than that
of saturated steam at the same pressure. Superheated steam is produced by, for example,
passing it over heated surfaces or coils. For the purposes of sterilization, saturated steam
is the most dependable, efficient and effective of the three types of steam. Superheated
steam behaves more like a gas than vapor and takes up water avidly. Although it has a
higher temperature than saturated steam at the same pressure, it does not sterilize to the
extent of saturated steam. This is because it lacks moisture which enables heat to kill
micro-organisms at considerably lower temperatures than dry air. Superheated steam,
like dry air, would require that the organisms be exposed for periods as long as glassware
is exposed in a dry air oven. For transportation over long distances steam is transported
in the superheated form in pipes in order to reduce heat losses; it is returned to saturated
steam at the end of the transportation and at the point of use by the introduction of water.
The temperature of steam sterilization is 121°C for media both in industry and in the
laboratory, although other time-temperature combinations are equally satisfactory (Table
11.2). When industrial media are sterilized by heat, steam is forced into the medium
which is gently agitated; heating is supplemented when necessary by passing steam
through coils running along the fermentor wall. The dilution resulting from steam
injection is calculated from the quantity of steam introduced. In some instances the
medium may be autoclaved in a much larger version of a laboratory autoclave known as
a retort.
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Modern Industrial Microbiology and Biotechnology
Table 11.2
Minimum time/temperature relationship arrangements
Time (min)
Temp oC
30
18
12
8
2
116
118
121
121
132
The major difference between sterilization of media in industry and in the laboratory is
the much greater scale of the former. Due to the greater scale it takes a much longer time to
attain the sterilizing temperature and to cool down than would be the case in the
laboratory. In the laboratory a liter of medium would probably require ten minutes to
attain the sterilizing temperature. It would remain there for 15 minutes and cool down
gradually over another 10-15 minutes, making a total of 40-45 minutes. With a 10,000 liter
medium the equivalent periods may well take several hours for each of the three periods.
11.2.1.4
Radiations
The electromagnetic spectrum is given in Fig. 11.2. The shorter the wavelength the more
powerful the radiation. Thus on the electromagnetic spectrum the most powerful
wavelengths are those of gamma rays, while the least powerful are radio waves. The
radiations used for sterilizing ultra violet light, x-rays and gamma rays.
Fig. 11.2 The Electromagnetic Spectrum
Ionizing radiations: These are extremely high frequency electromagnetic waves (X-rays
and gamma rays), which have enough photon energy to produce ionization (create
positive and negative electrically charged atoms or parts of molecules) by knocking off
the electrons on the outer orbits of atoms of the materials through which they pass. The
atoms knocked out are accepted by other atoms. The atoms losing the electrons and those
accepting them become ionized on account of the electron changes. It is this ability of xrays and gamma rays to create ions that has earned them the name ionizing radiations.
Gamma rays are generated from x-ray machines such as those used in hospitals to take xray pictures. Gamma rays are also produced by the spontaneous decay of radioactive
metals such as cobalt 60 (Co60). Ionizing radiations can be used to sterilize plastic
syringes, rubber gloves, and other materials which are liable to damage by heat or
chemicals.
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Ultraviolet light: Visible light falls between wavelengths of 400 and 700 nm. Ultraviolet
light (UV) ranges from 100 to 400 nm. Not all uv is germicidal. The ‘germicidal range’ is
approximately 200 – 300 nm, with a peak germicidal effectiveness at 254 nm. The process
of the killing of microbes by UV involves absorption of a UV photon by DNA chains. This
causes a disruption in the DNA chain by causing adjacent thymine bases to dimerize or
become linked. The organism’s metabolism is disrupted and it may eventually die.
Unfortunately, ultraviolet light does not penetrate, and acts mainly on the surface.
Therefore its use would be limited to laboratory work such as sterilizing the laboratory
air, for creating mutations in culture improvement. In industry it is used for sterilizing the
air in fermentation halls and other such large open spaces.
11.2.2
Chemical Methods
These can be divided into two groups: chemosterilants (which kill both vegetative cells as
well as spores of bacteria, fungi, viruses, and protozoa) and disinfectants which may nokill spores, or even some vegetative cells, but at least kill unwanted (pathogenic or
spoilage) organisms.
11.2.2.1
Chemosterilants
For a chemical to be useful as a sterilant it should have the following properties:
(i) It should be effective at low concentrations.
(ii) The components of the medium should not be affected, when used for media.
(iii) Any breakdown products resulting from its use should be easily removed or be
innocuous.
(iv) It should be effective under ambient conditions.
(v) It should act rapidly, be inexpensive and be readily available.
(vi) It should be non-flammable, non-explosive, and non-toxic.
The discussion on chemosterilants will focus on gaseous sterilants because they have
special advantages when parts of the materials to be sterilized are difficult to reach or
when they are of heat-labile.
11.2.2.2
Gaseous Sterilants
(i) Ethylene oxide: Ethylene oxide CH2 – CH2 has become accepted as a gaseous sterilant
and a lot of information about it has accumulated. It reacts with water, alcohol, ammonia,
amines, organic acids and mineral acids. Above 10.7oC it is gaseous. It is very penetrating
and is widely used in the food and pharmaceutical industries where it is capable of
killing all forms of microorganisms. Bacterial spores are however 3-10 more resistant
than vegetative cells.
Spores of some bacteria e.g. the thermophilic Bacillus stearothermophilus are in fact less
resistant than vegetative cells of some bacteria e.g. Staphylocous aureus, Micrococcus
radiodurans, and Streptococcus faecalis.
Relative humidity is very important in deciding the bacterial activity of ethylene oxide;
it is most effective in the range of 28-33% relative humidity. At humidities higher than
33% it is converted to ethylene glycol which has a weaker anti-bacterial activity. For
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effectiveness ethylene oxide requires a much longer time of exposure than steam
sterilization.
It is widely used in the pharmaceutical industry for sterilizing rubber and plastic
bottles, vials, catheters and sometimes, sutures, syringes and needles and some
antibiotics and microbiological media. Residual ethylene oxide must however be
removed by allowing it to evaporate and this takes some time.
One of the main disadvantages of the sterilant is that the liquid (which form it assumes
below 107°C is highly inflammable; the gas also forms explosive mixtures with air from 3
to 80 by volume. For this reason it is mixed with inert gases such as CO2 often in a ratio of
10% ethylene oxide and 90% CO2. The explosive nature of ethylene oxide is made even
worse by the fact that the pure ethylene gas has an unpleasant odor. For use it is
introduced into large containers constructed like autoclaves.
(ii) Propylene oxide: This is only about half as active as ethylene oxide. It is liquid at room
temperature. It hydrolyzes less slowly than ethylene oxide in the presence of moisture. It
is used for room fumigation, and for food because some countries discourage the use of
ethylene oxide for this purpose. Propylene oxide has been used in industry for sterilizing
culture media, powdered and flaked foods, barley seeds and dried fruits. For these dried
foods an exposure of 1,000-2,000 mg/liter of the sterilant for 2-4 hours resulted in 90–99%
kill of various microorganisms, including bacteria and fungi. Like ethylene oxide it is an
alkylating agent and should be handled carefully since it is a potential carcinogen.
(iii) b-propiolactone: b-propiolactone is a heterocyclic colorless pungent liquid. It is highly
active as an anti-bacteria agent, but it has a low penetrative power. Its probable
carcinogenicity has lowered its general use, although it has been used to fumigate
houses. It is used in the pharmaceutical industry to sterilize plasma and vaccines; when
it was used to sterilize bacterial medium all the spores introduced were killed.
Subsequently, E. coli grew indicating that no residual toxicity resulted. Indeed bpropioplactone breaks down to the non-toxic and less carcinogenic b-hydroxypropionic
acid. Under maximum operating conditions (temperature, humidity, etc.) it has been
claimed that b-propiolactone in the vapor phase is 25 times more effective than
formaldehyde, 4000 times more than ethylene oxide and 50,000 more active than methyl
bromide. The relative humidity for maximum activity is 75%.
(iv) Formaldehyde: Formaldehyde is a gas which is highly soluble in water. Like other
gaeous sterilants relative humidity is important, but it is most active between 60-90%
humidity. It does not penetrate deeply and it should be used at 22oC or above to be
effective. An exposure of at least 12 hours is necessary. Formaldehyde oxidizes to formic
acid and this breakdown product could be corrosive to metals. It is used in the
pharmaceutical industries where it is used to preserve pathological specimens of
animals used for tests.
(v) Methylbromide: Methyl bromide is widely used for fumigation and disinfection in
cereal mills, warehouses, granaries, seed houses, and food processing plants. As it is
highly toxic ethylene oxide is sometimes preferred to it. Furthermore, it has been reported
to be only about one tenth as effective as ethylene oxide.
(vi) Sulfur dioxide: This is a colorless pungent gas. Due to its corrosiveness it is of limited
use, but it is used in the food industries; in wineries, it is used to partially ‘sterilize’ the
grape must before fermentation, to destroy wild yeasts and other unwanted organisms.
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11.2.2.3
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Other sterilants
(i) Chorine: is widely used in industry as solutions of hypochloride. It is used for washing
pipes in breweries and other establishments and in the dairy industry for sterilizing
utensils.
(ii) Phenol: Phenol and phenol-derivatives are widely used as disinfectants. Other
compounds which could find use in some aspects of industry include ozone, hydrogen
peroxide, and quaternary ammonium compounds.
11.3
ASPECTS OF STERILIZATION IN INDUSTRY
In the foregoing, principles of dealing with unwanted organisms have been stressed;
where it was possible some aspects of practice were discussed. In this section the
practical methods of dealing with contaminations and the potential for contaminations
to occur in industry will be discussed.
11.3.1
The Sterilization of the Fermentor and its Accessories
The fermentor itself, unless sterilized, is a source of contamination. Of the various
methods discussed above, steam is the most practical for fermentor sterilization. Steam is
used to sterilize the medium in situ in the fermentor but sometimes the medium may be
sterilized separately in a retort or autoclave and subsequently transferred aseptically to a
fermentor. In order to avoid microbial growth within the fermentor when not in use,
crevices and rough edges are avoided in the construction of fermentors, because these
provide pockets of media in which undesirable microorganisms can grow. These crevices
and rough edges may also protect any such organisms from the lethal effects of
sterilization. For the reasons discussed earlier, saturated steam should be used and
should remain in contact with all parts of the fermentor for at least half an hour. Pipes
which lead into the fermentor should be steam-sealed using saturated steam. The various
probes used for monitoring fermentor activities, namely probes for dissolved oxygen,
CO2, pH, foam, etc., should also be sterilized.
11.3.2
Media Sterilization
The following should be borne in mind when sterilizing industrial media with steam:
(i) Breakdown products may result from heating and may render the medium less
available to the microorganisms; some of the breakdown products may even be
toxic;
(ii) pH usually falls with sterilization and the usual laboratory practice of making the
pH slightly higher than the expected final pH should be followed;
(iii) Most media would have been sterilized if heat was available to all parts at a
temperature of 120-125oC for 15-20 minutes. Oils (sometimes used as anti-foams)
are generally more difficult to sterilize. If immiscible with water they may need to be
sterilized separately at a much higher temperature than the above and/or for a
longer period.
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(iv) The order and number of the addition of the various components of the medium
could be important. Thus, when powders such as corn starch are to be added it is
advisable to dissolve them separately and to add the slurry into the fermentor with
vigorous stirring; otherwise lumps could form. Such lumps may not only protect
some organisms, but may even render the powdered material unavailable as
nourishment for the target organisms. Some commercial autoclaves therefore have
an arrangement for stirring the medium to break up clumps of medium as well as
distribute the heat.
Sterilization of heat labile medium: Thermolabile media may be sterilized by
tyndalization. For this procedure the temperature of the medium is raised to boiling on
three consecutive days. The theory behind tydalization is that while boiling destroys the
vegetative cells, the bacterial spores survive. After the first day’s boiling the vegetative
cells are killed and the spores germinate. On the second day’s boiling the vegetative cells
resulting from the germinated spores surviving the first day’s boiling, are killed. In the
unlikely event that any spores still survive – after two days of boiling–they will germinate
and the resulting vegetative cells will be killed with the third day’s boiling. With the third
day’s boiling the medium in all likelihood will be sterile.
Chemical sterilization of the medium may be done with b-propiolactone. Filtration
may also be used. Filtration is especially useful in the pharmaceutical industry where in
addition to sterilization it also removes pyrogens (fever-producing agents resulting from
walls of Gram-negative bacteria), when filtration is combined with charcoal adsorption.
Batch vs. continuous Sterilization: The various advantages of continuous over batch
fermentation (Section 7.4) can be extended, with appropriate modifications, to
sterilization. Exposure to sterilization temperature and cooling thereafter are achieved in
continuous sterilization in much shorter periods than with batch sterilization (Fig. 11.3).
The two methods generally used for continuous sterilization are shown in Fig. 11.4
11.4
VIRUSES (PHAGES) IN INDUSTRIAL MICROBIOLOGY
Viruses are non-cellular entities which consist basically of protein and either DNA or
RNA and replicate only within specific living cells. They have no cellular metabolism of
their own and their genomes direct the genetic apparatuses of their hosts once they are
within them. Viruses are important in the industrial microbiology for at least two
reasons:
(i) Those that are pathogenic to man and animals are used to make vaccines against
disease caused by the viruses.
(ii) Viruses can cause economic losses by destroying microorganisms used in a
fermentations.
It is this second aspect which will be considered in this section. We will therefore look
at those viruses which attack organisms of industrial importance, namely bacteria
(including actinomycetes) and fungi. Such viruses are known as bacterophages,
actinophages, or mycophages depending on whether they attack bacteria, actinomycetes,
or fungi. Most of the discussion will center around baceriophages since more information
exist on them than on the other two groups.
Sterility in Industrial Microbiology
Fig. 11.3
Temperature-time Relationships in Continuous and Batch Sterilization
Top:
Direct injection of steam into medium
Bottom: Medium sterilization via heated plates, steam is not injected directly into the medium
Note that in both methods the medium is held in a holding section for a period of time
Fig. 11.4 Methods of Continuous Sterilization
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11.4.1
Morphological Grouping of Bacteriophages
Bacteriophages can be divided into six broad morphological groups (Table 11.3). Most
phages attacking industrial organisms are to be found in groups A, B, C. Groups D, E, and
F attack industrial organisms less frequently if at all.
11.4.2
Lysis of Hosts by Phages
The growth cycle of a phage has three steps: adsorption onto the host cell, multiplication
within the cell, and liberation of the prepotency phages by the lysis of the host cell.
Phages may be classified as virulent or temperate according to how they react to their
host. In temperate phages, the phage genome (known as a prophage) integrates with the
genetic apparatus of the host, replicates with it and can be lysogenic and are known as
lysogons.
Temperate phages may become virulent and lyse their hosts, either spontaneously or
after induction by various agents e.g. mitomyin C or UV light. They may also mutate to
lytic phages, complete the virus growth cycle and lyse their hosts. It is for this reason that
lysogenic phages should be avoided in industrial microorganisms.
The nature of the disturbance caused by phages in an industrial fermentation
depends on a number of factors.
(i)
(ii)
(iii)
(iv)
The kinds of phages.
The time and period of phage infect.
The medium composition.
The general physical and chemical conditions of the fermentor.
The manifestation of infection is variable and the same phage does not always cause
the same symptom. In general the symptom of infection could be a slowing down of the
process resulting in poor yield, this being the case when the infection is light. When it is
heavy, the cells may be completely lysed. The length of time taken before a phage
manifests itself is variable depending on its latent period (i.e., the period before the cell is
lysed) and the number of phages released. In a continuously operated fermentation,
phages may take up to three months to manifest themselves. In general, however, the
period is much shorter, being noticeable in a matter of days.
11.4.3
Prevention of Phage Contamination
Phages are as ubiquitous as microorganisms in general and are present in the air, water,
soil, etc. The first cardinal rule in avoiding phage contamination therefore is routine
general cleanliness and asepsis. Pipes, fermentors, utensils, and media, should all be
well sterilized. The culture should be protected from aerial phage contamination, an
insidious situation, which unlike bacterial or fungal contamination cannot be observed
on agar. Air filters should be replaced or sterilized regularly.
Aerosol sterilization of the factory with chlorine compounds, and other disinfectants,
as well as UV irradiation of fermentation halls should be done routinely.
Table 11.3 Morphology, nucleic acid composition and examples of the six groups of phages (II)
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11.4.4
Use of Phage Resistant Mutants
Phages may of course be introduced as direct contaminants or be lysogenic in the
organism being used in the fermentation. Mutants as productive as the original parent
but resistant to various contaminating phages should be developed. Such mutants
should have no tendency to revert to the phage-sensitive type. Freedom from particular
phages can be checked by treating the organisms with antisera against phages normally
or likely to attach to the surfaces of the organisms. Lysogenic bacteria which are resistant
to some phages even when high yielding should be avoided.
It must be remembered that phage-resistant mutants may become infected by new
phages to which the organisms have no resistance.
11.4.5
Inhibition of Phage Multiplication with Chemicals
Specific chemicals selectively active on phages and which spare bacteria may be used in
the fermentation medium.
(i) To prevent infection by phages requiring divalent cations (Mg2+; Ca2+) for
adsorption to host cell or for DNA injection into the host cell, chelating agents have
been used. These sequester the cations from the medium and hence the phage
cannot adsorb onto its host. Examples of the chelating agents are 0.2-0.3%
tripolyphosphate and 0.1-0.2% citrate.
(ii) Non-ionic detergents e.g. tween 20, tween 60, polyethylene glycol monoester also
inhibit the adsorption of some phages or the multiplication of the phages in the
cell. The above two agents usually have no effect on the growth of many industrial
organisms.
(iii) The addition of Fe2+ suppresses cell lysis by phages.
(iv) Certain antibiotics may be added to prevent growth of phages, but only the
selective ones should be used. Chloramphenicol has been used. It has no direct
action on the phage, but it inhibits protein replication in phage-infected cells,
probably due to selective absorption of the antibiotic by phage-infected cells.
11.4.6
Use of Adequate Media Conditions and Other Practices
Fermentation conditions and practices which adversely affect phage should be selected.
Media unfavorable to phages (high pH, low Ca2+, citrates, salts with cations reacting
with –SH groups) should be developed. Pasteurization of the final beer and high
temperature of incubation consistent with production should be used; both of these
adversely affect phage development.
SUGGESTED READINGS
Flickinger, M.C., Drew, S.W. (eds) 1999. Encyclopedia of Bioprocess Technology - Fermentation,
Biocatalysis, and Bioseparation, Vol 1-5. John Wiley, New York, USA.
Soares, C. 2002. Process Engineering Equipment Handbook Publisher. McGraw-Hill.
Zeng, A. 1999. Continuous culture. In: Manual of Industrial Microbiology and Biotechnology.
A.L. Demain, J.E. Davies (eds) 2nd Ed. ASM Press. Washington, DC, USA, pp. 151–164.
Vogel, H.C., Tadaro, C.L. 1997. Fermentation and Biochemical Engineering Handbook Principles, Process Design, and Equipment. (2nd Ed) Noyes.
Section
,
Alcohol-based
Fermentation Industries
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12
Production of Beer
12.1
BARLEY BEERS
The word beer derives from the Latin word bibere meaning to drink. The process of
producing beer is known as brewing. Beer brewing from barley was practiced by the
ancient Egyptians as far back as 4,000 years ago, but investigations suggest Egyptians
learnt the art from the peoples of the Tigris and Euphrates where man’s civilization is
said to have originated. The use of hops is however much more recent and can be traced
back to a few hundred years ago.
12.1.1
Types of Barley Beers
Barley beers can be divided into two broad groups: top-fermented beers and bottomfermented beers. This distinction is based on whether the yeast remains at the top of brew
(top-fermented beers) or sediments to the bottom (bottom-fermented beers) at the end of
the fermentation.
12.1.1.1
Bottom-fermented beers
Bottom-fermented beers are also known as lager beers because they were stored or
‘lagered’ (from German lagern = to store) in cold cellars after fermentation for clarification
and maturation. Yeasts used in bottom-fermented beers are strains of Saccharomyces
uvarum (formerly Saccharomyces carlsbergensis). Several types of lager beers are known.
They are Pilsener, Dortumund and Munich, and named after Pilsen (former
Czechoslovakia) Dortmund and Munich (Germany), the cities where they originated.
Most of the lager (70%-80%) beers drunk in the world is of the Pilsener type.
Bottom-fermentation was a closely guarded secret in the Bavarian region of Germany,
of which Munich is the capital. Legend has it that 1842 a monk passed the technique and
the yeasts to Pilsen. Three years later they found their way to Copenhagen, Denmark.
Shortly after, German immigrants transported bottom brewing to the US.
(i) Pilsener beer: This is a pale beer with a medium hop taste. Its alcohol content is 3.03.8% by weight. Classically it is lagered for two to three months, but modern
breweries have substantially reduced the lagering time, which has been cut down
to about two weeks in many breweries around the world. The water for Pilsener
brew is soft, containing comparatively little calcium and magnesium ions.
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(ii) Dortmund beer: This is a pale beer, but it contains less hops (and therefore is less
bitter) than Pilsener. However it has more body (i.e., it is thicker) and aroma. The
alcohol content is also 3.0-3.8%, and is classically lagered for slightly longer: 3-4
months. The brewing water is hard, containing large amounts of carbonates,
sulphates and chlorides.
(iii) Munich: This is a dark, aromatic and full-bodied beer with a slightly sweet taste,
because it is only slightly hopped. The alcohol content could be quite high, varying
from 2 to 5% alcohol. The brewing water is high in carbonates but low in other ions.
(iv) Weiss: Weiss beer of Germany made from wheat and steam beer of California, USA
are both bottom fermented beers which are characterized by being highly
effervescent.
12.1.1.2
Top-fermented beers
Top fermented beers are brewed with strains of Saccharomyces cerevisiae.
(i) Ale: Whereas lager beer can be said to be of German or continental European origin,
ale (Pale ale) is England’s own beer. Unless the term ‘lager?’ is specifically used,
beer always used to refer to ale in England. Lager is now becoming known in the
UK especially since the UK joined European Economic Community. English ale is
a pale, highly hopped beer with an alcohol content of 4.0 to 5.0% (w/v) and
sometimes as high as 8.0% Hops are added during and sometimes after
fermentation. It is therefore very bitter and has a sharp acid taste and an aroma of
wine because of its high ester content. Mild ale is sweeter because it is less strongly
hopped than the standard Pale ale. In Burton-on-Trent where the best ales are
made, the water is rich in gypsum (calcium sulfate). When ale is produced in
places with less suitable water, such water may be ‘burtonized’? by the addition of
calcium sulfate.
(ii) Porter: This is a dark-brown, heavy bodied, strongly foaming beer produced from
dark malts. It contains less hops than ale and consequently is sweeter. It has an
alcohol content of about 5.0%.
(iii) Stout: Stout is a very dark heavily bodied and highly hopped beer with a strong
malt aroma. It is produced from dark or caramelized malt; sometimes caramel may
be added. It has a comparatively high alcohol content, 5.0-6.5% (w/v) and is
classically stored for up to six months, fermentation sometimes proceeding in the
bottle. Some stouts are sweet, being less hopped than usual.
12.1.2
Raw Materials for Brewing
The raw materials used in brewing are: barley, malt, adjuncts, yeasts, hops, and water.
12.1.2.1
Barley malt
As a brewing cereal, barley has the following advantages. Its husks are thick, difficult to
crush and adhere to the kernel. This makes malting as well as filtration after mashing,
much easier than with other cereals, such as wheat. The second advantage is that the
thick husk is a protection against fungal attack during storage. Thirdly, the
gelatinization temperature (i.e., the temperature at which the starch is converted into a
Production of Beer
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water soluble gel) is 52-59°C much lower than the optimum temperature of
alpha-amylase (70°C) as well as of beta-amylase (65°C) of barley malt. The effect of this is
that it is possible to bring the starch into solution and to hydrolyze it in one operation.
Finally, the barley grain even before malting contains very high amounts of beta-amylase
unlike wheat, rice and sorghum. (Alpha-amylase is produced only in the germinated seed).
Two distinct barley types are known. One with six rows of fertile kernel (Hordeum
vulgare) and the other with two rows of fertile kernels (Hordeum distichon). These differ in
many other properties and as a result there are thousands of varieties. The six-row variety
is used extensively in the United States, whereas the two-row variety is used in Europe as
well as in parts of the US. The six-row varieties are richer in protein and enzyme content
than the two-row varieties. This high enzymic content is one of the reasons why adjuncts
are so widely used in breweries in the United States. Adjuncts dilute out the proteins i.e.
increase the carbohydrate/protein ratio. If an all-malt beer were brewed from malts as
rich in protein as the six-row varieties, this protein would find its way into the beer and
give rise to hazes. The process of malting, during which enzymes (amylases and
proteases) are produced by the germinating seedling will be discussed later.
2
10.1.1.2
Adjuncts
Adjuncts are starchy materials which were originally introduced because the six-row
barley varieties grown in the United States produced a malt that had more diastatic
power (i.e. amylases) than was required to hydrolyze the starch in the malt. The term has
since come to include materials other than would be hydrolyzed by amylase. For example
the term now includes sugars (e.g. sucrose) added to increase the alcoholic content of the
beer. Starchy adjuncts, which usually contain little protein contribute, after their
hydrolysis, to fermentable sugars which in turn increase the alcoholic content of the
beverage.
Adjuncts thus help bring down the cost of brewing because they are much cheaper
than malt. They do not play much part in imparting aroma, color, or taste. Starch sources
such as sorghum, maize, rice, unmalted barley, cassava, potatoes can or have been used,
depending on the price. Corn grits (defatted and ground), corn syrup, and rice are most
widely used in the United States.
When corn is used as an adjunct it is so milled as to remove as much as possible of the
germ and the husk which contain most of the oil of maize, which could form 7% of the
maize grain. The oil may become rancid in the beer aid thus adversely affecting the flavor
of the beverage if it were not removed. The de-fatted ground maize is known as corn grits.
Corn syrups produced by enzymic or acid hydrolysis, are also used in brewing. Since
adjuncts contain little nitrogen, all the needs for the growth of the yeast must come from
the malt. The malt/adjunct ratio hardly exceeds 60/40. Soy bean powder (preferably
defatted) may be added to brews to help nourish the yeast. It is rich nitrogen and in B
vitamins.
12.1.2.3
Hops
Hops are the dried cone-shaped female flower of hop-plant Humulus lupulus (synomyn:
H. americanus, H. heomexicams, H. cordifolius). It is a temperate climate crop and grows wild
in northern parts of Europe, Asia and North America. It is botanically related to the genus
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Cannabis, whose only representative is Cannabis sativa (Indian hemp, marijuana, or
hashish). Nowadays hop extracts are becoming favored in place of the dried hops. The
importance of hops in brewing lies in its resins which provide the precursors of the bitter
principles in beer and the essential (volatile) oils which provide the hop aroma. Both the
resin and the essential oils are lodged in lupulin glands borne on the flower.
In the original Pilsener beer the amount of hops added is about 4 g/liter, but smaller
amounts varying 0.4-4.0 g/liter are used elsewhere. The addition of hops has several effects:
(a) Originally it was to replace the flat taste of unhopped beer with the characteristic
bitterness and pleasant aroma of hops.
(b) Hops have some anti-microbial effects especially against beer sarcina (Pediococus
damnosus) and other beer spoiling bacteria.
(c) Due to the colloidal nature of the bitter substances they contribute to the body,
colloidal stability and foam head retention of beer.
(d) The tannins in the hops help precipitate proteins during the boiling of the wort;
these proteins if not removed cause a haze (chill haze) in the beer at low
temperature. This is further discussed under beer defects later in this chapter.
12.1.2.4
Water
The mineral and ionic content and the pH of the water have profound effects on the type
of beer produced. Some ions are undesirable in brewing water: nitrates slow down
fermentation, while iron destroys the colloidal stability of the beer. In general calcium
ions lead to a better flavor than magnesium and sodium ions. The pH of the water and
that of malt extract produced with it control the various enzyme systems in malt, the
degree of extraction of soluble materials from the malt, the solution of tannins and other
coloring components, isomerization rate of hop humulone and the stability of the beer
itself and the foam on it. Calcium and bicarbonate ions are most important because of
their effect on pH. Water is so important that the natural water available in great brewing
centers of the world lent special character to beers peculiar to these centers. Water with a
large calcium and bicarbonate ions content as is the case with Munich, Copenhagen,
Dublin, and Burton-on-Trent are suited to the production of the darker, sweeter beers. The
reason for this is not clear but carbonates in particular tend to increase the pH, a
condition which appears to enhance the extraction of dark colored components of the
malt. Water low in minerals such as that of Pilsen (Table 12.1) is suitable for the
production of a pale, light colored beer, such as Pilsen has made famous.
Water of a composition ideal for brewing may not always be naturally available. If the
production is of a pale beer without too heavy a taste of hops, and the water is rich in
carbonates then it is treated in one of the following ways:
(a) The water may be ‘burtonized’ by the addition of calcium sulfate (gypsum).
Addition of gypsum neutralizes the alkalinity of the carbonates in an equation
which probably runs thus:
2Ca (HCO3) 2 + 2Ca(SO4) ® 2Ca++ + 2H2SO4 + 4CO2
(b) An acid may be added: lactic acid, phosphoric, sulfuric or hydrochloric. CO2 is
released, but there is an undesirable chance that the resulting salt may remain. The
CO2 released is removed by gas stripping.
Production of Beer
"
Table 12.1 Mineral content of water in some cities with breweries
Mineral content in ppm
Total
Solids
Place
Miwaukee
New York
St. Louis
Pilsen
Munich
Dublin
Copenhangen
Burton-on-trent
148
28
201
63
270
3
480
1,206
2+
Ca
Mg2+
34
6
22
9
71
100
114
268
11
1
12
3
19
4
16
62
SO42–
NO3–
Cl –
20
8
77
3
18
45
62
638
0.8
0.5
4
6.6
0.5
10
5
2
16
60
36
31
HCO3–
11
65
37
283
266
347
287
(c) The water may be decarbonated by boiling or by the addition of lime calcium
hydroxide.
(d) The water may be improved by ion exchange, which may if it is so desired remove
all the ions.
One or more of the above methods may be used simultaneously.
12.1.2.5
Brewer’s yeasts
Yeasts in general will produce alcohol from sugars under anaerobic conditions, but not
all yeasts are necessarily suitable for brewing. Brewing yeasts are able, besides
producing alcohol, to produce from wort sugars and proteins a balanced proportion of
esters, acids, higher alcohols, and ketones which contribute to the peculiar flavor of beer.
A number of characteristics distinguish the two types of brewers’ yeasts (i.e. the top and
the bottom-fermenting yeasts).
(a) Under the microscope Sacch. uvarum (Sacch. carlsbergensis) usually occurs singly or
in pairs. Sacch. cerevisiae usually forms chains and occasionally cross-chains as
well. These characteristics must however be taken together with other more
diagnostic (particularly the biochemical) tests given below.
(b) Sacch. cerevisiae sporulates more readily than does Sacch. uvarum.
(c) Perhaps the most diagnostic distinction between them is that Sacch. uvarum is able
to ferment the trisaccharide, raffinose, made up of galactose, glucose, and fructose.
Sacch. cereisiae is capable of fermenting only the fructose moiety; in other words, it
lacks the enzyme system needed to ferment melibiose which is formed from
galactose and glucose.
(d) Sacch. cerevisiae strains have a stronger respiratory system than Sacch uvarum and
this is reflected in the different cytochrome spectra of the two groups.
(e) Bottom-fermenters are able to flocculate and sink to the bottom of the brew, a characteristic lacking in most strains of Sacch. cerevisiae. Bottom ferments are classified
into rapid settling or slow-settling (powdery); settling characteristics affect the rate
of production, some secondary yeast metabolites, and hence beer quality.
Yeasts are reused after fermentation for a number of times which depend on the
practice of the particular brewery. Mutation and contamination are two hazards in this
practice, but they are inherent in all inocula.
"
Modern Industrial Microbiology and Biotechnology
12.1.3
Brewery Processes
The processes involved in the conversion of barley malt to beer may be divided into the
following:
1.
2.
3.
4.
5.
6.
7.
8.
Malting
Cleaning and milling of the malt
Mashing
Mash operation
Wort boiling treatment
Fermentation
Storage or lagering
Packaging
Of the above processes, malting is specialized and is not carried out in the brew house.
Rather, breweries purchase their malt from specialized malsters (or malt producers). The
description to be given will in general relate to lager beers and where the processes differ
from those of ales this will be pointed out.
12.1.3.1
Malting
The purpose of malting is to develop amylases and proteases in the grain. These enzymes
are produced by the germinated barley to enable it to break down the carbohydrates and
proteins in the grain to nourish the germinated seedling before its photosynthetic
systems are developed enough to support the plant. However, as soon as the enzymes are
formed and before the young seedling has made any appreciable in-road into the nutrient
reserve of the grain, the development of the seedling is halted by drying, but at
temperatures which will not completely inactivate the enzymes in the grain. These
enzymes are reactivated during mashing and used to hydrolyze starch and proteins and
release nutrients for the nourishment of the yeasts.
Not all barley strains are suitable for brewing; some are better used for fodder. During
malting, barley grains are cleaned; broken barley grains as well as foreign seeds, sand,
bits of metal etc. are removed. The grains are then steeped in water at 10-15°C. The grain
absorbs water and increases in volume ultimately by about 4%. Respiration of the embryo
commences as soon as water is absorbed. Microorganisms grow in the steep and in order
not to allow grain deterioration the steep water is changed approximately at 12-hourly
intervals until the moisture content of the grain is about 45%. Steeping takes two to three
days.
The grains are then drained of the moisture and may be transferred to a malting floor
or a revolving drum to germinate. The heat generated by the sprouts further hastens
germination. Sometimes moist warm air is blown through beds of germinating seedlings
about 30 cm deep. Water may also be sprinkled on them. The plant hormone gibberellic
acid is sometimes added to the grains to shorten germination time. The grain itself
synthesizes gibberellic acid and it is this acid which triggers off the synthesis of various
hydrolytic enzymes by the aleurone layer situated on the periphery of the grain. The
enzymes so formed diffuse into the center of the grain where the endosperm is located.
In the endosperm, the starch granules are harbored within cells. These cell walls are
made up of hemicellulose, which is broken down by hemicellulases before amylases can
Production of Beer
Pericarp
"!
Husk
Starch
granules
in cells
Embryo
Starchy
endosperm
Aleurone
(Protein)
Layer
Fig. 12.1 Structure of the Barley Grain
attack the starch. Alpha-amylase (see discussion on mashing below) is also synthesized
by the grain. Beta-amylase is already present and is not synthesized but is bound to
proteins and is released by proteolytic enzymes.
‘Modification’ or production of enzymes is complete in four to five days of the growth
of the seedling; the extent being tested roughly by the sweet taste developed in the grain
and by the length of the young plumule. The various enzymes formed break down some
quantities of their respective substrates but the major breakdown takes place during
mashing.
Further reactions in the grain are halted by kilning, which consists of heating the
‘green’ malt in an oven, first with a relatively mild temperature until the moisture content
is reduced from about 40% to about 6%. Subsequently the temperature of heating depends
on the type of beer to be produced. For beer of the Pilsener type the malt is pale and has no
pronounced aroma and kilning takes 20-24 hours at 80–90°C. For the darker Munich
beers with a strong aroma drying takes up to 48 hours at 100 – 110°C. For the caramelized
malts used for stout and other very dark beers, kilning temperature can be as high as
120°C. Such malts contain little enzymic activity.
At the end of malting, some changes occur in the gross composition of the barley grain
as seen in Table 12.2. The rootlets are removed and used as cattle feed.
Weight loss known as malting loss occurs at each stage of malting and the
accumulated loss may be as high 15%. The barley malt with its rich enzyme content
resembles swollen grains of unthreshed rice and can be stored for considerable periods
before being used.
10.1.3.2
2
Cleaning and milling of malt
The barley is transported to the top of the brewing tower. Subsequent processes in the
brewery process occur at progressively lower floors. Lagering and bottling are usually
done on the ground level floor. In this way gravity is used to transport the materials and
the expense of pumping is eliminated. At the top of the brewing tower, the barley malt is
""
Modern Industrial Microbiology and Biotechnology
Table 12.2
Composition of barley grain before and after malting
Fraction
Proportion (% dry weight)
Starch
Sucrose
Reducing sugars
Other sugars
Soluble gums
Hemicelluloses
Cellulose
Lipids
Crude protein (N x 6.25)
Albumin
Globulin
Hordein-protein
Glutelin-protein
Amino acids and peptides
Nucleic acids
Minerals
Others
Barley
Malt
63-65
1-2
0.1-0.2
1
1-1.5
8-10
4-5
2-3
8-11
0.5
3
3-4
3-4
0.5
0.2-0.3
2
5-6
58-60
3-5
3-4
2
2-4
6-8
5
2-3
8-11
5
2
3-4
1-2
0.2-0.3
3
6-7
cleaned of dirt and passed over a magnet to remove pieces of metals, particularly iron. It
is then milled.
The purpose of milling is to expose particles of the malt to the hydrolytic effects of malt
enzymes during the mashing process. The finer the particles therefore the greater the
extract from the malt. However, very fine particles hinder filtration and prolong it
unduly. The brewer has therefore to find a compromise particle size which will give him
maximum extraction, and yet permit reasonably rapid filtration rate. No matter what is
chosen the crushing is so done as to preserve the husks which contribute to filtration,
while reducing the endosperm to fine grits.
12.1.3.3
Mashing
Mashing is the central part of brewing. It determines the nature of the wort, hence the
nature of the nutrients available to the yeasts and therefore the type of beer produced. The
purpose of mashing is to extract as much as possible the soluble portion of the malt and
to enzymatically hydrolyze insoluble portions of the malt and adjuncts. In the sense of
the latter objective, mashing may be regarded as an extension of malting. In essence
mashing consists of mixing the ground malt and adjuncts at temperatures optimal for
amylases and proteases derived from the malt. The aqueous solution resulting from
mashing is known as wort.
The two largest components in terms of dry weight of the grain are starch (55%) and
protein (10-12%). The controlled breakdown of these two components has tremendous
influence on beer character and will be considered below.
Production of Beer
"#
12.1.3.3.1 Starch breakdown during mashing
Starch forms about 55% of the dry weight of barley malt. Of the malt starch 20-25% is
made up of amylose. The key enzymes in the break down of malt starch are the alpha and
beta-amylases. The temperature of optimal activity and destruction of these enzymes as
well as their optimum pH are given in Table 12.3 (Starch and its breakdown are also
discussed in Chapter 4).
Table 12.3 Temperature optima of alpha- and beta-amylases
Enzyme
Alpha-amylase
Beta-amylase
Optimum temperature
Temperature of destruction
Optimal pH
70°C
60-65°C
80°C
75°C
5.8
5.4
12.1.3.3.2 Protein breakdown during mashing
The breakdown of the malt proteins, albumins, globulins, hordeins, and gluteins starts
during malting and continues during mashing by proteases which breakdown proteins
through peptones to polypeptides and polypeitidases which breakdown the polypetides
to amino acids. Protein breakdown has no pronounced optimum temperature, but during
mashing it occurs evenly up to 60°C, beyond which temperature proteases and
polypeptidases are greatly retarded. Proteoloytic activity in wort is however dependent
on pH and for this reason wort pH is maintained at 5.2-5.5 with lactic acid, mineral acids,
or calcium sulphate.
12.1.3.3.3 General environmental conditions affecting mashing
The progress of mashing is affected by a combination of temperature, pH, time, and
concentration of the wort. When the temperature is held at 60-65°C for long periods a
wort rich in maltose occurs because beta amylase activity is at its optimum and this
enzyme yields mainly maltose. On the other hand, when a higher temperature around
70°C is employed dextrins predominate. Dextrins contribute to the body of the beer but
are not utilized by yeast. Mash exposed to too high a temperature will therefore be low in
alcohol due to insufficient maltose production.
The pH optima for amylases and proteolytic enzymes have already been discussed.
The optimum pH for beta-amylase activity is about the same as that of proteolysis and as
can be seen in Table 12.3, a fortunate coincidence for the maximum production of maltose
and the breakdown of protein.
The concentration of the mash is important. The thinner the mash the higher the
extract (i.e., the materials dissolved from the malt) and the maltose content.
12.1.3.3.4 Mashing methods
There are three broad mashing methods:
(a) Decoction methods, where part of the mash is transferred from the mash tun to the
mash kettle where it is boiled.
"$
Modern Industrial Microbiology and Biotechnology
(b) Infusion methods, where the mash is never boiled, but the temperature is gradually
raised.
(c) The double mash method in where the starchy adjuncts are boiled and added to the
malt.
(i) Decoction methods: In these methods the mash is mixed at an initial temperature of
35-37°C and the temperature is raised in steps to about 75°C. About one-third of the initial
mash is withdrawn, transferred to the mash kettle, and heated slowly to boil, and
returned to the mash tun, the temperature of the mash becoming raised in the process. The
enzymes in the heated portion become destroyed but the starch grains are cooked,
gelatinized and exposed. Another portion may be removed, boiled and returned. In this
way the process may be a one, two or three-mash process. In a three-mash process (Fig.
12.2) the initial temperature of 35-40°C favors proteolysis; the mash is held for about half
hour at 50°C for full proteolysis, for about one hour at 60-65°C for saccharification and
production of maltose, and at 70-75°C for two or three hours for dextrin production. The
three-mash method is the oldest and best known and it was originated in Bavaria, West
Germany. Figure 12.2 shows the temperature relations in a three-mash decoction. The
decoction is used in continental Europe.
(ii) Infusion method: The infusion method is the one used in Britain and is typically used
to produce top-fermenting beers. It is carried out in a mash tun, which resembles a lauter
tub of lager beer, but it is deeper. The method involves grinding malt and a smaller
amount of unmalted cereal, which may sometimes be precooked. The ground material, or
grist, is mixed thoroughly with hot water (2:1 by weight) to produce a thick porridge-like
mash and the temperature is carefully raised to about 65°C. It is then held at this
temperature for a period varying from 30 minutes to several hours. On the average the
holding is for 1-2 hours. The enzyme acts principally on the starch and its degradation
products in both the malted and unmalted cereal, and only a little protein breakdown
occurs. Further hot water at 75-78°C is sprayed on the mash to obtain as much extract as
possible and to halt the enzyme action. It is believed by some authors that this method is
not as efficient as the double mash or decoction method in extracting materials from the
malt. No part of the mash is boiled from mashing-in to mashing-off. It is, however, more
easily automated, but a malt in which the proteins are already well degraded must be
used since the high temperature of mashing rapidly destroys the proteolytic enzymes.
(iii) The double-mash (also called the cooker method): This method was developed in the
US because of its use of adjuncts. It has features in common with the infusion and the
decoction method. Indeed some authors have described it as the downward infusion
method whilst describing the infusion method mentioned above as an upward infusion
method. In a typical US double mash method ground malt is mashed with water at a
temperature of 35°C. It is then held for an hour during the ‘protein rest’ for proteolysis.
Adjuncts are then cooked in an adjunct cooker for 60-90 minutes. Sometimes about 10%
malt is added during the cooking. Hot cooked adjunct is then added to the mash of
ground malt to raise the temperature to 65-68°C for starch hydrolysis and maintained at
this level for about half hour. The temperature is then increased to 75°C-80°C after which
the mashing is terminated. During starch hydrolysis completion of the process is tested
with the iodine test.
Production of Beer
"%
100
Temperature (oC)
90
80
70
60
50
40
30
1
2
3
4
Time (hrs)
5
6
Broken lines indicate temperature of main mash
Unbroken lines indicate temperature of added portion of mash
Fig. 12.2 Three-stage Decoction Method
Various combinations of the above methods may be used, depending on the type of
beer, the type of malt, and the nature of the adjunct.
12.1.3.3.5 Mash separation
At the end of mashing, husks and other insoluble materials are removed from the wort in
two steps. First, the wort is separated from the solids. Second, the solids themselves are
freed of any further extractable material by washing or sparging with hot water.
The conventional method of separating the husks and other solids from the mash is to
strain the mash in a lauter (German for clarifying) tub which is a vessel with a perforated
false-bottom about 10 mm above the real bottom on which the husks themselves form a
bed through which the filtration takes place. In recent times in large breweries, especially
"&
Modern Industrial Microbiology and Biotechnology
in the United States, the Nooter strain master has come into use. Like the Lauter tub,
filtration is through a bed formed by the husks, but instead of a false bottom, straining is
through a series of triangular perforated pipes placed at different heights of the bed. The
strain master itself is rectangular with a conical bottom whereas the Lauter tub is
cylindrical. Its advantage among others is that it can handle larger quantities than the
Lauter tub. Besides the Lauter tub and the strainmaster, cloth filters located in plate filters
and screening centrifuges are also used.
The sparging (or washing with hot water) of the mash solids is done with water at
about 80°C and is continued till the extraction is deemed complete. The material which is
left after sparging is known as spent grain and is used as animal feed. Sometimes liquid
is extracted from the spent grain by centrifuging, the extract being used to cook the
adjuncts.
12.1.3.3.6 Wort boiling
The wort is boiled for 1-1½ hours in a brew kettle (or copper) which used to be made of
copper (hence the name) but which, in many modern breweries, is now made of stainless
steel. When corn syrup or sucrose is used as an adjunct it is added at the beginning of the
boiling. Hops are also added, some before and some at the end of the boiling. The purpose
of boiling is as follows.
(a) To concentrate the wort, which loses 5-8% of its volume by evaporation during the
boiling;
(b) To sterilize the wort to reduce its microbial load before its introduction into the
fermentor.
(c) To inactivate any enzymes so that no change occurs in the composition of the wort.
(d) To extract soluble materials from the hops, which not only aid in protein removal,
but also in introducing the bitterness of hops.
(e) To precipitate protein, which forms large flocs because of heat denaturation and
complexing with tannins extracted from the hops and malt husks. Unprecipitated
proteins form hazes in the beer, but too little protein leads to poor foam head
formation.
(f) To develop color in the beer; some of the color in beer comes from malting but the
bulk develops during wort boiling. Color is formed by several chemical reactions
including caramelization of sugars, oxidation of phenolic compounds, and
reactions between amino acids and reducing sugars.
(g) Removal of volatile compounds: volatile compounds such as fatty acids which
could lead to rancidity in the beer are removed.
During the boiling, agitation and circulation of the wort help increase the amount of
precipitation and flock formation.
Pre-fermentation treatment of wort: The hot wort is not sent directly to the fermentation
tanks. If dried hops are used then they are usually removed in a hop strainer. During
boiling proteins and tannins are precipitated while the liquid is still warm. Some more
precipitation takes place when it has cooled to about 50°C. The warm precipitate is
known as “trub” and consists of 50-60% protein, 16-20% hop resins, 20-30%
polyphenols and about 3% ash. Trub is removed either with a centrifuge, or a whirlpool
Production of Beer
"'
separator which is now more common. In this equipment the wort which is fed into a flat
centrifuge, is thrown at the side of the equipment and finds its way out through an outlet
on the periphery. The heavier particles (the trub) are thrown to the center and withdrawn
through a centrally located outlet. The separated wort is cooled in a heat exchanger.
When the temperature has fallen to about 50°C further sludge known as ‘cold break’
begins to settle, but it cannot be separated in a centrifuge because it is too fine. In many
breweries the wort is filtered at this stage with kieselghur, a white distomaceous earth.
The cooled wort is now ready for fermentation. It contains no enzymes but it is a rich
medium for fermentation. It has therefore to be protected from contamination. During the
transfer to the fermentor the wort is oxygenated at about 8 mg/liter of wort in order to
provide the yeasts with the necessary oxygen for initial growth.
12.1.3.4
Fermentation
The cooled wort is pumped or allowed to flow by gravity into fermentation tanks and
yeast is inoculated or ‘pitched in’ at a rate of 7-15 x 106 yeast cells/ml, usually collected
from a previous brew.
12.1.3.4.1 Top fermentation
This is used in the UK for the production of stout and ale, using strains of Saccharomyces
cerevisiae. Traditionally an open fermentor is used. Wort is introduced by a fish tail spray
so that it becomes aerated to the tune of 5-10 ml/liter of oxygen for the initial growth of the
yeasts. Yeast is pitched in at the rate of 0.15 to 0.30 kg/hl at a temperature of 15-16°C. The
temperature is allowed to rise gradually to 20°C over a period of about three days. At this
point it is cooled to a constant temperature. The entire primary fermentation takes about
six days. Yeasts float to the top during this period, they are scooped off and used for
future pitching. In the last three days the yeasts turn to a hard leathery layer, which is also
skimmed off. Sometimes the wort is transferred to another vessel in the so-called
dropping system after the first 24-36 hours. The transfer helps aerate the system and also
enables the discarding of the cold-break sediments. Sometimes the aeration is also
achieved by circulation with paddles and by the means of pumps. Nowadays
cyclindrical vertical closed tanks are replacing the traditional open tanks. A typical top
fermentation cycle is shown in Fig. 12.3.
12.1.3.4.2 Bottom fermentation
Wort is inoculated to the tune of 7-15 x 106 yeast cells per ml of wort. The yeasts then
increase four to five times in number over three to four days. Yeast is pitched in at 6-10°C
and is allowed to rise to 10-12°C, which takes some three to four days; it is cooled to about
5°C at the end of the fermentation. CO2 is released and this creates a head called Krausen,
which begins to collapse after four to five days as the yeasts begin to settle. The total
fermentation period may last from 7-12 days (Fig. 12.4).
12.1.3.4.3 Formation of some beer components
During wort fermentation in both top and bottom fermentation anaerobic conditions
predominate; the initial oxygen is only required for cell growth. Fermentable sugars are
converted to alcohol, CO2 and heat which must be removed by cooling. Dextrins and
#
Modern Industrial Microbiology and Biotechnology
Fig. 12.3 Typical Fermentation with Top-fermenting Yeast
Fig. 12.4 Typical Fermentation with Bottom-fermenting Yeast
Production of Beer
#
maltoteraose are not fermented. Higher alcohols (sometimes known as fusel oils)
including propanol and isobutanol are generated from amino acids. Organic acids such
as acetic, lactic, pyruvic, citric, and malic are also derived from carbohydrates via the
tricarboxylie acid cycle.
12.1.3.4.4 Monitoring following fermentation progress
The progress of fermentation is followed by wort specific gravity. During fermentation
the gravity of the wort gradually decreases because yeasts are using up the extract.
However alcohol is also being formed. As alcohol has a lower gravity than wort the
reading of the special hydrometer (known as a saccharometer) is even lower. The
saccharometer reading does not therefore reflect the real extract, but an apparent extract,
which is always lower than the real extract because of the presence of alcohol. In the UK
and some other countries the extract is measured as the direct specific gravity as 60°F
(15.5°C) x 1000. Hence, with wort with sp. Gr. of 1.053 the extract would be 1053°. Outside
the UK extract is measured in °Balling or °Plato. Both systems measure the percentage of
sucrose required to give solutions of the same specific gravity. The original tables were
designed by Von Balling. Improvements and greater accuracy were made on Von
Balling’s tables first by Brix and later by Plato but the figures were not changed
drastically. For this reason °Balling, °Brix, °Plato are the same except for the fifth and
sixth decimal places (Table 12.4). °Brix is used in the sugar industry, whereas Balling
(United States) and °Plato (continental Europe) are used in the brewing industry.
Table 12.4 Comparison between original gravity and percent extract
Original gravity
1.01968
1.02370
1.02774
1.03180
1.03591
1.04003
1.04419
1.04837
1.05260
1.05684
o
B
o
4.925
5.931
6.920
7.913
8.917
9.925
10.921
11.920
12.928
13.943
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
P
The apparent extract, real, extract, and alcohol content are related to each other as well
as to the original extract, i.e., the solids in the original worts and may be read from tables.
The degree of attenuation is the amount of extract fermented, measured as a percentage of
the original or total extract, hence an apparent and a real degree of attenuation both exist.
12.1.3.5
Lagering (bottom-fermented beers) and treatment
(top-fermented beers)
(a) Lagering: At the end of the primary fermentation above, the beer, known as ‘green’
beer, is harsh and bitter. It has a yeasty taste arising probably from higher alcohols and
aldehydes.
#
Modern Industrial Microbiology and Biotechnology
The green beer is stored in closed vats at a low temperature (around O°C), for periods
which used to be as long as six months in some cases to mature and make it ready for
drinking.
During lagering secondary fermentation occurs. Yeasts are sometimes added to
induce this secondary fermentation, utilizing some sugars in the green beer. The
secondary fermentation saturates the beer with CO2, indeed the progress of secondary
fermentation is followed by the rate of CO2 escape from a safety valve. Sometimes actively
fermenting wort or Kraeusen may be added. At other times CO2 may be added artificially
into the lagering beer. Materials which might undesirably affect flavor and which are
present in green beer e.g. diacetyl, hydrogen sulfide, mercaptans and acetaldehyde are
decreased by evaporation during secondary fermentation. An increase occurs in the
desirable components of the beer such as esters. Any tannins, proteins, and hop resins
still left are precipitated during the lagering period.
Lagering used to take up to nine months in some cases. The time is now considerably
shorter and in some countries the turnover time from brewing, lagering, and
consumption could be as short as three weeks. This reduction has been achieved by
artificial carbonation and by the manipulation of the beer due to greater understanding of
the lagering processes. Thus, in one method used to reduce lagering time, beer is stored at
high temperature (14°C) to drive off volatile compounds e.g. H2S, and acetaldehyde. The
beer is then chilled at – 2°C to remove chill haze materials, and thereafter it is carbonated.
In this way lagering could be reduced from 2 months to 10 days.
Lagering gives the beer its final desirable organoleptic qualities, but it is hazy due to
protein-tannin complexes and yeast cells. The beer is filtered through kieselghur or
through membrane filters to remove these. Some properties of lager beer are given in Table
12.5.
Table 12.5 Some properties of lager beer
Property
Pilsener
United
States
lager beer
Danish
Pilsener
English
ale
English
stout
Mounich
Lowenbrau
Dortmund
Original
12.1
extract
content opc
Real extract
5.3
content opc
Alcoholic
3.5
content, wt %
Protein
0.28-0.35
content, wt %
CO2 content %
Color, EBC
10
Air in
bottle, mL
pH
Real degree of
attenuation, %
11.5-12.0
10.6
15.0
21.1
13.3
13.6
5.5
3.1
5.0
8.7
6.4
5.5
3.4-3.8
3.9
5.2
6.7
3.6
4.2
0.3
0.6
0.6
0.5
0.8
0.53
2.7
1.5
0.5
5
2
0.4
0.41
4.2-4.50
60-75
4
69
8
10
66
59
40
0.42
8
6
48
60
Production of Beer
#!
(b) Beer treatment (for top-fermented beers): Top-fermented beers do not undergo the
extensive lagering of bottom-fermented beers. They are treated in casks or bottles in
various ways. In some processes the beer is transferred to casks at the end of fermentation
with a load of 0.2-4.00 million yeast cells/ml. It is ‘primed’ to improve its taste and
appearance by the addition of a small amount of sugar mixed with caramel. The yeasts
grow in the sugar and carbonate the beer. Hops are also sometimes added at this stage. It
is stored for seven days or less at about 15°C. After ‘priming’, the beer is ‘fined’ by the
addition of isinglass. Isinglass, a gelatinous material from the swim bladder of fish,
precipitates yeast cells, tannins and protein-tannin complexes. The beer is thereafter
pasteurized and distributed.
12.1.3.6
Packaging
The beer is transferred to pressure tanks from where it is distributed to cans, bottles and
other containers. The beer is not allowed to come in contact with oxygen during this
operation; it is also not allowed to lose CO2, or to become contaminated with microorganisms. To achieve these objectives, the beer is added to the tanks under a CO2,
atmosphere, bottled under a counter pressure of CO2, and all the equipment is cleaned
and disinfected regularly.
Bottles are thoroughly washed with hot water and sodium hydroxide before being
filled. The filled and crowned bottles are passed through a pasteurizer, set to heat the
bottles at 60°C for half hour. The bottles take about half hour to attain the pasteurizing
temperature, remain in the pasteurizer for half hour and take another half hour to cool
down. This method of pasteurization sometimes causes hazes and some of the larger
breweries now carry out bulk pasteurization and fill containers aseptically.
12.1.4
Beer Defects
The most important beer defect is the presence of haze or turbidity, which can be of
biological or physico-chemical origin.
12.1.4.1
Biological turbidities
Biological turbidities are caused by spoilage organisms and arise because of poor
brewery hygiene (i.e. poorly washed pipes) and poor pasteurization. Spoilage organisms
in beer must be able to survive the following stringent conditions found in beer: low pH,
the antiseptic substances in hops, pasteurization of beer, and anaerobic conditions.
Yeasts and certain bacteria are responsible for biological spoilage because they can
withstand these. Wild or unwanted yeasts which have been identified in beer spoilage
are spread into many genera including Kloeckera, Hansenula, and Brettanomyces, but
Saccharomyces spp appear to be commonest, particularly in top-fermented beers. These
include Sacch. cerevisiae var. turblidans, and Sacch. diastaticus. The latter is important
because of its ability to grow on dextrins in beer, thereby causing hazes and off flavors.
Among the bacteria, Acetobacter, and the lactic acid bacteria, Lactobacillus and
Streptococcus are the most important. The latter are tolerant of low pH and hop antiseptics
and are micro-aerophilic hence they grow well in beer. Acetobacter is an acetic acid
bacterium and produces acetic acid from alcohol thereby giving rise to sourness in beer.
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Modern Industrial Microbiology and Biotechnology
Lactobacillus pastorianus is the typical beer spoiling lactobacilli, in top-fermented beers,
where it produces sourness and a silky type of turbidity. Streptococcus damnosus
(Pediococcus damnosus, Pediococcus cerevisiae) is known as ‘beer sarcina’ and gives rise to
‘sarcina sickness’ or beer which is characterized by a honey-like odor.
12.1.4.2
Physico-chemical turbidities
Non-biological hazes developing beer may be due to one or more of the following:
(i)
(ii)
(iii)
(iv)
Hazes induced by metals.
Protein-tannin hazes.
Polysaccharide sediments.
Oxalate hazes and sediments.
(i) Hazes induced by metals: Tin, iron, copper have all been identified as causing hazes
in beer. An amount of only 0.1 ppm of tin will immediately produce haze in beer. It does
not unlike other metals, acts as an oxidation catalyst, but precipitates haze precursors
directly. It may occur in some canned beers.
Copper and iron act as catalysts in the oxidation of the polyphenolic moiety of the
protein-haze precursors of beer. They appear to be derived from both malt and hop (from
copper insecticides) and also from the brewing plant. It has been suggested that EDTA
(ethylenediaminetetraacetic acid) be used to form chelates with copper and iron and
thereby prevent their deleterious action.
(ii) Protein-tannin hazes: The polyphenols of beer have often been solely and incorrectly
referred to as tannins. Tannins proper are used to convert hides to leather but beer
polyphenols cannot be so used. Polyphenols are widely distributed in plants. Beer
tannings or polyphenols (Fig. 12.5) are derived from hops and barley husks. They react
with proteins to form complex molecules which become insoluble in the form of haze.
Hazes contain polypeptides, polyphenols, carbohydrates and a small amount of
minerals.
Beer hazes are divided into two: Chill hazes (0.1–2 nm diameter particles) form at O°C
and re-dissolve at 20°C. Permanent hazes (1.0–10 nm) remains above 20°C.
(I)
(II)
OH
OH
HOOC
CO-O
HO
OH
gallic acid
HO
HO
HO
COOH
OH
gallic tannic acid
tunnic acid
Fig. 12.5 Some Barley Polyphenols (‘Tannins’)
or
Production of Beer
##
Protein-tannin hazes may be removed by:
(a) addition of papain which hydrolyzes the polypetides to low molecular weight
components which cannot form hazes;
(b) adsorption of the polypeptides by silica gel and bentonite;
(c) precipitation of polypetides by tannic acid;
(d) adsorption of the polyphenols by polyamide resins e.g. Nylon 66.
(iii) Polysaccharide sediments: Freezing and thawing of beer may cause an
unpredictable haze which can appear in the form of flakes. This haze differs from chill
haze in being distinctly carbohydrate in nature. They were found in lager chilled to –
10°C and consisted mainly of Beta glucans derived from malt.
(iv) Oxalate sediments: Oxalate sediments may appear after several week’s storage in
beers rich in oxalate as a result of a low calcium content.
(v) Other beer defects: Wild or gushing beer is a defect observed as a violent overfoaming when a bottle of beer is opened. The taste is unaffected. Gushing is due to the
formation of micro-bubbles; excess pressure may force the micro-bubbles back into
solution. Gushing beers have been identified with malt made from old barley and trial
brews have shown them to be associated with the presence of mycelia of Fusarium during
the steeping.
The off-flavor developed when beer is exposed to sunlight is due to the formation of
mercaptans by photochemical reaction in the blue-green region (420-520 nm) of visible
light.
12.1.5
Some Developments in Beer Brewing
The description made above is of conventional beer brewing. Some developments have
taken place both in the manner of the production of beer as well as in the type of beer
produced: This section will look briefly at some of these.
12.1.5.1
Continuous brewing
Although it is not yet widely used, continuous brewing is gaining gradual acceptance in
many countries. In the current commercial continuous brewing systems, it is mainly
fermentation that is continuous, secondary fermentation and lagering are usually batch.
Two systems of continuous fermentation are known: the open and the partially closed.
(i) The open system of continuous fermentation: In the open system wort is fed
continuously into the fermentor, while beer flows out at the same rate. The yeast is
allowed to attain its natural concentration or steady state. In the system described here
wort is collected batch wise from the brew house and may be stored for up to 14 days at
2°C before use. The wort is sterilized in a heat-exchanger prior to oxygenation. It is then
passed through the bottom into the first tank, which is continuously stirred and where
aerobic growth occurs. It is later passed into a second tank where conditions are
anaerobic; alcohol and CO2, are formed in this tank. From there the beer with its
suspended yeasts overflows into a third vessel for sedimentation. Finished beer is
removed from the top and yeast cells from the bottom. The amount of yeast in the beer is
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Modern Industrial Microbiology and Biotechnology
just adequate for secondary fermentation. CO2 is collected from the top. The yeast
employed is a special one which apart from imparting the right flavor, must be able to
remain in active fermenting condition in suspension in the anaerobic vessel and yet be
able to flocculate rapidly once in the cooled sedimentation tank. It is possible
theoretically to use one tank, or more than two tanks for sedimentation. Indeed in another
system three tanks are used, but two afford flexibility of design and use (Fig. 12.6).
1 = Pump; 2 = Flow regulator; 3 = Sterilization; 4 = Perforated plates;
5 = Control of temperature; 6 = Yeast separator
Fig. 12.6 The Open System of Continuous Brewing
(ii) Partially closed system of continuous fermentation: In the closed system, yeast in
held at a given concentration instead of allowing it to grow at its own steady state as in
the open system. (The open system itself may indeed be modified to achieve a higher yeast
concentration by recycling yeasts from the sedimentation tank into the first tank. The
disadvantage of the modification is the possibility of contamination. Secondly, the
returned yeasts are in a different physiological state of growth from those actively
involved in fermentation, hence the wort and the beer quality may suffer).
In the closed system, typified by the tower fermentor (Fig. 12.6), sterilized wort is
pumped into the base of the cylindrical tower with aeration, if necessary, and the beer is
drawn off at the top at the same rate.
Yeasts attain a very high density, in excess of 350 gm/liter and wort becomes almost
ready beer. The upper regions have a lower yeast concentration and serve partly as a final
fermentation stage but especially as a means of separating the yeasts. Baffles enable the
diversion of the rising CO2 and beer from the beer outlet. Its over-riding advantage is that
beer can be produced in four hours if the lower regions have the optimum yeast
concentration of 350-400 gm/liter. Special yeasts able to maintain the high mass at the
lower level and yet able to pass out of the fermentor if adequate amount must be used.
Although continuous brewing has not been generally adopted, its emergence forced
brewers to make batch brewing more efficient and to find ‘batch’ answers to the
advantages offered by continuous brewing.
Production of Beer
#%
1 = Pump; 2 = Flow regulator; 3 = Sterilization; 4 = Perforated plates;
5 = Control of temperature; 6 = Yeast separator
Fig. 12.7 The Tower Fermentor (Closed) System of Continuous Brewing
12.1.5.2
Use of enzymes
A number of firms now market enzymes isolated from bacteria and fungi which can carry
out the functions of malt. The advantage of the use of these enzymes is to greatly reduce
costs since malting can be eliminated entirely. Despite the great potentials offered by this
#&
Modern Industrial Microbiology and Biotechnology
method, brewers are yet unwilling to accept it. The consequences of eliminating or
reducing the need for malt from the barley farmer and the malting industry, two longstanding establishments, would pose great difficulties in adopting this method. When
enzymes to become generally used, care must be taken to ensure that not only the major
enzymes, amylases, and proteases, are included but that others such as Beta-glucanases
which hydrolyze the gums of barley are also present in the enzyme mixture. It must also
be certain that toxic microbial products are eliminated from the enzyme preparations.
12.2
SORGHUM BEERS
12.2.1
Kaffir Beer and Other Traditional Sorghum Beers
Barley is a temperate crop. In many parts of tropical Africa beer has been brewed for
generations with locally available cereals. The commonest cereal used is Sorghum bicolor
(= Sorghum vulgare) known in the United States as milo, in South Africa as kaffir corn and
in some parts of West Africa as Guinea corn. The cereal which is indigenous to Africa is
highly resistant to drought. Sorghum is often mixed with maize (Zea mays) or millets,
(Pennisetum spp). In some cases such as in Central Africa e.g. Zimbabwe, maize may form
the major cereal. Outside Africa sorghum is not used normally for brewing except in the
United States where it is occasionally used as an adjunct.
The method for producing these sorghum beers of the African continent as well as
their natures are remarkably similar. They
(i) are all pinkish in color; sour in taste; and of fairly heavy consistency imposed
partly by starch particles, and also because they are
(ii) consumed without the removal of the organisms;
(iii) are not aged, or clarified, and
(iv) include a lactic fermentation.
The tropical beers are known by different names in different parts of the world: ‘burukutu’, ‘otika’, and ‘pito’ in Nigeria, , ‘maujek’ among the Nandi’s in Kenya, ‘mowa’ in
Malawi, ‘kaffir beer’ in South Africa, ‘merisa’ in Sudan, ‘bouza’ in Ethiopia and ‘pombe’
in many parts of East Africa.
It is only in South Africa that production has been undertaken in large breweries;
elsewhere although considerable quantities are produced, this is done by small holders
to satisfy small local clientele. In South Africa, in fact, it is reported that three or four times
more kaffir beer is produced and drunk than is the case with barley beers. The processes
of producing the beer include malting, mashing and fermentation.
12.2.1.1
Malting
For malting, sorghum grains are steeped in water for periods varying from 16-46 hours.
They are then drained and allowed to germinate for five to seven days, water being
sprinkled on the spread-out grains. At the end of this period, the grains are usually dried,
often in the sun or in the South African system at 50°-60°C in driers. Kilning is however
not done. In some parts, the dried malt may be stored and used over several months.
Contrary to opinions previously held by many, sorghum malt is rich in amylases,
particularly a-amylase, although the ungerminated grain does not contain b-amylase as
Production of Beer
#'
is the case with barley. Sorghum has not received much attention as a brewing material,
except occasionally as an adjunct in the United States. However in recent times interest
has grown in West Africa in its use for malting and it may be that strains which perform
in malting as well as barley does may be found.
It has been suggested that the saccharification of sorghum starch is brought about
partly by the fungi which grow on the grains during their germination as well as by the
germinated sprout. This, however, has been disputed vehemently by some workers. The
fungi so implicated are Rhizopus oryzae, Botryodiplodia theobromae, Aspergillus flavus,
Penicillium funiculosum, and P. citrinum.
12.2.1.2
Mashing
The malt is ground coarsely and mixed in a rough 6:1 (v/v) proportion with water and
boiled for about 2 hours. During the boiling starchy adjunct in the form of dried powder
of plantains, cassava (‘gari’) or unmalted cereal may be added so that an approximate
1:2:6 proportion of the adjunct malt and water is attained. It is filtered and is then ready
for fermentation. In South African kaffir beer breweries the adjunct consisting of boiled
sorghum or maize grits is added after the initial souring of the mash.
12.2.1.3
Fermentation
Two fermentations take place during sorghum beer production: a lactic acid
fermentation, and an alcoholic fermentation. In traditional fermentation, the dregs of a
previous fermentation are inoculated into the boiled, filtered, and cooled wort. This
inoculum consists of a mixture of yeasts, lactic acid and acetic acid bacteria. The first
phase of the fermentation is brought about by lactic bacteria mainly Lactobacillus
mesenteroides, and Lactobacillus plantarum.
In the sorghum beer breweries in South Africa, the temperature of the mash is held
initially at 48-50°C to encourage the growth of thermophilic lactic acid bacteria which
occur naturally on the grain, for 16-24 hours. The pH then drops to about 3-4. The sour
malt is added to the previously cooked adjunct of unmalted sorghum or maize, and
sometimes some more malt may be added. It is then cooled to 38°C and pitched with the
top fermenting yeasts.
In the traditional method yeasts and lactic acid bacteria are present in the dregs. The
yeasts which have been identified in Nigerian sorghum beer fermentation are: Candida
spp, Saccharomyces cervisiae, and Sacch. chevalieri.
Fermentation is for about 48 hours during which lactic acid bacteria proliferate.
Thereafter it is ready for distribution and consumption. No secondary fermentation of the
kind seen in lager beer, lagering, or clarification is done. The live yeasts, and the lactic
acid bacteria are consumed in much the same ways as they are done in palm wine. In
some localities the fermentation lasts a little longer and the flavor is influenced by a slight
vinegary taste introduced by the release of acetic acid by acetic acid bacteria.
Sorghum beers usually contain large amounts of solids (Table 12.6) mainly starch
apart from the microorganisms. For this reason some authors have regarded them as
much as foods as they are alcholic beverages an alcoholic beverage.
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Modern Industrial Microbiology and Biotechnology
Table 12.6
Properties of South African sorghum beer
Properties
pH
Alcohol
Solids (%w/v)
Total
Insoluble
Nitrogen (%w/v)
Total
Soluble
Small scale
Factory
3.5
0.1
3.4
3.0
4.9
2.3
5.4
3.7
0.084
-
0.093
0.014
SUGGESTED READINGS
Amerine, M.A., Berg, H.W., Cruess, W.V. 1972. The Technology of Wine Making 3rd Ed. Avi
Publications. West Port, USA, pp. 357-644.
Aniche, G.N. 1982. Brewing of Lager Beer from Sorghum. Ph.D Thesis, University of Nigeria,
Nsukka, Nigeria.
Ault, R.G., Newton, R. 1971. In: Modern Brewing Technology, W.P.K. Findlay, (ed) Macmillan,
London, UK. pp. 164-197.
Battcock, M., Azam-Ali, S. 2001. Fermented Fruits and Vegetables: A Global Perspective. FAO
Agric. Services Bull. 134, Rome, Italy, pp. 96.
Doyle, M.P., Beuchat, L.R., Montville, T.J. 2004. Food Microbiology: Fundamentals and Frontiers,
2nd edit., ASM Press, Washington DC, USA.
Flickinger, M.C., Drew, S.W. (eds) 1999. Encyclopedia of Bioprocess Technology - Fermentation,
Biocatalysis, and Bioseparation, Vol 1-5. John Wiley, New York, USA.
Gastineau, C.F., Darby, W.J., Turner, T.B. 1979. Fermented Food Beverages in Nutrition.
Academic Press; New York, USA, pp. 133-186.
Gutcho, M.H. 1976. Alcoholic Beverages Processes. Noyes Data Corporation New Jersey and
London. pp. 10-106.
Hammond, J.R.M., Bamforth, C.W. 1993. Progress in the development of new barley, hop, and
yeast variants for malting and brewing. Biotechnology and Genetic Engineering Reviews 11,
147-169.
Hanum, H., Blumberg, S. 1976. Brandies and Liqeuers of the World. Doubleday Inc. New York
USA.
Hough, J.S. 1985. The Biotechnology of Malting and Brewing. Cambridge University Press;
Cambridge, UK. pp. 15-188.
Hough, J.S., Briggs, D.E., Stevens, R. 1971. Malting and Brewing Science, Chapman and Hall,
London, UK.
Hoyrup, H.E. 1978. Beer Kirk-Othmer’s Encyclopaedia of Chemical Technology, Wiley, New
York, USA. Microbiology 24: 173-200.
Okafor, N. 1972. Palm-wine yeasts of Nigeria. Journal of the Science of Food and Agriculture 23,
1399-1407.
Okafor, N. 1978. Microbiology and biochemistry of oil palm wine. Advances in Applied.
Okafor, N. 1987. Industrial Microbiology. University of Ife Press Ile-Ife: Nigeria. pp. 201-210.
Okafor, N. 1990. Traditional alcoholic beverages of tropical Africa: strategies for scale-up. Process
Biochemistry Intemational 25, 213-220.
Production of Beer
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Okafor, N., Aniche, G.N. 1980. Lager beer from Nigerian Sorghum. Brewing and Distilling
International. 10, 32-33, 35.
Packowski, G.W. 1978. Distilled Alcoholic Beverages, Encyclopadeia of Chemical Technology, 3rd
Edit. Wiley, New York, USA. pp. 824-863.
Singleton, V.L., Butzke, C.E. 1998 Wine. Kirk-Othmer Encyclopedia of Chemical Technology.
John Wiley & Sons, Inc. Article Online Posting Date: December 4, 2000.
Soares, C. 2002. Process Engineering Equipment Handbook Publisher. McGraw-Hill.
Taylor, J.R.N., Dewar, J. 2001. Developments in Sorghum Food Technologies. Advances in Food
and Nutrition Research 43, 217-264.
Vogel, H.C., Tadaro, C.L. 1997. Fermentation and Biochemical Engineering Handbook Principles, Process Design, and Equipment. 2nd Edit Noyes.
Zeng, A. 1999. Continuous culture. In: Manual of Industrial Microbiology and Biotechnology.
A.L. Demain, J.E. Davies (eds) 2nd Edit. ASM Press. Washington, DC, USA. pp. 151-164.
Modern Industrial Microbiology and Biotechnology
13
Production of
Wines and Spirits
13.1
GRAPE WINES
Wine is by common usage defined as a product of the “normal alcoholic fermentation of
the juice of sound ripe grapes”. Nevertheless any fruit with a good proportion of sugar
may be used for wine production. Thus, citrus, bananas, apples, pineapples,
strawberries etc., may all be used to produce wine. Such wines are always qualified as
fruit wines. If the term is not qualified then it is regarded as being derived from grapes,
Vitis vinifera. The production of wine is simpler than that of beer in that no need exists for
malting since sugars are already present in the fruit juice being used. This however
exposes wine making to greater contamination hazards.
Wine is today principally produced in countries or regions with mild winters, cool
summers, and an even distribution of rainfall throughout the year. In North America, the
United States is the leading producer, most of the wine coming from the State of California
and some from New York. In Europe the principal producers are Italy, Spain and France.
In South America, Argentina, Chile, and Brazil are the major producers; and in Africa,
they are Algeria, Morocco, and South Africa. Other producers are Turkey, Syria, Iran, and
Australia.
13.1.1
13.1.1.1
Processes in Wine Making
Crushing of Grapes
Selected ripe grapes of 21° to 23° Balling (Chapter 12) are crushed to release the juice
which is known as ‘must’, after the stalks which support the fruits have been removed.
These stalks contain tannins which would give the wine a harsh taste if left in the must.
The skin contains most of the materials which give wine its aroma and color. For the
production of red wines the skins of black grapes are included, to impart the color.
Grapes for sweet wines must have a sugar content of 24 to 28 Balling so that a residual
sugar content is maintained after fermentation. The chief sugars in grapes are glocuse
and fructose; in ripe fruits they occur in about the same proportion. Grape juice has an
acidity of 0.60-0.65% and a pH of 3.0-4.0 due mainly to malic and tartaric acids with a
little citric acid. During ripening both the levulose content and the tartaric acid contents
rise.
Production of Wines and Spirits
$!
Nitrogen is present in the form of amino acids, peptides, purines, small amounts of
ammonium compounds and nitrates.
13.1.2
Fermentation
(i) Yeast used: The grapes themselves harbor a natural flora of microorganisms (the
bloom) which in previous times brought about the fermentation and contributed to the
special characters of various wines. Nowadays the must is partially ‘sterilized’ by the
use of sulphur dioxide, a bisulphate or a metabisulphite which eliminates most microorganisms in the must leaving wine yeasts. Yeasts are then inoculated into the must. The
yeast which is used is Saccaromyces cerevisiae var, ellipsoideus (synonyms: Sacch. cerevisiae,
Sacch. ellipsoideus, Sacch, vini.) Other yeasts which have been used for special wines are
Sacch. fermentati, Sacch. oyiformis and Sacch. bayanus.
Wine yeasts have the following characteristics: (a) growth at the relatively high acidity
(i.e., low pH) of grape juice; (b) resistance to high alcohol content (higher than 10%); (c)
resistance to sulfite.
(ii) Control of fermentation
(a) Temperature: Heat is released during the fermentations. It has been calculated
that on the basis of 24 Cal per 180 gm of sugar the temperature of a must containing
22% sugar would rise 52°F (11°C) if all the heat were stopped from escaping. If the
initial temperature were 60°F (16°C) the temperature would be 100°F (38°C) and
fermentation would halt while only 5% alcohol has been accumulated. For this
reason the fermentation is cooled and the temperature is maintained at around
24°C with cooling coils mounted in the fermentor.
(b) Yeast Nutrition: Yeasts normally ferment the glucose preferentially although some
yeasts e.g. Sacch. elegans prefer fructose. To produce sweet wine glucose-fermenting
wine yeasts are used leaving the fructose which is much sweeter than glucose.
Most nutrients including macro- and micro-nutrients are usually abundant in
must; occasionally, however, nitrogeneous compounds are limiting. They are then
made adequate with small amounts of (NH4)2 SO4 or (NH4)2 HPO4.
(c) Oxygen: As with beer, oxygen is required in the earlier stage of fermentation when
yeast multiplication is occurring. In the second stage when alcohol is produced the
growth is anaerobic and this forces the yeasts to utilize such intermediate products
as acetaldehydes as hydrogen acceptors and hence encourage alcohol production.
(iii) Flavor development: Although some flavor materials come from the grape most of it
come from yeast action. The flavor of wine has been elucidated with gas chromatography
and has been shown to be due to alcohols, esters, fatty acids, and carbonyl compounds,
the esters being the most important. Diacetyl, acetonin, fusel oils, volatile esters, and
hydrogen sulfide have received special attention. Autolysates from yeasts also have a
special influence on flavor.
13.1.3
Ageing and Storage
The fermentation is usually over in three to five days. At this time ‘pomace’ formed from
grape skins (in red wines) will have risen to the top of the brew. As has been indicated
Modern Industrial Microbiology and Biotechnology
earlier for white wine, the skin is not allowed in the fermentation. At the end of this
fermentation the wine is allowed to flow through a perforated bottom if pomace had been
allowed. When the pomace has been separated from wine and the fermentation is
complete or stopped, the next stage is ‘racking’. The wine is allowed to stand until a major
portion of the yeast cells and other fine suspended materials have collected at the bottom
of the container as sediment or ‘lees’. It is then ‘racked’, during which process the clear
wine is carefully pumped or siphoned off without disturbing the lees.
The wine is then transferred to wooden casks (100-1,000 gallons), barrels (about
50 gallons) or tanks (several thousand gallons). The wood allows the wine only slow
access to oxygen. Water and ethanol evaporate slowly leading to air pockets which
permit the growth of aerobic wine spoilers e.g. acetic acid bacteria and some yeasts. The
casks are therefore regularly topped up to prevent the pockets. In modern tanks made of
stainless steel the problem of air pockets is tackled by filling the airspace with an inert gas
such as carbon dioxide or nitrogen.
During ageing desirable changes occur in the wine. These changes are due to a
number of factors:
(a) Slow oxidation, since oxygen can only diffuse slowly through the wood. Small
amounts of oxygen also enter during the filling up. Alcohols react with acids to
form esters; tannins are oxidized.
(b) Wood extractives also affect ageing by affecting the flavor.
(c) In some wines microbial malo-lactic fermentation occurs. In this fermentation,
malic acid is first converted to pyruvic acid and then to lactic acid. (Fig. 13.1)
Fig. 13.1 Chemical Reactions Involved in Flavor Development in Grape Wines
The reaction is responsible for the rich flavor developed during the ageing of some
wines e.g. Bordeaux. Cultures which have been implicated in this fermentation are
Lactobacillus sp and Leuconostoc sp. A temperature of 11-16°C is best for ageing wines,
High temperature probably functions by accelerating oxidation.
13.1.4
Clarification
The wine is allowed to age in a period ranging from two years to five years, depending on
the type of wine. At the end of the period some will have cleared naturally. For others
Production of Wines and Spirits
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artificial clarification may be necessary. The addition of a fining agent is often practiced
to help clarification. Fining agents react with the tannin, acid, protein or with some
added substance to give heavy quick-settling coagulums. In the process of setting various
suspended materials are adsorbed. The usual fining agents for wine are gelatin, casein,
tannin, isinglass, egg albumin, and bentonite. In some countries the removal of metal ions
is accomplished with potassium ferrocyanide known as ‘blue fining’; it removes excess
ions of copper, iron, manganese, and zinc from wines.
13.1.5
Packaging
Before packing in bottles the wine from various sources is sometimes blended and then
pasteurized. In some wineries, the wine is not pasteurized, rather it is sterilized by
filtration. In many countries the wine is packaged and distributed in casks.
13.1.6
Wine Defects
The most important cause of wine spoilage is microbial; less important defects are acidity
and cloudiness. Factors which influence spoilage by bacteria and yeasts include the
following (a) wine composition, specifically the sugar, alcohol, and sulfur dioxide
content; (b) storage conditions e.g. high temperature and the amount of air space in the
container; (c) the extent of the initial contamination by microorganism during the bottling
process.
When proper hygiene is practiced bacterial spoilage is rare. When it does occur the
microorganisms concerned are acetic acid bacteria which cause sourness in the wine.
Lactic acid bacteria especially Leuconostoc, and sometimes Lactobacillus also spoil wines.
Various spoilage yeasts may also grow in wine. The most prevalent is Brettanomyces, slow
growing yeasts which grow in wine causing turbidities and off-flavors. Other wine
spoilage yeasts are Saccharomyces oviformis, which may use up residual sugars in a sweet
wine and Saccharomyces bayanus which may cause turbidity and sedimentation in dry
wines with some residual sugar. Pichia membanaefaciens is an aerobic yeast which grows
especially in young wines with sufficient oxygen.
Other defects of wine include cloudiness and acidity.
13.1.7 Wine Preservation
Wine is preserved either by chemicals or by some physical means. The chemicals which
have been used include bisulphites, diethyl pyrocarbonate and sorbic acid. Physical
means include pasteurization and sterile filtration. Pasteurization is avoided when
possible because of its deleterious effect on wine flavor.
13.1.8
Classification of Wines
Grape wines may be classified in several ways. Some of the criteria include place of
origin, color, alcohol content and sweetness. The one being adopted here is primarily
used in the United States and is shown in Table 13.1. This system classifies wine into two
groups: natural wines and fortified wines.
$$
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Table 13.1
Broad classification of grape wines
A. Natural wines: 9-14% alcohol; nature and keeping quality mostly dependent on
‘complete’ yeast fermentation and protection from air
1. Still wines (known as ‘Table’ wines intended as part of meal); no carbon
dioxide added.
(a) Dry table wines: (no noticeable sweetness)
(i) White; (ii) Rose (pink); (iii) Red
(b) Sweet table wines
(i) White (ii) Rose
Further naming of above depends on grape type, or region of origin.
2. Sparkling wines (appreciable CO2 under pressure)
(a) White (Champagne); (b) Rose (Pink Champagne); (c) Red (Sparkling
burgundy; cold duck)
B. Fortified (Dessert and appetizer) wines: Contain 15 to 21% alcohol; nature and
keeping quality depends heavily on addition of alcohol distilled from grape wine.
1. Sweet wines
(a) White (Muscatel, White port, angelica)
(b) Pink (California tokay, tawny port)
(c) Red (Port, black Muscat)
2. Sherries: (White sweet or dry wines with oxidized flavors)
(a) Aged types
(b) Flor types
(c) Baked types
3. Flavored specialty wines (usually white Port base)
(a) Vermouth (pale dry, French; Italian sweet types)
(b) Proprietary brands
13.1.8.1
The natural wines
These result from complete natural fermentation. Further fermentation is prevented
because the sugar is to a large extent exhausted. Spoilage organisms such as acetic acid
bacteria do not grow if air is excluded. Owing to the natural limit of sugar in grapes, the
alcohol content does not usually exceed 12%. They are sub-divided into still (without
added CO2) and sparkling (with added CO2). Color and sweetness also subdivide the
wines. The color, pink or red, is derived from the color of the grape; white wine comes
from a grade whose skin is light-green, but whose juice is clear. White wine can also
result from black (or deep-red) grapes if the skin is removed immediately after pressing
before fermentation. Red wine results when the skin of black grapes are allowed in the
fermenting must. Pink wine results when the skin is left just long enough for some
material to extract some skin coloring.
Table wines: In general the natural wines are usually consumed at one sitting once they
are opened. For this reason they are called ‘table’ wines and intended to be part of a meal.
Production of Wines and Spirits
They are usually served in generous amounts, partly because they contain less alcohol
than the desserts and appetizers and partly because they do not have a high keeping
quality once opened compared with appetizers and dessert wines.
Dessert and appetizer wines: The second broad group of wines are dessert or appetizer
wines. As can be seen from their names they are served at the beginning (appetizers) or at
the end (dessert) of meals. They contain extra alcohol from distilled wines, partly to make
them more potent, but also to preserve them from yeast spoilage. These are divided into
three categories: (a) Sweet e.g. port; (b) Sherries – sweet or dry, they originated from
Portugal and are characterized by flavors induced by various degrees of oxidation; (c)
Flavored wines e.g. vermouth; these are flavored with herbs and other components which
are secrets of the producing firms (Table 13.1).
Sparkling wines (especially champagne), sherry, and flavored wines will be
discussed briefly.
Sparkling Wines: Sparkling wines contain CO2 under pressure before they are opened.
They are called sparkling because the gentle release of carbon dioxide from the wine after
the bottle is opened gives the wine a sparkle. The best known of the sparkling wines is
produced in Champagne region in northern France which has given its name to the wine.
Champagne is produced either in a bottle or in bulk.
(a) Bottle Champagne: Champagne is usually a clear sparkling wine made from white
wine. Pink or red champagne made from wines of the same color are preferred by
some. Champagne is produced by a second fermentation in the bottle of an already
good wine. Not only does its production take a long time, it also requires a
complicated method which is difficult to automate and hence must be handled
manually. For these reasons the drink is expensive. The usual process is described
below. The parlance of champagne manufacture is understandably French. The
method Champenoise to be described is the one used for making the best sparkling
wines. After the must is fully fermented, the wines to be used for champagne are
racked, clarified, stabilized and fined. A blend is then made from several different
wines to give the desired aroma. The blend is known as cuvèe. The cuvèe should
have an alcohol content of 9.5 to 11.5%, adequate acidity (0.7 to 0.9% titrable as
tartaric acid) and light straw or light yellow color. The SO2 content should be low
otherwise SO2 odor would show when the wine is poured, or worse still, the yeasts
might convert SO2 to forms of hydrogen sulphide, which would give a rotten egg
odor.
The cuvee is placed into thick walled bottles able to withstand the high pressure
of CO2 to be built up later in the bottle. For the secondary fermentation in the bottle,
more yeast, more fermentable sugar, usually sucrose and nowadays sometimes a
small amount (0.05 to 0.1%) of ammonium phosphate is added. The yeast usually
used is Saccharomyces bayanus. It is selected because it meets the following
requirements encountered in secondary fermentation for champagne production:
it must grow at a fairly high alcohol content (10-12%), at high pressures (see below)
and relatively low temperatures; the yeast must die or become inactive within a
short time in order to prevent further fermentation after sugar (known as the dosage)
is added once again before the final corking. Finally the yeast must be able to form
a compact granulated sediment after fermentation.
Modern Industrial Microbiology and Biotechnology
The amount of sugar added depends on the expected CO2 pressure of the
sparkling wine. Sparkling wines have a pressure of about six atmospheres but not
less than four. As a rough rule, 4 gm of sugar per liter will produce one atmosphere
of CO2 pressure. Therefore the sugar added is about 24 gm/liter. Account is taken
of any sugar present in the wine. Although the bottles are thick, if the fermentation
is too rapid, temperature too high or sugar too high the bottle may burst.
Champagne bottles are therefore not reused since a scratched bottle may burst.
The bottle with its mixture of wine, sugar and yeast is placed horizontally and
allowed to ferment at a temperature of about 15-16°C; this secondary fermentation
takes two to three months. The secondarily fermented wine now known as triage is
then stored still horizontally at a temperature of 10°C and remains undisturbed in
that position for at least a year and sometimes up to five years. Much of the aroma
of well-aged champagne appears to come from the reactions involving materials
released from yeast autolysis.
The next stage is getting the sediment from the side to the neck of the bottle.
Several methods are used by individual handlers. This transfer or remuage is
achieved by first placing the bottle neck downwards at an angle in a rack with
varying degrees of jolting. The bottles are next rotated clockwise and anticlockwise on alternate days during which the bottle is gradually straightened to
the perpendicular. The process may take anything from two or six weeks at the end
of which the sediment finds it way to the neck of the bottle. To remove the sediment
of yeast cells, the neck of the bottle is frozen to 1°C to 15°C; an ice plug which
includes the sediment forms therein. The bottle is turned to an angle of about 45°
and opened. The pressure in the bottle forces out the ice plug. During this process
of disgorging some CO2 is lost but sufficient is left to give the usual pop which
launches a bride, a ship or a graduating student into the future! The lost wine is
replaced from another bottle and the dosage is added. The dosage is a syrup
consisting of about 60 gm of sugar in 100 ml of well-aged wine. All champagnes,
even those labeled dry contain dosage; otherwise it would taste sour. Sweet
champagne contains up to 10% sugar.
(b) Bulk production of Champagne: Champagne is sometimes produced in bulk in a
large tank rather than in individual bottles. Bulk production is known as the
Charmat process named after its inventor. When this is so produced it is usually
declared on the label to save the more labor-intensive bottle-made versions from
unfair competition. The tank which has a lining of a inert material such as glass
holds 500 to 25,000 gallons and is built to withstand 10-20 atmospheres as a safety
measure. Valves control the pressure and cooling coils the temperature. Since the
tanks are aerated a rapid turnover is possible and 6-12 fermentations per year are
made. Another reason for the rapid turnover is that a heavy yeast growth occurs
which could lead to the production of off flavors especially H2S if allowed
prolonged contact with the wine.
Tank fermented champagne is usually given a cold-stabilizing treatment to
remove excess tartarates. It is filtered still cold and under pressure to remove yeast
cells. The wine is then filled into bottles with dosage of the right kind. It is usual to
introduce some sulfur dioxide into it just in case some yeasts were not removed by
filtration. Sulfur dioxide also helps to prevent darkening oxidation which may
Production of Wines and Spirits
$'
occur as the wine takes up oxygen during the transfer process. The sulphur
dioxide odor is usually noticeable when Charmat-prepared champagne bottles are
opened. Furthermore, they lack the aroma conferred on well-aged bottle brands by
the autolysis of yeast. Bulk champagnes are amenable to bulk production because
they are more difficult to ferment owing to their higher tannin contents; they may
also require higher fermentation temperatures.
13.1.8.2 Fortified wines
The fortified wines can be divided into three main groups, which derive their names from
the warmer more southern portion of the Iberian (Spain-Portugal) peninsula in Europe,
and an island off that peninsula, where they were originally produced: sherry (Jerez de la
Frontera area in Spain); port (Douro Valley in Portugal); Madeira (the Island of Madeira).
In other wines contact with oxygen no matter how small is undesirable. The fortified
wines are however produced by the deliberate but controlled oxidation of wine. The
oxidation is achieved by prolonged ageing in the pressure of air, by the growth of an
aerobic yeast or by heating. The consequence of this oxidation is a product which has a
dark, reddish-brown color with a characteristic flavor, whether the starting wine is white
or red. For sherry therefore a white wine is used but for port or Madeira a red or a white
wine may be used.
All the three fortified wines have a high alcohol content of ranging from 15-20% (v/v),
derived from added alcohol hence their name. They are usually separated into two
groups: (a) Vermouth (b) Other flavor wines (Special natural wines).
Table 13.2 Flow Sheet for the Production of Sherry, Port, and Madeira
1. Type of wine used
2. Grape sun dried to increase sugar
content of juice
3. Skin of grapes left in contact with
must during initial wine
4. fermentation
5. SO2 added to juice
6. Juice fermented to dry wine
Freshly fermented wine fortified to
7. alcohol content 15% given (% v/v)
8. Matured in contact with air for
flor film
9. Maturation with heating
Adjusted to given alcohol content
10. About six months after fermentation
(% v/v)
11. Matured in wood for several months
Fortified wine
Key: + = Yes, — = No.
Sherry
Port
Madeira
White
White/red
White/red
+ +
-
-
_ _
+ +
+ +
+
-
+
15% 15%
17%
18%
—
-
+
20%
+
Port
18%
+
Madeira
+
—
_ _
15% 18%
+ +
Fino, Oloros
Amantillado
%
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Vermouth may be of Italian (sweet) or French (dry) type. Vermouth comes from the
German ‘wermut’ (wormwood, Artemesia absinthium a common herb) a frequent
component of vermouth. The other flavored fortified wines, such as ‘Campari’,
‘Dubonnet’, ‘Byrrh’, like vermouth, contain 15-21% alcohol. In both cases the aromatic
components of the herbs are extracted by immersing the herbs in wine or alcohol, or by
distilling them. The nature and proportion of many of the components of flavored
fortified wines are kept secret.
13.1.8.3
Fruit wines: cider and perry
Often fruits do not contain enough sugar to make a potent alcoholic beverage. Under such
conditions, extra sugar in the form of sucrose is added to encourage fermentation. Fruit
wines are popular in some countries where grapes cannot thrive.
Cider is derived from apples, (Malus pumila) and perry from pears or a mixture of pears
and apples. They differ from other fruit wines in that their alcohol content is low (4-5%
with a maximum of 7-8% v/v) because sugar is not usually added. The basic processes
are similar to those of grape wine: pressing out the juice, fermenting, maturing, and
bottling. Fruit wines have been made from cashew, pineapples, and other fruits.
13.2
PALM WINE
Palm wine is a general name for alcoholic beverages produced from the saps of palm
trees. It differs from the grape wines in that it is opaque. It is drunk all over the tropical
world in Africa, Asia, South America. Table 13.3 shows the palms from which palm wine
is derived in the various areas.
Palm wine is usually a whitish and effervescent liquid both of which properties derive
from the fact that the fermenting organisms are numerous and alive when the beverage is
consumed. More information appears to exist on wine derived from the oil palm, Elaeis
guinieensis than on any other and this will be discussed.
The sap of the palm is obtained from a variety of positions: the stem of the standing
tree, the tip or trunk of the felled tree and the base of the immature male inflorescence.
Which method is favored depends on the country concerned but most studies have
centered on inflorescence wine. The sap produced by this method in Elaeis guiniensis
contains about 12% sucrose, about 1% each of fructose, glucose, and raffinose, and small
quantities of protein and some vitamins and is a clear, sweet, syrupy liquid.
To produce palm wine a succession of microorganisms occurs roughly: Gramnegative bacteria, lactic acid bacteria and yeasts and finally acetic acid bacteria. Yeasts in
palm wine have been identified as coming from various genera (Table 13.4).
The organisms are not artificially inoculated and find their way into the wine from a
variety of sources including the air, the tapping utensils including previous brews and
the tree. The wine contains about 3% (v/v) alcohol and since the bacteria and yeasts are
consumed live, it is a source of (single cell) protein and various vitamins.
The great problem with palm wine is that its shelf life is extremely short. It is best
consumed within about 48 hours, but certainly not beyond about five days after tapping.
For this reason various methods have been devised to preserve it. Pasteurization has met
with some success, but methods which lower the microbial load of the wine by
Production of Wines and Spirits
Table 13.3
Palms from which palm wine is obtained
Name of palm
Acromia vinifera Oerst
Arenga pinnata (Wurmb.) Merr.
(Syn. A. sacoharifera Labill.)
Attalea speciosa Mart.
Borassus aethipum Mart.
Broassus flabelifer Linn.
Caryota urens Linn.
Cocos nucifera Linn.
Corypha umbraculifera L.
Elaesis guineensis Jacq.
Hyospathe elegans Mart.
Hyphaenae guineensis Thonn.
Jubaea chilensis (Molina) Baillon
Mauritiella aculeate (H.B. and K.)
Burret (Syn. Lepidococcus aculeatus H.
Wendl and Drude)
Marenia montana (Hump. and Bonpl.)
Burret (Syn. Kunthia Montana
Humb. and Bonpl.)
Nypa fruticans Wurmb.
Orbignya cohune (Mart.) Dahlgreen ex
Standley (Sun. Attalea cohune Mart.)
Phoenix dactylifera Linn.
Phoenix reclinata Jacq. (Syn. Phoenix)
Spinosa Schum. and Thonn.)
Phoenix sylvestris (L) Roxb.
Raphia hookeri Mann and Wendl.
Raphia sundanica A. Chev.
Raphia vinifera Beauv.
Scheelea princeps (Marts.) Karsten
(Syn. Attalea princeps Mart.)
Location
Nicargua, Panama, Costa Rica
Far East
Brazil, Guyana
Tropical Africa
India, Cambodia, Java
India
India, Sri Lanka, Africa
Sri Lanka
Africa
Brazil, Guyana
West Africa
Chile
Brazil, Venezuela
Brazil
Sir Lanka, Bay of Bengal, The Phillippines,
Carolines, Salmonon Islands
Hondruras, Mexico, Gautemala
North Africa, Middle East
Central Africa
India
Africa
Africa
Africa
Brazil, Bolivia
centrifugation or filtration permit the use of milder pasteurization temperatures and
lower quantities of chemicals (Table 13.5). Of the chemicals tried, sorbate and sulphite
were found best. Fully fermented palm wine has 5% to 8% alcohol and is distilled for kaikai, a gin with a distinct fruity flavor.
Other African alcoholic beverages
(i) Bouza is an alcoholic beverage produced in Egypt since the time of the Pharoahs.
Formerly drunk by all classes, it is now drunk mainly among the lower income groups.
Egyptian Bouza is prepared either from wheat or maize but the most popular is from
wheat. To prepare bouza, coarsely ground wheat (about 20% of total) is placed in a large
wooden basin and kneaded with water to form a dough which is cut into thick loaves and
Modern Industrial Microbiology and Biotechnology
Table 13.4
Yeasts identified in palm wines
Yeast
Saccharomyces pastorianus
Saccharomyces ellipsoids
Saccharomyces cereviside
Saccharomyces cerevisiae
Saccharomyces chevalieri
Pichia sp.
Schizosaccharomyces pombe
Saccharomyces vafer
Endomycopsis sp.
Saccharomyces markii
Kloekera apculata
Saccharomyces chevalieri
Saccharomyces rosei
Candida spp.
Saccharomycoides luduigii
Type of Wine
Oil palm
Oil palm,
Arenga palm
Oil palm,
Oil palm,
Oil palm,
Oil palm,
Oil palm,
Palmyra palm
Oil palm,
Oil palm,
Oil palm,
Palmyra palm
Raphia
Oil palm
Oil palm
Palmyra palm
Table 13.5 Counts of Bacteria on whole palm wine and on supernatant of centrifuged palm
wine after various treatments
Treatment
Whole palm wine
Untreated
0.5% potassium sorbate
0.10%
0.15%
0.15%
0.15% sodium metaisulfie
0.10%
0.15%
Supernatant after centrifuging
Untreated
Pasteurized at 60oC for ½ hour
Pasteurization plus 0.05%
sorbate
Pasteurization plus 0.05%
sodium metabisulfite
Number
Percent Survival
´ 101
2.96 ´ 109
2.32 ´ 109
2.24 ´ 109
1.40 ´ 109
1.20 ´ 108
4.80 ´ 105
100.00
1.0571
0.8286
0.8000
0.5000
0.4285
0.00017
4.00 ´ 104
2.00 ´ 102
0.00001
0.0000007
2.00 ´ 102
0.00000007
0.4 ´ 102
Virtually sterile
lightly baked. The rest of the wheat (about 80% of the total) is germinated for three to five
days, sun dried and coarsely ground. The malted wheat and the crumbled bread are
mixed in water in a wooden barrel. Bouza from a previous fermentation is added and the
whole mixture is fermented for 24 hours at room temperature. The mash is sieved to
remove large particles and the beverage is ready for drinking. The beverage has a pH of
Production of Wines and Spirits
about 3.6 and an alcohol content of about 4%: but the pH drops while alcohol increases
with further fermentation.
(ii) Talla (tella) is an Ethiopian small-producer beer with a smokey flavor derived from
inverting the fermentation containers and talla collection pots over smouldering olive
wood. Talla also acquires some smoke flavor from the toasted, milled and boiled cereal
grains. During the toasting the grains are roasted until they begin to smoke slightly. In the
production of talla, of which various types exist, powdered hop leaves and water are put
in a fermentation vessel and allowed to stand for about three days. Ground barley or
wheat malt and pieces of flat bread baked to burning on the outside. are added. On the
fifth day hop stems are added in addition to cereal flour made by milling sorghum which
is first boiled and then toasted. Water is added and the fermentation allowed for two days
(i.e., to the seventh day). It is filtered and is ready to drink.
(iii) Busaa is an acidic alcoholic beverage drunk among the Luo. Abuluhya and Maragoli
ethnic groups of Kenya. It is porridge-like and light-brown in color and is warmed to 3540°C before being consumed. A stiff dough made from maize flour and water is incubated
at room temperature for three to four days. The fermented dough is pounded and then
heated on a metal plate till it turns brown.
Malt is made from millet, allowed to germinate for three to four days, sun dried, and
ground to powder. A slurry is made in water of the millet malt powder and roasted maize
flour dough and left to ferment for two to four days. It is filtered through cloth. The filtrate
is busaa.
The organisms responsible for fermenting the uncooked maize dough include Candida
krusei. Saccharomyces cerevisiae. Lactobacillus helveticus, L. salivarus. L. brevis. L viridescens. L.
plantarurn and Pediococcus damnosus. The final product is the result of alcohol produced
mainly by S. cerevisiae: the lactic acid in the beverage is produced by several lactobacilli:
(iv) Merissa is a sour Sudanese alcoholic (up to 6%) beverage made from sorghum. It has
a pH of about four and a lactic acid content of about 2.5%. Sorghum grains are malted,
dried, and ground into a coarse powder. Unmalted sorghum is milled into a fine powder.
One third of this powder is mixed with a little water and allowed to ferment at room
temperature for 24-36 hours. The resulting fermented sour dough is heated without
further water addition until the product caramelizes to give rise to soorji. The cooled soorji
is allowed to ferment for four in five hours in a mixture of malt, to which previous merissa
is added as inoculum. The two are mixed together and portions of these are allowed to
cool resulting in a product called ‘futtura. Futtura is mixed from with malt flour (about
5%) and added to the bedoba from time to time. Fermentation lasts from 8 to 10 hours after
which it is filtered to give rise to the drink, merissa.
(v) Tej is a mead (i.e., a wine made by fermenting honey) of Ethiopian origin. It is yellow,
sweet, effervescent, and cloudy due to its yeast content. As it is expensive, it is beyond the
reach of most Ethiopians and used only on special occasions. The wine may be flavored
with spices or hops and also by passing smoke into the fermentation pot before it is used.
To prepare Tej the honey is diluted with water by between 1:2 to 1:5 i.e., to a liquid of
between 13 and 27% sugar since honey contains about 80% sugar. Yeasts of the genus
Saccharomyces spontaneously ferment the brew in about five days to give a yellow wine of
7-14% alcohol (v/v).
Modern Industrial Microbiology and Biotechnology
(vi) Agadagidi wines are made from bananas and plantains and have the opaque,
effervescent sweet-sour nature typical of African traditional alcoholic beverages. In
Nigeria the best-known agadagidi is found in the cocoa-growing areas of south western
Nigeria where plantains provide shade for the young cocoa trees. The ripe fruits are
peeled and soaked in water where the sugars dissolving from the preparation permit the
development of yeasts and lactic acid and giving rise to a typical opaque effervescent
wine. The alcohol content is about l%.
(vii) Mbege is consumed in Tanzania mainly by those living near Mount Kilimanjaro. It is
not a wholly banana/plantain wine as is the case with Nigerian agadagidi. Rather it is
produced from a mixture of malted millet and fermented banana juice. The juice is
produced by boiling the ripe banana followed by decantation. The banana infusion is
mixed with cooked and cooled millet malt and allowed to ferment for four to five days:
13.3
THE DISTILLED ALCOHOLIC (OR SPIRIT) BEVERAGES
The distilled alcoholic or spirit beverages are those potable products whose alcohol
contents are increased by distillation. In the process of distillation volatile materials
emanating directly from the fermented substrate or after microbial (especially yeast)
metabolism introduce materials which have a great influence on the nature of beverage.
The character of the beverage is also influenced by such post-distillation processes as
ageing, blending, etc. The components of spirit beverages which confer specific aromas
on them are known as congeners.
13.3.1 Measurement of the Alcoholic Strength of
Distilled Beverages
(i) Proof: In English-speaking countries, such as the United States, Canada, the United
Kingdom, and Australia, the alcoholic content of spirit beverages (and also of nonpotable alcohol) is given by the term ‘proof’. The reason for the term is historical. Before
the advent of the use of the hydrometer in alcoholic measurements, an estimate of the
alcoholic content of a spirit beverage was obtained by mixing it with gunpowder. If the
gunpowder still ignited then it was satisfactory because it contained less than 50% (v/v)
of alcohol. If it did not ignite it was ‘under proof’ because it contained less than 50%
water. Proof has nowadays become more clearly defined, although slight differences
occur among countries. In the United States proof spirit (i.e., 100 degrees proof, written
100°) shall be held to be that alcoholic liquor which contains one half its volume of
alcohol of a specific gravity of 0.7939 at 15.6°C. In other words the proof is always exactly
twice the alcoholic content by volume. Thus, 100 proof spirit contains 50% alcohol. In the
British system proof spirit contains 57.1% by volume and 49.28% by weight of alcohol at
15.6°C.
A conversion factor of 1.142
FG Volume of alcohol of British Pr oof at 15.6° C = 57.1 = 1.14IJ
H Volume of alcohol of United States Pr oof at 15.6° C 50
K
is applied to convert United States proof to a British proof.
Production of Wines and Spirits
It is customary to quantify large amounts of distilled alcoholic beverages or alcohol in
proof gallons for tax and other purposes. This term simply specifies the amount of alcohol
in a gallon of spirits. Thus a United States proof gallon contains 50% ethyl alcohol by
volume; a gallon of liquor at 120° proof is 1.2 proof gallon and a gallon at 75° proof is 0.75
proof gallon. However, the ordinary United States gallon is 3.785 liters whereas the
British (imperial) gallon is larger, 4.546 liters. The British proof gallon is multiplied by
1.37 to convert it to a United States gallon:
Volume of British gallon
4.546
=
´ 1.142 = 1.375
Volume of United States gallon ´ Conversion factor
3.785
The proof is read off a special hydrometer.
(ii) Percentage by weight: This is used in Germany for the ethanol content of a beverage or
other liquid. The hydrometers are graduated at 15°C and the reference is with
(Mendeleaf’s) table of density. This results in a scale independent of the ambient
temperature.
(iii) Percentage by volume: Many countries especially in Europe use the percentage by
volume system. For most of them the hydrometer is calibrated at 15°C. France, Belgium,
Spain, Sweden, Norway, Finland, Switzerland (which also used weight %), Brazil and
Egypt, Russia use 20°C, Denmark and Italy 15.6°C whereas many South American
countries use 12.5°C. In many of these countries the specific gravity of alcohol used as
reference differ slightly.
13.3.2 General Principles in the Production of
Spirit Beverages
In general the following steps are involved in the preparation of the above beverages. The
details differ according to beverage.
(i) Preparation of the medium: In the grain beverages (whisky, vodka, gin) the grain
starch is hydrolyzed to sugars with microbial enzymes or with the enzymes of
barley malt. In all the others no hydrolysis is necessary as sugars are present in the
fermenting substrate as in brandy (grape sugar) and rum (cane sugar).
(ii) Propagation of yeast inoculum: Large distilleries produce hundreds of liters of
spirits daily for which fermentation broths many more times in volume are
required. These broths are inoculated with up to 5% (v/v) of thick yeast broth.
Although yeast is re-used there is still a need for regular inocula. In general the
inocula are made of selected alcohol-tolerant yeast strains usually Sacch. cerevisiae
grown aerobically with agitation and in a molasses base. Progressively larger
volumes of culture may be developed before the desired volume is attained.
(iii) Fermentation: When the nitrogen content of the medium is insufficient nitrogen is
added usually in the form of an ammonium salt. As in all alcohol fermentations the
heat released must be reduced by cooling and temperatures are generally not
permitted to exceed 35-37°C. The pH is usually in the range 4.5-4.7, when the
buffering capacity of the medium is high. Higher pH values tend to lead to higher
glycerol formation. When the buffering capacity is lower, the initial pH is 5.5 but
this usually falls to about 3.5. During the fermentation contaminations can have
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serious effects on the process: sugars are used up leading to reduced yields;
metabolic products from the contaminants may not only alter the flavor of the
finished product, but metabolites such as acids affect the function of the yeast. The
most important contaminants in distilling industries are lactic acid which affects
the flavor of the product.
(iv) Distillation: Distillation is the separation of more volatile materials from less
volatile ones by a process of vaporization and condensation. Three systems used
in spirit distillation will be discussed.
(a) Rectifying Stills: If the condensate is repeatedly distilled, the successive
distillates will contain components which are more and more volatile. The
process of repeated distillation is known as rectification. Rectification is done
in columns, towers, or stills containing a series of plates at which contact
occurs i.e., returned to the system. The alcohol-water mixture flows
downwards and is stripped of alcohol by steam which is introduced from the
bottom and flows upwards. Alcohol-rich distillate is withdrawn at the top of
the column. Fusel oils higher alcohols separate out just above the point of
entry of the mixture and are drawn off to another column. Volatile fractions
are composed of esters and aldehydes. Whisky and brandy may be distilled
successfully in a two-column still, but for high-strength distillates, at least
three columns and possibly four or five may be required. The above
description is of the modern still a modification of which is also used for
producing industrial alcohol. Much older-type stills, such as are described
below, continue to be used in some parts of the world.
(b) Pot Still: These are traditional stills, usually made of copper. They are
spherical at the lower-portion which is connected to a cooling coil. They are
operated batchwise. The first portion, or ‘heads’ and the latter portion, ‘tails’,
of the ‘low wines’ are usually discarded and only the middle portion is
collected. Malt whisky, rum, and brandy are made in the post still. Its
advantage is that most of the lesser aroma conferring compounds are
collected in the beverage thereby conferring a rich aroma to it.
(c) Coffey (patent) Still: The Coffey still was patented in 1830 and the various
modifications since then have not added much to the original design of the
still. Its main feature is that it has a rectifying column besides the wash
column in which the beer is first distilled.
(v) Maturation: Some of the distilled alcoholic beverages are aged for some years, often
prescribed by legislation.
(vi) Blending: Before packaging, samples of various batches of different types of a given
beverage are blended together to develop a particular aroma.
13.3.3
The Spirit Beverages
The beverages to be discussed are whisky, brandy, rum, vodka, kai-kai (or akpeteshi),
schnapps, and cordials.
Production of Wines and Spirits
13.3.3.1 Whisky
Whisky is the alcoholic beverage derived from the distillation of fermented cereal.
Various types of whiskies are produced; they differ principally in the cereal used.
Although many countries including Japan and Australia now produce whisky for
export, the countries best associated with whisky are first and foremost, Scotland
followed by Ireland, the USA and Canada.
In all whisky-producing countries the alcoholic content, the materials and the method
of preparation are controlled by government regulations. Whiskies from various
countries differ. In Scottish Malt Whisky the barley is malted just as in beer making, but
during the kilning smoke from peat is allowed to permeate the green (fresh) malt, that the
whisky made from the malt has a strong aroma of peat smoke, derived mainly from
phenol. In the United States the principal types of whisky are rye and bourbon whiskies.
Rye whisky is prepared from rye and rye malt, or rye and barley and barley malt. Bourbon
whisky is prepared from preferably yellow maize, barley malt or wheat malt. A typical
mash which will contain 51% corn, may have a composition of this type: 70% corn, 15%
rye, and 15% barley malt.
The unmalted rye or corn is cooked to gelatinize it and hence to facilitate
saccharification (or conversion to sugar) by the enzymes in the malt. The solids are not
removed from the mash and the inoculating yeast sometimes contains Lactobacillus,
whose lactic acid is said to improve the flavor of the whisky. Fermentation is usually in a
two-column coffey-type still.
All whisky is matured in wooden casks for a number of years, usually three or more.
They may then be blended with various types (usually controlled by law) before bottling.
13.3.3.2
Brandy
Brandy is a distillate of fermented fruit juice. Thus, brandy can be produced from any
fruit-strawberries, paw-paw, or cashew. However, when it is unqualified, the word
brandy refers to the distillate from fermented grape juice. It is subject to a distillation
limitation of 170° proof (85%). The fermented liquor is double distilled, without previous
storage, in pot stills. A minimum of two years maturation in oak casks is required for
maturation.
Some of the best brandies come from the cognac region of France. Brandies produced in
other parts of France are merely eau de vie (water of life) and are not called brandy. Certain
parts of Europe (e.g., Spain, where brandy is distilled from Jerez sherry) and South
America as well as the USA produce special brandies.
13.3.3.3 Rum
Rum is produced from cane or sugar by products especially molasses or cane juice. Rum
production is associated with the Carribean especially Jamaica, Cuba, and Puerto Rico. It
is also produced in the eastern USA. Rum with a heavy body is produced from molasses;
while light rum is produced from cane syrup using continuous distillation.
During the fermentation the molasses is clarified to remove colloidal material which
could block the still by the addition of sulphuric acid. The pH is adjusted to about 5.5 and
a nitrogen source ammonium sulphate or urea may be added. For the heavier rums
Modern Industrial Microbiology and Biotechnology
Schizosaccharomyces pombe is used while Saccharomyces cerevisiae is used for the lighter
types. For maturation rum is stored in oak casks for two to fifteen years.
13.3.3.4
Gin, Vodka, and Schnapps
These beverages differ from whisky, rum and brandy in the following ways:
(a) Brandy, rum and whisky are pale-yellow colored straw to deep brown by
extractives from wooden casks in which they are aged and which have sometimes
been used to store molasses or sherry. To obtain consistent color caramel is
sometimes added. Gin, vodka, and schnapps are water clear.
(b) The flavor of brandy and whisky is due to congenerics present in the fermented
mash or must. For gin, vodka, and schnapps the congenerics derived from
fermentation are removed and flavoring is provided (except in vodka) with plant
parts.
(c) The raw materials for their production is usually a cereal but potatoes or molasses
may be used. For gin, maize is used, while for vodka rye is used. The cereals are
gelatinized by cooking and mashed with malted barley. In recent times amylases
produced by fungi or bacilli have been used since the flavor of malt is not necessary
in the beverage. Congeners are removed by continuous distillation in multicolumn stills.
In gin production, the grain-spirits (i.e., without the congeners) are distilled over
juniper berries, Juniperus communis, dried angelica roots, Angelica officinalis and others
including citrus peels, cinnamon, nutmeg, etc. Russian vodka is produced from rye spirit,
which is passed over specially activated wood charcoal. In other countries it is
sometimes produced from potatoes or molasses. Schnapps are gin flavored with herbs.
13.3.3.5
Cordials (Liqueurs)
Cordials are the American name for what are known as liqueurs in Europe. They are
obtained by soaking herbs and other plants in grain spirits, brandy, or gin or by distilling
these beverages over the plant parts mentioned above. The are usually very sweet, being
required to contain 10% sugar. Some well-known brand names of cordials are Drambui,
Crème de menthe, Triple Sac, Benedictine, and Anisete.
13.3.3.6
Kai-kai, Akpeteshi, or Ogogoro
Kai-kai is an alcoholic beverage widely drunk in West Africa. It is produced by distilling
fermented palm-wine. It is the base for preparing some of the better known brands such as
schnapps.
SUGGESTED READINGS
Amerine M.A., Berg H.W., Cruess, W.V. 1972. The Technology of Wine Making. 3rd Edit Avi
Publications. West Port, USA. pp. 357–644.
Battcock, M., Azam-Ali, S. 2001. Fermented Fruits and Vegetables: A Global Perspective. FAO
Agric. Services Bull. 134, Rome, Italy. pp. 96.
Doyle, M.P., Beuchat, L.R., Montville, T.J. 2004. Food Microbiology: Fundamentals and Frontiers,
2nd edit., ASM Press, Washington DC, USA.
Production of Wines and Spirits
Flickinger, M.C., Drew, S.W. (eds) 1999. Encyclopedia of Bioprocess Technology - Fermentation,
Biocatalysis, and Bioseparation, Vol 1-5. John Wiley, New York, USA.
Gastineau, C.F., Darby, W.J., Turner T.B. 1979. Fermented Food Beverages in Nutrition.
Academic Press; New York, USA. pp. 133-186.
Gutcho, M.H. 1976. Alcoholic Beverages Processes. Noyes Data Corporation, New Jersey and
London, pp. 10 -106.
Hammond, J.R.M., Bamforth, C.W. 1993. Progress in the development of new barley, hop, and
yeast variants for malting and brewing. Biotechnology and Genetic Engineering Reviews 11,
147-169.
Hanum, H., Blumberg, S. 1976. Brandies and Liqeuers of the World. Doubleday Inc. New York,
USA.
Hough, J.S. 1985. The Biotechnology of Malting and Brewing. Cambridge University Press;
Cambridge, UK. pp. 15-188.
Okafor, N. 1972. Palm-wine yeasts of Nigeria. Journal of the Science of Food and Agriculture 23,
1399-1407.
Okafor, N. 1978. Microbiology and biochemistry of oil palm wine. Advances in Applied
Microbiology. 24: 173-200.
Okafor, N. 1987. Industrial Microbiology. University of Ife Press; Ile-Ife Nigeria. pp. 201-210.
Okafor, N. 1990. Traditional alcoholic beverages of tropical Africa: strategies for scale-up. Process
Biochemistry Intemational 25, 213-220.
Okafor, N., Aniche, G.N. 1980. Lager beer from Nigerian Sorghum. Brewing and Distilling
International. 10, 32-33, 35.
Packowski, G.W. 1978. Distilled Alcoholic Beverages, Encyclopadeia of Chemical Technology, 3rd
Edit. Wiley, New York, USA. pp. 824-863.
Singleton, V.L., Butzke, C.E. 1998. Wine. Kirk-Othmer Encyclopedia of Chemical Technology.
John Wiley & Sons, Inc. Article Online Posting Date: December 4, 2000.
Soares, C. 2002. Process Engineering Equipment Handbook Publisher. McGraw-Hill.
Taylor, J.R.N., Dewar, J. 2001. Developments in Sorghum Food Technologies. Advances in Food
and Nutrition Research 43, 217-264.
Vogel, H.C., Tadaro, C.L. 1997. Fermentation and Biochemical Engineering Handbook Principles, Process Design, and Equipment. 2nd Edit Noyes.
Zeng, A. 1999. Continuous culture. In: Manual of Industrial Microbiology and Biotechnology.
A.L. Demain, J.E. Davies (eds) 2nd Edit. ASM Press. Washington, DC, USA. pp. 151–164.
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+0)26-4
14
Production of Vinegar
Vinegar is a product resulting from the conversion of alcohol to acetic acid by acetic acid
bacteria, Acetobacter spp. The name is derived from French (Vin = wine; Aigre-sour or
sharp). With the ubiquity of acetic acid bacteria and the consequent ease with which
wine is spoilt, vinegar must have been known to man for thousands of years since he
apparently learnt to produce alcoholic beverages some 10,000 years ago. The Bible has
many references to vinegar both in the Old and New Testaments, the best known of
which, probably is: “It is consummated” which according to John (19, 29-30), was uttered
by Christ before He bowed his head and died, after he had been offered vinegar while he
was crucified on the cross. Vinegar may be regarded as wine spoilt by acetic acid bacteria,
but for which other uses have been found.
Although acetic acid is the major component of vinegar, the material cannot be
produced simply by dissolving acetic acid in water. When alcoholic fermentation occurs
and later during acidifications many other compounds are produced, depending mostly
on the nature of the material fermented and some of these find their way into vinegar.
Furthermore, reactions also occur between these fermentation products. Ethyl acetate, for
example, is formed from the reaction between acetic acid and ethanol. It is these other
compounds which give the various vinegars their bouquets or organoleptic properties.
The other compounds include non-volatile organic acids such as malic, citric, succinic
and lactic acids; unfermented and unfermentable sugars; oxidized alcohol and
acetaldelyde, acetoin, phosphate, chloride, and other ions.
14.1
USES
(i) Ancient uses: The ancient uses of vinegar which can be seen from various records
include a wide variety of uses including use as a food condiment, treatment of
wounds, and a wide variety of illnesses such as plague, ringworms, burns,
lameness, variocose veins. It was also used as a general cleansing agent. Finally, it
was used as a cosmetic aid.
(ii) Modern uses: Vinegar is used today mainly in the food industry as; (a) a food
condiment, sprinkled on certain foods such as fish at the table; (b) for pickling and
preserving meats and vegetables; vinegar is particularly useful in this respect as it
can reduce the pH of food below that which even sporeformers may not survive; (c)
It is an important component of sauces especially renowned French sauces many
Production of Vinegar
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of which contain vinegar; (d) Nearly 70% of the vinegar produced today is
supplied to various arms of the food industry where it finds use in the manufacture
of sauces, salad dressings, mayonnaise, tomato productions, cheese dressings,
mustard, and soft drinks. Most of the vinegar used in industry is the distilled or
concentrated type (see below).
14.2
MEASUREMENT OF ACETIC ACID IN VINEGAR
Just as the alcoholic content of distilled alcoholic beverages is measured in proof, the
acetic acid content is usually measured in ‘grain’. Originally the strength of acetic acid
was expressed in terms of the grains of sodium bicarbonate neutralized by one fluid
ounce measure of vinegar and this was measured by the CO2 evolved during the reaction
of the two substances. The ‘grain strength’ is now measured differently and one-grain
vinegar is now defined as that containing 0.1 gm of acetic acid in 100 ml at 20°C. Grain is
derived by multiplying the acetic acid content (w/v) of a sample of vinegar by 10 or by
moving the decimal point one place to the right. Thus, vinegar containing 8% acetic acid
is 80 grain. Sometimes the percentage (w/v) is used.
14.3
TYPES OF VINEGAR
Vinegar is normally a product of two fermentations: alcoholic fermentation with a yeast
and the production of acetic acid from the alcohol by acetic acid bacteria (Chapter 2).
There is no distillation between the two fermentations, except in the production of spirit
vinegar, which is described below. The vinegar may or may not be flavored. The substrate
for the alcoholic fermentation for vinegar productions varies from one locality to the
other. Thus, while wine vinegar made from grapes is common in continental Europe and
other vine growing countries, malt vinegar is common in the United Kingdom; the United
States on account of its great variety of climatic regions uses both malt and wine vinegars.
Rice vinegar is common in the far Eastern countries of Japan and China and pineapple
vinegar is used in Malaysia. In some tropical countries vinegar has been manufactured
from palm wine derived from oil or raffia palm.
The composition and specifications of various types of vinegars are defined by
regulations set up by the governments of different countries . In the United States, for
example, vinegar should not contain less than 4.0% (w/v) acetic acid and not more than
0.5% ethanol (v/v). The various major vinegars are defined briefly
(i) Cider vinegar, apple vinegar: Vinegar produced from fermented apple justice (US)
and non-grape fruits.
(ii) Wine vinegar, grape vinegar: Fermented grape juice malt.
(iii) Malt vinegar: Produced from a fermented infusion of barley malt with or without
adjuncts.
(iv) Sugar, glucose, dried fruits: In the US vinegar from sugar syrup or molasses should
be labeled sugar vinegar, while that from glucose (which should be dextroserotatory) and dried fruits should be labeled with ‘glucose’ or the particular fried
fruit involved.
(v) Spirit vinegar: Vinegar made from distilled alcohol. In the US synonyms for spirit
vinegar are ‘white distilled vinegar’ and ‘grain vinegar’. The alcohol used in the
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distillation is denatured for tax reasons with ethyl acetate. One gallon of ethyl
acetate is usually added to 100 gal of 95% alcohol. The ethyl acetate is not
deleterious and in any case is present in vinegar by the alcohol acetic acid reaction.
It should be noted that in the Unites States the term ‘distilled’ refers to the ethanol
used; in the United Kingdom, however, ‘distilled’ vinegar refers to a distillate of
malt vinegar.
(vi) Some specialty vinegars: Specialty vinegars make up a category of vinegar
products that are formulated or flavored to provide a special or unusual taste when
added to foods.
Specialty vinegars are favorites in the gourmet market:
(a) Herbal vinegars: Wine or white distilled vinegars are sometimes flavored with the
addition of herbs, spices or other seasonings. Popular flavorings are garlic, basil,
and tarragon, but cinnamon, clove, and nutmeg flavored vinegars can be tasty and
aromatic addition to dressings.
(b) Fruit vinegars: Fruit or fruit juice can also be infused with wine or white vinegar.
Raspberry flavored vinegars, for example, create a sweetened vinegar with a sweetsour taste.
(c) Balsamic vinegar: Traditional balsamic vinegar of Modena, Italy is made from
white and sugary Trebbiano grapes grown on the hills around Modena. The
grapes are harvested as late as possible to take advantage of the warmth of the
weather. The traditional vinegar is made from the cooked grape ‘must’ (juice)
matured by a long and slow process of natural fermentation, followed by
progressive concentration by aging in a series of casks made from different types of
wood and without the addition of any other spices or flavorings. The color is dark
brown and the fragrance is distinct. Production of traditional balsamic vinegar is
governed by the stringent standards imposed by the quasi-governmental
Consortium of Producers of the Traditional Balsamic Vinegar of Modena.
(d) Raspberry red wine vinegar: Natural raspberry flavor is added to red wine vinegar,
which is the aged and filtered product obtained from the acetous fermentation of
select red wine. Raspberry red wine vinegar has a characteristic dark red color and
a piquant, yet delicate raspberry flavor.
(e) Other specialty vinegars: Coconut and cane vinegars are common in India, the
Phillipines and Indonesia with date vinegar being popular in the Middle East.
International definitions and standards are set by the joint efforts of the Food and
Agriculture (FAO) as well as the World Health Organization (WHO) of the United
Nations. Apart from these, various professional bodies such as the Vinegar Institute (a
manufacturing association) also set standards.
14.4
ORGANISMS INVOLVED
Although vinegar had been known to man from time immemorial, like many other
fermentative processes, the identity of the organism concerned is recent. Even then much
more needs to be known about them, mainly because of the difficulty of cultivating them.
The bacteria converting alcohol to acetic acid under natural conditions are filmforming organisms on the surface of wine and beer. The film was known as ‘mother of
Production of Vinegar
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vinegar’ before its bacteriological nature became known. The bacteria were first described
as Mycoderma (viscous film) in 1822. Later other workers classified them in M. vini
(forming film on wine) an M. acetic (forming film on beer). Pasteur confirmed that acetic
acid is produced only in the presence of the bacteria, but he did not identify them. The
genus name Acetobacter was put forward by Beijerinck in 1900. Suffice it to state that
although Acetobacter spp are responsible for vinegar production, pure cultures are hardly
used, except in submerged fermentation because of the difficulty of isolating and
maintaining the organisms. The only member of the genus which is not useful, if not
positively harmful in vinegar production is Acetobacter xylinum which tends to produce
slime (Chapter 2). Recently a new species, Acetobacter europaeus, was described. Its
distinguishing features are its strong tolerance of acetic acid of 4 to 8% in A–E agar, and
its absolute requirement of acetic acid for growth.
Strains of acetic acid bacteria to be used in industrial production should a) tolerate
high concentrations of acetic acid; b) require small amounts of nutrient; c) not overoxidize the acetic acid formed; and d) be high yielding in terms of the acetic acid
produced.
The biochemical processes are simple and are shown below:
CH3CH 2OH + (O)
CH 3CHO + H2O
Ethyl alcohol oxygen
Acetaldedyde Water
CH 3CH + (OH)2
CH3CHO + H2 O
Hydrated acetaldehyde
(Aldehyde)
CH 3COOH + H2O
CH3CH(OH)2 + (O)
Dehydrogenase
Acetic acid
Theoretically, 1 gm of alcohol should yield 1.304 gm of acetic acid but this is hardly
achieved and only in unusual cases is a yield of 1.1 attained. From the reactions one mole
of ethanol will yield one more of acetic acid and more of water. It can be calculated that
1 gallon of 12% alcohol will yield 1 gal. of 12.4% acetic acid.
Over-oxidation can occur and it is undesirable. In over-oxidation acetic acid is
converted to CO2 and H2O. It occurs when there is a lack or low level of alcohol. It occurs
more frequently in submerged fermentations that in the trickle processes.
14.5
MANUFACTURE OF VINEGAR
The three methods used for the production of vinegar are the Orleans Method (also
known as the slow method), the Trickling (or quick) Method and Submerged
Fermentation. The last two are the most widely used in modern times.
14.5.1
The Orleans (or Slow) Method
The oldest method of vinegar production is the ‘let alone’ method in which wine left in
open vats became converted to vinegar by acetic acid bacteria entering it from the
atmosphere. Later the wine was put in casks and left in the open field in the ‘fielding
process’. A small amount of vinegar was introduced into a cask of wine to help initiate
fermentation. The introduced vinegar not only lowered the pH to the disadvantage of
many other organisms but also introduced an inoculum of acetic acid bacteria.
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The casks were wooden and of approximately 200 liter capacity. It was never filled
beyond about two-thirds of its capacity so that there was always a large amount of air
available above the wine. A thick film of acetic acid bacteria formed on the wine and
converted it in to vinegar in about five weeks. About 10-20% of the vinegar was drawn off
at weekly intervals and replaced with new wine. The withdrawal and replenishment
were done from the bottom of the cask so that the film would not be disturbed. Often a
series of casks was present and the transfer was done from one cask to another.
Often due to its thickness and consequent weight and sometimes due to disturbance,
the film sank. When this happened the whole process had to be restarted, since acetic
acid bacteria are aerobic. Following Pasteur’s (1868) suggestion, the film of bacteria often
developed in wooden rafts is placed in the cask for this purpose. Later on the casks were
stored, especially in the Orleans district of France, in a heated building or in an
underground cellar to speed up the process. The process now derives its name from the
district. The process had a number of disadvantages:
(a) It was slow in comparison with later methods, taking up to five weeks sometimes
as against days, hence it is also known as the slow method.
(b) It was inefficient, yielding 75-85% of the theoretical amount.
(c) The ‘mother of vinegar’ usually gradually filled the cask and effectively killed the
process.
Despite these disadvantages the product was of good quality and it continued to be
used in many European countries long after the introduction of the Quick Process,
described below. Modern vinegar production uses mainly the Trickling (quick) and
submerged methods to be described below. There are fewer and fewer of the Orleans
equipment in use today.
14.5.2
The Trickling Generators (Quick) Method
Credit for devising the fore-runner of the modern trickling generator is usually given to
the Dutch Boerhaave who in 1732 devised the first trickling generator in which he used
branches of vines, and grape stems as packing. Improvements were made by a number of
other people from time to time. Later ventilation holes were drilled at the bottom of the
generator and provided a mechanical means for the repeated distribution of the alcohol
acetic acid mixture over the packing. The heat generated by the exothermic reaction in the
generator caused a draft which provided oxygen for the aerobic conversion of alcohol to
acetic acid. This latter model of the quick method (sometimes called the German method)
enabled the production of vinegar in days instead of in weeks. It remained in vogue
unmodified for just over a century when several modifications were introduced in the
Frings method, including: (a) forced aeration, (b) temperature control, and (c) semicontinuous operation.
The modern vinegar generator consists of a tank constructed usually of wood
preferably of cypress and occasionally of stainless steel. A false bottom supports the coils
of birchwood shavings and separates them from the collection chamber which occupies
about one fifth of the total capacity of the generator (Fig. 14.1). A pump circulates the
alcohol-acetic acid mixture from the reservoir through a heat exchanger to the top of the
generator where a spray mechanism distributes it over the packing in much the same way
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Fig. 14.1 Trickling Generator for Vinegar Manufacture
as a trickling filter functions in waste-water treatment (Chapter 29). Air is forced through
the false bottom up through the set-up. The cooling water in the heat exchanger is used to
regulate the temperature in the generator so that it is between 29°C and 35°C; this is
determined with thermometers placed at different levels of the generator.
The top of the generator is covered but provision exists for exhaust air to be let out.
Meters measure three parameters: (a) the circulation of the mash, (b) the flow of cooling
water through the heat exchange, and (c) the amount of air delivered through the system.
If the air flow rate is too high alcohol and vinegar are lost in effluent air.
Operation of the generator: The trickling or circulating Frings generator is reasonably
efficient, achieving, when operating maximally, an efficiency of 91-92% and it is capable
of producing 500–1000 gallons of 100-grain (i.e. 10%) vinegar every 24 hours. Although
the wood shavings soften with age, well-maintained generators can proceed without
much attention for twenty to thirty years. They are easy to maintain once airflow and
recirculation rates as well as temperatures are maintained at the required level. The level
of ethyl alcohol must be maintained so that it does not fall below 0.3-0.5% at any time.
Complete exhaustion of the alcohol will lead to the death of the bacteria.
When wine and cider vinegar are made no nutrients need be added to the charge (i.e.,
the alcohol-containing material). However, when white vinegar (produced from
synthetic alcohol is used) nutrients e.g. simple low concentration sugar-mineral salts
solution sometimes containing a little yeast extraction may be added. Growth of the
slime-forming Acetobacter xylinum is less with white vinegar (from pure alcohol) than
with wine and cider vinegar. Generators for producing white vinegar therefore become
blocked by slime much less quickly than those used for wine and cider vinegar, and can
last far in excess of 20 to 30 years before the wood shavings are changed.
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The finished acidity of the vinegar is about 12%; when it is higher, production drops
off. In order not to exceed this level of acidity, when drawing off vinegar, the amount of
alcohol in the replacement should be such that the total amount of alcohol is less than 5%.
14.5.3
Submerged Generators
With knowledge in submerged fermentation gained from the antibiotics and yeast
industry it is not surprising that vinegar production was soon produced by the method.
Several submerged growth vinegar generators have been described or are in operation.
The common feature in all submerged vinegar production is that the aeration must be
very vigorous as shortage of oxygen because of the highly acid conditions of submerged
production, would result in the death of the bacteria within 30 seconds. Furthermore,
because a lot of heat is released (over 30,000 calories are released per gallon of ethanol) an
efficient cooling system must be provided. All submerged vinegar is turbid because of the
high bacterial content and have to be filtered. Some submerged generators will be
discussed below.
14.5.3.1
Frings acetator
First publicized in 1949, most of the world’s vinegar is now produced with this
fermentor. It consists of a stainless steel tank fitted with internal cooling coils and a highspeed agitator fitted through the bottom. Air is sucked in through an air-meter located at
the top. It is then finely dispersed by the agitator and distributed throughout the liquid.
Temperature is maintained at 30°C, although some strains can grow at a higher
temperature. Foaming is interrupted with an automatic foam breaker. Essentially it is
shaped like the typical aerated stirred tank fermentor described in Chapter 9).
It is operated batchwise and the cycle time for producing 12% vinegar is about
35 hours. Details of the parts of the Frings acetator are shown in Fig. 14.2. It is selfaspirating, no compressed air being needed. The hollow rotor is installed on the shaft of
a motor mounted under the fermentor, connected to an air suction pipe and surrounded
by a stator. It pumps liquid that enters the rotor from above outward through the channels
of the stator that are formed by the wedges, thereby sucking air through the openings of
the rotor and creating an air–liquid emulsion that is ejected outward at a given speed.
This speed must be chosen adequately so that the turbulence of the stream causes a
uniform distribution of the air over the whole cross section of the fermentor.
The Frings alkograph automatically monitors the alcohol content and signals the end
of the batch when the alcohol content falls to 0.2% (v/v). At this stage about one third of
the product is pumped out and fresh feed pumped to the original level. The aeration must
continue throughout the period of the unloading and loading. The Frings Alkograph is
an automatic instrument for measuring the amount of ethanol in the fermenting liquid.
Small amounts of liquid flow through the analyzer continuously, at first through a
heating vessel and then through three boiling vessels. The boiling temperature of the
incoming liquid is measured in the first boiling vessel. While alcohol is distilled off
continuously from the second and the third boiling vessel, the higher boiling point of the
liquid from which ethanol has been removed is measured in the third boiling vessel. The
difference in temperature corresponds to the ethanol content and is recorded
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a = hollow rotor; b = stator;
c = air suction pipe; d = openings for air exit;
e = wedges to form the channels;
f = channels to form the beams of air–liquid emulsion
Fig. 14.2
The Frings Acetator
automatically. As the flow through the vessels takes some time, there is a delay of about
15 minutes between the beginning of the inflow of a sample and the appearance of the
correct value on the recorder. However, because alcohol concentration is decreasing
slowly during fermentation, this delay has no disadvantage on fermentation control. In
more modern alkographs there is no time gap.
The more recent Frings acetators can be run on a semi-continuous basis. To carry out
the single-stage semi-continuous process at a defined alcohol content, a contact in the
Alkograph activates the vinegar discharge pump. As soon as a preset level has been
reached, the mash pump starts adding fresh mash. This pump is controlled by the
fermentation temperature in order to refill under constant temperature. The pump is
stopped when the desired level is reached and an automatic cooling system is activated.
A fermentation cycle takes 24 to 48 hours Since its first description, improvements and
modifications have been made on the Frings acetator. One in recent operation is shown in
Fig. 14.2 .
Advantages
(a) The efficiency of the acetator is much higher than that of the trickling generator; the
production rate of the acetator may be 10-fold higher than a trickling unit. Values of
94% and 85% of the theoretical have been recorded for both the acetator and the
trickling filter.
(b) The quality is more uniform and the inexplicable variability in quality noted for the
trickling generator is absent.
(c) A much smaller space is occupied (about one-sixth) in comparison with the
trickling generator.
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Modern Industrial Microbiology and Biotechnology
(d) It is easy and cheap to change from one type of vinegar to another.
(e) Continuous production and automation can take place more easily with Frings
acetator than with trickling.
Disadvantages
(a) A risk exists of complete stoppage following death of bacteria from power failure
even for a short time. Automatic stand-by generators have helped to solve this
problem.
(b) It has a high rate of power consumption. Some authors have however argued that
in fact in terms of power consumed per gallon of acetic produced the acetator is less
power consuming.
14.5.3.2
The cavitators
The cavitator was originally designed to treat sewage: it was then modified for vinegar
production. In many ways it resembled the acetator. However, the agitator was fixed to
the top and finely dispersed air bubbles are introduced into the liquid. It operated on a
continuous basis and was quite successful in producing cider and other vinegars as long
as the grain strength was low. It was not successful with high grain vinegar and the
manufacture of the ‘cavitator’ was discontinued in 1969. It is mentioned here only for its
historical interest, although some are still being used in Japan and the US.
14.5.3.3
The tower fermentor
The tubular (tower) fermentor developed in the UK has been used on a commercial scale
for the production of beer, vinegar, and citric acid. The fermentor is two feet in diameter,
about 20 feet tall in the tubular section with an expansion chamber of about four feet in
diameter and six feet high. It has a working volume of 3,000 liters and aeration is
achieved by a stainless steel perforated plate covering the cross section of the tower and
holding up the liquid. The charging wort is fed at the bottom. The vinegar overflows in a
quiet Y-shaped area free of rising gas. The unit can produce up to 1 million gallons
(450,000 liters) of 5% acetic acid per annum. The Acetobacter sp requires a month to adapt
to the new system. The system can be batch, semi or fully-continuous without noticeable
differences in the quality of the product.
14.6
PROCESSING OF VINEGAR
(a) Clarification and bottling: Irrespective of the method of manufacture, vinegar for
retailing is clarified by careful filtration using a filter aid such as diatomaceous
earth. Vinegar from trickling generators are however less turbid than those from
submerged fermentations because a high proportion of the bacterial population
responsible for the acetification is held back on the shavings. After clarification it is
pasteurized at 60-65°C for 30 minutes.
(b) Concentration of vinegar: Vinegar can be concentrated by freezing; thereafter the
resulting slurry is centrifuged to separate the ice and produce the concentrate.
With this method 200° grain (i.e., 20% w/v) acetic acid can be produced.
Concentration is necessitated by two considerations. One is the consequent
Production of Vinegar
&'
reduction in transportation costs. The other is the need to prevent loss of activity of
the vinegar when cucumbers were picked in it after first being soaked in brine.
SUGGESTED READINGS
Asai, T. 1968. Acetic Acid Bacteria University of Tokyo Press/University Park Press, Baltimore.
Ebner, H., Sellmer-Wilsberg, S. 1997. Vinegar, Acetic Acid Production. Kirk Othmer’s
Encyclopedia of Science and Technology. John Wiley, New York, USA.
Flickinger, M.C., Drew, S.W. (eds) 1999. Encyclopedia of Bioprocess Technology - Fermentation,
Biocatalysis, and Bioseparation, Vol 1-5. John Wiley, New York, USA.
Greenshilds, R.N. 1978. In: Primary Products of Metabolism. A.H. Rose, (ed). Academic Press
New York, USA. pp. 121-186.
Wagner, F.S. 2002. Acetic Acid. Kirk-Othmer Encyclopedia of Chemical Technology
John Wiley & Sons, Inc. Article Online Posting Date: July 19, 2002.
Webb, A.D. 1997. Vinegar. Kirk-Othmer Encyclopedia of Chemical Technology John Wiley &
Sons, Inc. All rights reserved. Article Online Posting Date: December 4, 2000.
Modern Industrial
Microbiology and Biotechnology
Section
Use of Whole Cells for
Food Related Purposes
-
Modern Industrial
Microbiology and Biotechnology
+0)26-4
15
Single Cell Protein (SCP)
The term ‘Single Cell Protein’ (SCP) is a euphemism for protein derived from microorganisms. It was coined by Professor Wilson of the Massachusetts Institute of
Technology to replace the less inviting ‘microbial’ or ‘bacterial’ protein or ‘petroprotein’
(for cells grown specifically on petroleum). The term has since become widely accepted.
In the 1950s and 1960s concern grew about the ‘food gap’ between the industrialized and
the less industrialized parts of the world, especially as there was rapid and continuing
population growth in the latter. As a result of this concern, alternate and unconventional
sources of food were sought. It was recognized that protein malnutrition is usually far
more severe than that of other foods. The hope was that microorganisms would help meet
this world protein deficiency. It was not thought that SCP would replace the need to
increase proteins from plants such as oil beans or from animals such as fish. However,
the limitations of conventional sources of proteins were recognized. These include: (a)
possible crop failure due to unfavorable climatic conditions in the case of plants; (b) the
need to allow a time lapse for the replenishment of stock in the case of fish; (c) the limited
land available for farming in the case of plant production. On the other hand the
production of SCP has a number of attractive features: (a) it was not subject to the
vicissitudes of the weather and can be produced every minute of the year. (b)
Microorganisms have a much more rapid growth than plants or animals. Thus a bullock
weighing 10 hundred weight would synthesize less than 1 lb (or 1 of its weight) of
10,000
protein a day, 10 hundred weight of yeasts would produce over 50 tons (or over 100
times) of their own weight of protein a day. Furthermore, (c) waste products can be turned
into food in the production of SCP.
SCP is itself not entirely lacking in disadvantages. One of the most obvious is that
many developing countries, where protein malnutrition actually exists, lack the expertise
and/or the financial resources to develop the highly capital intensive fermentation
industries involved. But this short-coming can be bridged by the use of improvised
fermentors and recovery methods which do not require sophisticated equipments. Other
criticisms of SCP are that microorganisms contain high levels of RNA and that its
consumption could lead to uric acid accumulation, kidney stone formation and gout.
These are discussed later.
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Modern Industrial Microbiology and Biotechnology
As had been stated earlier microorganisms began receiving attention as food on a
worldwide basis in the late 1950s and early 1960s. Nevertheless they have for centuries
been consumed in large amounts, either wholly or as part of a meal or alcoholic beverage
by man, although he did not always recognize this. In many tropical countries, palm
wine and sorghum beers which have high suspensions of bacteria and yeast have been
consumed for centuries. Fermented milks and yoghurts which have been consumed from
ancient times right up to the present day contain large amounts of bacteria and yeasts
(1012 -1014 /ml). In Chad (Central Africa) the blue-green alga, Spirulina has been eaten for
centuries as did also the Aztecs of South America.
The organized and deliberate cultivation of micro-organisms for food, however, is
relatively recent. During the First World War (1914-1918) baker’s yeasts, Saccharomyces
cerevisiae, were grown on a molasses-ammonium medium. Development continued in
between the wars and in the second world war (1939-1945), Geotrichum lactis, (Odium
lactis), Endomyces vernalis, and Candida utilis were grown for food.
15.1 SUBSTRATES FOR SINGLE CELL PROTEIN
PRODUCTION
A wide variety of substrates have been used for SCP production and include hydrocarbons, alcohols, and wastes from various sources.
15.1.1
Hydrocarbons
Patterns in the utilization of hydrocarbons by microorganisms have been summarized in
the so-called Zobell’s rules and shown in a modified form below:
a. Aliphatic hydrocarbons are assimilated by strains of yeasts in many genera. Other
classes of hydrocarbons, including aromatics may be oxidized but are not usually
efficiently assimilated, if at all.
b. n-Alkanes of chain length shorter than n-nonane are not usually assimilated, but
may be oxidized. Yield factors increase but the rate of oxidation decreases with
increasing chain length from n-nonane.
c. Unsaturated compounds are degraded less readily than saturated ones.
d. Branched chain compounds are degraded less readily than straight chain
chemical compounds.
15.1.1.1 Gaseous hydrocarbons
Among the gaseous hydrocarbons, methane has been most widely studied as a source of
SCP. Others which have been studied include propane and butane. Methane is the
predominant gas in natural gas, (Table 15.1) whether such natural gas is associated with
oil wells (‘casinghead gas’) or not. Natural gas is plentiful over the world and when
present, is cheap. Indeed in many oil fields, it is flared. Perhaps its greatest advantage is
the absence of residual hydrocarbon in the single cell protein produced from it, unlike the
case with liquid hydrocarbons. One of its major disadvantages is that it is highly
inflammable.
Single Cell Protein
'#
Table 15.1 Composition of natural gas
Gas
Methane
Ethane
Propane
%
82-90
4-8
2-3
Others
iso-butane, n-Butane, iso-pentane,
n-Potone, Heptanes plus CO2,
Nitrogen
Less than 1%
Methane is the most widely studied gaseous hydrocarbon for SCP production. Single
cultures in methane are usually very slow growing. Single cell protein prodduction from
methane has used continuous cultures and a mixed population of microorganisms. The
advantages of a mixed methane are higher growth rate, higher yield coefficient, greater
stability resistance to contaminations and a reduction in foam production. It has been
suggested that the various members of a four-organism mixture had the following
functions (Fig. 15.1): the unnamed methane bacterium utilizes methane slowly alone and
produces methanol. Hyphomicrobium utilizes the methanol, whereas the other members,
Flavobacterium and Acinetobacter (which do not grow on methane) remove waste products.
The result is a fast growing mixture.
15.1.1.2
Liquid hydrocarbons
The major source of liquid hydrocarbons is crude petroleum. These hydrocarbons were
first studied as a source of microbial vitamins and lipids. Later these studies were
extended in the late 1940s to the feeding of whole paraffin-grown bacterial and yeast cells
to rats. The first important move to grow cells on paraffin on a commercial scale was for
‘dewaxing’ i.e., removal of higher n-alkanes from crude petroleum fractions (see below).
With the concern for world shortage of food, protein production soon became the goal.
Crude petroleum is highly variable in composition, differing from one part of the
world to the other. However, most crude petroleum oils are made up of 90-95%
hydrocarbons, which are most often saturated. During petroleum refining, the crude oil is
first distilled at atmospheric pressure in a process known as ‘topping’. The products of
this primary distillation for various temperatures during topping and use of these
fractions are shown in Table 15.2. The components left after topping contain large
quantities of normal alkanes with carbon atoms longer than C8. Such higher alkanes are
crystalline solids at room temperature. It was this removal (or dewaxing of solid nparaffins or waxes which first attracted the use of microorganisms. (After topping further
distillation is done under vacuum). The petroleum hydrocarbons which have been used
to grow SCP are diesel oil, gas oil, fuel oil, n-alkanes (C10 - C30 and C14 – C18, C11 – C18, C10
- C18) n-hexadecane, n-dodecane.
British Petroleum (BP) pioneered the use of petroleum fractions in SCP production and
by 1973 had the largest number of patents in the field. Soon after, many other oil
companies and governments all over the world set-up research and pilot plants. Plans to
build production plants were made by some.
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Modern Industrial Microbiology and Biotechnology
Methane Utilising bacterium
CH4
CH4
CH30H
Cell biomass
metabolites
Metabolites removed by
Flavobacterium sp.
Acinetobacter sp.
CH3 0H
Hyphomicro bium sp.
CH3 0H
cell biomass
Fig. 15.1 Schematic Outline of Postulated Interactions of Methane Utilizing Organisms
Table 15.2 Products of the primary distillation of crude petroleum
Primary Fraction
Light gasoline
Medium naphtha
Heavy naphtha
Light gas oil
Heavy gas oil
Residue
Cut Point
100°C
150°C
200°C
300°C
360°C
Final Product and Boiling Range
Gasoline 20-15°C
Kerosene 120–200oC
Gas oil 200-350oC
Blends of the appropriate
Primary fractions
In BP’s plant in Scotland a material from the distillation column is passed through a
molecular sieve so that only the part readily assimilated by micro-organism i.e. nparraffins (specifically 97.5-99% nC10 – C33 boiling range 30-33°C) is allowed into the
fermentor under aseptic conditions. In the other plant in Lavera in the South of France,
Single Cell Protein
'%
which was not aseptic gasoil was used untreated (i.e., not passed through a molecular
sieve). About 10% of the gas oil was used and the remaining 90% returned to the refinery.
Solvent extraction was used to remove the last traces of oil from the yeast creams (Candida
tropicalis) including the 0.5 ton yeast lipids. By 1963, five tons dry weight of yeast was
produced by this method per day. There was little difference between the two in terms of
yeast composition. In terms of economics, marginal advantage accrued to the Lavera
(dewaxing) process.
Since the oil boycott of 1973-1974 crude oil prices have risen sharply and the initial
attraction to the use of crude oil as a substrate for SCP has been eroded. Consequently it is
doubtful that the greatly raised expectations of SCP from petroleum is likely to be
achieved. Indeed many of the plans announced by many oil companies for production
stage fermentors were soon abandoned.
15.1.2
Alcohols
While work on SCP production from n-paraffin and gas oil was in progress, alternatives
to petroleum based substrates were sought. Methanol and ethanol are such alternatives.
15.1.2.1
Methanol
Methanol is produced by the oxidation of paraffins in the gas or liquid phase or by the
catalytic reduction by hydrogen of CO and CO2, either singly or mixed. The catalysts are
mixed zinc and chromium oxides. The source of the feed gas is natural gas, fermentation
or fuel gas.
Methanol is suitable as a substrate for SCP for the following reasons: (a) it is highly
soluble in water and this avoids the three-phase (water-paraffin-cell) transfer problems
inherent in the use of paraffins; (b) the explosion hazard of methanol is minimized in
comparison with methane-oxygen mixtures; (c) it is readily available in a wide range of
hydrocarbon sources ranging from methane to naphtha; (d) it can be readily purified in a
process which avoids the carry over of the most toxic polycyclic aromatic compounds; (e)
it requires less oxygen than methane for metabolism by micro-organisms and hence a
lower cooling load; (f) it is not utilized by many organisms.
The use of methanol as a SCP substrate has received attention by oil companies in
Italy, West Germany, Norway, Sweden, Israel, the United Kingdom, and the United
States. One of the most advanced in all these countries is the project of the Imperial
Chemical Industries (ICI) which using the bacterium, Methylophilus methylotropha was
due to start the annual production of several tons of proprietary ‘Pruteen’ in Billingham,
the UK, using the loop fermentor, (‘pressure cycle fermentor’).
Over 20 species from the genera Hansenula, (Hansenula polymorpha Pichia, Torulopsis
and Candida have been shown to grow on methanol.
15.1.2.2
Ethanol
Ethanol may, of course, be produced by the fermentative activity of yeasts. In the synthetic
process however, it is produced by the hydration of ethylene which itself is obtained
during petroleum refining from coke oven gas, the vapor-phase cracking of petroleum or
the propane-butane cut of stabilizer gas. Ethylene is absorbed by concentrated H2 SO4 to
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Modern Industrial Microbiology and Biotechnology
form ethyl hydrogen sulfate. The dilution of the acid with water causes hydrolysis to
ethyl alcohol and H2SO4 The alcohol is then distilled off.
Although ethanol can be utilized ordinarily by many bacteria and yeasts, as a
substrate for SCP, it is largely used by yeasts. Ethanol has the following advantages:
(a) Since it is already consumed in alcoholic beverages it is not quite as suspect a
substrate for SCP as are gas oil and n-paraffins. (b) It is like methanol, highly miscible
with water and hence more easily available than the three-phase paraffin system. (c)
Ethanol in contrast with methane can be more safely stored and transported (d) As,
unlike methanol, it is non-toxic it can be more easily handled. (e) Ethanol is partially
oxidized. For these reasons, the fermentation of ethanol for SCP production requires
comparatively less oxygen and hence releases considerably less heat than if it were
unsaturated.
The major disadvantage in using ethanol for SCP production is that it is expensive,
even when produced by the catalytic method described above. Despite this advantage
yeast produced from ethanol is being produced and marketed as a flavor enhancer in
baked foods, pizzas, sauces, etc., in the United States by the Amoco Oil Company in a
plant which will ultimately produce 15 million lbs per annum. The yeast used is Candida
utilis.
In Japan the Mitsubishi Oil Company has developed strains of Candida
acidothermophilum which grow at a lower pH value and higher temperature than Candida
utilis. These properties should help reduce costs by minimizing the need for asepticity
and cooling, as also is the use of unpurified ethanol derived from the process described
above. The pilot plant production is 100 tons per annum. In Spain Hansenula anomala is
used. Ethanol-based SCP is also produced in Czechoslovakia and the USSR. In
Switzerland a joint project between Nestles (the food Company) and Exxon (the US Oil
Company) utilizes a bacterium Acinetobacter caloaceticum rather than a yeast. Unlike many
other plants it is directed primarily towards human consumption hence reduction in the
nucleic acid content is important.
15.1.3
Waste Products
In recent times petroleum prices have continued to soar; it is therefore unlikely that
petroleum-based substrates such as synthetically produced methanol and ethanol, gas
oil, etc., will be much less used in the future. Indeed many projects designed to operate on
them are already being shelved. It is not however the end of the SCP story, because
attention is being turned more and more to substrates derived from plants which are
renewable during photosynthesis. Usually however these are obtained as waste
products from various sources.
A large number of reports of SCP production from waste material lies scattered in the
literature. They may be discussed under the following general headings:
(i) Plant/wood wastes: These are cellulose containing materials. The major difficulty with
them is that cellulose is crystalline and highly resistant to fermentation without prior
treatment. When lignin is present as is usually the case the resistance is even greater as it
protects cellulose from direct attack. This is why wastes from manufacturing processes,
such as sulfite pulping which must necessarily break down lignin, yield wastes which
Single Cell Protein
''
are comparatively easy to handle. Methods which convert lignocellulosic materials to
fermentable sugars were discussed in Chapter 4.
Plant wastes containing cellulose include corn cobs, plant stems, leaves, stalks, husks,
etc. For them to be used for SCP production, they usually have to be treated in some form
such as ball-milling, acid, alkali, sodium chlorate or liquid ammonia treatment. The
material may then be digested by a chemical means or by the use of microorganisms.
Cellulosic agricultural wastes are available in large amounts all over the world; they are
usually of little economic value, and are non-toxic. However, they are usually widely
scattered and any process which aims at utilizing them must take into account the cost of
collecting and storing them, as well as the fact that they vary widely in their content of
cellulose and other materials. It is ironic that the tropical countries of the world which
may be expected to have large amounts of plant wastes and which are also the areas most
critically hit by protein shortage usually do not have the manpower, finance to purchase,
or expertise to run, these fermentation equipments. It is encouraging that some studies
aimed specifically at developing countries exist. For example the high points of the
procedure being pursued by Tate and Lyle Ltd, the British sugar manufacturing
Company, is the use of labor-intensive methods employing fermentor and other
equipments fashioned from relatively cheap materials. Many developing countries in
Africa/Asia and South American can indeed adopt these methods and produce SCP
locally from agricultural wastes.
(ii) Starch-wastes: Starch-containing wastes from rice, potatoes, or cassava manufacturing
industry are relatively easy to utilize in SCP production in comparison with cellulosic
agricultural wastes. Starch hydrolysis is relatively easily achieved with either whole
microbial cells or enzyme. A very interesting procedure is the Symba Process developed
by the Swedish Sugar Corporation. In this process two yeasts are used symbiotically:
Endomycopsis fibuligera hydrolyses starch to the sugars glucose and maltose with alpha
and beta amylases. Candida utilis then utilizes these sugars for growth.
(iii) Dairy wastes: Whey is a by-product of the diary industry resulting from the removal of
proteins (and fat) in cheese manufacture. It is a liquid rich in lactose which can be
obtained in concentrated forms from cheese manufacturers and can then be suitably
diluted to give the desired lactose concentration. Saccharomyces fragilis is grown in it for a
high-quality edible food yeast. The process can be adjusted to produce either SCP or
alcohol. Due to the cost of aeration, the authors recommend the concomitant manufacture
of SCP and alcohol under anaerobic conditions. In the closed-loop continuous system
described by the authors no effluent results.
(iv) Wastes from chemical industries: Various substrates from chemical industries can be
utilized for SCP production, provided they contain sufficient amounts of utilizable
carbon sources. Thus, C. lipolutica or Trichosporon cutaneum can be used for SCP
production in oxanone water, a waste mixture of organic acids from the copralactam
used for the manufacture of nylon.
(v) Miscellaneous substrates: Molasses the by-product of the sugar industry is a well-known
raw material for microbial industries (Chapter 4). Its use for food yeast production, a form
of SCP, will be described in the next chapter.
A wide variety of substrates may be, and have been, used for SCP production. These
include coffee wastes, coconut wastes, palm-oil wastes, citrus waste, etc. In the study of
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Modern Industrial Microbiology and Biotechnology
any hitherto unexplored waste source, what is required is to determine what
pretreatment, if any, is required and search for the appropriate organism which will grow
in the hydrolysate.
15.2
MICROORGANISMS USED IN SCP PRODUCTION
A list of selected organisms currently used in SCP production is given in Table 15.3.
Obviously that list is not exhaustive; new organisms may be discovered or new strains
may be developed from existing strains.
The properties required in industrial organisms in general have been described in
Chapter 1. In addition organisms to be used in SCP production should have the following
properties:
(a) Absence of pathogenicity and toxicity: It is obvious that the large-scale cultivation of
organisms which are pathogenic to animals or plants could pose a great threat to
health and therefore should be avoided. The organisms should also not contain or
produce toxic or carcinogenic materials.
(b) Protein quality and content: The amount of protein in the organisms should not only
be high but should contain as much as possible of the amino acids required by
man.
(c) Digestibility and organoleptic qualities: The organism should not only be digestible,
but it should possess acceptable taste and aroma.
(d) Growth rate: It must grow rapidly in a cheap, easily available medium.
(e) Adaptability to unusual environmental conditions: In order to eliminate contaminants
and hence reduce the cost of production, environmental conditions which are
antagonistic to possible contaminants are often advantageous. Thus, strains
which grow at low pH conditions or at high temperature are often chosen.
The heterotrophic microorganisms currently used are bacteria (and actinomycetes
and fungi (moulds and yeasts); protozoa have not been used in SCP production. Of the
substrates currently in use, the gaseous hydrocarbons (methane, propane, butane) are
almost exclusively attacked by bacteria. Liquid hydrocarbons (n-paraffins, gas oil, diesel
oil) and alcohols are utilized by both bacteria and yeasts. Of the carbohydrates sugar is
readily utilized by all the classes of microorganisms; a large number of them can also
utilize starch. Cellulose is not generally utilized directly by many microorganisms save
after treatment. Cellulose and other materials in peanut shells, carob beans, spoiled
fruits, corn and pea wastes, sugarcane bagasse, palm, cassava wastes have been used to
make SCP using the moulds Trichoderma sp., Glicladium sp., Geotrichum sp., Fusarium, and
Aspergilus. Paecilomyces variotii is used in the Pekilo sulfite liquor SCP method. Fungi
have the advantage that they are lower in RNA content and are easily harvested.
15.3 USE OF AUTOTROPHIC MICROORGANISMS IN
SCP PRODUCTION
Autotrophic organisms include the photosynthetic bacteria and algae. Most of the work
on SCP production by autotrophic microbes seems to be limited to the algae. It does not
Single Cell Protein
!
Table 15.3 Organisms and substrates which have been used for single cell protein production
Gaseous hydrocarbons
i) Methane
ii) Propane
iii) Butane
Liquid Hydrocarbons:
i) n-Alkanes (C10 –C30)
Bacteria
Methanomones sp.
Methylococius capsulatus
Pseudononas sp.
Hyphomisobium sp. Mixed
Acinetobacter sp.
Flavobacterium sp.
Arthrobacter simplex
Nocardia paraffinica
Nocardia paraffinica
Mycobacterium phlei
Nocardia sp.
carbon length
ii) n-Alkanes (unspecified)
iii) Liquified petroleum gas
iv) Gas oil
v) Diesel oil
Alcohols
Methanol
Ethanol
Plant/Wood Wastes
Sulphite liquor
Cellulose pulping fines
Mesquite wood
Wheat bran
Wastes from carb,
Palms, papaya, etc.
Starch Wastes
Potato hydrolysate
Tapioca (Cassava) starch
Diary Wastes
Whey
Sugar Wastes
Molasses
Chemical Industries Wastes:
Oxanone Waste Water
Waste polyethylene
Fungi
Acinetobacter
Pseudomonas
Acromobacter delcavate
Methylomonas Methanolica
Methyliphilus (Pseudomonas)
methylotrophus
Candida guillermondi
Candida lipolytica
Candida Kofuensis
Candida tropicals
Candida lipolylica
Candida rigida
Candida tropicalis
Candida lipolytica
Torulopsis methansoba
Toralopsis methanolove
Candida boidini
Methylomonas clara
Hansemula polymorpha
Candid ethanomorphium
Candida tropicalis
Thermomonespore fusca
Brevibacterium sp.
Paecylomyces variotii
Candida utilis
Rhodopseudomonas glatinosa
Fusarium sp
Aspergillus sp
Rhodotromla rubra
Candida tropicalis
Endomycopsis
Libuligera
Kluyveromyces fragilis
Trichosporon cutaneum
Candida utilis
Trichosporon cutaneum
Candida pseudotropicalis
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Modern Industrial Microbiology and Biotechnology
appear that photosynthetic bacteria hold out much hope for use as SCP sources because
they require anaerobic conditions for photosynthesis. These conditions are difficult to
provide and maintain.
The annual production of the oceans and seas of the world which harbor the bulk of
the world’s algae is very high – some 550 x 109 tons – about 100 tons per annum for every
human being alive. Man consumes algae through fish for which the algae serve as food.
The algae themselves are rich in protein and could be harvested for this purpose.
However, the concentration of algae in marine water is only 3 mg/litre whereas for
economic viability the harvest should be at least 250 mg/litre. This is the first reason why
algae need to be cultivated and produced in high concentrations.
The second reason is that, when given adequate conditions, 20 tons (dry weight) of
algae having a protein concentration of 50% can be produced per acre of pond per
annum. In terms of the yield of digestible protein, this is 10-15 times greater than the soya
bean and 25-50 times more than the same area planted with corn. From the view point of
good energy the yield per acre of algae in terms of dietary energy is eight times as great as
sugar beet, and between 22 and 45 times as great as that of corn and potato respectively.
Third, in terms of water use for the same amount of protein, much less water is required
(Table 15.4).
Table 15.4
Comparison of water use for food production by some conventional crops and by
algae
Annual Protein
Yield (Ib/Acre)
Soya bean
Corn
Wheat
Algae
576
240
135
20,000
Annual Water
Consumption Acre/Feet/Acre
2.0
2.0
1.5
4.0
Ib Protein/
Acre Food
288
120
90
5,000
Capital investment involving various facets of the development of land, energy
consumption, and manpower utilization are about the same when conventional and
algal farming are compared.
Feeding tests in animals (using the green algae Scendemus and Cholorella) showed that
in general algae had beneficial effects if fed to animals in small amounts at a time. Feeding
in large amounts for long periods was more successful if they were supplemented by
proteins from other sources. Humans consuming foods containing algae, did so for
periods of up to 20 days with only minor abdominal upset due, probably, to the novelty of
the foods. However, due to the peculiar taste of the algae, such foods would probably be
more immediately acceptable in communities such as in Chad or in Mexico which have
developed a taste for the blue-green algae Spirulina through generations of consumption.
For the highest algal yields carbon dioxide is supplied to algae growing in day light;
where natural saline water rich in bicarbonates is available, such as is found in Chad
Republic or in Lake Texicoco in Mexico, supplementation with CO2 is not necessary.
Effluents emanating from sewage treatment with their rich content of minerals would
be ideal for growing algae for animal feeding. The resulting algae should be heat-treated
to avoid any possibility of pathogen transmission.
Single Cell Protein
!!
With the advantages of algae cultivation mentioned earlier, particularly the
comparatively low capital investment, the digestibility by ruminants and other animals
the possibility of integrating waste disposal with algae production and above all the
availability of sunlight and warm temperatures throughout the year, the tropics should
be the place for algae cultivation for animal feeding.
As can also be seen from Table 15.5 the production cost of protein per kilogram is very
favorable for SCP when compared with other protein sources.
Table 15.5
Production of various proteins (1980 figures, USA, $ dollars)
Beef
Pork
Poultry
Cheese
Soy flour
Peanut flour
Yeast from n-alkanes
Yeast from molasses
Fungi from celluloses
Algae
15.4
$ kg-1
% Protein
$ per kg protein
1.54
1.10
0.66
0.78
0.15
0.15
0.42
0.33
0.15
0.66
15
12
20
24
52
59
53
53
43
46
20.3
19.1
6.6
6.5
0.6
0.5
1.4
1.3
0.7
2.8
SAFETY OF SINGLE CELL PROTEIN
Probably on account of the novelty of SCP as food it has met with very strong opposition
especially in some countries, notably Japan and Italy. The public in the former country
had become aware of the health hazards of environmental pollution and in particular the
‘minimata disease’ which was due to the consumption of mercury from sea food
contaminated with it. The government was concerned with the possibility of the presence
of carcinogenic compounds in petroleum-grown SCP, with its limited and nonrenewable nature and because it was not conventional. The oil companies which had
been working on the SCP from petroleum-derived substrates switched over to working
with non-petroleum substrates. In Italy the concern was over the safe content of nucleic
acid in SCP, the polycyclic aromatic hydrocarbons, fatty acids containing odd-numbered
carbon skeletons and the presence of n-paraffins carried over from protein-grown yeasts
fed to farm animals. Evidence in support of the overall safety of SCP has been however,
presented and it is likely that SCP will eventually receive official approval.
The two examples given above are typical of the concern shown by the public and
organizations in many parts of the world, including some specialized agencies of the
United Nations, namely the World Health Organization (WHO), the Food and
Agriculture Organization (FAO) and the United Nations International Children’s
Emergency Fund (UNICEF). The concern for the nutritional completeness and
toxicological safety of novel protein foods designed for developing countries (solvent and
heat-extracted soy proteins, synthetic amino acids, flours from ground nut and cotton
seed, fish and leaf proteins, and microbial protein, etc.) led the WHO in 1955 to form the
Protein Advisory Group (PAG). WHO was joined by the two other above-mentioned
!"
Modern Industrial Microbiology and Biotechnology
bodies in sponsoring the Protein Advisory Group (PAG) in 1960. The PAG appointed a
number of ad hoc working groups including an Ad hoc Working Group of SCP which
was formed in 1969. The PAG on SCP concluded that low levels of residual alkanes, and
the presence of odd-number fatty acids, or polycyclic hydrocarbons which are all derived
from petroleum do not constitute a danger in terms of carcinogenicity or toxicity. It has
also developed guidelines for the production, and nutritional and safety standards, of
SCP for human consumption. The International Union of Pure and Applied Chemists
(IUPAC) has a Fermentation Section which has prepared a set of standards and
specifications relating to the feeding of SCP to farm animals since these are ultimately
consumed by man. The two groups mentioned have similar protocols for determining
safety. These include microbiological examination for pathogens and toxin producers,
chemical analyses for heavy metals, nucleic acid content, presence of hydrocarbons,
safety tests on animals and protein quality studies.
15.4.1
Nucleic Acids and their Removal from SCP
Apart from the fears of carcinogenicity and toxicity from petroleum derivatives
mentioned above, both of which fears have been allayed in extensive studies, another
area of concern in SCP feeding is the consumption of high levels of nucleic acid. Man has
lost the enzyme uricase which oxidizes uric acid to the soluble and excretable allantoin.
When nucleic acid is eaten by man, it is broken up by nucleases present in the pancreatic
juice, and converted into nucleosides by intestinal juices before absorption. Guanine and
adenine are converted to uric acid, which as had been pointed out earlier cannot be
converted to the soluble and excretable allantoin. As a result when foods rich in nucleic
acid are consumed in large amounts, an unusually high level of uric acid occurs in the
blood plasma. Owing to the low solubility of uric acid, uricates may be deposited in
various tissues in the body including the kidneys and the joints when the diseases
known as kidney stones and gout may respectively result. In June, 1970 the PAG working
group of SCP established the upper limit of 2 gm nucleic acid per day in addition to the
quantity present in the usual diet for adults.
Some ordinary foods are high in nucleic acid (mostly RNA): liver, sardines, and fish
roe (caviar) contain 2.2. and 5.7 gm of nucleic acids per 100 gm of proteins respectively.
With SCP, comparable figures vary from 8 to 25. The proportion of nucleic acids in total
cell content of various micro-organisms is as follows: moulds, 2.5-6%; algae, 4-6%; yeasts,
6-11%; bacteria, up to 16%.
Various ways have been devised for the removal of nucleic acids from SCP.
(a) Growth and cell physiology method: The RNA content of cell is dependent on growth
rate: the higher the dilution rate (in continuous cultures) the higher the RNA/
protein ratio. In other words the higher the growth rate the higher the RNA content.
The growth rate is therefore reduced as a means of reducing nucleic acid. It must
however be borne in mind that high growth is one of the requirements of reducing
costs in SCP, hence the method may have only limited usefulness.
(b) Extraction with chemicals: Dilute bases such as NaOH or KOH will hydrolyze RNA
easily. Hot 10% sodium chloride may also be used to extract RNA. The cells
usually have to be disrupted before using these methods. In some cases the protein
may then be extracted, purified and concentrated.
Single Cell Protein
!#
(c) Use of pancreatic juice: RNAase from bovine pancreatic juice, which is heat-stable,
has been used to hydrolyze yeast RNA at 80°C at which temperature the cells are
more permeable.
(d) Activation of endogenous RNA: The RNAase of the organism itself may be activated
by heat-shock or by chemicals. The RNA content of yeasts have been reduced in
this way.
15.5
NUTRITIONAL VALUE OF SINGLE CELL PROTEIN
The nutritional value of SCP depends on the composition of the microbial cells used
especially their protein, amino acid, vitamin, and mineral contents. These to some extent
also depend on the conditions of growth of the organism. The FAO has set up reference
values for the amino acid content of proteins. On this basis, SCP derived from bacteria
and yeasts is deficient in methionine. Glycine and methionine are sometimes deficient in
molds. These can be improved by supplementation with small amounts of animal
proteins.
SUGGESTED READINGS
Boze, H., Moulin, G., Galzy, P. 1991. Production of Microbila Biomass. In: Biotechnology G., Reed
T.W. Nagodawitana, (eds). VCH Weinheim Germany. pp. 167-220.
Caron, C. 1991. Commercial Production of Bakers Yeast and Wine Yeast In: Biotechnology
G. Reed, T.W. Nagodawitana (eds). VCH Weinheim Germany. pp. 321-350.
Flickinger, M.C., Drew, S.W. (eds) 1999. Encyclopedia of Bioprocess Technology - Fermentation,
Biocatalysis, and Bioseparation, Vol 1-5. John Wiley.
Litch Field, J. 1994. Foods, Nonconventional. Kirk-Othmer Encyclopedia of Chemical
Technology 10.
Scrimshaw, N.S., Murray, E.B. 1991. Nutritional Value and Safety of “Single Cell Protein”. In: H.J.
Rehm, (ed) Biotechnology. 2nd Edit VCH Weinheim Germany. pp. 221-240.
!$
Modern Industrial Microbiology and Biotechnology
+0)26-4
16
Yeast Production
Yeasts have interacted with man from time immemorial – from the time when he first
learnt that fruit juices developed into intoxicating drinks and that the dough produced
from his ground cereal can be leavened, although he did not understand these two
phenomena. Today yeasts which are produced and used in all the six continents of the
world form the single most produced micro-organisms in terms of weight. The estimated
world production (excluding the Eastern European countries) for 1977 is given in Table
16.1, which are clearly underestimates in such areas as Middle East/Asia and Africa. In
the United States.
It would be safe to double the figures in the table as today’s estimated production.
Baker’s yeast is manufactured by six major companies in the United States. These
companies are Universal Foods (Red Star Yeast), Fleischmanns, Gist-brocades, Lallemand
(American Yeast), Minn-dak, and Columbia. There are 13 manufacturing plants owned by
these companies. Table 16.2 lists the locations of these plants by manufacturer.
Table 16.1
Estimated yeast production (dry weight, tons) in 1977
Region
Europe (excluding Eastern)
North America
Middle east/Asia Countries
United Kingdom
South America
Africa
Baker’s Yeast
Food and Fodder Yeast
74,000
73,000
15,000
15,000
7,500
2,700
160,000
53,000
25,000
2,000
2,500
The purpose for which yeasts are used and the types of yeasts employed for each
purpose are given in Table 16.2. Of these, the production of baker’s yeasts has received
the greatest attention, followed by food and fodder yeasts. Due to recent interest in the
production of single cell protein, food and fodder yeasts may become as important as
baker’s yeasts in terms of total quantity produced. The chapter will discuss the largescale production of baker’s, food and fodder yeasts.
16.1
PRODUCTION OF BAKER’S YEAST
The use of yeasts in bread making is an ancient art, although man did not always
recognize the mechanism of the rise of dough. It is of interest to give a brief historical
Yeast Production
!%
Table 16.2 Yeast manufacturing plants in the United States
Company
Lallemand (American yeast)
Columbia
Fleischmanns
Gist-brocades
Minn-dak
Location of Plant
Baltimore, Maryland
Headland, Alabama
Gastonia, North Carolina
Memphis, Tennessee
Oakland, California
Sumner, Washington
Bakersfield, California
East Brunswick, New Jersey
Wahpeton, North Dakota
Table 16.3 Major uses of yeasts
Use
1. Bakery
2. Beer brewing
3. Food yeasts and feed yeasts
4.
5.
6.
7.
8.
Feed yeasts
Wine making
Wine making (sparkling wines)
Industrial alcohol/spirits
Yeast-products (antolysates, biochemicals)
Yeasts involved
Saccheromyces cerevisiae
Sacch. uvarum (Sacch cerevisiae) Scch. cerevisiae
Candida tropicalis Candida pseudotropicalis
Candida utilis Sacch. cerevisiae
Kluyveromyces fragilis (Sacch. fragilis
Candida lipolytica
Saccharomyces cerevisiae var. elliposoides
Sacch. bayanus
Sacch. cerevisiae (Sacch. fragilis with whey)
Sacch. cerevisiae
account of the development of the yeast industry. The dough of leavened bread whose
antiquity is testified by biblical records, was probably raised by a mixture of yeasts and
lactic acid bacteria. A small piece of successful dough was used as the inoculum for the
next batch, providing a type of early continuous culture. This system of course has been
largely abandoned except in a special type of sour bread produced in San Francisco,
California, United States.
From about the Middle Ages, bakery yeasts were obtained from winemaking and
brewing. But the quality of the yeast was variable and in the case of yeast obtained from
beer the product was bitter because of the hops in the beer. This period lasted until the
latter part of the 19th century when the work of Pasteur from 1855 to 1857 elucidated the
nature of yeasts.
The first major step in the development of baker’s yeast technology can be said to be the
so-called Vienna process introduced about 1860 in which grain mash meant for
anaerobic alcohol production was gently aerated so that a good quantity of yeast was
obtained. Thus two birds were killed with one stone – yeasts and alcohol being obtained
in one operation even if the quantities were less than would be optimal if either one or the
other alone were sought. The work of Pasteur later led to more vigorous aeration, thus
yielding more cells and less alcohol. As a result of grain shortages resulting from World
War I a shift was made from the use of grain to the use of beet molasses, supplemented by
ammonia and phosphate.
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Modern Industrial Microbiology and Biotechnology
The next major step in the development of baker’s yeast technology was the
introduction of fed-batch or incremental addition of nutrients rather than the introduction
of all nutrients at the beginning, as is the case in the classical batch method. The essence
of this system known as the Zulaut method is still used today in baker’s yeast
manufacture and ensures that an excess of molasses sugar which might lead to alcohol
production is avoided.
Another important development, the production of dried active yeast, was
necessitated by the need to provide troops fighting in far off lands a means of producing
bread, instead of the compressed yeast normally used in temperate countries.
Today’s production methods for baker’s yeast do not allow alcohol production
because of the vigorous aeration used. Furthermore the yield has increased from 3% in the
mid-19th century through 13% early in this century to the present-day yield of over 50%
dry weight of yeast.
16.1.1
Yeast Strain Used
Non-sporulating ‘torula’ yeasts have occasionally been used for baking; nowadays
however specially selected strains of Saccharomyces cerevisiae are used. For some time two
strains of baker’s yeasts were available: one was highly active but had poor stability
during storage; the other had poor activity but was highly stable in storage. Successful
breeding program were then undertaken to produce new strains from them.
New large-scale factory process for bread-making used in Western countries (the
Chorleywood Bread Process) involving sophisticated plants and computerization have
led to new demands on traditionally used yeasts. These new demands include the
fermentation of more complex sugars, high initial fermentation ability, faster adaptation
to maltose fermentation, and ability to reconstitute rapidly when prepared in the active
dry form. Yeast strains used for the modern fast-rising dough have been developed with
the following traditional and new physiological properties in mind.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
ability to grow rapidly at room temperature of about 20-25°C;
easy dispensability in water;
ability to produce large amounts of CO2 in flour dough, rather than alcohol;
good keeping quality, i.e., ability to resist autolysis when stored at 20°C;
high potential glycolytic activity;
ability to adapt rapidly to changing substrates;
high invertase and other enzyme activity to hydrolyze the higher glucofructans
rapidly;
(h) ability to grow and synthesize enzymes and coenzymes under the anaerobic
conditions of the dough;
(i) ability to resist the osmotic effect of salts and sugars in the dough;
(j) high competitiveness i.e. high yielding in terms of dry weight per unit of substrate
used.
In many Eastern European countries no special yeasts are produced to cope with the
newer baking techniques mentioned above. Baking yeasts are not therefore produced
specially and a type of Vienna process is used, the yeast being obtained from grain mash
fermented for alcohol distillation.
Yeast Production
16.1.2
!'
Culture Maintenance
The specially selected baking strains of Saccharomyces cerevisiae are apt to mutate and
therefore proper storage is most important. Of the various methods used, storage in liquid
nitrogen and the oil culture method in which sterile oil is placed over a slant of yeast and
refrigerated at 4°C are most widely used. Freeze drying is not highly used as it induces
loss of viability in many yeasts and a tendency towards mutation.
16.1.3
16.1.3.1
Factory Production
Substrate
The substrate usually used for baker’ yeast production is molasses. Where these are not
available or are too expensive any suitable sugar-containing substrate e.g. corn steep
liquor may of course be used. In the Soviet Union for example sulphite liquor is used for
both alcohol and baker’s yeast production. Ethanol has been used in laboratory studies,
but is yet to be used on a large scale.
Beet and cane molasses, when they are simultaneously available, are treated
separately: clarified, pH adjusted and sterilized. They are then mixed in equal amounts
so that the nutritional deficiency of one type is made up by the other (Chapter 4). Cane
sugar is particularly richer in biotin, panthothenic acid, thiamin and magnesium and
calcium; while beet molasses is much richer in nitrogen. Molasses composition is
however not constant and varies with the geographical area of growth, the factory
extraction of the sugar and other factors. When only one type of molasses is available,
deficiencies are made up by adding appropriate nutrients.
The molasses is clarified to remove inert colored material arising from colloidal
particles and which can impart undesirable color to the yeast. Clarification may be
achieved by precipitation with alum or calcium phosphate or by poly-electrolyte
flocculating agents such as alginates and polyacrylamides. Clarification also helps
reduce foaming. ‘Sterilization’ is achieved by heating at 100°–110°C for about an hour,
after the pH has been adjusted to pH 6-8 to prevent caramelization of the sugar.
Phosphorous, ammonium and smaller amounts of magnesium, potassium, zinc, and
thiamin are added for maximum productivity to the mixed molasses. Antifoam is
sometimes added.
16.1.3.2
Fermentor processes
The fermentor for baker’s yeast propagation is nowadays made of stainless steel. The
trade fermentor (i.e. the final fermentor) may be anything from 75 to 225 cubic meters. Of
this about 75% is occupied by the medium, the unused space being allowed for foaming.
The typical stirred tank fermentor with agitator baffles and sparging is not often used in
yeast growth because of the high initial and operating cost. Generally, baker’s yeast
fermentors are aerated only by spargers which are so arranged that large volumes of air
pass through per unit time: about one volume of air per volume of broth per minute.
Spargers of different types are available. It is most important that aeration be high and
constant. When the oxygen falls below 0.2 ppm anaerobic conditions set in and alcohol is
formed.
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Modern Industrial Microbiology and Biotechnology
The aeration through sparger holes is started as soon as mixing begins in the steam
sterilized fermentor. Water, mineral nutrients, yeasts and the blended molasses
containing 1% glucose are mixed. The amount of blended molasses added is calculated
so that the total sugar in the fermentor does not exceed 0.1%. Molasses is added
incrementally during the course of the fermentation as it is used up by the yeast, beyond
the 0.1% ceiling. The pH is maintained at pH 4-6 by the addition of alkali and the
temperature at 30°C by cooling. The amount of molasses to be added at predetermined
intervals is arrived at by experimentation. Automatic sensoring and self-adjusting
equipment for temperature pH, aeration, sugar, etc., are built into some modern
fermentors. Large amounts of heat are evolved and the cooling of the fermentor is very
important. 1-Ib dry wt. of yeast would require 4.3 Ib of molasses, 0.9 Ib of ammonia, 0.3 lb
of NH4H2PO4, 1.1 lb of (NH4) 2 SO4 and 60 lb of air. Continuous fermentation has not been
widely used in baker’s yeast production. It is used (see below) for feed yeast production
from sulfite liquor.
16.1.3.3
Harvesting the yeast
The period of fermentation in the trade or production fermentor varies from 10 to 20 hours
depending on how much yeast is pitched into it; cells form from 3.5% to 5% dry wt. of the
broth. In some processes aeration is allowed to continue for 30-60 minutes at the end of
the feeding to allow unused nutrients to be used up, budding cells to divide so that most
cells are ‘resting’ at the beginning of the budding cycle. This ensures that the cells divide
somewhat ‘synchronously’ when growth resumes.
The fermentation broth is cooled and cells concentrated in centrifugal separators; they
are washed by resuspension in water and centrifugation until they are lighter in color.
The yeast cream resulting from this treatment contains 15-20% yeast cells. It is further
concentrated by passing over a rotary vacuum filter or through a filter press. Sometimes
the Mautner process is used to ensure a friable dry cream during vacuum filtration. This
latter process consists of adding before filtration 0.2-0.6% (w/v) sodium chloride, which
causes cell shrinking by osmosis. Excess salt is removed during filtration by spraying
water over the filtered yeast, so that the cells swell again. The resulting product has a dry
matter content of 28-30%.
The yeast may then be packaged as compressed yeast or active dry yeast. It may also be
converted into dried yeast for human or animal feeding as described further on in this
chapter.
16.1.3.4
Packaging
Baker’s yeasts may be packaged as moist (compressed) yeasts or as dried active yeast.
(i) Compressed yeast: The yeast product obtained after harvesting, is mixed with fine
particles of ice, starch, fungal inhibitors and processed vegetable oils (e.g. glyceryl
monostearate) which all help to stabilize it. It is then compressed into blocks of
small (1-5 Ib) blocks for household use or large (up to 50 Ib) for factory bakery
operations, stored at – 7 to 0°C and transported in refrigerated vans.
(ii) Active dry yeast: Dry yeast is more stable in that it can be used in areas or countries
where refrigeration is not available. In many developing countries baker’s yeast is
imported from abroad in the form of active dry yeast. For active dry yeast
Yeast Production
!
production special strains better suited for use and dry conditions may be used. It
has been found that when regular strains are used they perform better as dry yeasts
when they are subjected to a number of treatments. These treatments include
raising the temperature to 36°C (from about 30°C) towards the end of the
fermentation, addition of alcohol-containing spent broth (resulting from
centrifugation or finished yeast fermentation), synchronization of budding by
alternate feeding and starving. The reason for the benefit is not known.
Yeast cream of 30-38% content from filter pressing is extruded through a screen to form
continuous thread-like forms. These are then chopped fine and dried, using a variety of
driers: tray driers, rotary drum driers, or fluidized bed driers. The final product has a
moisture content of about 8% and may be packaged in nitrogen-filled tins. Sometimes
anti-oxidants may be added to the yeast emulsion to further ensure stability.
Fig. 16.1 Various Methods for Packaging Yeasts
16.2
FOOD YEASTS
Yeasts are used for food by man for the following reasons: to provide protein; to impart
flavor and to supply vitamins especially B-vitamins. Food yeasts are sometimes
prescribed medically when a deficiency of B-vitamins exists in a patient. Food yeasts
have several synonyms: dried yeast, inactive dried yeast, dry yeast, dry inactive yeast,
dried torula yeast, Saccaromyces siccum, sulfite yeast, wood sugar yeast, and xylose yeast.
The link between wood and the production of yeast for animal consumption is shown by
the last three names.
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Modern Industrial Microbiology and Biotechnology
As food yeasts are consumed by man, stringent standards are imposed on the product
by governmental agencies, professional bodies and manufacturers. The standards of the
International Union of Pure and Applied Chemists (IUPAC) will be quoted here only as
an example, because they are the most comprehensive. The IUPAC requires that any
organism to be used as a food yeast belong to the family Cryptococcaceae, should be
unextracted, should have a fat content of not more than 20%, should contain no inert
fillers not indigenous to the yeasts, and should be free of Salmonella. The IUPAC has also
set upper limits for bacterial and fungal counts, lead, arsenic and lower limits for protein,
thiamin, riboflavin and niacin.
Too high a consumption of yeasts is detrimental to health because of the high RNA
content of yeasts, which the kidneys are unable to dispose of. This was discussed in the
previous chapter.
16.2.1
Production of Food Yeast
While baker’s yeasts are usually produced from molasses using special strains of Sacch.
cerevisiae food yeasts are produced from a wide variety of yeasts and substrates.
16.2.1.1
Yeasts used as food yeasts
Yeasts used as food yeasts are Saccharomyces cerevisiae, Saccharomyces carlbergiensis,
Saccaromyces fragilis, Candida utilis, and Candida tropicalis. Only Saccharomyces fragilis
(imperfect stage Candida pseudotropicalis) can utilize lactose hence it is used for the
fermentation of whey. Ethanol may be used as substrate for food yeast production; it is
however used only by Saccharomyces fragilis and Candida utilis. Candida utilis is the most
versatile of all the yeasts and will utilize a wider range of carbon and nitrogen sources
than any other, hence it is most widely used in food yeast preparations.
16.2.1.2
Substrates used for food yeast production
The most commonly used substrates are molasses, sulphite liquor, wood hydrolysate,
and whey. Since interest developed in single cell protein other unconventional sources
have been developed. These include hydrocarbons, alcohol and wastes of various types.
These have been discussed in the previous chapter. Only molasses, sulphite liquor, wood
hydrolysate and whey will be discussed.
(i) Molasses: Bakers yeast grown on molasses as described above may, after separation
from the spent liquor by centrifugation, be dried to yield food yeast. Drum-drying,
spray-drying or fluidized bed drying may be used to reduce the moisture content to
only about 5%. Sometimes food yeast is grown on molasses for that purpose per se.
Thus Candida utilis is grown fed-batch in Taiwan in Waldhof fermentors. The fedbatch method using molasses is also used in South Africa.
Recently food yeasts using Candida utilis in continuous culture in molasses has
been grown in Cuba and Eastern Europe.
(ii) Sulfite liquor: The impetus to produce food and fodder yeast from sulfite liquor
(Chapter 4) derived from an attempt to reduce the pollution which would arise if
the wastes containing fermentable substrates were discharged directly into a
Yeast Production
!!
stream. The use of continuous fermentation was attractive because the sulfite is
produced almost continuously in the operation of the pulp factory. In general a
Waldhof-type fermentor is used for the continuous production of yeasts from
sulfite waste. Liquors from various sources are usually blended. Thereafter, the
sulfite containing compounds are removed either by precipation with lime, by
aeration or by passing steam through it (steam stripping). The pH is adjusted from
about 2 to 5.5 using ammonia. The lowest pH consistent with high yield is usually
preferred in order to lessen the chances of contamination.
Ammonium, phosphate and potassium are monitored and supplied
continuously. The versatile and hardy yeast Candida utilis is usually used so that
biotin is not added. The yeast is harvested continuously and recovered by
removing liquor at the same rate as it is introduced. The effluent liquor containing
about 1% cell is concentrated to an 8% concentration by centrifuging. It is usually
washed with water by diluting and centrifuging to remove lignosulfnic acid. Yield
from sulfite liquor, whose assimilable matter content is usually low may be
increased by the addition of new carbon sources e.g. acetic acid and ethanol. The
liquor may be re-hydrolysed with H2SO4 thereby increasing the sugar content from
4% to about 24%. In some cases, addition of nutrients to the liquor e.g. yeast
hydrolysate or corn steep liquor leads to an increased yield of about 5%.
Simultaneously, more efficient organisms are usually also sought.
(iii) Production of food yeast from whey: The effluent which drains from the coagulum
from milk during cheese manufacture is known as whey. It contains
approximately 4% sugar (lactose), 1% mineral and some of the lactic acid which
enabled the coagulation of the milk protein. In countries where a lot of cheese is
produced, whey is a waste product but it is sometimes turned into good use in the
production of alcohol or yeasts. Very few yeasts metabolize lactose. Those which
do include Saccharomyces lactis, Kluyveromyces fragilis and its imperfect or
asporogeneous stage Candida pseudotropicalis. The whey is diluted, fortified with
ammonia, phosphate, minerals, yeast extract and then pasteurized at 80°C for
about 45 minutes. It is then inoculated with yeasts at pH 4.5 at an incubation
temperature of 30°. Any of the above yeasts could be used but in the United States
the preference is for K. fragilis. In many establishments the fermentation is
continuous and sugar, pH and minerals are monitored automatically. The yeast is
recovered by centrifugation and may be drum or spray dried.
16.3
FEED YEASTS
Feed yeasts are the same as food yeasts described above. The only difference is that less
rigid standards are imposed on the production of feed yeasts. Thus, feed yeasts intended
for animal feeding are usually obtained by drying out the whole fermentation broth, often
without washing.
Several thousands tons of yeasts are recovered from breweries around the world
annually. To be used as food yeasts, such yeast is ‘debittered’ of hop resins by repeated
washing with dilute alkali until the bitterness no longer exists. It is then slightly acidified
to about pH 5.5. Cells are recovered by centrifugation and spray – or drum-dried.
!"
16.4
Modern Industrial Microbiology and Biotechnology
ALCOHOL YEASTS
Alcohol yeasts are those to be used in beer brewing wineries and distilleries for spirits of
industrial alcohol. In the production of alcohol yeasts, the aim is cell production. The
methods are generally similar to those already described for baker’s yeasts. Beginning
from a lypholized vial or tube, contamination is checked in a plate. A single colony (or
prefereably a single spore by micromanipulation) is picked and multiplied in
sequentially increasing amounts.
The yeasts used are specially selected strains of the following:
Brewing: Saccaromyces cerevisiae, Saccharomyces uvarum carlbergensis S. uvarum.
Wine: Saccharomyces cerevisiae, Sacch. bayanus, Sacch. beticus, Sacch. elipsoides.
Distillery Yeasts: Saccharomyces cerevisiae.
The medium used in the multiplication of the yeast is made of materials to be found in
the final fermentation. Thus for growing brewery yeasts wort is used, for distiller’s yeast
a rye-malt medium is used, and for wine grape juice is used.
Alcohol yeasts are usually recovered and reused for several rounds of fermentation
before being discarded.
16.5
YEAST PRODUCTS
Various products used in the food, pharmaceutical and related industries may be
produced from yeasts.
Yeast extracts are used in the preparation of soups, sausages, gravies, to which they
impart a meaty flavor. A well-known example is marketed in certain parts of the world as
‘marmite’. The extracts may be obtained by autolysing the yeasts and thereafter spraydrying or drum-drying with or without extracting soluble materials from the autolysate.
The extract may also be obtained by hydrolyzing the yeast cells in acid solution. It is
neutralysed with sodium hydroxide, filtered, decolorized through charcoal and
concentrated to a syrup or spray-dried. Yeast products are usually fortified with the
flavoring compound, mono-sodium glutamate, extracts of animal or vegetable protein or
with yeast cells.
Yeast extracts are consumed for dietary purposes on pharmaceutical grounds as a
source of vitamins, mainly vitamin B12.
SUGGESTED READINGS
Boze, H., Moulin, G., Galzy, P. 1991. Production of Microbila Biomass. In: Biotechnology G. Reed,
T.W. Nagodawitana, (eds). VCH Weinheim Germany. pp. 167-220.
Burrows, S. 1979. In: Microbial Biomass. A.H. Rose, (ed.) Academic Press, New York, USA. pp. 3264.
Caron, C. 1991. Commercial Production of Bakers Yeast and Wine Yeast In: Biotechnology
G. Reed, T.W. Nagodawitana, (eds). VCH Weinheim Germany. pp. 321-350.
Flickinger, M.C., Drew, S.W. (eds) 1999. Encyclopedia of Bioprocess Technology - Fermentation,
Biocatalysis, and Bioseparation, Vol 1-5. John Wiley, New York, USA.
Scrimshaw, N.S., Murray, E.B. 1991. Nutritional value and Safety of “Single Cell Protein”. In:
Biotechnology G. Reed, T.W. Nagodawitana, (eds). VCH Weinheim Germany. pp. 221-240.
+0)26-4
17
Production of
Microbial Insecticides
Insects are major pests of crops. Enormous losses occur when they attack various plant
parts, often transmitting disease in the process. Even after harvest insects attack stored
foods; this attack of stored foods is not limited to plant foods, but also includes animal
foods such as dried fish. Besides the loss they cause in agriculture and food, insects are
also vectors of various animal and human diseases.
In modern times insects have been controlled mainly with the use of chemicals. Over
the past decade or so there has been a move away from the sole use of chemical control,
and towards integrated control, which employs other methods as well as chemical
control. The reasons for this include non-specificity of chemical insecticides leading to
the destruction of pests as well as their natural predators, resistance to chemical
insecticides, concern for the environment and human health since the insecticides enter
drinking water from soil, and since some are toxic or carcinogenic. Finally due to
increased cost of petroleum on which many of these insecticides are based, their cost has
also increased.
17.1
ALTERNATIVES TO CHEMICAL INSECTICIDES
The alternatives to the use of chemicals include the following:
(a) Predators: Among vertebrates one of the best known is the use of fish especially
Gambusia affinis to eat mosquito larvae. Invertebrate predators include other larger
insects e.g. wasps, while plant predators include Utricularia (a bladder wort).
(b) Genetic manipulations: These include the production (by chemicals or by
irradiation) of large numbers of sterile males, whose mating does not result in
fertile eggs.
(c) The use of hormones or hormone analogs: Pheromones are synthetic compounds
which act as sex attractants. The insects attracted are destroyed.
(d) The use of pathogens: Pathogens of insects are found among bacteria, fungi,
protozoa, viruses and nematodes. The idea of using pathogens to control insects
originated from studies of the diseases of the silkworm Bombyx mori. The pioneer
work of Bassi was followed by those of Le Conte, Pasteur, Hagen until Metchnikoff
actually tested the control of sugar beet pests with the fungus Metarrhizium
anisopliae in South Russia.
!$
17.2
Modern Industrial Microbiology and Biotechnology
BIOLOGICAL CONTROL OF INSECTS
Biological control has been studied or practiced to a large extent in relation to agriculture,
food production and forestry. Its study and use in the control of insect vectors of disease
such as mosquitoes has been small in comparison. In 1976 the World Bank in
collaboration with the United Nations Development Programme (UNDP) and the World
Health Organization (WHO) put into operation a Special Programme for Research and
Training in Tropical Diseases. The diseases were malaria, trypanosomiasis, filariasis,
leishmaniasis, schistosomiasis and leprosy. Of these the first four are transmitted by
insect vectors. The WHO which administered the Programme also studied biological
control with respect to the insect-borne disease and ranked the organisms to be used in
order of effectiveness from time to time (Table 17.1). The organisms included bacterial,
fungi, nematodes, and fish, and targets are mainly mosquitoes-vectors of malaria and
yellow fever and black flies (Simulium spp), vectors of oncocerchiasis (river blindness).
For agricultural biological control however some viruses pathogenic to insects are also
used (Table 17.2). In this chapter only control using microorganisms will be discussed.
Table 17.1
Priority 1
Priority 2
Priority 3
Priority 4
Biological control agents as ranked in order of priority by the special program of the
World Health Organization 1980
Bacillus thuringiensis, serotype H-14 (bacterium)
Bacillus sphaericus, strain 1593 (bacterium)
Culicinomyces sp. (fungus)
Poecilia reticulata (fish)
Romanomermis culicivorax (nematode)
Toxorhynchites (predatory mosquitoes)
Zacco platypus (fish)
Aphanius dispar (fish)
Coelomomyces (fungi)
Lagenidium (fungus)
Leptolegnia (fungus)
Metarrhizium anisopliae (fungus)
Parasitoids in general (insects)
Romanomermis iyengari (nematode)
Stenopharyngodon idella (fish)
Group A
Aplocheilus (fish)
Baculoviruses (viruses)
Dimorphic Microspordia (protozoa)
Dugesia (Planaria)
Lutzia (predatory mosquito)
Octomyomermis muspratt (nematode)
Protozoa of snails (protozoan)
Tolypocladium
Group B
Entomophthorales (fungi)
Nosema algerae (protozoan)
Vavraia culicis (protozoa)
Production of Microbial Insecticides
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Table 17.2 Pathogenic viruses found in insects
Family
Nucleic
Acida
Particle
Symmetry
Vertebrate and plant viruses resembling
families of insect viruses
Vertebrate
Plant
viruses
viruses
Baculoviridae
(Baculovirus
groups A,B,C)
Poxviridae
(Entomopox
viruses)
DNA
Rod
(occluded)
None
None
DNA
‘Brick’
(occluded)
None
Reoviruses
Reoviridae
(Cytoplasmic
polyhedrosis
viruses)
Irodioviridae
(Iridovirus)
dsRNA
Isometric
(occluded)
Orthopoxvirus
Avipoxvirus
Capripoxvirus
Leporipoxvirus
Parapoxvirus
Reovirus
Orbivirus
DNA
Isometric
5.
Parvoviridae
(Densovirus)
ssDNA
Isometric
6.
Picornaviridae
(Enterovirus;
unclassified
groups)
Rhabdoviridae
(Sigmavirus)
RNA
Isometric
African Swine Fever
Frog Viruses 1-3
Lumphocystis virus
Parvovirus
Adeno-associated
Group
Enterovirus
RNA
Bullet/
bacciliform
Vesiculovirus
Lyssavirus
1.
2.
3.
4.
7.
Plant
Fungal
Algal
None
Small RNA
Viruses
(single
polypeptide)
Plant rhabdovirus
a
ds RNA, double stranded RNA, ss, single stranded DNA
17.2.1
Desirable Properties in Organisms to be Used for
Biological Control
The following are desirable in microorganisms to be used in the biological control of
insects:
(a) The agent should be highly virulent for the target insect, but should kill no other
insects.
(b) The killing should be done quickly so that in the case of crops, damage is kept as
low as possible, and in the case of vectors of disease before extensive transmission
of the disease occurs.
(c) The killing ability should be predictable.
(d) The agent should not be harmful to man, animals or crops; in other words it should
be safe to use.
!&
Modern Industrial Microbiology and Biotechnology
(e) It should be technically amenable to cheap industrial production.
(f) When produced, it should be stable under the conditions of use such as under the
high temperature and ultra violet light of ordinary sunlight.
(g) It should be viable over reasonably long periods to permit storage and
transportation as necessary.
(h) It should ideally persist or recycle and/or be able to search for its host.
17.2.2
Candidates Which have been Considered as
Biological Control Agents
(i) Bacteria A large number of bacteria are pathogenic to insects including Bacillus spp.,
Pseudomonas sp. Klebsiella sp., Serratia marcescens. In practice, spore formers have been
developed commercially because they survive more easily in the environment then
vegetative cells, but especially because they are amenable mass production. The four
bacilli which have been produced for control purposes are:
(a) Bacillus thuringiensis: B. thuringiensis (commonly known as ‘Bt’) is an insecticidal
bacterium, marketed worldwide for control of many important plant pests–mainly
caterpillars of the Lepidoptera (butterflies and moths) but also mosquito larvae,
and simuliid blackflies that vector river blindness in Africa. Bt products represent
about 1% of the total ‘agrochemical’ market (fungicides, herbicides, and
insecticides) across the world. The commercial Bt products are powders
containing a mixture of dried spores and toxin crystals. They are applied to leaves
or other environments where the insect larvae feed. The toxin genes have also been
genetically engineered into several crop plants. The method of use, mode of action,
and host range of this biocontrol agent differ markedly from those of Bacillus
popilliae.
It is a complex of several organisms regarded by some as being variants of B.
cereus. There are 19 serotypes based on some flagellar or H-antigens. Serotype H3
and H3A are used in the United States on alfalfa, cotton, tobacco, spinach,
potatoes, tomatoes, oranges, and grapes. Serotype H14 attacks mosquitoes and
blackflies and will be discussed below. Bacilus thuringiensis produces at least three
toxins, a Phospholipase C, a water-soluble heat stable B-exotoxin potentially toxic
to mammals, and a crystalline, d-toxin or the parasporal body which is enclosed
within the sporangium (this will be discussed further below).
The crystalline d-toxin is the active principle against most insects. The spores
and crystals are released into the medium in most strains of B. thuringiensis
following the lysis of the sporangium.
(b) Bacillus moritai: This is used in Japan for the same purpose as B. thuringiensis
serotypes H3 and H3A.
(c) Bacillus popilliae: This is an obligate pathogen of the Japanese beetle Popilla japonica
against which it is used. Since it is an obligate parasite it is produced in the larvae
of the beetle.
(d) Bacillus thuringinensis var. israelensis (also known as serotype H14). This was
isolated in 1976 by Goldberg and Margalit from a mosquito breeding site in Israel.
It has proved very effective in killing mosquito larvae and the black fly (Simulium
Production of Microbial Insecticides
!'
spp). So promising is it from results of various projects sponsored by the Special
Program of the WHO that it was expected that it would be produced on a large
scale in the US and Europe and probably on smaller scales in tropical countries. It
has a (nearly 100%) kill of mosquito larvae and shows no adverse effect on nontarget organisms. Unlike classical Bacillus thuringiensis it does not produce a betatoxin. Its killing effect is therefore based principally on its crystalline delta-toxin,
(d-toxin) which is resistant to both heat (surviving 80°C for 10 minutes and 60°C
for 20 minutes) and ultra violet light.
(e) Bacillus sphericus: Bacillus sphericus is an highly specific for mosquito larvae as
Bacillus thuringiensis, var israelensis (B.t.i.). However, whereas the lethality of B.t.i.
resides in toxic protein crystals formed during the spores of the organism, the toxin
of B. sphericus resides in the cell wall of the organism. The toxin of B. sphericus works
slowly (8-40 hours) compared with that of B.t.i. (2-10 hours). Bacillus sphericus
however, has the advantage of being able to lay dormant in muds or sewage ponds
and to recycle as susceptible mosquito larvae appear. Like B.t.i., it had reached
stage 4 of had WHO scheme for screening and evaluating biological agents for
control of disease vectors shown in Table 17.3.
(ii) Viruses: A large number of viruses has been isolated from insects. The advantages of
viruses as biological control agents is that they are specific. Seven groups of insectpathogentic viruses have been identified (Table 17.2). The most useful of them for
biological control purposes are the baculoviruses, which are easily recognizable because
the virus particles are included within a proteinaceous inclusion body large enough to be
seen under a light microscope. (These inclusion bodies, polyhedrons and granules, are
found in the nucleus of the host cell – hence they are nuclear polyhedrosis and
granuloses).
The baculoviruses are the best candidates for insect control because they are (a)
effective in controlling insect populations, (b) restricted to a host range of invertebrates,
(c) relatively easy to produce in large quantities and (d) stable under specific conditions
because of the inclusion bodies.
Several experimental preparations are available and at least two (one each in the USA
and Japan) have been produced on a commercial scale. The preparations are ingested
when the insects consume leaves and other plant parts on which the virus particles have
been sprayed. After ingestion the polyhedral inclusion bodies dissolve within the midgut; the released virions pass through the mid-gut epithelial cells into the haemocoel.
Death of the larvae occurs four to nine days after ingestion.
(iii) Fungi: All the four major groups of fungi, Phycomycetes, Ascomycetes, Fungi Imperfecti
and Basidiomycetes contain members pathogenic to insects. The great difficulty with
using fungi for biological control is that environmental conditions including
temperature and humidity must be adequate for spore germination and insect cuticle
penetration by the hyphae. Since these environmental conditions are not always assured
the result is that fungi are used for biological control only in a few countries especially the
USSR. Fungi which have been most widely used as Beauvaria bassiana and Metarrhizium
anisopliae. Others are Hirsutella thompsonii Verticillium and Aschersonia aleyrodis. H.
thompsonii is being developed commercially as acaricide, for killing mites which attack
plants, although a large number of other fungi attack mites. H. thomposonii has been
!
Scheme for screening and evaluating the efficacy, safety, and environmental impact of biological agents for control of disease
vectors designed by the WHO
Stage I
Stage II
Laboratory
Laboratory
A. Identification A. Mammalian Results of
and charateriinfectivity
Review of
zation
tests to
stages I and II
ensure safet
to laboratory
and field
personnel
B. Assessment
against
selected
target
vectors
species
C. Preliminary
evaluation
of ease of
rearing in
B. Preliminary
assessment
against
certain
non-target
Stage III
Preliminary
field trials
Strictly
regulated
ponds tests under
WHO supervision
to determine
efficacy against
disease vectors
under natural
conditions
Stage IV
Laboratory
A. More detailed
tests on
mammalian
infectivity using
appropriate
techniques
Laboratory and
field trials
B. Detailed studies
on non-target
range especially
other fauna in
habitats where
stage V trials may
be conducted
Formulation
C. Studies on
stability of suitable
formulations and
delivery system
Stage V
Review of stages
I, II, III and IV by
by informal
consultation group
Large-scale field trials
To be conducted
under WHO auspices.
Not presently defined,
and will vary according
to target vector,
habitat(s), mode of
application, etc.
Modern Industrial Microbiology and Biotechnology
Table 17.3
Production of Microbial Insecticides
!
found particularly active against mites which attack citrus. It is applied as the conidial
powder and maximum effectiveness occurs at 27°C and under moist conditions or at
relative humidities of 79-100%. Coelomomyces sp. is very effective against mosquitoes but
its production is difficult because of the need of a secondary host. Most effective and
specific against mosquitoes are Culicinomyces sp. which was isolated in Australia and
produced a mortality rate on mosquitoes of 90-100%. Tolypocladium cylindrosporum is
essentially like Culicinomyces in being highly specific for mosquitoes. Lagenidium
giganteum and Leptolegnia sp have been shown to have high mortality for mosquitoes. All
the above (except Coelomomyces) can be mass-produced by fermentation. Beauvaria and
Metarrhizium already discussed have broad activity against mosquitoes.
(iv) Protozoa: In constrast to the rapid action of viruses and spore-forming bacteria, killing
by protozoa is slow and may take weeks. Furthermore they are difficult to produce, being
accomplished only in vivo. Nevertheless they have been produced and successfully used
experimentally for stored-product pests (Matosia trogoderina) mosquitoes (Nosema algerae)
and grasshoppers (Nosema pyrasta).
Vavra vilivis is also effective against mosquitoes and has properties similar to those of
Nogema algerae. Studies sponsored by the WHO have shown that N. algerae does not seem
to constitute a safety hazard for man. Factors favoring the use of N. algerae are sporelongevity, ease of spore-production under laboratory and especially cottage industry
conditions and the probable impact on disease transmission by reducing the longevity of
infected female mosquitoes.
So far however protozoa have not been produced on an industrial scale for biological
control.
17.2.3
Bacillus thuringiensis Insecticidal Toxin
B. thuringiensis strains produce two types of toxin. The main types are the Cry (crystal)
toxins, encoded by different cry genes, and this is how different types of Bt are classified.
The second types are the Cyt (cytolytic) toxins, which can augment the Cry toxins,
enhancing the effectiveness of insect control. Over 50 of the genes that encode the Cry
toxins have now been sequenced and enable the toxins to be assigned to more than 15
groups on the basis of sequence similarities. The table below shows the state of such a
classification in 1995. An alternative classification has recently been proposed based on
the degree of evolutionary diversity of the amino acid sequences of the toxins, but this has
not yet been widely adopted.
Cry toxins are encoded by genes on plasmids of B. thuringiensis. There can be five or six
different plasmids in a single Bt strain, and these plasmids can encode different toxin
genes. The plasmids can be exchanged between Bt strains by a conjugation-like process,
so there is a potentially wide variety of strains with different combinations of Cry toxins.
In addition to this, Bt contains transposons (transposable genetic elements that flank
genes and that can be excised from one part of the genome and inserted elsewhere). All
these properties increase the variety of toxins produced naturally by Bt strains, and
provide the basis for commercial companies to create genetically engineered strains with
novel toxin combinations.
!
Modern Industrial Microbiology and Biotechnology
Table 17.4
Bt toxins and their classification
Gene
Crystal shape
Protein size (kDa) Insect activity
cry I [several subgroups:
A(a), A(b), A(c), B, C, D, E, F, G]
cry II [subgroups A, B, C]
cry III [subgroups A, B, C]
cry IV [subgroups A, B, C, D]
cry V-IX
bipyramidal
130-138
lepidoptera larvae
cuboidal
flat/irregular
bipyramidal
various
69-71
73-74
73-134
35-129
lepidoptera and diptera
coleoptera
diptera
various
Mode of Action of Bt Toxin
The toxin of Bt is lodges in a large structure, the parasporal structure, which is produced
during sporulation. The parasporal crystal is not the toxin. However, once it is
solubulized a protoxin is released.
The crystals are aggregates of a large protein (about 130-140 kDa) that is actually a
protoxin, which must be activated before it has any effect. The crystal protein is highly
insoluble in normal conditions, so it is entirely safe to humans, higher animals and most
insects. However, it is solubilised in reducing conditions of high pH (above about pH
9.5), the conditions commonly found in the mid-gut of lepidopteran larvae. For this
reason, Bt is a highly specific insecticidal agent.
Once it has been solubilized in the insect gut, the protoxin is cleaved by a gut protease
to produce an active toxin of about 60 kDa. This toxin is termed delta-endotoxin. It binds
to the mid-gut epithelial cells, creating pores in the cell membranes and leading to
equilibration of ions. As a result, the gut is rapidly immobilized, the epithelial cells lyse,
the larva stops feeding, and the gut pH is lowered by equilibration with the blood pH.
This lower pH enables the bacterial spores to germinate, and the bacterium can then
invade the host, causing lethal septicaemia.
17.3
PRODUCTION OF BIOLOGICAL INSECTICIDES
Microbiological insecticides are produced in one of three ways: submerged fermentation;
surface or semi-solid fermentation; and in vivo production. The first two are for
facultative pathogens and the third is for obligate pathogens.
17.3.1
Submerged Fermentations
These have been used for the production of Bacillus spp. (excluding production of B.
poppillae which is produced in vivo) and to a lesser extent, fungi.
Medium: In fermentation for Bacillus thuringiensis the active principle sought is the delta
toxin found in the crystals. Media for submerged fermentation have been compounded by
various workers in a number of patents. In one such preparation, the initial growth in a
shake flask occurred in nutrient broth; in the second shake flask, and in the seed
fermentor best molasses (1%), corn steep liquour (0.85%) and CaCO3 (0.1%) were used. A
typical medium for production would be beet molasses (1.86%), pharmamedia (1.4%)
and CaCO3 (0.1%). Other production media contain corn starch (6.8%), sucrose (0.64%),
casein (9.94%), corn steep liquor (4.7%), yeast extract (0.6%) and phosphate buffer (0.6%).
Production of Microbial Insecticides
! !
A third medium contained soya bean meal (15%), dextrose (5%), corn starch (5%), MgSO4
(0.3%), FeSO4 (0.02%), ZnSO4 (0.02%) and CaCO3 (1.0%).
The above media were used for agricultural strains of B. thuringiensis but could no
doubt be used also for B. thuringiensis var israelensis.
Bacillus thuringiensis var insraelensis and Bacillus sphericus do not require carbohydrates
for growth and can grow well and produce materials which will kill the larvae of
mosquitoes in a variety of proteinaceous materials such as commercial powders of soy
products, dried milk products, blood and even materials from primary sewage tanks.
Effective powders of B. sphericus 1593 and B. thuringiensis var israelensis have been
produced using discarded cow blood from abattoirs and various legumes.
Extraction: At the end of the fermentation, the active components of the broth are recovered
by centrifugation, vacuum filtration with filter aid or by precipitation. Precipitation has
been done with CaCl2 and the acetone method yields products of very high potency. The
fermentation beer may readily be diluted and used directly.
17.3.2
Surface Culture
Surface culture techniques are used for fungi and for spore formers. The organisms after
shake-flask growth are cultured in a seed tank from where the broth is transferred to flat
bins with perforated bottoms. The semi-solid medium is a mixture of an agricultural byproduct such as bran, an inert product such as kisselghur, soy bean meal, dextrose, and
mineral salts. The use of this medium increases the surface area and hence aeration
because of the thinness of its spread in the bins. Hot air is passed through the
perforations to dry the material. It is ground, assayed and compounded to any required
strength with inert material. Submerged, culture in which the hyphae are used have been
carried out with good results in the United States using Hirsutella thompsonii.
17.3.3
In vivo Culture
In vivo culture methods are used for producing caterpillar viruses, mosquito protozoa
and Bacillus popillae. The method is labor-intensive and could be easily applied for
suitable candidates in developing countries where expertise for submerged culture
production is usually lacking.
Once the organism has been obtained in a sufficient quantity to last for several years it
is lyophilized and stored at low temperature. The viruses are introduced into the food of
the larvae and the dead larvae are crushed, centrifuged to remove large particles and the
rest are dried. The amount of viruses in each larva is variable but the virus content of
between one and one hundred caterpillars should be sufficient to treat one acre in the
case of cotton moths. Usually separate facilities are used for rearing the caterpillars, for
infecting them and for the extraction of the virus particles. The preparation is then bioassayed and mixed with a suitable carrier.
17.4
BIOASSAY OF BIOLOGICAL INSECTICIDES
It is obvious that a reference standard must be set up against which various preparations
can be compared. The standard will differ with each particular bioinsecticide. Thus,
! "
Modern Industrial Microbiology and Biotechnology
standards do exist for Bacillus thuringiensis serotypes H3 and H3A used against
caterpillars and a standard for B. thuringiensis var israelensis against mosquito ‘IPS82’
exists. Both standards are prepared and deposited at the Institute Pasteur in Paris. In the
simplest terms a standard is based on the LD50, the dose of the insecticide which will kill
50% of the population must be clearly defined; the age and type of insect to be used; the
food of the insect; the temperature conditions and a host of other parameters.
17.5
FORMULATION AND USE OF BIOINSECTICIDES
The formulation of the bioinsecticides is extremely important. An insecticide shown to be
highly potent under laboratory experimental conditions may prove valueless in the field
unless the formulation has been correctly done. Since microorganisms cannot by
themselves be patented, industrial firms producing bioinsecticides depend for their
profits on the efficiency of their formulation (i.e., the inert material which ensures
adequate presentation of the larvicide to the target insect). The inert material is referred to
as a carrier or an extender. Carriers or extenders are the solids or liquids in which the active
principle is diluted. When the carrier is a liquid and the active principle is suitable in it
the application is a spray. There are thus two types of formulation: (a) powders and dusts
(b) flowable liquid; which of the two is manufactured depends to a large extent on the
method of production and intended use of the insecticide.
17.5.1
Dusts
Semi-solid preparations based on waste plant products usually are compounded as
dusts or powders because making them into liquid causes the bran to absorb water and
prevent free flow thus leading to the clogging of conventional liquid applicators. The
advantage of dusts is greater stability of the preparation. They are also useful when the
insecticide is intended to reach the underside of low lying crops such as cabbages. Heavy
rains unfortunately wash off dusts. They may also lead to inhalation of the
bioinsecticides by the persons applying them. Diluents which have been used in
commercial dust of Bacillus thuringiensis are celite, chalk, kaolin, bentonite, starch, and
lactose. Lactose has also been used for diluting virus insecticide dusts. When the active
principle is absorbed on to the extender (or filler), the extender is referred to as a carrier.
If the extender or carrier is attractive to the insect as a food, oviposition site etc., then the
extender or filler is known as a bait. Baits for Bacillus thuringiensis include ground corn
meal, and for protozoa, cotton seed oil, honey, hydroxyethyl cellulose.
17.5.2
Liquid Formulation
Liquid formulations are usually made from water in which both the crystal and spores
are stable. Sometimes oils and water/oil emulsions may be used. When liquids other
than water are used it must be ascertained that they do not inactivate the active agent.
Emulsifiers may be added to stabilize emulsions when these are used. Some emulsifiers
which have been used for B. thuringiensis and viruses are Tween 80, Triton B 1956, and
Span 20.
The nature of the surface on which the insecticide is applied and which may be oily,
smooth or waxy may prevent the liquid from wetting the sprayed surface. Spreaders or
Production of Microbial Insecticides
! #
wetting agents which are surface-tension reducers may be added. Wetting agents may be
added to dusts to produce wettable-powders which are more easily suspended in water.
Some wetting agents and spreaders which have been used for agricultural Bacillus
thuringienses include alkyl phenols Tween 20, Triton X114 and for viruses Triton X100
and Arlacel ‘C’ which are all commercial surface-tension reducing agents.
To prevent run-off of liquids or wettable powders, stickers or adhesives are added to
hold the insecticide to the surface. Stickers which have been used for bacteria and viruses
include skim milk, dried blood, corn syrup, casein, molasses, and polyvinyl chloride
latexes.
Protectants are often added to insecticides which protect the active agent from the effect
of ultra violet light, oxidation, desiccation, heat and other environmental factors which
reduce the effectiveness of the active agent. These are usually trade secrets and their
composition is not disclosed. Dyes combined with proteins such as brewers yeast plus
charcoal, skin milk plus charcoal, and albumin plus charcoal have also proved effective
in protecting virus preparations from the effect of the ultraviolet light of the sun. Microencapsulation of bioinsecticides with carbon also affords protection.
17.6
SAFETY TESTING OF BIOINSECTICIDES
Many individuals on first learning of the use of microorganisms to control insect pests
and vectors of disease express fear about the effect of these entomopathogens or their
effective components (e.g., crystals of B. thuringiensis). For this reason animal tests
including feeding by mouth, inhalation, intraperitoneal, intradermal and intravenous
inoculations, and teratogenicity and carcinogenicity tests are done. Test animals
include rats, mice, monkeys, rabbits, fish, and sometimes when appropriate, human
volunteers.
Tests conducted on the following agricultural entomopathogens in the United States,
Russia and Japan have shown them non-toxic for man, other animals or plants: Bacteria
(Bacillus popillae, B. thuringiensis, B. moritai), five viruses (Heliothus, Orgyia, Lymantria,
Autographa, Dendrolimus), three protozoa (Nosema locustae, N. algerae, N. troqodermae) and
two fungi (Beauveria bassiana, Hirsutella thompsonii).
Tests sponsored by the WHO and carried out in France and the United State have
shown the following useful or potentially useful entomopathogens to be safe. Bacillus
sphericus stain SS11-1, B. sphericus strain 1593-4; B. sphericus strain 1404-9, Bacillus
thuringiensis, var. israelensis (serotype H14) strain WHO/CCBC 1897; Metarrhuzium
anisopliae, Nosema algerae.
17.7 SEARCH AND DEVELOPMENT OF NEW
BIOINSECTICIDES
There are a number of stages in the development of a new bioinsecticide. The World
Health Organization has for some years followed the scheme given in Table 17.3 for the
screening, evaluation, safety, and environmental impact of entomopathogens to be used
for biological control.
Except where the material can be produced on a small scale, cottage industry, level,
production and sale of the final material will have to be done by industry, with its
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Modern Industrial Microbiology and Biotechnology
experience of formulation and sale distribution. It has been estimated that it will take five
to seven years to develop an entomopathogen into a biological insecticide; it will take less
than five years if some information on safety already exists on safety of a related
bioinsecticide. The cost of developing a biological insecticide is far less than that of
developing a chemical insecticide by between 20% and 50%.
SUGGESTED READINGS
Glare, R.E., Callaghan, M. 2000. Bacillus thuringiensis: Biology, Ecology and Safety. Wiley
Chichester UK.
Knowles, B.H. 1994. Mechanism of action of Bacillus thuringiensis insecticidal delta-endotoxins. In:
Advances in Insect Physiology, P.D. Evans, (ed.) Vol 24 Academic Press. London: UK. pp. 275308.
Obeta, J.A.N., Okafor, N. 1984. Medium for the production of the primary powder of Bacillus
thuringiensis sub-species israelensis. Appl. Environ. Microbiol. 47, 863-867.
Obeta, J.A.N., Okafor, N. 1983. Production of Bacillus sphaericus strain 1593 primary powder on
media made from locally obtainable Nigerian agricultural products. Canadian J. Microbiol. 29,
704-709.
World Health Organization. 1979a. Report of a meeting on standardization and industrial
development of Microbial Control Agents. TDR/BCV/79.01.
World Health Organization. 1979b. Biological Control Data Sheet: Bacillus thuringiensis de Barjac,
1978. VBC/BCDS/79.01.
World Health Organization. 1979c. Progress Report: Mammalian Safety Tests on the
Entomocidal Microbials Contract V2/181/113/(A): WHO/TDR/VBC.
World Health Organization. 1979d. Proposals for the adoption of a standardized bioassay
method for the evaluation of insecticidal formulations derived from serotype H14 of Bacillus
thuringiensis (Barjac de. H., and Larget, I.) WHO/VBC/79.744. Geneva.
World Health Organization. 1980. Annual Rept. Scientific Working Group on Biological Control
of Vectors, July 1979-June, 1980. WHO Geneva, Switzerland.
Yousten, A.Y., Federici, B., Roberts, D.W. 1991. Microbial Insecticides. In: Encyclopedia of
Microbiology Vol 2, Academic Press Sandiego, USA. pp. 521-531.
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18
The Manufacture of
Rhizobium Inoculants
Nitrogen is a key element in the nutrition of living things because of its importance in
nucleic acids, which are concerned with heredity, and in proteins, which inter alia
provide the bases for enzymes. Gaseous nitrogen is present in abundance in the Earth’s
atmosphere forming about 80% of atmospheric gases. Indeed it has been estimated that
each acre of land has about 3,500 tons of N2 above it. Unfortunately, most living
organisms cannot utilize gaseous nitrogen but require it in a fixed form; that is, when it
forms a compound with other elements. Nitrogen can be fixed both chemically and
biologically. Chemical fixation is employed in the production of nitrogenous chemical
fertilizers, which are used to replace nitrogen removed from the soil by plants. The ability
to carry out biological fixation is found only in the bacteria and blue-green algae. Some of
these organisms fix nitrogen in the free-living state and thereby contribute to the
improvement of the nitrogen status of the soil. Others do so closely associated (in
symbiosis) with higher plants. In some of these associations such as with some tropical
cereals, the organisms live on the surface of the plant roots and fix the nitrogen there. In
some others the microorganism penetrates the roots and forms outgrowths known as
nodules within which the nitrogen is fixed. Of the nodule-forming nitrogen fixing
associations between plant and micro-organisms, the most important are the legumebacteria associations. There are about 1,200 legumes species and their nodulation is
important because about 100 of them are used for food in various parts of the world.
Apart from serving as food for man and animals the legumes provide nitrogenous
fertilization to the soil for the better growth of crops in general; the importance of soil
fertilization can be seen from the fact that 100 kg of symbiotically-fixed nitrogen has been
estimated to be equivalent to an application of 50 kg of ammonium sulfate fertilizer.
The bacteria which form nitrogen-fixing nodules with legumes are members of the
genus Rhizobium. Inoculation of legumes with rhizobia started as long ago as 1896, eight
years after the beneficial association between the legumes and rhizobia was discovered.
Today there are thriving industries producing rhizobia inoculants in most parts of the
world. The inoculation of rhizobia into soils or on seeds is done where the specific
rhizobia which will form nodules with a given legume are absent in the soil because the
legume is new to the area or where because of a lapse of many years without the legume
the soil may become deficient in effective strains of rhizobia.
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The need for legume inoculation has become more urgent in recent years because of the
rise in the cost of chemical fertilizers, the inefficient use of chemical fertilizers by
agricultural crops and the short-term and long-term environmental consequences of
unused nitrate fertilizers which find their way into, and cause the pollution of, drinking
water.
Finally the problem of providing more protein for an ever-rising world population has
been compounded by the fact that areas of the world where protein shortage is most acute
are just those least able to afford the plants for the manufacture of chemical nitrogenous
fertilizers. By contrast investment in rhizobium inoculant production is relatively cheap
and the manufacture unsophisticated in comparison with chemical factories.
18.1
18.1.1
BIOLOGY OF RHIZOBIUM
General Properties
Members of the genus Rhizobium are aerobic, Gram-negative relatively large rods which
are motile and have peritrichuous flagellation. Older cells contain prominent granules of
poly->-hydroxybutyrate which give them a banded appearance. Within nodules they
form irregularly shaped cells known as ‘bacteroids”; no fixation occurs in the absence of
bacteroids. At the end of the growing season, the nodules occur in the absence of
bacteroids. At this time, the nodules disintegrate, releasing bacterioids which, once in the
soil, revert to rod-shaped cells and can survive there, sometimes for years. They invade
roots of appropriate legumes and form new modules when appropriate legumes are
planted.
18.1.2
Cross-inoculation Groups of Rhizobium
The most distinctive feature of Rhizobium spp. is their ability to form nodules with
legumes. Different rhizobia will form nodules only with some legumes. A group of
legumes with which a particular rhizobia bacterium will form nodules is known as a
cross-inoculation group. Over 20 cross-inoculation groups have been set-up out of which
seven (Table 18.1) are well-known. As will be seen from Table 18.1, rhizobia which lack a
more suitable group get lumped into the ‘cowpea rhizobia’ group. The group has also
been reclassified using numerical taxonomy, DNA base composition and homology,
serology phage susceptibility, patterns of isoenzymes, etc.
18.1.3
Properties Desirable in Strains to be Selected for
use as Rhizobium Inoculants
Before the production of a rhizobial inoculant is done the strain of the organism which
will yield the highest amount of fixed nitrogen in a given legume and a given
environment (or the most effective strain) is selected on the following basis.
(i) Effectiveness: ‘Effectiveness’ is a term used to describe the overall ability of a given
rhizobial strain to form nodules with a particular legume in a given environment
and fix useful quantities of nitrogen. Effectiveness itself is based on nitrogen-fixing
ability, invasiveness and competitiveness.
The Manufacture of Rhizobium Inoculants
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Table 18.1 Cross-inoculation groups of the genus Rhizobium
Rhizobium species
Cross
Inoculation Group
Legume host included Trigonella
(Fenugreak)
Trifolium (clover)
Medicago (alfalfa)
Melilotus (sweet clover)
Phaseolus (Beans)
Pisum (pea); Vivia (Vetch)
Lathyrus (sweet pea)
Lens (Lentil)
Lupinus (lupines)
Ornithopus (Serradella)
Glycine (soy bean)
Vigna (cowpea); Grotolaria
(Crotolaria) Pueraria
(Kudzu) Arachis (Peanut)
Phaseolus (lima beans)
1.
2.
Clover group
Alfalfa group
R. Trifolii
Rhizobium meliloti
3.
4.
Bean group
Pea group
R. Phaseoli
R. leguminosarum
5.
Lupine group
R. lupini
6.
7.
Soybean group
Cowpea group
R. japonium
Nitrogen-fixing ability is an important factor as not all nodules fix nitrogen, or do
so to the same extent. Nitrogen-fixing nodules are usually comparatively few in
number, relatively large, and are pink in color. Invasiveness (also referred to as
infectiveness) is a measure of the ability of the legume to invade and nodulate the
roots of a high proportion of the plants to which it is applied. Competitiveness is
related to invasiness and is a measure of the ability of the rhizobium strain to
produce a large number of nodules in the presence of other infective or invasive
strains.
(ii) Ability to perform in the environment of a particular soil: When effectiveness is a
measure of properties inherent in the bacterium itself other factors relate to
performance in the soil. These are:
(a)
(b)
(c)
(d)
ability to fix nitrogen in the presence of fertilizers used in the soil;
tolerance of insecticide and seed disinfectants;
resistance to bacteriophages common in the soil;
adequate performance under the pH, aeration, and the mineral status of the
soil.
(iii) Growth and Survival in the carrier: The ability of the organism to survive and
multiply in the carrier (i.e., the inert support for distributing the bacteria) is
important; an otherwise adequate organism may be inhibited by the carrier.
18.1.4
Selection of Strains for Use as Rhizobial Inoculants
Methods available for assessing the performance of a rhizobium strain before choosing it
as an inoculant are discussed below:
(i) The agar method: Seeds sterilized with mercuric chloride, sulphuric acid and
alcohol are washed with sterile water are allowed to germinate in contact with the
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rhizobia being assessed. One seedling each is placed on nitrogen free slants of agar
containing appropriate mineral salts in a large test tube. (approx. 5 cm x 20 cm).
The surface of the agar is flooded with a heavy suspension of the rhizobium to be
tested. Control slants with nitrate and without rhizobia are prepared. The
nitrogen-fixing ability of the system is assessed by harvesting the seedlings and
determining their dry weight and nitrogen contents over the control after an
appropriate period of growth. Alternatively all the nodules may be excised from
the various test groups and assessed indirectly for nitrogen fixation by the
acetylene reduction method. This is a rapid and convenient method for assessing
nitrogen fixation. Nodules are exposed to acetylene and thereafter the gases are
checked in a gas chromatograph for ethylene production. The ability of nodules,
free-living bacteria or other biological systems to fix nitrogen is determined from
the extent of acetylene-ethylene conversion.
(ii) Soil cores: Undisturbed soil cores from the field may be planted with germinated
seedlings and inoculated with a culture of rhizobium in a glass house. Assessment
of nitrogen fixation may then be done as described above.
(iii) Field assessment: For field assessment the material in which the rhizobium is carried
should have been selected and the experiment laid out in such a way that; (a)
uninoculated plots are tested for the presence of naturally occurring rhizobia; (b)
plots are inoculated with the organism being tested; (c) plots are inoculated with
the test organism and simultaneously supplied with nitrogen fertilizers. The
plants are then harvested and checked for dry weight and nitrogen content.
While soil agar and soil tube tests are useful for rapid screening, there is no substitute
for field testing using the expected final carrier in soil as similar as possible to that in
which the rhizobium is destined to grow.
18.2
FERMENTATION FOR RHIZOBIA
(i) The inoculum: The inoculum is prepared from a stock culture preferably stored in
sterile soil or on agar overlain with oil. The organisms used in preparing the
inoculum are preferably those able to form nitrogen-fixing nodules with several
legumes (i.e., the so-called ‘broad-spectrum’ strains). Where these are not available
a mixture of several strains effective in a wide range of legumes are used. In this
way the need to prepare a large number of small amounts of the inoculant is
obviated. The inoculum added is preferably about 1.0% of the total volume and
should have a density of 106-107 organisms per ml.
(ii) Medium: Rhizobia are not very demanding of nutritional requirements. Most
industrial media used consist of yeast extract or yeast hydrolysate, a carbohydrate
source and mineral salts. In some media yeast extracts supply all the nitrogen,
vitamins (especially biotin) and minerals required by the bacteria. Corn steep
liquour and hydrolyzed casein are sometimes used to supplement yeast extract. In
some media, one or more of potassium phosphate, magnesim sulfate, ferric
chloride, and sodium chloride may be added. For the fast growers the carbohydrate
source is usually sucrose; for the slow growers it may be mannitol, galactose or
arabinose. Fast growers (e.g. Rhizobium meliloti) have large gummy colonies which
form in three to five days. Slow growers (e.g. Rhizobium japonium) have small
The Manufacture of Rhizobium Inoculants
!!
colonies taking 7 to 10 days to develop. The former are petrichuously flagellated
while the latter are sub-polarly flagellated.
(iii) Aeration: Rhizobia are aerobic organisms; nevertheless the type of intensive
agitation and aeration used for the production of yeasts or some antibiotics does
not seem necessary. Indeed rhizobia will grow quite well in an unaerated
fermentor if there is a broad enough surface area to permit oxygen diffusion. Very
low aeration, as low as 0.5 liters/hour, has been found satisfactory. In large
fermentors air sparging without agitation is usually satisfactory.
(iv) Time and temperature: The temperature used is about 20°C and while fast growers
attain high numbers (in excess of 140 x 109/ml) in about two to three days, slow
growers in medium specially designed to facilitate their growth attain slightly less
than this number in three to five days.
(v) The fermentor: Fermentors used for rhizobium culture are small in comparison with
those used for antibiotics. The larger sizes range from 1,000 to 2,000 liters.
Ordinarily they range from 5 liters through 40 liters to about 200 liters and are also
usually quite unsophisticated compared with those used for producing
antibiotics.
18.3
INOCULANT PACKAGING FOR USE
After the fermentation of the organism it is packaged for delivery in either of two forms: (a)
as a coating on the seeds, (or seed inoculants) from which the rhizobia develop at
planting and invade the roots; (b) direct application into soil with the seeds introduced
shortly before or after soil inoculants.
18.3.1
Seed Inoculants
Seed inoculants are more commonly used than soil inoculants. In seed inoculation the
rhizobia may be offered as a liquid or broth, frozen concentrates, freeze-dried or oil-dried
preparation. No specific carrier is used for these preparations. Although gum Arabic,
milk, and sucrose are sometimes added to these essentially liquid preparations, they still
do not offer sufficient protection to the bacterial from the environment. The bacteria
therefore die out quickly. By far the commonest preparations are offered with carriers.
A carrier is the material which binds the rhizobium to the seed. Carriers should have
a high water-holding capacity provided a nutritive medium for the growth of rhizobia
protect the bacteria from harmful environmental effect e.g. sunlight and favor their
survival on the seeds and in the soil, and in particular they should not be toxic to the
bacteria.
Agar may sometimes be used as a carrier but by far the most widely used carrier is peat.
Other locally available materials may be used.
(i) The use of peat as a carrier
Peat is the first stage in the formation of coal, which when freshly obtained is moist. It
must be dried and milled or shredded. Peats vary in their properties and each must be
studied and undesirable properties rectified before final use. For example peats mined in
some parts of the world have a high content of sodium chloride, which must be removed
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Modern Industrial Microbiology and Biotechnology
by leaching with water before the peat is used. Peats are also usually acid, and finely
ground CaCO3 up to about 5% is used to raise the pH to about 6.8.
The survival rate of rhizobia in unsterilized carriers is low because of competition and
antagonism from resident organisms. The peat is therefore usually sterilized by hot air,
steam (including autoclaving), gamma irradiation and chemical sterilants. Hot air and
steam seem to be favored, as gumma-irradiation facilities are not always easily accessible
and the post-treatment removal of chemical sterilants may sometimes be difficult. Care
must be taken to ensure that materials toxic to the rhizobia are not released by the use of
too high a temperature.
In order to introduce the organism into a carrier two methods are used. In the United
States the broth containing a heavy growth of rhizobium in excess of 109 /ml, is mixed
with CaCO3 and sprayed onto sterilized ground peat. It is then incubated in thin layers at
26-28°C for two to three days to allow the heat generated during the wetting of the peat to
dissipate. Thereafter it is ground and bagged: adequate numbers are reached in the peat
in three to five weeks for fast growers and in about twice as long for slow growers. At the
end of this period the preparation is refrigerated till used. In other parts of the world, the
broth is inoculated into bottles or polythene bags containing sterile peat or a peat/soil
mixture. They are shaken after 24 hours and allowed to grow for one to two weeks at
26-30°C and thereafter stored at 2-4°C. For seed inoculation the rhizobium in broth or
with a carrier is brought in contact with the seeds, which then become coated with the
organisms.
(ii) Use of other carriers
Peat is not available in some parts of the world. Any carrier which meets the requirements
indicated above would do. A wide range of materials have in fact been tried with success
including lignite, coal, charcoal, bagasse, coir dust, composted straw plus charcoal,
ground wheat straw, rice husk, and ground talc.
The essential thing is that the carrier, be it peat or any other substance(s) must be
shown to be suitable both in laboratory experimentation, and also in the field.
18.3.2
Soil Inoculants
When seed inoculation is not practicable, soil inoculation is resorted to. The following
are conditions when seed inoculation is not efficient:
(a) In some epigeal legumes for example soybean, the seed coat of the seedling is lifted
out of the ground during the emergence of the cotyledons (seed leaves). Under this
condition, the rhizobia clinging to the seed coat are not deposited in the soil.
(b) Seeds coated with fungicides or insecticides cannot be successfully inoculated
with rhizobia.
(c) With legumes having small seeds and therefore only a limited number of rhizobia
are introduced, heavy soil inoculation is practiced, especially if very aggressive but
ineffective rhizobia exist in the soil.
(d) Finally fragile seeds such as peanuts (groundnuts) may break following prior
wetting during seed inoculation.
The Manufacture of Rhizobium Inoculants
!!!
Besides the above difficulties soil inoculation has the additional advantage of relative
ease of application; furthermore, it may be used to inoculate the growing plant in
situations where earlier inoculation failed.
Rhizobia may be inoculated into soil using one of two ways:
(a) Frozen concentrate obtained from centrifugation is thawed and diluted with water
and applied to soil;
(b) Free flowing granules made from peat-rhizobium preparations may be applied to
soil.
18.4
QUALITY CONTROL
It is important that the number and nitrogen-fixing qualities of the inoculant meet the
expectation of the user at the time of application. The number of rhizobia required for
effective nodulation depends on a large number of factors including the size of the seed,
the presence or absence of competing rhizobia in the soil weather conditions; the
temperature moisture, and type of soil. Nevertheless the accepted standard is a minimum
of 1,000 cells of Rhizobium per seed, except in the case of soybean where the minimum is
10.
In some countries, such as Australia a government or university body supervises all
stages of the production of inoculants including the testing and selection of the strains,
maintenance and issuing of stock cultures to manufacturers, assessing the quality of the
broth and the final preparation of the peat-carried inoculum. In other countries notably
the United States the control is left to the producing companies. In either case the
performance of the inoculants in the hands of the farmer is the ultimate test. Wherever
rhizobia are inoculated some means of ensuring the quality of the product must exist.
Once the material has been prepared and the maximum number possible in the
preparation attained quality is most easily maintained by storing it under refrigeration
until use, provided that adequate content and some aeration is maintained in the
package. A system of expiry date based on experience is often indicated on the package.
SUGGESTED READINGS
Brockwell, J. 1977. In: A Treatise on Dinitrogen Fixation: Section IV: Agronomy and Ecology.
R.W.F. Hardy, A.H. Gibson, (eds). Wiley, New York, USA. pp. 277-309.
Flickinger, M.C., Drew, S.W. (eds), 1999. Encyclopedia of Bioprocess Technology - Fermentation,
Biocatalysis, and Bioseparation, Vol 1-5. John Wiley, New York, USA.
Temprano, F.J., Albareda, M., Camacho, M., Daza, A., Santamaria, C., Rodrýguez-Navarro. 2002.
Survival of several Rhizobium/Bradyrhizobium strains on different inoculant formulations and
inoculated seeds. International Microbioliology. 5, 81–86.
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Modern Industrial Microbiology and Biotechnology
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19
Production of
Fermented Foods
19.1
INTRODUCTION
Fermented foods may be defined as foods which are processed through the activities of
microorganisms but in which the weight of the microorganisms in the food is usually
small. The influence of microbial activity on the nature of the food, especially in terms of
flavor and other organoleptic properties, is profound. In terms of this definition,
mushrooms cannot properly be described as fermented foods as they form the bulk of the
food and do not act on a substrate which is consumed along with the organism. In
contrast, yeasts form a small proportion by weight on bread, but are responsible for the
flavor of bread; hence bread is a fermented food.
Fermented foods have been known from the earliest period of human existence, and
exist in all societies. Fermented foods have several advantages:
(a) Fermentation serves as a means of preserving foods in a low cost manner; thus
cheese keeps longer than the milk from which it is produced;
(b) The organoleptic properties of fermented foods are improved in comparison with
the raw materials from which they are prepared; cheese for example, tastes very
different from milk from which it is produced;
(c) Fermentation sometimes removes unwanted or harmful properties in the raw
material; thus fermentation removes flatulence factors in soybeans, and reduces
the poisonous cyanide content of cassava during garri preparation (see below);
(d) The nutritive content of the food is improved in many items by the presence of the
microorganisms; thus the lactic acid bacteria and yeasts in garri and the yeasts in
bread add to the nutritive quality of these foods;
(e) Fermentation often reduces the cooking time of the food as in the case of fermented
soy bean products, or ogi the weaning West African food produced from fermented
maize.
Fermented foods are influenced mainly by the nature of the substrate and the
organisms involved in the fermentation, the length of the fermentation and the treatment
of the food during the processing.
Production of Fermented Foods
!!#
The fermented foods discussed in this chapter are arranged according to the
substrates used:
Wheat
Bread
Milk
Cheese
Yoghurt
Maize
Ogi, Akamu, Kokonte
Cassava
Garri
Foo-foo, Akpu, Lafun
Vegetables
Sauerkraut
Pickled cucumbers
Stimulant beverages
Coffee, Tea and Cocoa
Legumes and oil seeds
Soy sauce, Miso, Sufu
Oncom. idli
Ogili, Dawa dawa, Ugba
Fish
Fish sauce
19.2
FERMENTED FOOD FROM WHEAT: BREAD
Bread has been known to man for many centuries and excavations have revealed that
bakers’ ovens were in use by the Babylonians, about 4,000 B.C. Today, bread supplies
over half of the caloric intake of the world’s population including a high proportion of the
intake of Vitamins B and E. Bread is therefore a major food of the world.
19.2.1
Ingredients for Modern Bread-making
The basic ingredients in bread-making are flour, water, salt, and yeasts. In modern breadmaking however a large number of other components and additives are used as
knowledge of the baking process has grown. These components depend on the type of
bread and on the practice and regulations operating in a country. They include ‘yeast
food’, sugar, milk, eggs, shortening (fat) emulsifiers, anti-fungal agents, anti-oxidants,
enzymes, flavoring, and enriching ingredients. The ingredients are mixed together to
form dough which is then baked.
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Modern Industrial Microbiology and Biotechnology
19.2.1.1
Flour
Flour is the chief ingredient of bread and is produced by milling the grains of wheat,
various species and varieties of which are known. For flour production most countries
use Triticum vulgare. A few countries use T. durum, but this yellow colored variety is more
familiarly used for semolina and macaroni in many countries. The chief constituents of
flour are starch (70%), protein (7-15%), sugar (1%), and lipids (1%).
In bread-making from T. vulgare the quality of the flour depends on the quality and
quantity of its proteins. Flour proteins are of two types. The first type forming about 15%
of the total is soluble in water and dilute salt solutions and is non-dough forming. It
consists of albumins, globulins, peptides, amino acids, and enzymes. The remaining 85%
are insoluble in aqueous media and are responsible for dough formation. They are
collectively known as gluten. It also contains lipids.
Gluten has the unique property of forming an elastic structure when moistened with
water. It forms the skeleton which holds the starch, yeasts, gases and other components of
dough. Gluten can be easily extracted, by adding enough water to flour and kneading it
into dough. After allowing the dough to stand for an hour the starch can be washed off
under a running tap water leaving a tough, elastic, sticky and viscous material which is
the gluten. Gluten is separable into an alcohol soluble fraction which forms one third of
the total and known as gladilins and a fraction (two thirds) that is not alcohol-soluble
and known as the glutenins. Gladilins are of lower molecules weight than glutenins; they
are more extensible, but less, elastic than glutenins. Glutelins are soluble in acids and
bases whereas glutenins are not. The latter will also complex with lipids, whereas
glutelins do not. ‘Hard’ wheat with a high content of protein (over 12%) are best for
making bread because the high content of glutenins enables a firm skeleton for holding
the gases released curing fermentation. ‘Soft’ wheat with low protein contents (9-11%)
are best for making cakes.
19.2.1.2
Yeast
The yeasts used for baking are strains of Saccharomyces cerevisiae. The ideal properties of
yeasts used in modern bakeries are as follows:
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
Ability to grow rapidly at room temperature of about 20-25°C;
Easy dispersability in water;
Ability to produce large amounts of CO2 rather than alcohol in flour dough;
Good keeping quality i.e., ability to resist autolysis when stored at 20°C;
Ability to adapt rapidly to changing substrates such as are available to the yeasts
during dough making.
High invertase and other enzyme activity to hydrolyze sucrose to higher
glucofructans rapidly;
Ability to grow and synthesize enzymes and coenzymes under the anaerobic
conditions of the dough;
Ability to resist the osmotic effect of salts and sugars in the dough;
High competitiveness i.e., high yielding in terms of dry weight per unit of substrate
used.
Production of Fermented Foods
!!%
The amount of yeasts used during baking depends on the flour type, the ingredients
used in the baking, and the system of baking used. Very ‘strong’ flours (i.e., with high
protein levels) require more yeast than softer ones. High amount of components
inhibitory to yeasts e.g., sugar (over 2%), antifungal agents and fat) usually require high
yeast additions. Baking systems which involve short periods for dough formation, need
more yeast than others. In general however yeast amounts vary from 2-2.75% (and
exceptionally to 3.0%) of flour weight. The roles of yeasts in bread-making are leavening,
flavor development and increased nutritiveness. These roles and the factors affecting
them are discussed more fully below.
Yeast ‘food’ The name yeast ‘food’ is something of a misnomer, because these
ingredients serve purposes outside merely nourishing the yeasts. In general the ‘foods’
contain a calcium salt, an ammonium salt and an oxidizing agent. The bivalent calcium
ion has a beneficial strengthening effect on the colloidal structure of the wheat gluten.
The ammonium is a nitrogen source for the yeast. The oxidizing agent strengthens gluten
by its reaction with the proteins’ sulfydryl groups to provide cross-links between protein
molecules and thus enhances its ability to hold gas releases during dough formation.
Oxidizing agents which have been used include iodates, bromates and peroxide. A wellused yeast food has the following composition: calcium sulfate, 30%, ammonium
chloride, 9.4%, sodium chloride, 35%, potassium bromate, 0.3%; starch (25.3%) is used as
a filler.
19.2.1.3 Sugar
Sugar is added (a) to provide carbon nourishment for the yeasts additional to the amount
available in flour sugar (b) to sweeten the bread; (c) to afford more rapid browning
(through sugar caramelization) of the crust and hence greater moisture retention within
the bread. Sugar is supplied by the use of sucrose, fructose corn syrups (regular and high
fructose), depending on availability.
19.2.1.4
Shortening (Fat)
Animal and vegetable fats are added as shortenings in bread-making at about 3% (w/w)
of flour in order to yield (a) increased loaf size; (b) a more tender crumb; and c) enhanced
slicing properties. While the desirable effects of fats have been clearly demonstrated their
mode of action is as yet a matter of controversy among bakery scientists and cereal
chemists. Butter is used only in the most expensive breads; lard (fat from pork) may be
used, but vegetable fats especially soy bean oil, because of its most assured supply is now
common.
19.2.1.5
Emulsifiers (Surfactants)
Emulsifiers are used in conjunction with shortening and ensure a better distribution of
the latter in the dough. Emulsifiers contain a fatty acid, palmitic, or stearic acid, which is
bound to one or more poly functional molecules with carboxylic, hydroxyl, and/or
amino groups e.g., glycerol, lactic acid, sorbic acid, or tartaric acid. Sometimes the
carboxylic group is converted to its sodium or calcium salt. Emulsifiers are added as 0.5%
flour weight. Commonly used surfactants include: calcium stearyl- 2-lactylate, lactylic
stearate, sodium stearyl fumarate.
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Modern Industrial Microbiology and Biotechnology
19.2.1.6
Milk
Milk to be used in bread-making must be heated to high temperatures before being dried;
otherwise for reasons not yet known the dough becomes sticky. Milk is added to make the
bread more nutritious, to help improve the crust color, presumably by sugar
cearamelization and because of its buffering value. Due to the rising cost of milk, skim
milk and blends made from various components including whey, buttermilk solids,
sodium or potassium caseinate, soy flour and/or corn flour are used. The milk
substitutes are added in the ratio of 1-2 parts per 100 parts of flour.
19.2.1.7
Salt
About 2% sodium chloride is usually added to bread. It serves the following purposes:
(a)
(b)
(c)
(d)
(e)
It improves taste;
It stabilizes yeast fermentation;
As a toughening effect on gluten;
Helps retard proteolytic activity, which may be related to its effect on gluten;
It participates in the lipid binding of dough.
Due to the retarding effect on fermentation, salt is preferably added towards the end of
the mixing. For this reason flake-salt which has enhanced solubility is used and is added
towards the end of the mixing. Fat-coated salt may also be used; the salt becomes
available only at the later stages of dough or at the early stages of baking.
19.2.1.8
Water
Water is needed to form gluten, to permit swelling of the starch, and to provide a medium
for the various reactions that take place in dough formation. Water is not softened for
bread-making because, as has been seen, calcium is even added for reasons already
discussed. Water with high sulphide content is undesirable because gluten is softened by
the sulphydryl groups.
19.2.1.9
Enzymes
Sufficient amylolytic enzymes must be present during bread-making to breakdown the
starch in flour into fermentable sugars. Since most flours are deficient in alpha-amylase
flour is supplemented during the milling of the wheat with malted barley or wheat to
provide this enzyme. Fungal or bacterial amylase preparations may be added during
dough mixing. Bacterial amy1ase from Bacillus subtilis is particularly useful because it is
heat-stable and partly survives the baking process. Proteolytic enzymes from Aspergillus
oryzae are used in dough making, particularly in flours with excessively high protein
contents. Ordinarily however, proteases have the effect of reducing the mixing time of the
dough.
19.2.1.10
Mold-inhibitors (antimycotics) and enriching additives
The spoilage of bread is caused mainly by the fungi Rhizopus, Mucor, Aspergillus and
Penincillium. Spoilage by Bacillus mesenteroides (ropes) rarely occurs. The chief antimycotic agent added to bread is calcium propionate. Others used to a much lesser extent
are sodium diacetate, vinegar, mono-calcium phosphate, and lactic acid.
Production of Fermented Foods
!!'
Bread is also often enriched with various vitamins and minerals including thiamin,
riboflavin, niacin and iron.
19.2.2
Systems of Bread-making
Large-scale bread-making is mechanized. The processes of yeast-leavened bread-making
may be divided into:
(a) Pre-fermentation (or sponge mixing): At this stage a portion of the ingredients is
mixed with yeast and with or without flour to produce an inoculum. During this
the yeast becomes adapted to the growth conditions of the dough and rapidly
multiplies. Gluten development is not sought at this stage.
(b) Dough mixing: The balance of the ingredients is mixed together with the inoculum
to form the dough. This is the stage when maximum gluten development is sought.
(c) Cutting and rounding: The dough formed above is cut into specific weights and
rounded by machines.
(d) First (intermediate) proofing: The dough is allowed to rest for about 15 minutes
usually at the same temperature as it has been previous to this time i.e., at about
27°C. This is done in equipment known as an overhead proofer.
(e) Molding: The dough is flattened to a sheet and then moulded into a spherical body
and placed in a baking pan which will confer shape to the loaf.
(f) Second proofing: This consists of holding the dough for about 1 hour at 35-43°C and
in an atmosphere of high humidity (89-95°C)
(g) Baking: During baking the proofed dough is transferred, still in the final pan, to the
oven where it is subjected to an average temperature of 215-225°C for 17-23
minutes. Baking is the final of the various baking processes. It is the point at which
the success or otherwise of all the previous inputs is determined.
(h) Cooling, slicing, and wrapping: The bread is depanned, cooled to 4-5°C sliced
(optional in some countries) and wrapped in waxed paper, or plastic bags.
The Three Basic Systems of Bread-making
There are three basic systems of baking. All three are essentially similar and differ only in
the presence or absence of a pre-fermentation. Where pre-fermentation is present, the
formulation of the pre-ferment may consists of a broth or it may be a sponge (i.e., includes
flour). All three basic types may be sponge i.e includes flour. All three basic types may
also be batch or continuous.
(i) Sponge doughs: This system or modification of it is the most widely used worldwide.
It has consequently been the most widely described. In the sponge-dough system of
baking a portion (60-70%) of the flour is mixed with water, yeast and yeast food in a slurry
tank (or ‘ingridator’) during the pre-fermentation to yield a spongy material due to
bubbles caused by alcohol and CO2 (hence the name). If enzymes are used they may be
added at this stage. The sponge is allowed to rest at about 27°C and a relative humidity of
75-80% for 3.5 to 5 hours. During this period the sponges rises five to six times because of
the volatile products released by this yeast and usually collapses spontaneously. During
the next (or dough) stage the sponge is mixed with the other ingredients. The result is a
dough which follows the rest of the scheme described above. The heat of the oven causes
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Modern Industrial Microbiology and Biotechnology
the metabolic products of the yeast – CO2, alcohol, and water vapor to expand to the final
size of the loaf. The protein becomes denatured beginning from about 70°C; the denatured
protein soon sets, and imposes fixed sizes to the air vesicles. The enzymes alpha and B
amylases are active for a while as the temperature passes through their optimum
temperatures, which are 55-65°C and 65-70°C respectively. At temperatures of about
10°C beyond their optima, these two enzymes become denatured. The temperature of the
outside of the bread is about 195°C but the internal temperature never exceeds 100°C. At
about 65-70°C the yeasts are killed. The higher outside temperature leads to browning of
the crust, a result of reactions between the reducing sugars and the free amino acids in the
dough. The starch granules which have become hydrated are broken down only slightly
by the amylolytic enzymes before they become denatured to dextrin and maltose by alpha
amylase and B amylase respectively.
(ii) The liquid ferment system. In this system water, yeast, food, malt, sugar, salt and,
sometimes, milk are mixed during the pre-fermentation at about 30°C and left for about 6
hours. After that, flour and other ingredients are added in mixed to form a dough. The rest
is as described above.
(iii) The straight dough system: In this system, all the components are mixed at the same
time until a dough is formed. The dough is then allowed to ferment at about 28-30°C for 24 hours. During this period .the risen dough is occasionally knocked down to cause it to
collapse. Thereafter, it follows the same process as those already described. The straight
dough is usually used for home bread making.
The Chorleywood Bread Process
The Chorleywood Bread Process is a unique modification of the straight dough process,
which is used in most bakeries in the United Kingdom and Australia. The process, also
know as CBP (Chorleywood Bread Process) was developed at the laboratories of the Flour
Milling & Baking Research Association (Chorleywood, Herefordshire, UK) as a means of
cutting down baking time. The essential components of the system are that:
(a) All the components are mixed together with a finite amount of energy at so high a
rate that mixing is complete in 3-5 minutes.
(b) Fast-acting oxidizing agents (potassium iodate or bromate, or more usually
ascorbic acid) are used.
(c) The level of yeast added is 50-100% of the normal level; often specially-developed
fast-acting yeasts are employed.
(d) No pre-fermentation time is allowed and the time required to produce bread from
flour is shortened from 6-7 hours to 1½-2 hours.
19.2.3
Role of Yeasts in Bread-making
Methods of Leavening: Leavening is the increase in the size of the dough induced by gases
during bread-making. Leavening may be brought about in a number of ways.
(a) Air or carbon dioxide may be forced into the dough; this method has not become
popular.
(b) Water vapor or steam which develops during baking has a leavening effect. This
has not been used in baking; it is however the major leavening gas in crackers.
Production of Fermented Foods
!"
(c) Oxygen has been used for leavening bread. Hydrogen peroxide was added to the
dough and oxygen was then released with catalase.
(d) It has been suggested that carbon-dioxide can be released in the dough by the use
of decarboxylases, enzymes which cleave off carbon dioxide from carboxylic acids.
This has not been tried in practice.
(e) The use of baking powder has been suggested. Baking powder consists of about
30% sodium bicarbonate mixed in the dry state with one of a number of leavening
acids, including sodium acid pyrophosphate, monocalcium phosphate, sodium
aluminum phosphate, monocalcium phosphate, glucono-delta-lactone. CO2 is
evolved on contact of the components with water: part of the CO2 is evolved during
dough making, but the bulk is evolved during baking. Baking powder is suitable
for cakes and other high-sugar leavened foods, whose osmotic pressure would be
too high for yeasts. Furthermore, weight for weight yeasts are vastly superior to
baking powder for leavening.
(f) Leavening by microorganisms, may be done by any facultative organism releasing
gas under anaerobic conditions such as heterofermentative lactic acid bacteria,
including Lactobacillus plantarum or pseudolactics such as Escherichia coli. In
practice however yeasts are used; even when it is desirable to produce bread
quickly such as for the military or for sportsmen and for other emergency
conditions the use of yeasts recommends itself over the use of baking powder.
The Process of Leavening: The events taking place in dough during primary fermentation
i.e. fermentation before the dough is introduced into the oven may be summarized as
follows. During bread making, yeasts ferment hexose sugars mainly into alcohol (0.48
gm) carbon dioxide (0.48 gm) and smaller amounts of glycerol (0.002-0.003 gm) and trace
compounds (0.0005 gm) of various other alcohols, esters aldehydes, and organic acids.
The figure given in parenthesis indicate the amount of the respective compounds
produced from 1 gm of hexose sugars. The CO2 dissolves continuously in the dough, until
the latter becomes saturated. Subsequently the excess CO2 in the gaseous state begins to
form bubbles in the dough. It is this formation of bubbles which causes the dough to rise
or to leaven. The total time taken for the yeast to act upon the dough varies from 2-6 hours
or longer depending on the method of baking used.
19.2.3.1
Factors which effect the leavening action of yeasts
(i) The nature of the sugar available: When no sugar is added to the dough such as in the
traditional method of bread-making, or in sponge of sponge-doughs and some liquid
ferments, the yeast utilizes the maltose in the flour. Such maltose is produced by the
action of the amylases of the wheat. When however glucose, fructose, or sucrose are
added these are utilized in preference to maltose. The formation of ‘Malto-zymase’ or the
group of enzymes responsible for maltose utilization is repressed by the presence of these
sugars. Malto-zymase is produced only at the exhaustion of the more easily utilizable
sugars. Malto-zymase is inducible and is produced readily in yeasts grown on grain and
which contain maltose. Sucrose is inverted into glucose and fructose by the saccharase of
the cell surface of bakers yeasts. While fructose and glucose are rather similarly
fermented, glucose ís the preferred substrate. Fermentation of the fructose moeity of
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Modern Industrial Microbiology and Biotechnology
sucrose is initiated after an induction period of about 1 hour. It is clear from the above that
the most rapid leavening is achievable by the use of glucose.
(ii) Osmotic pressure: High osmotic pressures inhibit yeast action. Baker’s yeast will
produce CO2 rapidly in doughs up to a maximum of about 5% glucose, sucrose or fructose
or in solutions of about 10%. Beyond that gas production drops off rapidly. Salt at levels
beyond about 2% (based on flour weight) is inhibitory on yeasts. In dough the amount
used is 2.0-2.5% (based on flour weight) and this is inhibitory on yeasts. The level of salt
addition is maintained as a compromise on account of its role in gluten formation. Salt is
therefore added as late as possible in the dough formation process.
(iii) Effect of nitrogen and other nutrients: Short fermentations require no nutrients but for
longer fermentation, the addition of minerals and a nitrogen source increases gas
production. Ammonium normally added as yeast food is rapidly utilized. Flour also
supplies amino acids and peptides and thiamine. Thiamine is required for the growth of
yeasts. When liquid pre-ferments containing no flour are prepared therefore thiamine is
added.
(iv) Effect on fungal inhibitors (anti-mycotic agents): Anti-mycotics added to bread are all
inhibitory to yeast. In all cases therefore a compromise must be worked between the
maximum level permitted by government regulations, the minimum level inhibitory to
yeasts and the minimum level inhibitory to fungi. A compromise level for calcium
propionate which is the most widely used anti-mycotic, is 0.19% (based on flour weight).
(v) Yeast concentration: The weight of yeast for baking rarely exceeds 3% of the flour weight.
A balance exists between the sugar concentration, the length of the fermentation and the
yeast concentration. Provided that enough sugar is available the higher the yeast
concentration the more rapid is the leavening. However, although the loaf may be bigger
the taste and in particular the texture may be adversely affected. Experimentation is
necessary before the optimum concentration of a new strain of yeast is chosen.
19.2.3.2
Flavor development
The aroma of fermented materials such as beer, wine, fruit wines, and dough exhibit some
resemblance. However, the aroma of bread is distinct from those of the substances
mentioned earlier because of the baking process. During baking the lower boiling point
materials escape with the oven gases; furthermore, new compounds result from the
chemical reactions taking place at the high temperature. The flavor compound found in
bread are organic acids, esters, alcohols, aldehydes, ketones and other carbonyl
compounds. The organic acids include formic, acetic, propionic, n-butyric, isobutyric,
isocapric, heptanoic, caprylic, pelargonic, capric, lactic, and pyruvic acids. The esters
include the ethyl esters of most of these acids as would be expected in their reaction with
ethanol. Beside ethanol, amyl alcohols, and isobutanol are the most abundant alcohols.
In oven vapor condensates ethanol constitutes 11-12 % while other alcohols collectively
make up only about 0.04%. Besides the three earlier-mentioned alcohols, others are npropanol, 2-3 butanediol, b-phenyl ethyl alcohol. At least one worker has found a
correlation between the concentration of amyl alcohols with the aroma of bread. Of the
aldehydes and ketones acetaldehyde appears to be the major component of prefermentation. Formaldehyde, acetone, propinaldehyde, isobutyraldehyde and methyl
Production of Fermented Foods
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ethyl ketone, 2-methyl butanol and isovaleradehyde are others. A good proportion of
many of these is lost during baking.
19.2.3.3 Baking
Bread is baked at a temperature of about 235°C for 45–60 minutes. As the baking
progresses and temperature rises gas production rises and various events occur as
below:
• At about 45°C the undamaged starch granules begin to gelatinize and are attacked
by alpha-amylase, yielding fermentable sugars;
• Between 50 and 60°C the yeast is killed;
• At about 65°C the beta-amylase is thermally inactivated;
• At about 75°C the fungal amylase is inactivated;
• At about 87°C the cereal alpha-amylase is inactivated;
• Finally, the gluten is denatured and coagulates, stabilizing the shape and size of
the loaf.
19.3
FERMENTED FOODS MADE FROM MILK
19.3.1
Composition of Milk
Milk is the fluid from the mammary glands of animals which is meant for feeding the
young of mammals. It is a complex liquid consisting of several hundred components of
which the most important are proteins, lactose, fat, minerals, enzymes, and vitamins in
which emusified fat globules and casein micelles are present. Its composition varies from
breed to breed, as shown in Table 19.1.
Proteins: Milk proteins are divided into two: caseins and whey proteins. Caseins consist
of carbohydrate, phosphorus, and protein ( glyco-phspho-protein) and make up 85% of
the total milk proteins. Casein exists in milk as the calcium salt, ie, as calcium caseinate in
globules (micelles) ranging from 40-300 mµ in diameter. Casein exists in four types
designated, a b, k and g and depending on their electric charges. The proportion of the
various types in milk depends on the breed of the cow producing the milk. The letter ‘s’
after as - caseins indicates its sensitivity to precipitation by calcium.
Table 19.1 Chemical composition (%) of milk from various mammals
Animal
Water
Fat
Ass
Buffalo
Camel
Cow
Goat
Mare
Reindeer
Sheep
89.0
82.1
87.1
87.6
87.0
89.0
63.3
81.6
2.5
8.0
4.2
3.8
4.5
1.5
22.5
7.5
Protein
2.0
4.2
3.7
3.3
3.3
2.6
10.3
5.6
Lactose
Ash
6.0
4.9
4.1
4.7
4.6
6.2
2.5
4.4
0.5
0.8
0.9
0.6
0.6
0.7
1.4
0.9
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Modern Industrial Microbiology and Biotechnology
Whey proteins consist of different components which are normally stable to acid, but
very sensitive to heat. b-Lactoglobulin forms about 66% of the total whey proteins,
followed by a-Lactabumin (22%). The immune globulins from about 10% of the the total,
and contribute towards the immunity derived by the young from the consumption of
colostrum.
Lactose: The main carbohydrate in milk is lactose, which is found only in milk. It is a
dissacharide of glucose and galactose and has a low sweetening ability, as well as low
solubility in water.
Fat: Fat consists of one molecule of glycerol and three of fatty acids. Over 60 different acids
are known in butter, many of them, being of low molecular weight of about 10 carbon
atoms or less, and include saturated and unsaturated fatty acids.
Enzymes: Enzymes found in milk include proteases, carbohydrases, esterases, oxidases/
reductases.
Minerals: Milk is a major source of calcium; other minerals in milk are phosphorous,
magnesium, sodium, potassium, as well as sulphate and chloride ions.
When fat is removed from milk such as during butter making, the remnant is skim milk.
On the other hand, when casein is removed such as during cheese manufacture, the
remnant is known as whey. Whey is high in lactose and its disposal sometimes poses
some problem as not all microorganisms can break down whey. It is however used in the
production of yeasts to be used as food or fodder.
19.3.2
Cheese
Cheese is a highly proteinaceous food made from the milk of some herbivores. Cheese is
believed to have originated in the warm climates of the Middle East some thousands of
years ago, and is said to have evolved when milk placed in goat stomach was found to
have curdled. The scientific study and manipulation of milk for cheese manufacture is
however just over a hundred years old. Most cheese in the temperate countries of the
world such as Western Europe and the USA is made from cow’s milk, the composition of
which varies according to the breed of the cattle, the stage of lactation, the adequacy of its
nutrition, the age of the cow, and the presence or absence of disease in the breasts
(udders), known as mastitis. In some subtropical countries milk from sheep, goats, the
lama, yak, or ass is also used. Sheep milk is used specifically for the production of certain
special cheese types in some parts of Europe (e.g. Roquefort in France, and Brinsen in
Hungary). Milk from the water buffalo may be used in India and other countries, while
milk from the reindeer and the mare may be used in northern parts of Scandinavia and in
Russia, respectively. Cheese made from the milk of goat and sheep has a much stronger
flavor than that made from cow’s milk. This is because the fat in goat and sheep milk
contain much lower amounts of the lower fatty acids, caproic, capryllic, and capric acids.
These acids confer a sharp taste (similar to that of Roquefort cheese) to cheese made from
these mammals. Future discussion of cheese in this chapter will however refer to that
made from cow’s milk.
About a thousand types of cheese have been described depending on the properties
and treatment of the milk, the method of production, conditions such as temperature, and
the properties of the coagulum, and the local preferences.
Production of Fermented Foods
19.3.2.1
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Stages in the manufacture of cheese
The manufacture of all the types of cheeses include all or some of the following processes:
(a) Standardization of milk
The quality of the milk has a decided effect on the nature of cheese. Cheese made from
skim milk is hard and leathery; the more fat a cheese contains the smoother its feel to the
palate. The fat/protein ratio is often adjusted through fat addition in order to yield a
cheese of consistent quality. In the US, pasteurization (High Temperature Short Time) or
(Long Temperature Short Time) must be given to milk to be in certain types of cheeses,
such as cottage or cream cheese. For others the milk need not be pasteurized. but must be
stored at for at least 60 days at 2°C. If however the ‘starter’ is slow acting or souring is
delayed, food-poisoning staphylococci could develop and produce toxins in the cheese.
Sub-pasteurization temperatures are often the legal compromise. Pasteurization gives a
better control over the processes of cheese production. However, the organisms present in
raw milk are important during the ripening processes.
The milk may also be homogenized by forcing it at high speed through small orifices to
reduce the milk fat globules for use in producing soft cheeses.
(b) Inoculation of pure cultures of lactic acid bacteria as starter cultures
In the past, lactic acid was produced by naturally occurring bacteria. Nowadays they are
inoculated artificially, by specially selected bacteria termed starters. Indeed lactic acid
formation is necessary in all kinds of cheese. The propagation and distribution of lactic
acid bacteria for use in cheese manufacture is an industry in its own right in the United
States. For cheese prepared at temperatures less than 40°C strains of Lactococcus lactis are
used. For those prepared at higher temperatures the more thermophilic Streptococcus
thermophilus, Lactobacillus bulgaricus, and Lact. helveticus are used.
Lactic acid has the following effects:
(i) It causes the coagulation of casein at pH 4.6, the isoelectric point of that protein,
which is used in the manufacture of some cheeses, e.g. cottage cheese.
(ii) It provides a favorably low pH for the action of rennin the enzyme which forms the
curd from casein in other types of cheeses.
(iii) The low pH eliminates proteolytic and other undesirable bacteria.
(iv) It causes the curd to shrink and thus promotes the drainage of whey.
(v) Metabolic products from the lactic acid bacteria such as ketones, esters and
aldehydes contribute to the flavor of the cheese.
Problems of lactic acid bacteria in cheese-making
(i) Attack by bacteriophages: Bacteriophages sometimes attack the lactic acid starters
and besides choosing strains that are resistant to phages, rotations (i.e., using
different lactic mixtures every three or four days) helps eliminate them.
(ii) Inhibition by penicillin and other antibiotics: Lactic acid bacteria, being Gram-positive
are particularly susceptible to penicillin used to treat diseased udder in mastitis if
the antibiotic finds its way into the milk; other antiobiotics also have an inhibitory
effect on them.
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Modern Industrial Microbiology and Biotechnology
(iii) Undesirable strains: Some strains of lactic acid bacteria are undesirable in cheese
making because they produce too much gas, undesirable flavors, or produce
antibiotics against other lactic acid bacteria. They arise by mutation.
(iv) Sterilant and detergent residues: Sterilant and detergent residues may inhibit the
growth of starter bacteria. The minimum concentration required for inhibition
varies with the different anti-microbial agents and between different strains of
starter bacteria. Residues gain entry to milk at the (a) farm, (b) during
transportation to the factory, and (c) the factory due to careless use of sterilants or
detergents, incomplete draining or inadequate rinsing of equipment. The
inhibitory effects of sterilant and detergent residues are prevented by the correct
and ethical use of these materials. Proper use includes the use of the chemical at
the correct concentration and adequate rinsing and draining. Their presence is
mitigated by dilution with uncontaminated milk.
(c) Adding of rennet for coagulum formation
The classical material used in the formation of the coagulum is ‘rennet’ which is derived
from the fourth stomach, abomasum or vell of freshly slaughtered milk-fed calves.
Besides those of calves, the abomasum of kids (young goats), lamb or other young
mammals have been used. Rennet is produced by soaking and/or shredding air-dried
vells under acid conditions with 12-20% salt. Extracts from young calves contain 94%
rennin and 6% pepsin and from older cows, 40% rennin and 60% pepsin. Rennin
(chymosin) is the enzyme responsible for the coagulation of the milk. Pepsin is proteolytic
and too high an amount of pepsin can result in the hydrolysis of the coagulum and a
resulting low yield of cheese, and a bitter taste may result from the amino acids. Due to the
high cost of animal rennet, other sources, mostly of microbial origins, have been found
(Table 19.2).
Table 19.2
Some commercial microbial rennets and their microbial sources
Commercial Rennet
Microbial source
Harmilase
Rermilase
Fromase
Emposase
Meito
Suparen
Surd curd
Mikrozyme
Mucor miehei
Mucor miehei
Mucor miehei
Mucor pusillus
Mucor pusillus
Endothia parasitica
Endothia parasitica
Bacillus subtilis
The major effect of the milk-clotting enzymes is the conversion of casein from a
colloidal to a fibrous form. First the pH of the milk is brought down from pH 6.8 -7 to pH
5.5 by the action of lactic acid bacteria which produce lactic acid from lactose in the milk.
On addition of rennet, the active component, rennin, catalyses the hydrolyses of k-casein
to release para-k-casein and k-casein macropeptide. The latter goes into whey, while the
para-k-casein remains part of casein micelles, which now bind together to form the curd
following the removal of carbohydrates with the k-casein macropeptide and the exposure
Production of Fermented Foods
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of binding surfaces. The events up this coagulation are aided by lowered pH and by
increasing temperatures up to 45°C. Most of the bacteria, fat, and other particulate matter
are entrapped in the curd. When casein is removed the remaining liquid containing
proteins, lactalbumin, globulin, and yellow-green riboflavin (vitamin B2) is whey. The
whey proteins may be precipitated by heat, but not acid or rennin and they are used in
making whey cheese. The enzymes used in cheese making are now obtained from
microorganisms, mainly fungi.
(d) Shrinkage of the curd
The removal of whey and further shrinkage of the curd is greatly facilitated by heating it,
cutting it into smaller pieces, applying some pressure on it and lowering the pH. In many
types of cheeses, such as Parmesan, Emmenthal and Gruyere, there is a stage known as
‘scalding’ in which the temperature can be as high as 56°C in the preparation. Acid
produced by the lactic starters introduce elasticity in the curd, a property desirable in the
final qualities of cheese.
(e) Sa1ting of the curd and pressing into shape
Salt is added to most cheese varieties at some stage in their manufacture. Salt is important
not only for the taste, but it also contributes to moisture and acidity control. Most
importantly however it he1ps limit the growth of proteiolytic bacteria which are
undesirable. The curd is pressed into shape before being allowed to mature.
(f) Cheese ripening
The ripening or maturing of cheese is a slow joint microbiological and biochemical
process which converts the brittle white curd or raw cheese to the final full-flavored
cheese. The agents responsible for the final change are enzymes in the milk, in the rennet
and those from the added starter microorganisms as well as other micro-organisms
which confer the special character of the cheese to it. Among the cheese whose peculiar
characteristics are dependent on particular microorganisms are the blue-veined cheese
Roqueforti, Gorgonzola, Stilton, conferred by Penicillium roquefort, Swiss cheese, with its
characteristic flavor and holes produced by the fermentation products and gases from
Propionibacterium spp. Yeasts, micrococci, and Brevibacterium linens impart the
characteristic flavor of Limburger cheese. In soft cheese, such as Camembert, the protein
is completely broken down to almost amino acids, whereas in the hard cheese, the protein
remains intact.
19.3.3
Yoghurt and Fermented Milk Foods
Many types of fermented milks are produced and drunk around the world (Table 19.3)
Yoghurt is a fermented milk traditionally believed to be an invention of the Turks of
Central Asia, in whose language the word yoghurt means to blend, a reference to how the
milk product is made. Although accidentally invented thousands of years ago, yoghurt
has only recently gained popularity in the United States. While yoghurt has been present
for many years, it is only recently (within the last 30-40 years) that it has become popular.
This is due to many factors including the introduction of fruit and other flavorings into
yoghurt, the convenience of it as a ready-made breakfast food and the image of yoghurt as
a low fat healthy food.
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Modern Industrial Microbiology and Biotechnology
Table 19.3 Fermented milks and their presumed countries of origin
Name
Presumed
country of
origin
Yoghurt Asia,
Balkans
Acidophilus
USA
milk
Kafir
Caucasus
Kumiss
Mongolia
Lassi
India
Dahi
India
Leben
Middle East
Filmjilk
Sweden
Villi
Finland
Description
Cultures
Acidic, set or stirred,
characteristic aroma
S. thermophilus, Lb. bulgaricus,
(and Lb. acidophilus, Bifidobacterium spp.) *
Set, stirred or liquid,
mild flavor
Stirred beverage,
creamy consistency,
characteristic taste
and aroma (CO2)
Frothy beverage, acid,
refreshing taste
Sour milk drink diluted
with water, consumed
salted, spicy or sweet
Set, stirred, or liquid
beverage pleasant
Set or stirred product,
pleasant taste and aroma
Viscous stirred beverage,
clean acid taste
Viscous stirred product,
mildly sour
Lb. acidophilus
Lc. lactis, Lc. cremoris, Lb. kefir, Lb. casei,Lb.
Lb. bulgaticus, Lb. acidophilus, yeasts
Lactococcus spp., Lactobacillus
spp., Leuconostoc
S. thermophilus, Lb. bulgaricus,
Lc. diacetylactis,
S. thermophilus, Lb. bulgaricus,
Lb. acidophilus,
Lc. lactis, Lc. cremoris, Lc. diacetylactis, Ln.
Lc. lactis, Lc. cremoris, Lc. diacetylactis, Lc.
* Depending on the country
In the manufacture of yoghurt, two kinds of lactic acid bacteria, Lactococcus spp. and
Lactobacillus spp., are generally used with usually unpasteurized milk. Most commonly
used are Lactococcus salivarius and thermophilus, and Lactobacillus spp., such as Lacto.
acidophilus, bulgaricus and bifidus.
The bacteria produce lactic acid from lactose in the milk causing the pH to drop to
about 4-5 from about 7.0. This drop in pH causes the milk to coagulate. The lactic acid
gives yoghurt its sour taste and limits the growth of spoilage bacteria. Yoghurt is flavored
usually with fruits.
19.4
FERMENTED FOODS FROM CORN
Corn is a tropical crop, but grows in the summer in temperate climates, which have
at least 90 days which are frost free. It is known as maize in some parts of the
world. Scientifically known as Zea mays L, it is used to make important fermented foods in
west Africa and it is sometimes mixed with sorghum, Sorghum bicolor Linn for this
purpose.
Production of Fermented Foods
19.4.1
!"'
Ogi, Koko, Mahewu
Ogi, also known as akamu, is a Nigerian sour gruel made from maize. In Ghana the
equivalent foods are known as koko. For ogi preparation, corn is soaked in water for
about two days. Thereafter the cereal is wet-milled and sieved to remove the fibrous portions
of the maize. The starchy sediment is allowed to settle and to ferment for another two
days. The water is decanted off and the starchy sediment is ogi. It is prepared by boiling
it to form a thick gruel which can be consumed sweetened with sugar, or eaten with foods
made from beans. It may also be heated to form a stiff gel, known as agidi or eko when cool.
Ogi or the stiff gel made from it are popular weaning and convalescent foods in Nigeria.
Studies of the microbiology of ogi production show that in the early stages of
fermentation, fungi such as Fusarium and Cephalosporium which are acquired from the
field, and which form the bulk of the organisms in the first 24 hours, soon disappear to be
replaced with lactic acid bacteria especially (Lactobacillus plantarum and Lact
mesenteroides) and yeasts (Saccharomyces cerevisiae, Rhodotorula spp., and Candida
mycoderma which become the dominant organisms at the time of the milling.
The flavor of ogi has been shown to be due to the activities of lactic acid bacteria and
occasionally to yeasts and acetic acid bacteria. The following acids were identified in that
quantitative order by gas chromatography: acetic, butyric, pentanoic, isohexanoic, and
isobutyric.
The nutritional quality of ogi suffers from its method of preparation: the soaking of the
grains and the discarding of the overtails before milling leads to loss of minerals as well
as a portion of the already low quantity of protein and amino acids present in the cereals.
The flow charts for ogi production and that of a similar food, koko, from Ghana are in Fig.
19.1
Koko
Soak grains
12 hours
Grind
Mix
Ferment
1-3 days
Disperse
Cook
Ogi
Soak grains
1/3 days
Grind
Sieve
Ferment
1-3 days
Decant
Cook
Sievates
discarded
Fig. 19.1
Supernatant
discarded
Flow Charts for Koko and Ogi Production
Mahewu, also known as mogou, is a South African sour food. Although the main
organism found in locally-made mahewu is Streptococcus lactis, mahewu is made on an
industrial scale by inoculating Leuconostoc delbruckii into autoclaved 8-10% maize slurry,
fermenting the mixture for about 12 hours and spray-drying the slurry. It is an acid food
of about pH 3.5, and in order to ensure that the proper level of lactic acid is produced to
attain this pH level, buffering salts such as CaHPO4 are somtimes added. It is a
convenient food consumed by miners and the dry powder needs only to be reconstituted
in cold water to get the food ready for consumption. The food is sometimes enriched with
vitamin and protein rich additives such as yeast extract.
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Modern Industrial Microbiology and Biotechnology
19.5 FERMENTED FOODS FROM CASSAVA: GARRI, FOO-FOO,
CHIKWUANGE, KOKONTE, BIKEDI, AND CINGUADA
Cassava is an important source of food all over the tropical world in South America,
Africa, India and the Far East. Botanically it is a member of the family Euphorbiaceae and
is classified as Manihot esculenta Crantz (formerly Manihot utillisaima Pohl). It has a
number of synonyms around the world: manioc (Madagascar and French-speaking
Africa) tapioca (India, Malaysia), ubi scetlela (Indonesia) manioca or yucca (Latin
America).The plant tolerates low soil fertility and drought, better than most crops and
needs little maintenance once planted. It has also been claimed to be a higher producer of
carbohydrate then commonly cultivated cereals and tuber crops and under favorable
conditions will yield above 90 ton/hectare. It is therefore not surprising that it is the
staple food in densely populated areas of the tropics such as Central Java in Indonesia,
the State of Kerala in India, south-eastern Nigeria and north-eastern Brazil and is
consumes by an estimated 400 million people around the world. Nigeria is currently the
world’s largest producer followed by Brazil, Indonesia, Zaire, Thailand, and Tanzania.
Periderm
Cortex
}
Peel
Central vascular fiber
Starchy inner flesh
Starch granules in cells in the starchy
inner flesh
Fig. 19.2 Transverse Section of the Cassava Root
A major shortcoming of cassava roots is that they are very low in protein. In addition
many varieties contain the cyanogenic glucosides, linamarin and (to a lesser extent)
lotaustralin. These glucosides can give rise to fatalities if cassava roots are consumed
unprocessed. Cassava may be processed by boiling, roasting, drying, leaching with cold
water, or by fermentation. By far the most popular method of processing cassava is by
fermentation. In producing fermented cassava products, the roots may first be grated
before fermentation, the whole root may be cut into large pieces and fermented in water
(retted). The best known example of foods produced from cassava pulp is garri, while
those produced from the retting of whole roots include foo-foo, chikwuangue, kokonte,
and cinguada.
Production of Fermented Foods
19.5.1
!#
Garri
Garri is a popular food for about 100 miilion people in West Africa; in Cote d’Ivoire, atieke,
a food very similar to garri, produced from cassava but is is not fried like garri. A similar
food known as farinha de manioc or farinha de mega is consumed in parts of Brazil.
Preparation of Garri: Garri is currently prepared mostly on small, house-hold scales. The
first stage is the peeling to remove the brownish thin outer covering (Fig. 19.2) to reveal the
white fleshy inner portion which is grated on a hand-held rasper or crushed in a grating
machine. The central pith and primary xylem provide some fibers in the grated material
some of which is removed by sieving, but which is appreciated by some garri consumers.
Peeling of
Cassava roots
Grating
Palm
-oil
Added
Optional
Bagging,
Dewatering,
Fermentation
24–96 hours
Sieving
Frying
Sieving
GARRI
Fig. 19.3 Flow Chart of Garri Production
The mash resulting from the grating is placed in cloth bags for between 18 and
48 hours and fermented. During the period of fermentation, the mash is dewatered by
placing heavy objects on the cloth bags. At the end of the fermentation period, the mash is
sieved through a coarse sieve and heated, sometimes with a little palm oil, in a flat iron
pot with stirring.
Microbiology of the fermentation of Garri: In 1959 Collard and Levi published their study
on the two-stage fermentation of cassava pulp. In the first stage which lasted for the first
48 hours a yellow-pigmented bacterium, Corynebacterium manihot, proliferated. This
organism broke down starch eventually to organic acids including lactic acid. The
resulting drop in pH led to the spontaneous breakdown of the linamarin and the
proliferation of the fungus Geotrichum candidum which produced the flavoring
aldehydes, ketones and other compounds. In 1977 Okafor re-examined fermenting pulp
and while there he found some Corynebacterium, the bulk of the organisms were lactic
acid bacteria, especially Lactobacillus, Leuconostoc and and yeasts. He suggested the
absence of lactic acid bacteria in the work of Collard and Levi was probably because they
used nutrient agar, a medium lacking sugars in which lactic acid bacteria grow better.
Furthermore, linamarin is fairly stable and the glucoside is probably broken down more
by the indigenous linamarase of the enlarged roots, which is released when the roots are
crushed than by a change of pH. Other workers have since confirmed the importance of
lactic acid bacteria and yeasts in the fermentation of cassava during garri production.
The current thinking on the microbiological processes of cassava fermentation for
garri production is that when cassava roots are grated to produce the mash which is
bagged and fried to produce garri, the indigenous linamarase present in the roots is
released and makes contact with the cyanogenic glucosides in the roots. The glucosides,
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Modern Industrial Microbiology and Biotechnology
mostly linamarin and some lotaustralin (about 5% of the total glucosides) are then broken
down into glucose and HCN (Fig. 19.4). The HCN release is characteristically noticed by
the pungent smell in evidence whenever cassava is being grated. As the amount of the
linamarase is insufficient to hydrolyze all the cyanogenic glucosides or because the
particles are not fine enough to ensure complete contact between the enzyme and the
substrate, there is always residual glucoside which enters the garri as cyanide. Many of
the organisms encountered in fermenting cassava mash are lactic acid bacteria and
yeasts. Lactobacillus, Leuconostoc, the yeast Candida and various other yeasts are
encountered in fermenting cassava mash, and many strains of these have been found to
produce linamarase. By inoculating one of such linamarase producing organisms into
fermenting cassava mash the group was able to almost totally remove the residual
cyanide in garri.
Fig. 19.4 Breakdown of Cassava Cyanogenic Glucosides
19.5.2
Foo-Foo, Chikwuangue, Lafun, Kokonte,
Bikedi, and Cinguada
The preparation of these foods, although eaten in different parts of Africa, is similar. Foofoo is eaten in parts of eastern Nigeria, while lafun is eaten in western Nigeria.
Chikwuangue is eaten in Democratic Republic of the Congo, bikedi in Congo
(Brazzaville), kokonte and cinguada are eaten in Ghana and East Africa respectively. In
the preparation of these foods, cassava roots are cut into large pieces and immersed in
still water in pots or in running stream water and allowed to ret for one to five days; for
foo-foo or chikwuangue fermentation retting takes between three and six days so that the
starch can be extracted from the retted roots by macerating with the hands. For kokonte
and cinguada, retting is only partial and hardly lasts more than two days; the material is
then sun-dried and pounded into a flour when it is known as lafun in Nigeria. For foo-foo
and bikedi the retted roots are macerated to extract the starch. The retting is a result of the
breakdown of the pectin in the cell walls of the cassava root brought about by pectinases
produced by bacteria of the genus Bacillus spp., while the lactic acid bacteria are
responsible for the flavor of these foods.
Production of Fermented Foods
19.6
!#!
FERMENTED VEGETABLES
Like the fermentation of other foods, vegetables have been preserved by fermentation from
time immemorial by lactic bacterial action. A wide range of vegetables and fruits
including cabbages, olives, cucumber, onions, peppers, green tomatoes, carrots, okra,
celery, and cauliflower have been preserved. Only sauerkraut and cucumbers will be
discussed, as the same general principles apply to the fermentation of all vegetables and
fruits. In general they are fermented in brine, which eliminates other organisms and
encourages the lactic acid bacteria.
19.6.1
Sauerkraut
Sauerkraut is produced by the fermentation of cabbages, Brassica oleracea, and has been
known for a long time. Specially selected varieties which are mild-flavored are used. The
cabbage is sliced into thin pieces known as slaw and preserved in salt water or brine
containing about 2.5% salt. The slaw must be completely immersed in brine to prevent it
from darkening. Kraut fermentation is initiated by Leuconostoc mesenteroides, a
heterofermentative lactic acid bacterium (i.e., it produces lactic acid as well as acetic acid
and CO2.) It grows over a wide range of pH and temperature conditions. CO2 creates
anaerobic conditions and eliminates organisms which might produce enzymes which
can cause the softening of the slaw. CO2 also encourages the growth of other lactic acid
bacteria. Gram negative coliforms and pseudomonads soon disappear, and give way to a
rapid proliferation of other lactic acid bacteria, including L. brevis, which is
heterofermentative, and the homofermentative L. plantarum; sometimes Pediococcus
cerevisiae also occurs. Compounds which contribute to the flavor of sauerkraut begin to
appear with the increasing growth of the lactics. These compounds include lactic and
acetic acids, ethanol, and volatile compounds such as diacetyl, acetaldehyde, acetal,
isoamyl alcohol, n-hexanol, ethyl lactate, ethyl butarate, and iso amyl acetate. Besides the
2.5% salt, it is important that a temperature of about 15°C be used. Higher temperatures
cause a deterioration of the kraut.
19.6.2
Cucumbers (pickling)
Cucumber (Cucumis sativus) is eaten raw as well after fermentation or pickling.
Cucumbers for pickling are best harvested before they are mature. Mature cucumbers are
too large, ripen easily and are full of mature seeds. Cucumbers may be pickled by dry
salting or by brine salting.
Dry salting is also generally used for cauliflower, peppers, okra, and carrots. It consists of
adding 10 to 12% salt to the water before the cucumbers are placed in the tank. This
prevents bruising or other damage to the vegetables.
Brine salting is more widely used. A lower amount of salt is added, between 5 and 8% salt
being used. Higher amounts were previously used to prevent spoilage. It has been found
that at this salt concentration, the succession of bacteria is similar to that in kraut.
However Leuconostoc spp. never dominate. During the primary fermentation lasting two
or three days, most of the unwanted bacteria disappear allowing the lactics and yeasts to
proliferate. In the final stages, after 10 to 14 days, Lactobacillus plantarum and L. brevis,
followed by Pediococcus, are the major organisms.
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Modern Industrial Microbiology and Biotechnology
19.7 FERMENTATIONS FOR THE PRODUCTION OF
THE STIMULANT BEVERAGES: TEA,
COFFEE, AND COCOA
Tea, coffee, and cocoa are produced mainly in the rainforest zones of the Indian subcontinent and in South America and West Africa respectively. Tea can also grow in the
cooler temperatures of mountains. The beverages are stimulating on account of their
content of either one or the other of two chemically similar stimulants, caffeine and
theobromine (Fig. 19.5). Of the three, only cocoa and coffee are produced by some form of
fermentation; the production of tea is strictly speaking a chemical reaction, but it is
included for completeness.
Fig. 19.5 Structure of Caffeine and Theobromine
19.7.1
Tea Production
Tea (Camellia sinensis; previously Thea) is believed to have originated from south-east
China. It has now spread to many parts of the world including India, Sri Lanka,
Malaysia, Kenya, Georgia in the former USSR, Turkey, Iran, Malawi, Cameroon,
Thailand, Vietnam, Mexico, and Argentina, to name some of the countries where varieties
of tea grow. Young tea leaves are harvested by hand and spread on trays to wither.
Thereafter the leaves are rolled to squeeze out juices from the leaves and spread the juices
over the surface of the leaves. This exposes the polyphenols to oxidation, and the green
color gradually begins to turn brownish. Rolling also breaks the leaves into smaller
pieces. The ‘fermentation’ stage follows, but this is a chemical reaction involving
polyphenols. After fermentation, the tea is ‘fired’, i.e. subjected to hot air of between 80
and 90°C. After firing, the tea is sorted and graded.
19.7.2
Coffee Fermentation
Coffee (Coffea arabica and C robusta) originated from Ethiopia. The main producers of
coffee today are Colombia, Brazil, Angola, and Indonesia, in that order. It takes from three
to five years of growth before the coffee tree is ready to bear fruit. The fruits grow slowly,
Production of Fermented Foods
!##
taking from 8 to 12 months to reach maturity (when they are bright red in color). Each
coffee fruit or berry contains two seeds covered by pulp.
There are two methods of processing coffee: the wet method and the dry method. In the
wet method, the fruits are passed through a pulping machine which removes the pulp
leaving by mucilage which is removed by pectinolytic enzymes of microbiological origin.
The coffee may also be dried by exposure to sunlight. When dry, the fruits are dehulled to
remove the dry outer portions. The studies carried on the microbiology of the coffee
fermentation showed that many of the organisms were pectinolytic organisms, including
spore-forming and non-spore forming ones. Other workers found lactic acid bacteria
(Leuconostoc spp. and Lactobacillus spp.) and yeasts (Saccharomyces spp and
Schizosaccharomyces spp.), and it would appear that these developed from the release of
the pectinolytic organisms.
19.7.3
Cocoa Fermentation
Cocoa (Theobroma cacao) is a native of South America, but today the major producers are
Ghana, Nigeria, Ivory Coast, Cameroon, and Malaysia. The tree produces pods which
contain from 40 to 60 seeds. The pods are opened and the seeds heaped and allowed to
ferment, often in baskets which permit liquid to drain out. During fermentation the
mucilagenous outer covering of the seeds is broken down by microbial action, while the
seeds themselves change from pinkish to black. It is believed that the lactic acid bacteria
play important roles in the development of the aroma of cocoa.
19.8 FERMENTED FOODS DERIVED FROM
LEGUMES AND OIL SEEDS
Legumes are members of the Leguminosae. Their seeds are rich in proteins and they are
fermented in various parts of the world for flavoring condiments or as major meals.
Fermented seeds of soybeans, beans (Phaeseolus) and the African oil bean, Pentaclethra
macrophylla Benth will be discussed.
19.8.1
Fermented Foods from Soybeans
Fermented soybean products have been made and consumed in large amounts in
countries of the Orient for thousands of years. It has been suggested that the Buddhist
religion which emphasizes the absence of meat from the diet may have been responsible
for the development of soy-based foods in China, Japan, Korea, and other oriental
countries. Table 19.4 shows some soy foods and where they are consumed. The use of
some of them has spread to other parts of the world including the US and parts of Europe.
The soybean plant itself Glycine max is a legume believed to have originated from
Eastern Asia. It is now grown around the world.
The soybean seed has an unusual composition. It is rich in protein and oil, and
comparatively low in carbohydrates. Its average composition is 42% protein 17%
carbohydrate, 18% oil, and 4.6 ash. Sucrose, raffinose, stachyose and pentosans are
among the carbohydrates. The beans are rich in phospholipids, nucleic acids, and
vitamins especially thiamin, riboflavin, and niacin. It should be noted that the
composition of soybeans varies from place to place. The amino acid composition of its
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Modern Industrial Microbiology and Biotechnology
Table 19.4
Fermented products of soybeans and countries of origin or of greatest use
Product
Country
Soy sauce
Miso
Natto
Fermented soy sauce
Sufu
Tempeh
China, Japan
China, Japan, Philippines
Japan, China
Japan
China, Taiwan
Indonesia
protein is also unusual among plant proteins in that it contains high amounts of
methionine which is more characteristic of animal than plant proteins.
Soybean is a very nutritious food. However it has shortcomings which are ameliorated
by fermentation. Soybeans contain compounds which make the legume unattractive
until they are removed by the various stages involved in their processing by fermentation.
First, they contain carbohydrates, which are not absorbed until they reach the colon,
where the gases produced when they are broken down by microorganisms give rise to
flatulence. These carbohydrates include the oligosacharides, raffinose and stachyose
and the polysaccharide, arabinogalactan. Second, soybeans have a bitter and ‘beany’
taste when crushed. This is because the lipoxygenase enzyme which helps produce this
taste and the substrate (oil) are held in separate compartments in the tissues of the seeds
until the latter are broken or crushed.
Third, soybeans contain anti-nutritional factors such as trypsin inhibitor,
hemagglutinins and saponins. Finally even after cooking, about 1/3 of the protein of
soybeans cannot be digested.
The soaking of the soybean preceding cooking leaches out a large proportion of the
flatulence producing carbohydrates. The ‘beany’ flavor is due to the presence of several
carbonyl compounds such as hexanol and pentanol. These are removed by the action of
microorganisms. Fermentation also reduces the carbohydrates of rice and proteins of the
bean to lower molecular weights, hence rendering them more digestible. Finally, the anti
nutritional factors are destroyed by boiling. In addition to all this, fermentation by R
oligosporous produces an anti-oxidative compound (41, 61, 7 trihydroxy-bisoflavane)
which is absent from raw soybean, and which helps preserve the fermented foods. The
fermented foods derived from soybean (soy sauce, miso, natto) will be considered in this
section.
19.8.1.1
Soy sauce
Soy sauce known as shoyu in Japan is a salty pleasantly tasting liquid with a distinct
aroma and which is made by fermenting soybeans, wheat, salt with a mixture of molds,
yeasts and bacteria. Five different types of shoyu are recognized by the Japanese
Government, depending on the proportions of the ingredients used and the method of
preparation. Koikuchi-shoyu is the most produced, forming 85% of the total produced. It
is this type which is also best known in countries outside the orient. Koikuchi-shoyu is
deep red-brown in color and is an all purpose seasoning, with a strong aroma and
myriad flavor. It is the only type to be discussed.
Production of Fermented Foods
!#%
Soy sauce manaufacture: The manufacture of koikuchi-shoyu can be divided into four
sections: i) the preparation of the ingredients; ii) koji preparation; iii) brine fermentation;
iv) refining process
Preparation of the ingredients: Whole wheat is roasted and then coarsely ground. Roasting
adds color and flavor to the resulting sauce and kill surface organisms as well as
facilitates enzymatic hydrolysis of the grain. Soybeans, usually defatted, are cooked
under high pressure and temperature for a short time after a previous soaking in water.
Koji preparation: Whole wheat and soy prepared as described above are used for the
preparation of koji. A koji starter, or seed mold or inoculum is first prepared from the
spores of several different strains of Aspergillus oryzae or Asp soyae by inoculating the
spores of the fungi on to a mixture of boiled rice and wood ash or mineral salts and
spreading the mixture thinly at 30°C for up to five days. The koji starter (also known as
tane koji) is used to inoculate equal amounts of the wheat and soy prepared as above. This
used to be turned manually in shallow trays, but is now also being done mechanically.
The mixture is put into large vats and aerated by forced aeration. The important
requirements of koji are that it should have high protease and amylase activities. As these
are dependent on temperature and humidity, the latter are strictly controlled. After two to
three days koji is harvestod as a greenish-yellow material due to the spores of Aspergillus.
Brine Fermentation: Koji is introduced into deep fermentation tanks to which an equal
volume of salt solution 20-23% is added. The resulting mixture, known as moroni is
allowed to ferment for 6-8 months. It is frequently mixed to distribute the material and to
eliminate undesirable anaerobic organisms.
During the period, koji enzymes hydrolyze proteins to amino acids and low molecular
weight peptides; much of the starch is converted to simple sugars which are then
ferrmented to lactic acid, alcohol and CO2. The pH drops from around 6.5-7.0 to 4.7-4.8.
The effective salt concentration is about 18 % (because of the dilution with added koji); it
is never allowed to fall below 16% otherwise putrefactive organisms might develop.
There are three stages in the fermentation of moromi, which is brought about by
osmophilic strains of microorganisms, after the release of simpler substances by the fungi
of the koji. In the first stage, Pediococcus halophilus produces lactic acid, causing a drop of
the pH. In the second stage Saccharomyces rouxii develops and produces alcohol. In the
last stage, Torulopsis yeasts develop. These produce phenolic compounds which are
important components of koichuki-shoyu flavor. The organisms are selected by the
conditions of the fermentation, but pure cultures as used more and more nowadays to
ensure a more consistent flavor.
Refining: The final state consists of pressing the fermented moromi to release the soy
sauce. Hydraulic presses are used in modern production. The raw soy sauce is heated to
70-.80°C to pasteurize it, to develop color and flavor and to inactivate the enzymes. After
clarification by sedimentation the sauce is bottled under aseptic conditions, sometimes
with the addition of preservatives as well.
In China ‘tamari-shoyu’ which forms less than 3% of Japanese sauce, is the main type
of shoyu. The two differ in that tamari has a higher proportion of soybeans (90% instead
of 50%). Furthermore, tamari sauce is not pasteurized. Due to the low quantity of rice,
little alcoholic fermentation occurs in tamari because of the paucity of sugars.
!#&
Modern Industrial Microbiology and Biotechnology
19.8.1.2
Miso
Miso, a fermented paste of soybean, wheat and salt is the most important of the soy
fermented products in Japan. There are many types of bean pastes. They are also popular
in China, Korea and other parts of the Orient, where the different types of paste produced
vary according to the proportions of wheat, soybean and salt used, and the lengths of the
fermentation and ageing. In Korea they are known as ‘jang’; ‘miso’, and ‘shoyu’ in Japan,
‘tao-tjo’ in Indonesia and Thailand and ‘tao-si’ in the Phillipines. In Japan the average
annual consumption is 7.2 kg per person, 80-85% being used in the miso group and the
rest as seasonings for various types of foods. Most miso in Japan has a consistency like
peanut butter, the color varying from a creamy yellowish white to very dark brown. The
darker the color in general, the stronger the flavor. It is distinctively salty and has a
pleasant aroma.
Manufacture of Miso: Miso production is basically similar to that of shoyu or soy sauce.
There are however two basic differences in the production of the two foods. First, the koji
or shoyu is made by using a mixture of soybeans and wheat. In koji-making for miso, only
the carbohydrate material (rice or barley) is used. Soybeans are not used for making koji
miso except in the case of soybean miso. Second, no pressing is done after miso
fermentation. Since the material is a paste; the absence of pressing affects the cost of miso.
The organisms involved in the fermentation are the same, but Streptococcus faecalis is also
included. After fermentation, the resulting koji is mixed with salt, cooked soybean, pure
cultured yeasts, and lactic acid bacteria and then fermented for a second time. It is then
aged and packaged as miso; sometimes it may be freeze-dried before packaging.
19.8.1.3
Natto (Fermented whole soybean)
Whereas soy sauce (shoyu) and miso (bean paste) originated from China, nato, fermented
whole soybean is an indigenous Japanese food, originating there more than
1,000 years ago. There are two types of natto, itohiki-natto and hamma-natto. Hama-natto
is produced by the action of Aspergil1us. It is produced only in limited quantities. Natto
therefore usually refers to the second and commoner type itohiki-natto. This second type
is fermented by Bacillus natto. The shape of cooked whole soybean grains is kept, but the
surface of each grain is covered with a viscous material consisting of glutamic acid
polymers produced by B natto.
The manufacture is uncomplicated. Cooked soybean grains are inoculated with the
Bacillus and put into a small tray, covered, and incubated at 40°C. After 14-18 hour, the
packed tray cooled to 2-7°C and then shipped to the market. It is cheap and nutritious and
natto is usually served with shoyu and mustard.
19.8.1.4
Tempeh: Oncom and related foods
Tempeh is a popular Indonesian food made by fermenting soybean with strains of
Rhizopus. Especially in the Indonesian Island of Java, tempeh is a key protein source and
30-120 gm is consumed daily per person. It therefore replaces meat in the grain-centered
local meal. It is also eaten in Surinam and New Guinea, but not in the colder regions of the
Orient.
Production of Fermented Foods
!#'
Traditional tempeh preparation varies in minor details. Essentially air-dried
soybeans are soaked in water and the seed coats are removed. The dehuued beans are
boiled in water, drained, cooled, and inoculated with one of the traditional mold inocula.
The beans are then packed in small parcels and incubated at room temperature of about
25°C for approximately 40 hour. Fermentation is regarded as complete when the beans
have become bound tightly by the mold mycelium into compact white cakes, which are
usually consumed within a day or two. It can then, after fermentation be deep-fried for 34 minutes or boiled for 10 minutes.
Although several species of Rhizopus may be used, Rhizopus oligosporous Saito has
been shown to be the species producing tempeh. The fungus is strongly proteolytic but
has only weak amylase activity, desirable qualities since soybean is high in proteins, but
relatively low in carbohydrate content. The proteolytic enzyme of Rhisopus oligosporous is
not inhibited by inhibitory factors in soy bean.
19.8.2
Fermented Foods from Beans: Idli
Idli is a popular fermented breakfast and hospital food which has been eaten in South
India for many years. It is prepared from rice grains and the seeds of the leguminous
mung grain, Phaeseolus mungo, or from black beans, Vigna mungo, which are also known
as dahl. When the material contains Bengal grain, Circer orientium, the product is known
as khaman. It has a spongy texture and a pleasant sour taste due to the lactic acid in the
food. It is often embellished with flavoring ingredients such as cashew nuts, pepper and
ginger.
Production of Idli
The seeds of the dahl (black gram) are soaked in water for 1-3 hours to soften them and to
facilitate decortication, after which the seeds are mixed and pounded with rice in a
proportion of three parts of the beans and one of rice. The mixture is allowed to ferment
overnight (20-22 hours). In the traditional system the fermentation is spontaneous and
the mixture is leavened up to approximately 2 or 3 times. The organisms involved in the
acidification have been identified as Streptococcus faecalis, and Pediococcus spp. The
leavening is brought about by Leuconostoc mesenteroides, although the yeasts, Torulopsis
candida and Trichosporon pulluloma have also been found in traditional Idli. The
fermented batter is steamed and served hot. Idli is highly nutritious, being rich in
nicotinic acid, thiamine, riboflavin, and methionine.
bbb
Rice
Soaked
5-10 hr
Ground
Mixed
Decorticated
black gram
Soaked
5-10 hr
Fermented
20-22 hr
Ground
Fig. 19.6 Flow chart of Idli Production
Fermented
batter steamed
and served hot
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Modern Industrial Microbiology and Biotechnology
19.8.3
Fermented Foods from Protein-rich Oil-seeds
Stew condiments made from oil-rich seeds are eaten in parts of West Africa, while
condiments from fish and fish products appear to be common in parts of Asia.
Stew condiments eaten in parts of Nigeria include dawadawa, know as iru in the
Southwest geopolitical zone of Nigeria which is produced from the seeds of Parkia
biglobosa. Cadbury Plc Nigeria now makes and markets dawadawa as Dadawa. A
condiment very similar to dawawa is okpeyi and comes from the Nsukka area of the
Southeast and is made from the seeds of Prosopsis africana. Another major soup
condiment popular in the Southeast zone is ogili which may be made from the seeds of
castor-oil seeds (Ricinus communis) or egusi (Citrullus lanatus sub-species colocynthoides).
Egusi, pumpkins and squashes are members of the family Cucurbitaceae or cucurbits and
their seeds contain about 50% oil and 35% protein after dehulling. Besides egusi, another
well-known cucurbit in Nigeria is ugu or Telfaria sp.; its seeds and those of soybeans are
sometimes used for making ogili.
A fermented delicacy and meat substitute, ugba or ukpaka is made from the seeds of the
African locust bean, Pentaclethra macrophylla Benth). This is generally eaten with
stockfish and it is very popular in the South-east zone of Nigeria. Bacillus spp. have been
implicated in the fermentation of the various oilseeds discussed above.
19.8.4
Food Condiments Made from Fish
Fish sauce is eaten in many parts of Asia including Japan, Thailand, Vietnam, the
Philippines, Indonesia and Malaysia, and some parts of Northern Europe, including
France.
Table 19.5
S/No
1
2
3
4
5
6
Methods of fish sauce preparation and countries of use
Country
Fish used
Method
France
Indonesia
Japan
Malaysia
The Philippines
Thailand
Gobius spp.
Clupea spp.
Clupeapilchardus
Stelophorus spp.
Stelophorus spp.
Stelophorus spp.
2-8 weeks in 4:1 salt solution
6 months in 6 :1 salt solution
5-6 months in 5 :1 salt to rice
4–12 months; 5 :1 salt to sugar
3–12 months; 4: 1 salt solution
6-12 months; 4:1 salt solution
The fish is fermented in a solution of salt. Sometimes, the fish is fermented whole, or
sometimes only the viscera are fermented. Sometimes carbohydrate sources such as
malted rice may be added, as in Japan.
SUGGESTED READINGS
Amund, O.O., Ogunsina, O.A. 1987. Extracellular amylase production by cassava-fermenting
bacteria. Journal of Industrial Microbiology. 2, 123-127.
Bamforth, C.W. 2005. Food, Fermentation and Microorganisms. Blackwell Oxford, UK.
Banigo, E.O.I., de Man, J.M., Duitschaever, C.L. 1974. Utilization of high-lysine corn for the
manufacture of ogi, using a new improved proceessing system. Cereal Chemistry 51: 559–
573.
Production of Fermented Foods
!$
Bankole, M.O., Okagbue, R.N. 1992. Properties of “nono”, a Nigerian fermented milk food.
Ecology of Food and Nuitrition. 27, 145-149.
Batock, M., Azam-Ali, S. 1998. Fermented Frutis and Vegetables. A Global Perspective. FAO.
Agricultural Services Bulletin No. 134. Food and Agriculture Organization of the United
Nations, Rome, Italy.
Enujiugha, V.N., Akanbi, C.T. 2005. Compositional Changes in African Oil Bean (Pentaclethra
macrophylla Benth) Pakistan Journal of Nutrition, 4. 27-31.
Ikediobi, C.O., Onyike, E. 1982. The use of linamarase in gari production. Process Biochemistry
17, 2-5.
Kobawila, S.C. Louembe, D., Keleke, S., Hounhouigan, J., Gamba, C. 2005. Reduction of the
cyanide content during fermentation of cassava roots and leaves to produce bikedi and ntoba
mbodi, two food products from Congo. African Journal of Biotechnology. 4 689-696.
Lei, V., Amoa-Awua, W.K.A., Brimer, L. 1999. Degradation of cyanogenic glycosides by
Lactobacillus plantarum strains from spontaneous cassava fermentation and other
microorganisms International Journal of Food Microbiology 53, 169-184.
Nwachukwu, S.U., Edwards, A.W.A. 1987. Microorganisms associated with cassava
fermentation for lafun production. Journal of Food and Agriculture. 1, 39-42.
Okafor, N., Okeke, B., Umeh, C., Ibenegbu, C. 1999. Secretion of Lysine by Lactic Bacteria and
Yeasts Associated with Garri Production Using a Synthetic Gene. Letters in Applied
Microbiology, 28, 419-422.
Okafor, N., Uzuegbu., J.O. 1993. Studies on the Contributions of Factors other than microorganisms to the Flavour of Garri. Journal of Agricultural Technology, 1, 36-38.
Okafor, N., Umeh, C., Ibenegbu, C. 1998. Carriers for starter cultures for the production of garri,
a fermented food derived from cassava. World Journal of Microbiology and Biotechnology.
15, 231-234.
Okafor, N., Umeh, C., Ibenegbu, C. 1998 Amelioration of garri, a fermented food derived from
cassava, Manihot esculenta Crantz, by the inoculation into cassava mash, of micro-organisms
simultaneously producing amylase, linamarase, and lysine. World Journal of Microbiology
and Biotechnology, 14, 835-838.
Okafor, N., Umeh, C., Ibenegbu, C, Obizoba., I.C., Nnam, P. 1998. Improvement in garri quality
by the inoculation of micro-organisms into cassava mash. International Journal of Food
Microbiology. 40, 43-49.
Okafor, N., Ejiofor, M.A.N. 1985. Microbial breakdown of linamarin in fermenting cassava pulp.
MIRCEN Journal of Applied Microbiology and Biotechnology. 2, 269-276.
Okafor, N., Ejiofor, M.A.N. 1985. The linamarase of Leuconostoc mesenteroides: Production,
isolation and some properties. J. Sci. Food Agric. 36, 668-678.
Okafor, N., Ejiofor, A.O. 1990. Rapid detoxification of cassava mash fermenting for garri
production following inoculation with a yeast simultaneously producing linamarase and
amylase Process Biochemistry International. 25, 82-86.
Okafor, N., Uzuegbu, J. 1987. Studies on the contributions of micro organisms on the organoleptic properties of garri, a fermented food derived from cassava (Manihot esculenta Crantz).
J. Food Agric. 2, 99-105.
Okafor, N. 1977. Microorganisms associated cassava fermentation for garri production. J. Appl.
Bact. 42, 279-284.
Okafor, N., Oyolu, C., Ijioma, B.C. 1984. Microbiology and biochemistry of foo-foo production.
J. Appl. Microbiol., 55, 1-13.
Omofuvbe, B.O. Abiose, S.H., Shonukan, O.O. 2002. Fermentation of soybean (Glycine max) for
soy-daddawa production by starter culturesof Bacillus. Food Microbiology, 19, 187-190.
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Modern Industrial Microbiology and Biotechnology
Oteng-Gyang, K., Anuonye, C.C. 1987. Biochemical studies on the fermentation of cassava
(Manihot utilissima Pohl). Acta Biotechnologica. 7, 289-292.
Oyewole, O.B. 2001. Characteristics and significance of yeasts’ involvement in cassava fermentation
for ‘fufu’ production. International Journal of Food Microbiology. 65, 213-218.
Oyewole, O.B., Odunfas 1990. Characterization and distribution of lactic acid bacteria in cassava
fermentation during fufu production. Journal of Applied Bacteriology, 68, 145-152.
Popoola, T.O.S., Akueshi, C.O. 1986. Nutritional value of dawadawa, a local spice made from
soybean (Glycine max) MIRCEN Journal. 2, 405-409.
Sanni, A.I., Onilude, A.A., Fadahunsi. S.T., Ogunbanwo, S.T., Afolabi, R.O. 2002. Selection of
starter cultures for the production of ugba, a fermented soup condiment. European Food
Research and Technology, 215, 176-180.
Tamime, A.Y., Robinson, R.K. 1999. Yoghurt Science and Technology. CRC Press.
Wood, B.J.R. (ed) 1985. Microbiology of Fermented Foods, Vol 1 Elsevier Applied Science
Publishers. London and New York.
Wood, B.J.R. (ed) 1985. Microbiology of Fermented Foods, Vol 2 Elsevier Applied Science
Publishers. London and New York.
Section
.
Production of Metabolites as
Bulk Chemicals or as Inputs in
Other Processes
Modern Industrial
Microbiology and Biotechnology
+0)26-4
20
Production of Organic Acids
and Industrial Alcohol
20.1
ORGANIC ACIDS
A large number of organic acids with actual or potential uses are produced by microorganisms. Citric, itaconic, lactic, malic, tartaric, gluconic, mevalonic, salicyclic,
gibberelic, diamino-pimelic, and propionic acids are some of the acids whose microbial
production have been patented. In this chapter the production of only citric and lactic
acids will be discussed.
20.1.1
Production of Citric Acid
Citric acid is a tribasic acid with the structure shown in Fig. 20.1.
H2C
- COOH
OH - C - COOH
H2C - COOH
Fig. 20.1 Structure of Citric Acid
It crystallizes with the large rhombic crystals containing one molecule of water of
crystallization, which is lost when it is heated to 130°C. At temperatures as high as 175°C
it is converted to itaconic acid, aconitic acid, and other compounds.
20.1.2
Uses of Citric Acid
Citric acid is used in the food industry, in medicine, pharmacy and in various other
industries.
Uses in the food industry
(i) Citric acid is the major food acidulant used in the manufacture of jellies, jams,
sweets, and soft drinks.
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Modern Industrial Microbiology and Biotechnology
(ii) It is used for artificial flavoring in various foods including soft drinks.
(iii) Sodium citrate is employed in processed cheese manufacture.
Uses in medicine and pharmacy
(iv) Sodium citrate is used in blood transfusion and bacteriology for the prevention of
blood clotting.
(v) The acid is used in efferverscent powers which depend for their efferverscence on
the CO2 produced from the reaction between citric acid and sodium bicarbonate.
(vi) Since it is almost universally present in living things, it is rapidly and completely
metabolized in the human body and can therefore serve as a source of energy.
Uses in the cosmetic industry
(vii) It is used in astringent lotions such as aftershave lotions because of its low pH.
(viii) Citric acid is used in hair rinses and hair and wig setting fluids.
Miscellaneous uses in industry
(ix) In neutral or low pH conditions the acid has a strong tendency to form complexes
hence it is widely used in electroplating, leather tanning, and in the removal of iron
clogging the pores of the sand face in old oil wells.
(x) Citric acid has recently formed the basis of manufacture of detergents in place of
phosphates, because the presence of the latter in effluents gives rise to eutrophication
(an increase in nutrients which encourages aquatic flora development).
20.1.3
Biochemical Basis of the Production of Citric Acid
Citric acid is an intermediate in the citric acid cycle (TCA) (Fig. 20.2). The acid can
therefore be caused to accumulate by one of the following methods:
(a) By mutation – giving rise to mutant organisms which may only use part of a
metabolic pathway, or regulatory mutants; that is using a mutant lacking an
enzyme of the cycle.
(b) By inhibiting the free-flow of the cycle through altering the environmental
conditions, e.g. temperature, pH, medium composition (especially the elimination
of ions and cofactors considered essential for particular enzymes). The following
are some of such environmental conditions which are applied to increase citric
acid production:
(i) The concentrations of iron, manganese, magnesium, zinc, and phosphate must
be limited. To ensure their removal the medium is treated with ferro-cyanide or
by ion exchange fresins. These metal ions are required as prosthetic groups in
the following enzymes of the TCA: Mn++ or Mg++ by oxalosuccinic
decarboxylase, Fe+++ is required for succinic dehydrogenase, while phosphate
is required for the conversion of GDP to GTP (Fig. 20.2).
(i) The dehydrogenases, especially isocitrate dehydrogenase, are inhibited by
anaerobiosis, hence limited aeration is done on the fermentation so as to
increase the yield of citric acid.
(ii) Low pH and especially the presence of citric acid itself inhibits the TCA and
hence encourages the production of more citric acid; the pH of the fermentation
Production of Organic Acids and Industrial Alcohol
!$%
Citric acid can be caused to accumulate by using a mutant lacking an enzyme of the cycle or
by inhibiting the flow of the cycle
Fig. 20.2
The Tricarboxylic Acid Cycle
must therefore be kept low throughout the fermentation by preventing the
precipitation of the citric acid formed.
(iii) Many of the enzymes of the TCA can be directly inhibited by various
compounds and this phenomenon is exploited to increase citric acid
production. Thus, isocitric dehydrogenase is inhibited by ferrocyanide as well
as citric acid; aconitase is inhibited by fluorocitrate and succinic
dehydrogenase by malonate. These at enzyme antagonists may be added to the
fermentation.
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Modern Industrial Microbiology and Biotechnology
20.1.4
Fermentation for Citric Acid Production
For a long time the production of citric acid has been based on the use of molasses and
various strains of Aspergillus niger and occasionally Asp. wenti. Although several reports
of citric acid production by Penicillium are available, in practice, organisms in this group
are not used because of their low productivity. In recent times yeasts, especially Candida
spp. (including Candida quillermondi) have been used to produce the acid from sugar.
Paraffins became used as substrate from about 1970. In the processes described mainly
by Japanese workers bacteria and yeasts have been used. Among the bacteria were
Arthrobacter paraffineus and corynebacteria; the yeasts include Candida lipolytica and
Candida oleiphila.
Fermentation with molasses and other sugar sources can be either surface or
submerged. Fermentation with paraffins however is submerged.
(a) Surface fermentation: Surface fermentation using Aspergillus niger may be done on
rice bran as is the case in Japan, or in liquid solution in flat aluminium or stainless
steel pans. Special strains of Asp. niger which can produce citric acid despite the
high content of trace metals in rice bran are used. The citric acid is extracted from
the bran by leaching and is then precipitated from the resulting solution as calcium
citrate.
(b) Submerged fermentation: As in all other processes where citric acid is made the
fermentation the fermentor is made of acid-resistant materials such as stainless
steel. The carbohydrate sources are molasses decationized by ion exchange,
sucrose or glucose. MgSO4, 7H2 O and KH2PO4 at about 1% and 0.05-2%
respectively are added (in submerged fermentation phosphate restriction is not
necessary). The pH is never allowed higher than 3.5. Copper is used at up to 500
ppm as an antagonist of the enzyme aconitase which requires iron. 1-5% of
methanol, isopranol or ethanol when added to fermentations containing
unpurified materials increase the yield; the yields are reduced in media with
purified materials.
As high aeration is deleterious to citric acid production, mechanical agitation is not
necessary and air may be bubbled through. Anti-form is added. The fungus occurs as a
uniform dispersal of pellets in the medium. The fermentation lasts for five to fourteen
days.
20.1.5
Extraction
The broth is filtered until clear. Calcium citrate is precipitated by the addition of
magnesium-free (Ca(OH) 2. Since magnesium is more soluble than calcium, some acid
may be lost in the solution as magnesium citrate if magnesium is added. Calcium citrate
is filtered and the filter cake is treated with sulfuric acid to precipitate the calcium. The
dilute solution containing citric acid is purified by treatment with activated carbon and
passing through iron exchange beds. The purified dilute acid is evaporated to yield
crystals of citric acid. Further purification may be required to meet pharmaceutical
stipulations.
Production of Organic Acids and Industrial Alcohol
20.1.6
!$'
Lactic Acid
Lactic acid is produced by many organisms: animals including man produce the acid in
muscle during work.
20.1.6.1
Properties and chemical reactions of lactic acid
(i) Lactic acid is a three carbon organic acid: one terminal carbon atom is part of an
acid or carboxyl group; the other terminal carbon atom is part of a methyl or
hydrocarbon group; and a central carbon atom having an alcohol carbon group.
Lactic acid exists in two optically active isomeric forms (Fig. 20.3).
L (+) Lactic acid
D (-) Lactic acid
Fig. 20.3 Optical Isomers of Lactic Acid
(ii) Lactic acid is soluble in water and water miscible organic solvents but insoluble in
other organic solvents.
(iii) It exhibits low volatility. Other properties of lactic acid are summarized in Table
20.1.
(iv) The various reactions characteristic of an alcohol which lactic acid (or it esters or
amides) may undergo are xanthation with carbon bisulphide, esterification with
organic acids and dehdrogenation or oxygenation to form pyruvic acid or its
derivatives.
(v) The acid reactions of lactic acid are those that form salts and undergo esterification
with various alcohols.
(v) Liquid chromatography and its various techniques can be used for quantitative
analysis and separation of its optical isomers
Technical grade lactic acid is used as an acidulant in vegetable and leather tanning
industries. Various textile finishing operations and acid dyeing of food require low cost
technical grade lactic acid to compete with cheaper inorganic acid. Lactic acid is being
used in many small scale applications like pH adjustment, hardening baths for
cellophanes used in food packaging, terminating agent for phenol formaldehyde resins,
alkyl resin modifier, solder flux, lithographic and textile printing developers, adhesive
formulations, electroplating and electropolishing baths, detergent builders.
Lactic acid has many pharmaceutical and cosmetic applications and formulations in
topical ointments, lotions, anti acne solutions, humectants, parenteral solutions and
dialysis applications, and anti carries agents. Calcium lactate can be used for calcium
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Modern Industrial Microbiology and Biotechnology
deficiency therapy, and as an anti caries agent. Its biodegradable polymer has medical
applications as sutures, orthopedic implants, controlled drug release, etc. Polymers of
lactic acids are biodegradable thermoplastics. These polymers are transparent and their
degradation can be controlled by adjusting the composition, and the molecular weight.
Their properties approach those of petroleum derived plastics. Lactic acid esters like
ethyl/butyl lactate can be used as green solvents. They are high boiling, non-toxic and
degradable components. Poly L-lactic acid with low degree of polymerization can help in
controlled release or degradable mulch films for large-scale agricultural applications.
Lactic acid was among the earliest materials to be produced commercially by
fermentation and the first organic acid to be produced by fermentation.
Table 20.1 Physical properties of ethyl alcohol
Boiling point
Explosive limit in air, vol %
Freezing point
Specific gravity at 20/20o C
Surface tension at 20o C dynes/cm
Vapor pressure at 20o C mg/HG
78.2
43-19.0
114.1oC
0.7905
22.3
44
Chemical processing has offered and continues to offer stiff competition to
fermentation lactic acid. Very few firms around the world produce it fermentatively, but
this could change when the hydrocarbon-based raw material, lactonitrile, used in the
chemical preparation becomes too expensive because of the increase in petroleum prices.
Lactic acid exists in two forms, the D-form and the L-form. When the symbols (+) or
(-) are used, they refer to the optical rotation of the acid in a refractometer. However optical
rotation in lactic acid is difficult to determine because the pure acid has low optical
properties. The acid also spontaneously polymerizes in aqueous solutions; furthermore,
salts, esters, and polymers have rotational properties opposite to that of the pure acid
from which they are derived. All this makes it difficult to use optical rotation for
characterizing lactic acid.
Many organisms produce either the D-or the L-form of the acid. However, a few
organisms such as Lactobacillus plantarum produce both. When both the D- and L- form of
lactic acid are mixed it is a racemic mixture. The DL form which is optically inactive is the
form in which lactic acid is commercially marketed.
20.1.6.2
Uses of lactic acid
(i) It is used in the baking industry. Originally fermentation lactic acid was produced
to replace tartarates in baking powder with calcium lactate. Later it was used to
produce calcium stearyl 2- lactylate, a bread additive.
(ii) In medicine it is sometimes used to introduce calcium in to the body in the form of
calcium lactate, in diseases of calcium deficiency.
(iii) Esters of lactic acid are also used in the food industry as emulsifiers.
(iv) Lactic acid is used in the manufacture of rye bread.
(v) It is used in the manufacture of plastics.
Production of Organic Acids and Industrial Alcohol
!%
(vi) Lactic acid is used as acidulant/ flavoring/ pH buffering agent or inhibitor of
bacterial spoilage in a wide variety of processed foods. It has the advantage, in
contrast to other food acids in having a mild acidic taste.
(vii) It is non-volatile odorless and is classified as GRAS (generally regarded as safe) by
the FDA.
(viii) It is a very good preservative and pickling agent. Addition of lactic acid aqueous
solution to the packaging of poultry and fish increases their shelf life.
(ix) The esters of lactic acid are used as emulsifying agents in baking foods (stearoyl-2lactylate, glyceryl lactostearate, glyceryl lactopalmitate). The manufacture of these
emulsifiers requires heat stable lactic acid, hence only the synthetic or the heat
stable fermentation grades can be used for this application.
(x) Lactic acid has many pharmaceutical and cosmetic applications and formulations
in topical ointments, lotions, anti acne solutions, humectants, parenteral solutions
and dialysis applications, for anti carries agent.
(xi) Calcium lactate can be used for calcium deficiency therapy and as anti caries
agent.
(xii) Its biodegradable polymer has medical applications as sutures, orthopaedic
implants, controlled drug release, etc.
(xiii) Polymers of lactic acids are biodegradable thermoplastics. These polymers are
transparent and their degradation can be controlled by adjusting the composition,
and the molecular weight. Their properties approach those of petroleum derived
plastics.
(xiv) Lactic acid esters like ethyl/butyl lactate can be used as environment-friendly
solvents. They are high boiling, non-toxic and degradable components.
(xv) Poly L-lactic acid with low degree of polymerization can help in controlled release
or degradable mulch films for large-scale agricultural applications.
Table 20.2 Physical properties of lactic acid
Appearance
Melting point
Relative density
Boiling point
Flash point
Solubility
20.1.6.3
Yellow to colorless crystals or syrupy 50% liquid
16.8°C
1.249 at 15°C
122° @ 15 millimeter
110°C
Soluble in water, alcohol, furfurol
Slightly soluble in ether
Insoluble in chloroform, petroleum ether, and carbon
disulfide
Fermentation for lactic acid
Although many organisms can produce lactic acid, the amount so produced is small: the
organisms which produce adequate amounts and are therefore used in industry are the
homofermentative lactic acid bacteria, Lactobacillus spp., especially L. delbruckii. In recent
times Rhizopus oryzae has been used. Both organisms produce the L- form of the acid, but
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Modern Industrial Microbiology and Biotechnology
Rhizopus fermentation has the advantage of being much shorter in duration; further, the
isolation of the acid is much easier when the fungus is used.
Lactic acid is very corrosive and the fermentor, which is usually between 25,000 and
110,000 liters in capacity is made of wood. Alternatively special stainless steel (type 316)
may be used. They are sterilized by steaming before the introduction of the broth as
contamination with thermophilic clostridia yielding butanol and butyric acid is
common. Such contamination drastically reduces the value of the product.
During the step-wise preparation of the inoculum, which forms about 5% of the total
beer, calcium carbonate is added to the medium to maintain the pH at around 5.5-6.5. The
carbon source used in the broth has varied widely and have included whey, sugars in
potato and corn hydrolysates, sulfite liquour, and molasses. However, because of the
problems of recovery for high quality lactic acid, purified sugar and a minimum of other
nutrients are used.
Lactobacillus requires the addition of vitamins and growth factors for growth. These
requirements along with that of nitrogen are often met with ground vegetable materials
such as ground malt sprouts or malt rootlets. To aid recovery the initial sugar content of
the broth is not more than 12% to enable its exhaustion at the end of 72 hours.
Fermentation with Lactobacillus delbruckii is usually for 5 to 10 days whereas with
Rhizopus oryzae, it is about two days.
Although lactic fermentation is anaerobic, the organisms involved are facultative and
while air is excluded as much as possible, complete anaerobiosis is not necessary.
The temperature of the fermentation is high in comparison with other fermentation,
and is around 45°C. Contamination is therefore not a problem, except by thermophilic
clostridia.
20.1.6.4
Extraction
The main problem in lactic acid production is not fermentation but the recovery of the
acid. Lactic acid is crystallized with great difficulty and in low yield. The purest forms are
usually colorless syrups which readily absorb water.
At the end of the fermentation when the sugar content is about 0.1%, the beer is
pumped into settling tanks. Calcium hydroxide at pH 10 is mixed in and the mixture is
allowed to settle. The clear calcium lactate is decanted off and combined with the filtrate
from the slurry. It is then treated with sodium sulfide, decolorized by adsorption with
activated charcoal, acidified to pH 6.2 with lactic acid and filtered. The calcium lactate
liquor may then be spray-dried.
For technical grade lactic acid the calcium is precipitated as CaSO4.2H2O which is
filtered off. It is 44-45% total acidity. Food grade acid has a total acidity of about 50%. It is
made from the fermentation of higher grade sugar and bleached with activated carbon.
Metals especially iron and copper are removed by treatment with ferrocyanide. It is then
filtered. Plastic grade is obtained by esterification with methanol after concentration.
High-grade lactic acid is made by various methods: steam distillation under high
vacuum, solvent extraction etc.
Production of Organic Acids and Industrial Alcohol
20.2
!%!
INDUSTRIAL ALCOHOL PRODUCTION
Ethyl alcohol, CH3 CH2 OH (synonyms: ethanol, methyl carbinol, grain alcohol, molasses
alcohol, grain neutral spirits, cologne spirit, wine spirit), is a colorless, neutral, mobile
flammable liquid with a molecular weight of 46.47, a boiling point of 78.3 and a sharp
burning taste. Although known from antiquity as the intoxicating component of
alcoholic beverages, its formula was worked out in 1808. It is rarely found in nature,
being found only in the unripe seeds of Heracleum giganteun and H. spondylium.
20.2.1
Properties of Ethanol
Some of the physical properties of ethanol are given in Table 20.1
Ethyl alcohol undergoes a wide range of reactions, which makes it useful as a raw
material in the chemical industry. Some of the reactions are as follow:
(i) Oxidation: Ethanol may be oxidized to acetaldehyde by oxidation with copper or
silver as a catalyst:
Cu, Ag
CH3 CHO + H2
CH3 CH2 OH
(ii) Halogenation: Halides of hydrogen, phosphorous and other compounds react with
ethanol to replace the – OH group with a halogen:
3CH3 CH2 OH + PCl3
3CH3 CH2 Cl + P (OH)3
(iii) Reaction with metals: Ethanol reacts with sodium, potassium and calcium to give the
alcoholates (alkoxides) of these metals:
2CH3 ONa + H2
2CH3 CH2 OH + 2Na
(iv) Haloform Reaction: Hypohalides will react with ethanol to yield first acetaldehyde
and finally the haloform reaction:
CH3 CH2 OH + NaOCl
CH3 CHO + 2NaOCl
CCl3 CHO + NaOH
CH3 CHO + NaCl + H2 O
CCl3 CHO + 3NaOH
CHCl3 + HCOONa
Chloroform
(v) Esters: Ethanol reacts with organic and inorganic acids to give esters:
CH3 CH2 Cl + H20
CH3 CH2 OH + HCl
Ethylchoride
(vi) Ethers: Ethanol may be dehydrated to give ethers:
Catalyst
2CH3 CH2 OH
CH3 CN2 OCH2 CH3 + H2 O
(vii) Alkylation: Ethanol alkylates (adds alkyl-group to) a large number of compounds:
H3 SO4 :
NH3 :
CH3 CH2 HSO4 (ethyl hydrogen sulfate)
CH3 CH2 NH2 (ethyl amine)
!%"
Modern Industrial Microbiology and Biotechnology
20.2.2
Uses of Ethanol
(i) Use as a chemical feed stock: In the chemical industry, ethanol is an intermediate in
many chemical processes because of its great reactivity as shown above. It is thus a
very important chemical feed stock.
(ii) Solvent use: Ethanol is widely used in industry as a solvent for dyes, oils, waxes,
explosives, cosmetics etc.
(iii) General utility: Alcohol is used as a disinfectant in hospitals, for cleaning and
lighting in the home, and in the laboratory second only to water as a solvent.
(iv) Fuel: Ethanol is mixed with petrol or gasoline up to 10% and known as gasohol and
used in automobiles.
20.2.3
Denatured Alcohol
All over the world and even in ancient times, governments have derived revenue from
potable alcohol. For this reason when alcohol is used in large quantities it is denatured or
rendered unpleasant to drink. The base of denatured alcohol is usually 95% alcohol with
5% water; for domestic burning or hospital use denatured alcohol is dispened as
methylated spirit, which contains a 10% solution of methanol, pyridine and coloring
material. For industrial purpose methanol is used as the denaturant. In the United States
alcohol may be completely denatured (C.D.A. – completely denatured alcohol) when it
cannot be used orally because of a foul taste or four smelling additives. It may be specially
denatured (S.D.A. – specially denatured alcohol) when it can still be used for special
purposes such as vinegar manufacture without being suitable for consumption.
20.2.4
Manufacture of Ethanol
Ethanol may be produced by either synthetic chemical method or by fermentation.
Fermentation was until about 1930 the main means of alcohol production. In 1939, for
example 75% of the ethanol produced in the US was by fermentation, in 1968 over 90%
was made by synthesis from ethylene. The production of alcohol from ethylene is
discussed in Chapter 13.
Due to the increase in price of crude petroleum, the source of ethylene used for alcohol
production, attention has turned worldwide to the production of alcohol by
fermentation. Fermentation alcohol has the potential to replace two important needs
currently satisfied by petroleum, namely the provision of fuel and that of feedstock in the
chemical industry.
The production of gasohol (gasoline – alcohol blend) appears to have received more
attention than alcohol use as a feed stock. Nevertheless, the latter will also surely assume
more importance if petroleum price continues to ride. Governments the world over have
set up programs designed to conserve petroleum and to seek other energy sources. One of
the most widely publicized programs designed to utilize a new source of energy is the
Brazilian National Ethanol Program. Set-up in 1975, the first phase of this program aims
at extending gasoline by blending it with ethanol to the extent of 20% by volume. The
United States government also introduced the gasoline programme based on corn
fermentation in 1980 following the embargo on grain sales to the then Soviet Union.
Production of Organic Acids and Industrial Alcohol
20.2.4.1
!%#
Substrates
The various substrates which may be used for ethanol production have been discussed in
Chapter 2. It is clear that the substrate used will vary among countries. Thus, in Brazil
sugar cane, already widely grown in the country, is the major source of fermentation
alcohol, while it is planned to use cassava and sweet sorghum. In the United States
enormous quantities of corn and other cereals are grown and these are the obvious
substrates. Cassava grows in many tropical countries and since it is high yielding it is an
important source in tropical countries where sugar cane is not grown. It is recognized
that two important conditions must be met before fermentation alcohol can play a major
role in the economy either as gasohol or as a chemical feedstock. First, the production of
the crop to be used must be available to produce the crop without extensive and excessive
deforestation. Secondly, the substrate should not compete with human food.
20.2.4.2
Fermentation
The conditions of fermentation for alcohol production are similar to those already
described for whisky or rum production. Alcohol-resistant yeasts, strains of
Saccharomyces cerevisiae are used, and nutrients such as nitrogen and phosphate lacking
in the broth are added.
20.2.4.3
Distillation
After fermentation the fermented liquor or ‘beer’ contains alcohol as well as low boiling
point volatile compounds such as acetaldeydes, esters and the higher boiling, fusel oils.
The alcohol is obtained by several operations. First, steam is passed through the beer
which is said to be steam-stripped. The result is a dilute alcohol solution which still
contains part of the undesirable volatile compounds. Secondly, the dilute alcohol
solution is passed into the center of a multi-plate aldehyde column in which the
following fractions are separated: esters and aldehydes, fusel oil, water, and an ethanol
solution containing about 25% ethanol. Thirdly, the dilute alcohol solution is passed into
a rectifying column where a constant boiling mixture, an azeotrope, distils off at 95.6%
alcohol concentration.
To obtain 200° proof alcohol, such as is used in gasohol blending, the 96.58% alcohol
is obtained by azeotropic distillation. The principle of this method is to add an organic
solvent which will form a ternary (three-membered) azeotrope with most of the water, but
with only a small proportion of the alcohol. Benzene, carbon tetrachloride, chloroform,
and cyclohezane may be used, but in practice, benzene is used. Azeotropes usually have
lower boiling point than their individual components and that of benzene-ethanol-water
is 64.6°C. On condensation, it separates into two layers. The upper layer, which has
about 84% of the condensate, has the following percentage composition: benzene 85%,
ethanol 18%, water 1%. The heavier, lower portion, constituting 16% of the condensate,
has the following composition: benzene 11%, ethanol 53%, and water 36%.
In practice, the condensate is not allowed to separate out, but the arrangement of plates
within the columns enable separation of the alcohol. Four columns are usually used. The
first and second columns remove aldehydes and fusel oils, respectively, while the last
two towers are for the concentration of the alcohol. A flow diagram of conventional
absolute alcohol production from molasses is given in Fig. 20.4
YEAST
CULTURE
PREPARATION
OF INOCULUM
BRIX
ADJUSTMENT
14o TO 22 o
WORT
RECUPERATION OF YEAST
FERMENTED
WORT
FERMENTATION
CENTRIFUGATION
(7 – 8% V/V)
CENTRIFUGED MEDIUM
BENZENE
ALCOHOL
(96% V/V)
ALCOHOL
(40% - 50% V/V)
STILLAGE
(DISTILLATION)
FUSEL
OIL
WATER
(RECTIFICATION)
DECANTATION
ABSOLUTE
ALCOHOL
(TO RECTIFICATION)
(DEHYDRATION)
Fig. 20.4 Flow Diagram of Conventional Alcohol Production
RECYCLE
(RECUPERATION
OF BENZENE)
Modern Industrial Microbiology and Biotechnology
WEIGHING
!%$
MOLASSES OR
CANE JUICE
Production of Organic Acids and Industrial Alcohol
20.2.5
!%%
Some Developments in Alcohol Production
Due to the current interest in the potential of ethanol as a fuel and a chemical feedstock,
research aimed at improving the conventional method of production has been
undertaken, and more will, most certainly, be undertaken. Some of the techniques aimed
at improving productivity are the following:
(i) Developments of new strains of yeast of Saccharomyces uvarum able to ferment sugar
rapidly, to tolerate high alcohol concentrations, flocculate rapidly, and whose
regulatory system permits it to produce alcohol during growth.
(ii) The use of continuous fermentation with recycle using the rapidly flocculating yeasts.
(iii) Continuous vacuum fermentation in which alcohol is continuously evaporated
under low pressure from the fermentation broth.
(iv) The use of immobilized Saccharomyces cerevisiae in a packed column, instead of in a
conventional stirred tank fermentor. Higher productivity consequent on a higher
cell concentration was said to be the advantage.
(v) In the ‘Ex-ferm’ process sugar cane chips are fermented directly with a yeast
without first expressing the cane juice. The chips may be dried and used in the offseason period of cane production. It is claimed that there is no need to add
nutrients as would be the case with molasses, since these are derived from the cane
itself. A more complete extraction of the sugar, resulting in a 10% increase in
alcohol yield, is also claimed.
(vi) The use of Zymomonas mobilis, a Gram-negative bacterium which is found in some
tropical alcoholic beverages, rather than yeast is advocated. The advantages
claimed for the use of Zymomonas are the following:
(a) Higher specific rates of glucose uptake and ethanol production than reported
for yeasts. Up to 300% more ethanol is claimed for Zymomonas than for yeasts
in continuous fermentation with all recycle.
(b) Higher ethanol yields and lower biomass than with yeasts. This deduction is
based on Fig. 20.5 where, although the same quantity of alcohol is produced by
the two organisms in 30-40 hours, the biomass of Zymomonas required for this
level of production is much less than with yeast. The lower biomass appears to
be due to the lower energy available for growth. Zymomonas utilized glucose by
the Enthner-Duodoroff pathway (Fig. 5.4) which yields one mole of ATP/mole
glucose, whereas yeasts utilize glucose anaerobically via the glycolytic
pathway (Fig. 5.1) to give two ATP/mole glucose. Its use does not appear to
have gained general acceptance.
(c) Ethanol tolerance is at least as high or even higher [up to 16% (v/v)] in some
strains of the bacterium than with yeast.
(d) Zymomonas also tolerates high glocuse concentration and many cultures grow
in sugar solutions of up to 40% (w/v) glucose which should lead to high
ethanol production.
(e) Zymomonas grows anaerobically and, unlike yeasts, does not require the
controlled addition of oxygen for viability at the high cell concentrations used
in cell recycle.
(f) The many techniques for genetic engineering already worked out in bacteria
can be easily applied to Zymomonas for greater productivity.
!%&
Modern Industrial Microbiology and Biotechnology
Fig. 20.5
Alcohol Production by Yeast and Zymomonas mobilis
Production of Organic Acids and Industrial Alcohol
!%'
SUGGESTED READINGS
Charrington, C.A, Hinton, M., Mead, G.C., Chopra, I. 1991. Organic Acids: Chemistry,
Antibacterial Activity and Practical Applications. Advances in Microbial Physiology. 32, 87 –
108.
Ho, N.W.Y. 1980. Ann. Repts. Ferm. Proc. 4, 235-266.
Kosaric, D.C.M., Ng, I.R., Steart, G.S. 1980. Adv. Appl. Microbiol. 26, 137-227.
Lockwood, L.B. 1979. In: Microbial Technology. H.J. Peppler, D. Perlman, (eds.) 2nd Edit.
Academic Press, New York, USA, pp. 256-288.
Narayanan, N., Pradip, K., Roychoudhury, P.K., Srivastava, A. 2004. L (+) lactic acid fermentation
and its product polymerization. Electronic Journal of Biotechnology 7, Electronic Journal of
Biotechnology [online]. 15 August 2004, vol. 7, no. 3 [cited 23 March 2006]. Available from:
http://www.ejbiotechnology.info/content/vol2/issue3/full/3/index.html. ISSN 0717-3458.
Ward, W.P., Singh, A. 2003. Bioethanol Technology: Developments and Perspectives. Advances
In Applied Microbiology, 51, 53-80.
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Modern Industrial Microbiology and Biotechnology
+0)26-4
21
Production of Amino Acids
by Fermentation
Amino acids have the general formula R. CH—COOH and are the main components of
|
NH2
of which proteins are made. The amino acids found in proteins number 20. Of these eight
are essential for animals and must be supplied in their food, since animals cannot
synthesize them.
Each of the 20 amino acids found in proteins can be distinguished by the R-group
substitution on the carbon atom and can be divided into the following groups on that
basis: amino acids with aliphatic R groups, non-aromatic amino acids with hydroxyl R
groups, amino acids with sulfur containing R groups, amino acids with acidic R groups,
amino acids with basic R groups, amino acids with aromatic R groups and amino acids
with imino acids as the R groups. The nature of the R group influences the activity of the
amino acid. Thus, the hydrophilic amino acids which have –OH groups in their R
substituent (e.g. serine) tend to interact with the aqueous environment, are often involved
in the formation of H-bonds and are predominantly found on the exterior surfaces
proteins or in the reactive centers of enzymes. On the other hand, the hydrophobic amino
acids (without –OH groups in the R substituent, for example methionine) tend to repel the
aqueous environment and, therefore, reside predominantly in the interior of proteins.
This class of amino acids does not ionize nor participate in the formation of H-bonds
(Table 21.1).
All the amino acids, except glycine have two optically active isomers, the D – or the Lform. Natural proteins are usually made up of L- (or the so-called natural amino acids.)
Outside the 20 amino acids found in protein, many other rare amino acids have been
reported in various metabolites such as some antibiotics, other microbiological products
and in non-proteinaceous materials in plants and animals.
21.1
USES OF AMINO ACIDS
Amino acids find use in a large number of activities, including human and animal
nutrition, medicine, cosmetics, and in the synthesis of chemicals.
!&
Production of Amino Acids by Fermentation
Table 21.1 Amino acids found in proteins
Amino Acid
Symbol
Structure
Amino Acids with Aliphatic R-Groups
Glycine
Gly - G
H–CH–COOH
NH2
Alanine
Ala - A
CH3–CH2–COOH
NH2
* Valine
Val - V
* Leucine
Leu - L
* Isoleucine
Ile - I
H3C
H3C
H3C
H3C
CH–CH–COOH
NH2
CH–CH2–CH–COOH
NH2
H3C CH
2 CH–CH–COOH
H3C
NH
2
Non-Aromatic Amino Acids with Hydroxyl R-Groups
Serine
Ser - S
* Threonine
Thr - T
HO–CH2–CH–COOH
NH2
H3C
HO
CH–CH–COOH
NH2
Amino Acids with Sulfur-Containing R-Groups
Cysteine
Cys - C
HS–CH2–CH–COOH
NH2
* Methionine
Met-M
H3C–S–(CH2)2–CH–COOH
NH2
Acidic Amino Acids and their Amides
Aspartic Acid
Asp - D
HOOC–CH2–CH–COOH
NH2
Asparagine
Asn - N
H2N–C–CH2–CH–COOH
NH2
O
Glutamic Acid
Glu - E
HOOC–CH2–CH2–CH–COOH
NH2
Glutamine
Gln - Q
H2N–C–CH2–CH2–CH–COOH
NH2
O
Basic Amino Acids
Arginine
Arg - R
HN–CH2–CH2–CH2–CH–COOH
NH2
C=NH
NH2
* Lysine
Lys - K
H2N–(CH2)4–CH–COOH
NH2
Histidine
His - H
HN
—CH2–CH–COOH
NH2
N:
Contd
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Modern Industrial Microbiology and Biotechnology
Table 21.1 Contd.
Amino Acid
Symbol
Structure
Amino Acids with Aromatic Rings
* Phenylalanine
Phe - F
Tyrosine
Tyr - Y
* Tryptophan
Trp-W
CH2–CH–COOH
NH2
HO
CH2–CH–COOH
NH2
CH2–CH–COOH
NH2
N
H
Imino Acids
Proline
+
N
Pro - P
H
COOH
H
*Essential
(i) Use in human and animal nutritional supplementation: Proteins are metabolized
constantly in the body and most of the amino acids absorbed are used to replace body
proteins. The remaining are metabolized into various body components including
hormones, and nucleic acid bases. Of the amino acids in protein eight are essential and
the diet must contain them. Foods such as plant proteins lacking in some essential amino
acids are fortified with their addition. Most cereals are particularly low in lysine, the
addition of which greatly improves the quality of the food as determined by the PER
(protein equivalency ration). The PER is a means of comparing the amino acid content of
protein with that of hen’s egg or human milk. It is usually best determined by feeding tests
to rats and mice.
Animal feeds made from inexpensive plant proteins can be greatly improved with
only a small quantity of the limiting amino acids, resulting in higher growth rates in the
animals. L – lysine and DL methionine are widely used as feed improvers.
(ii) Flavor and taste enhancement in foods: Amino acids are important in deciding the
taste of meats and such foods. Mono-sodium glutamate well-known as a flavoring agent,
will be discussed later.
Amino acids influence the taste of foods. Some are very sweet; for example glycine is as
sweet as sugar and is sometimes used in soft drinks and soups. The amino acid is present
in large amounts in shrimps to which it confers its sweet taste. The peptide, L- aspartyl –
L – phenylalanine methyl ester is particularly sweet. Well-known sweetners such as
Aspartame, Sweet and Low, and Splenda contain a dipetide formed from aspartic acid
and phenylalanine Other amino acids e.g. valine are bitter. It is interesting that while the
L- isomers of leucine, phenylalanine, tyrosine, and tryptophane are bitter, the D-isomers
are sweet. The combination of various amino acids influence the taste and flavor of foods.
Thus cheese flavor derives from the combined effect of glutamic acid as well as that of
bitter amino acids such as valine, leucine and methionine.
Production of Amino Acids by Fermentation
!&!
(iii) Medical uses: The greatest application of amino acids in medicine is in transfusion;
which is administered when the oral consumption of proteinaceous food is not possible
such as after an operation. In the past only essential amino acids were used in
transfusion; nowadays non-essential ones are added.
Various amino acids are used for ammonia detoxification in blood in liver diseases, in
the treatment of heart failure, in cases of peptic ulcer and male sterility, etc. Table 21.2
summarizes some of these uses.
In addition derivatives of amino acids are widely used in medicine as discussed
below. Methyldopa (L-methy1-3, 4 dihydroxy-phenylalanin) is widely used as an antihypertensive with relatively few side effects. Dopa is used in treating Parkinson’s disease
(Fig. 21.1):
OH
OH
CH3
OH
CH2 CCOOH
OH
CH2 CHCOOH
NH2
Methyl dopa
Dopa
Fig. 21.1 Methyl Dopa and Dopa
A derivative of serine, cycloserine is an antibiotic produced by a streptomycete; it is
used for the treatment of tuberculosis.
(iv) Use as an industrial synthetic raw materials: Although numerous studies have been
concluded on the use of amino acids as raw materials in the chemical industry, very few
of these have been put into actual practice. Thus, the manufacture of artificial silk was
considered and dropped. However amino acids are used in the following:
(a) Surface-active agents: A surface-active agent has a water solube (or hydrophilic end)
as well as a water-repellent) or hydrophobic end. The hydrophilic end is dissolved
in the water and as a consequence the surface tension of the water is lowered.
Surface action agents can be prepared from amino acids by introducing long-chain
lipophilic groups to one of the two hydrophilic groups (- COOH, or – NH2) of
amino acids. The resulting surface-active agents is either cationic (if it has a
positive charge) or anionic (negative charge). As they lower the surface tension like
soap, they foam just like soap. Some are even more effective than soap as cleansing
agents. Many of them also have strong bacteriostatic action. Thus sodium lauryl
sarcosinate is used in toothpaste and shampoo because it has a bacteriocidal as
well as foaming action. These derivatives are also used as fungicides and
pesticides.
(b) Production of polymers from amino acids: Polymers derived from amino acids are
used in making synthetic leather, fire-resistant fabrics and anti-static materials.
(c) Use as cosmetics: Amino acids exhibit a buffering action that help maintain normal
skin function by regulating pH and a protective action against bacteria. Detergents
(surface action agents) derived from amino acids are less irritating than soaps
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Modern Industrial Microbiology and Biotechnology
Table 21.2 Therapeutic uses of some amino acids
Amino Acid
Ornithine
Arginine
Aspartic acid
Cysteine and cystine
Gluatamic acid
Glycine
Histidine
Methionine
Tryptophan
Use
Used for treating cases of hyperammonemia (excessive
ammonia in blood) because it increases urease activity in the
liver, thus enhancing urease production.
More common than ornithine.
1. Use for ammonia detoxication described above.
2. Arginine is the main component of spermatic proteins and
hence administered in cases of male sterility due to low
number or weak spermatozoa (Sterilitas virilis).
1. Used for ammonia detoxification combined with ornithine.
2. Used as carrier for K+ and Mg++ in form of potassium or
magnesium aspartate in cases of heart failure, fatigue, etc.
1. Protect SH-enzymes in the liver from enzyme inhibitors;
used for dealing with poisons generally including cyanide
poisoning.
2. L- cysteine is used in bronchitis and nasal catarrh.
Important in brain metabolism hence various analogues of
glutamic acid are used in treating various neuropathic diseases.
Rarely used as a drug, but only as a sweetener in medicines.
An amino acid essential for infants but not for adults, it is used in
adults for gastric and duodenal ulcers and is administered in cases
of anaemia because it helps in haemoglobin regeneration.
A sulfur-containing amino acid, it is important in the metabolism
of various sulfur-containing compounds in the body. It is also
used for detoxification in poisoning by arsenic, chloroform, and
benzene derivaties. A derivative of methionine, Vitamin U, is
used as an anti-ulcer drug, because it neutralizes histamine which
is known to induce ulcer formation.
Used as an anti-depressant.
because the pH of 5.5-6.0 is closer to that of the skin, whereas soap is slightly
alkaline. The addition of different amino acids to shampoo is practiced to achieve
different ends: anti-dandruff shampoos contain cysteine; thioglycolic acid is
employed as a reducing agent for the cold waving of hair.
21.2
METHODS FOR THE MANUFACTURE OF
AMINO ACIDS
The beginning of the development of the amino acid industry can be put at 1908 when
Kikunae Ikeda identified and isolated monosodium glutamate (MSG), the sodium salt of
glutamic acid, as the flavoring agent in ‘kombu’, a traditional seasoning agent used in
Japan, and derived from some marine algae. The Ajinomoto company the following year
started producing MSG by extraction from the acid hydrolysate of wheat gluten or
defatted soy. Glutamic acid, lysine and methionine are the most produced amino acids
globally (Fig. 21.2). Today amino acids are produced by a number of methods.
Production of Amino Acids by Fermentation
!&#
Fig. 21. 2 World Production of Amino Acids, 1996 (from Mueller and Huebner, 2003)
(i) Protein hydrolysis: Protein hydrolysis was the original method of amino manufacture.
Hair, keratin, blood meal and feathers are hydrolyzed using acid and the amino acid
extracted. It is not very popular because it depends on the availability of hair, feathers
and the other raw materials. However, cysteine and cystine are still produced by
isolating them from chemically hydrolyzed keratin protein in hair and feathers while
proline and hydroxyproline are precipitated from gelatin hydrolysates.
(ii) Chemical synthesis: Glycine, L-alanine, and DL- methionine are produced by
chemical synthesis. Chemical synthesis can only produce the D,L- (recemic) forms of
amino acids and an additional step involving the use of an immobilized enzyme,
aminoacylase, produced by Aspergillus niger is necessary to obtain the biologically active
L-form. (Fig. 21. 3). This step is expensive and on account of this, few amino acids are
prepared by chemical synthesis. Amino acids produced by chemical synthesis are
glycine and methionine; methionine is said to have the same effect as an animal feed
additive whether in the L- or in the D, L- form.
(iii) Microbiological methods: Microbiological methods are of three types:
1. Semi-fermentation;
2. Use of microbial enzymes or immobilized cells;
3. Direct fermentation.
Fig. 21.3 Action of Aminoacylase on Racemic Mixtures of Amino Acids
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Modern Industrial Microbiology and Biotechnology
Fig. 21.4
21.2.1
Examples of Enzyme Conversions for Producing Amino Acids
Semi-fermentation
In this process, the metabolic intermediate in the amino acid biosynthesis or its precursor
is added to the medium, which contains carbon and nitrogen sources, and other
nutrients required for growth and production; the metabolite is converted to the amino
acid during fermentation. Sometimes the intermediate could be another amino acid.
Examples of the commercial production of amino acids by semi-fermentation are L-serine
production from glycine and methanol using the methane-utilizing bacterium
Hyphomicrobium sp. or Pseudomonas sp. Other examples are the production of Ltryptophan from anthranillic acid or indole using E. coli and B. subtilis L-isoleucine
production from DL-=-aminobutyric acid and ethanol by Brevibacterium sp. has been
done commercially by this process (Table 21. 3).
21.2.2
Enzymatic Process
Chemically synthesized substrates can be converted to the corresponding amino acids by
the catalytic action of an enzyme or microbial cells as an enzyme source. Often the
enzymes or the cells may be immobilized. L-alanine production from L-aspartic acid, Laspartic acid production from fumaric acid, L-cysteine production from DL-2aminothiazoline-4-carboxylic acid (Fig. 21. 5). Others are D-phenylglycine (and D-phydroxyphenylglycine) production from DL-phenylhydantoin (and DL-phydroxyphenylhydantoin), and L-tryptophan production from indole and DL-serine
have been in operation as commercial processes.
Production of Amino Acids by Fermentation
Table 21.3
!&%
Amino acid production by semi-fermentation process (from Araki, 2003)
Amino acid produced
Precursor added to the medium
D-alanine
L-isoleucine
DL-alanine
D-threonine
DL-=-aminobutyric acid
DL-=-hydroxybutyric acid
DL-=-bromobutyric acid
L-hydroxy-4-methylthiobutyric acid
DL-5-(2-methylthioethyl)hydantoin
D-threonine
acetoamidocinnamic acid
L-glutamic acid
glycine
L-methionine
L-phenylalanine
L-proline
L-serine
L-threonine
L-tryptophan
L-homoserine
anthranilic acid
indole
Amount, mg/mL
48.8
15
15.7
10.9
34
75.9
108.3
16
54.5
16
40
16.7
Fig. 21.5 Metabolic Pathways Involved in the Biosynthesis of Amino Acids from Glucose
!&&
Modern Industrial Microbiology and Biotechnology
21.2.3
Production of Amino Acids by the Direct Fermentation
Although other microbiological methods for the commercial production of amino acids
exist, such as the biosynthesis of amino acids using intermediates, and the use of
enzymes, by far the most important method for producing amino acids microbiologically
is by direct fermentation. What method is used in any particular situation depends on
factors such as process economics, the available raw materials, market size, the
environmental regulation operating in the place of production, etc. Nevertheless, the
fermentation method appears to be the dominant one and will be discussed.
The production of amino acids by fermentation was stimulated by the discovery of an
efficient L- glutamic acid producer Corynebacterium glutamincum. Many microorganisms
have been reported to produce amino acids. They are mainly bacteria, but they also
include some molds and yeasts. The four most widely reported bacteria belong to the
following four genera, the typical species of which are given in parenthesis.
Corynebacterium spp. (C. glutamicum; C. lilum)
Brevibacterium spp. (B. divericartum: B. alanicum)
Microbacterium spp. (M. flavum var. glutamicum)
Arthrobacter spp. (A. globiformis; A. aminofaciens)
Auxotrophic and regulatory mutants of glutamic acid producing bacteria are used for
the commercial production of all amino acids outside L- glutamic acid and L-glutamine,
which are produced by the wild type of these organisms (Table 21.4).
Table 21.4
Amino acids produced from wild type and mutant strains of bacteria
Wild-type
L- glutamic acid
L-valine,
Auxotrophic Mutants
1
1
L- citruline
L- leucine
L- lysine
L- ornithine
L- proline
L- threonine
L- tyrosine
1 = Corynebacterium glutamicum
3 = Brevibacterium flavum
5 = Serratia marcescens
21.3
1
1
1
1
3
4
1
Regulatory Mutants
LLLLLLLLLLL-
arginine
histidine
isoleucine
leucine
lysine
methionine
phenylalanine
thereonine
tryptophane
tyrosine
valine
1,2,3
1,3,5
1,3,5
5,6
3
1
1,3
1,3
1,3
1,3
6
2 = Bacillus subtilis
4 = Escherichia coli
6 = Brevibacterium lactofermentum
PRODUCTION OF GLUTAMIC ACID BY
WILD TYPE BACTERIA
(i) Organisms: Wild type strains of the organisms of the four genera mentioned above are
now used for the production of glutamic acid. The preferred organism is however
Corynebacterium glutamicum. The properties common to the glutamic acid bacteria are: (a)
Production of Amino Acids by Fermentation
!&'
they are all Gram-positive and non-motile; (b) they require biotin to grow; (c) they lack or
have very low amounts of the enzyme =-ketoglutarate, which is formed by removal of CO2
from isocitrate formed in TCA cycle (citric acid cylce). Since =-ketoglutarate is not
dehydrogenated it is available to form glutarate by reacting with ammonia (Fig. 21.1).
(ii) Conditions of the fermentation: The composition of a medium which has been used for
the production of glutamic acid is as follows (%): glucose, 10; corn steep liquor 0.25;
enzymatic casein hydrolysate 0.25; K2HPO4 0.1, Mg. SO4, 7H2 O, 0.25; urea, 0.5. It should
be noted that besides glucose, hydrocarbons have served as carbon sources for glutamic
acid production. The optimal temperature is 30° to 35° and a high degree of aeration is
necessary.
(iii) Biochemical basis for glutamic acid production: Studies by several workers have clarified
the basis for glutamic acid production as summarized below.
(a) Glutamic acid production is greatest when biotin is limiting; that is, when it is suboptimal. When biotin is optimal, growth is luxuriant and lactic acid, not glutamic
acid, is excreted. The optimal level of biotin is 0.5 mg per gm of dry cells; with
higher amounts glutamic acid production falls.
(b) The isocitrate-succinate part of the TCA cycle (Fig. 21.5) is needed for growth. It is
only after the growth phase that glutamic acid production becomes optimal.
(c) An increase in the permeability of the cell is necessary so as to permit the outward
diffusion of glutamic acid, essential for high glutamic acid productivity. This
increased permeability to the acid can be achieved in the following ways: (i)
ensuring biotin deficiency in the medium (ii) treatment with fatty acid derivatives,
(iii) ensuring oleic acid deficiency in mutants requiring oleic acid (C16 - C18). (iv)
addition of penicillin during growth of glutamic acid bacteria, Cells treated in one
of the first three ways above have cell membranes in which the saturated to
unsaturated fatty acid ratio is abnormal, therefore the permeability barrier is
destroyed and glutamic acid accumulates in the medium. The major factor in
glutamic acid production by wild type organism is thus altered permeability.
Treatment with penicillin prevents cell-wall formation. Cell wall inhibiting
antibiotics such as penicillin and cephalosporin have enabled the use of molasses
which are rich in biotin for glutamic acid production.
21.4
PRODUCTION OF AMINO ACIDS BY MUTANTS
After wild type strains of C. glutamicum and of other bacteria were found to accumulate
glutamic acid, efforts to find in nature bacteria able to yield high amounts of other amino
acids failed. The reason for this is that microorganisms avoid over-production of amino
acids, producing only the quantity they require. To induce the organism to over produce,
regulatory mechanisms must be disorganized as discussed in Chapter 6. Two major
means of regulating amino acid synthesis are feedback inhibition and repression.
Auxotrotrophic mutants and regulatory mutants are two means by which the organisms’
tendency not to overproduce can be disorganized. In order to over produce an amino acid
which is an intermediate in a synthetic pathway, a mutant auxotroph is produced whose
pathway in the synthesis is blocked. When this mutant is cultivated, limiting nutrient
feedback and/or repression would have been removed and an overproduction of the
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amino acid will occur. The mutants used for the production of aminoacids other than
glutamic acid are produced from L- glutamic acid producing bacteria. This is because
these bacteria assimilate carbon efficiently and also because they do not degrade the
amino acid which they excrete.
21.4.1
Production of Amino Acids by Auxotrophic Mutants
Table 21.4 shows the amino acids produced by the use of auxotrophic mutants. The first
to be produced was L- lysine using limiting concentrations of either L- homoserine or Lmethionine plus L- threonine with a mutant strain of Corynebacterium glutamicum. In the
wild type of this organism concerted feed back inhibition is by both lysine and threonine.
Inhibition does not occur when only one is present. In this particular mutant absence of
biosynthetic homoserine derived from aspartic acid causes lysine to accumulate. This is
illustrated in Fig. 21.6.
L-aspartic
Acid
aspartyl
aspartyl
phosphate semialdehyde
L-homoserino
dihydrodopicolinate
L-threonine
a-ketobutyrate
diaminopimelate
L- lysine
L-methionine
L-isoleucine
--------------------------------- Feed back inhibition
______________________ Biosynthetic pathway
Fig. 21.6
21.4.2
Accumulation of Lysine in a Mutant Strain of Corynebacterium glutamicum
Production of Amino Acids by Regulatory Mutants
Regulatory mutants have a feed-back insensitive key enzyme and hence continues to over
produce the required amino acid. Examples are given in Table 21.4. In order to obtain
such mutants mutations are induced to produce organisms whose growth is not
inhibited by analogues of the amino-acid to be overproduced. A good example is the case
of lysine production by Brevibacterium flavum. In this organism the L- lysine pathway is
regulated at aspartate kinase which is the only enzyme sensitive to feed back inhibitation
by lysine. Mutants resistant to lysine analogues therefore over produce the amino acid
(Fig. 21.7).
Production of Amino Acids by Fermentation
Aspartate
Kinase
Homosertine
dehydro
genase
Kinase
!'
Homoserine
kinase
Enzyme reaction
Repression
Feedback inhibition
ASA = aspartate semino-aldehyde; DADP = dihydrodipicolinate;
Hse = Homoserne; DAP = diaminopimelate
Fig. 21.7
21.5
Lysine Biosynthesis in Brevibacteium flavum and Corynebacterium glutamicum
IMPROVEMENTS IN THE PRODUCTION OF AMINO
ACIDS USING METABOLICALLY ENGINEERED
ORGANISMS
The improvements of the microorganisms discussed above used classical mutation
techniques and screening procedures which relied on deleting competing pathways and
eliminating feed back regulations on the biosynthetic pathways. The mutagenic
procedure cannot however totally eliminate deregulation. The use of recombinant DNA
technology has enabled genetic modifications which have further improved existing
production strains through metabolic engineering (Chapter 7). As indicated in Chapter 7,
metabolic engineering involves the introduction of genes which will enhance the
production of a metabolic pathway. The pathways through which amino acids are made
by the organism are shown in Fig. 21.5. The genes limiting the production of the amino
acid are enhanced by gene amplication thus leading to a more rational improvement of
the organism.
Many examples exist of improvements in amino acid production through cloning of
genes (Table 21.5). Among the pathways which have been targeted for improvement
through gene cloning are:
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Modern Industrial Microbiology and Biotechnology
Table 21.5 Improvements in amino acid production through the cloning of different genes
Amino acid
produced
Microorganisms
Gene donor
Cloned gene
or enzyme
L-alanine
D-alanine
L-histidine
E. coli
E. coli
C. glutamicum
S. marcescens
C. glutamicum
B. flavum
C. glutamicum
C. glutamicum
C. glutamicum
B. stearothermophilus
Ochrobactrum anthropi
C. glutamicum
S. marcescens
C. glutamicum
E. coli
C. glutamicum
E. coli
C. glutamicum
C. glutamicum
B. lactofermentum
S. marcescens
E. coli
C. glutamicum
E. coli
B. lactofermentum
S. marcescens
E. coli
C. glutamicum
B. lactofermentum
B. lactofermentum
B. flavum
S. marcescens
E. coli
C. glutamicum
E. coli
E. coli
E. coli
C. glutamicum
C. glutamicum
E. coli
Ala dehydrogenase
D-aminopeptidase
His G, D, C, B
His G, D, B
Hom dehydrogenase
ilv A
Lys A, dap A, B, D, Y
Asp A
aro F, chorismate
mutase, PRDH
aro G, Phe A
aro F, E, L, PRDH
Pro A, B
Thr A, B, C
hom dehydrogenase,
hom kinase, Thr C
ppc, hom
dehydrogenase,
hom kinase
Thr B, C
ppc
Trp A, E, R, tna A
Trp E, aro F,
chorismate mutase,
PRDH
Aro F
L-isoleucine
L-lysine
L-phenylalanine
L-proline
L-threonine
L-tryptophan
L-tyrosine
Yield
mg/mL
200
15
43
11
21
28
21
75
55
51
33
27
60
40
45
9
(i) the terminal pathways of the amino acid synthesis
(ii) the central metabolic pathway for producing the amino acid
(iii) the transport process for secreting amino acid
21.5.1
Strategies to Modify the Terminal Pathways
The strategies for modifying the terminal pathways are indicated in Fig. 21.8.
1. Amplification of rate limiting enzyme: The gene coding for the rate limting enzyme in
the biosynthetic pathway is amplified. Large increases have been observed when
this technique was applied to L-phenyl alanine production in Corynebacterium
glutamicum.
2. Amplification of branch-point enzyme: The gene coding for the branch-point enzyme
is amplified to redirect the common intermediate to another amino acid. It has been
used successfully in converting L-lysine to L-tryptophane and L-tyrosine to Lphenylalanine.
Production of Amino Acids by Fermentation
!'!
Fig. 21.8 Strategies to Modify Terminal Pathways for the Improved Production of Amino
Acids, (from Ikeda, 2003)
3. Introduction of a different enzyme able to produce the same end amino acid: The gene for
a different enzyme for the same end amino acid is introduced. The enzyme creating
the bottle neck is thus bypassed. This has been used for increased L-isoleucine
production in Corynebacterium glutamicum.
4. Introduction of a more functional enzyme than the native one: Introduction of an enzyme
which is more active than the native one thereby enhancing the production of the
amino acid. This has enhanced the production of L-alanine production by
Corynebacterium glutamicum when L-alanine dehydrogenase from Arthrobacter
oxydans was engineered into it .
5. Amplification of the first enzyme in the terminal pathway: The first enzyme in a
pathway diverging from central metabolism is amplified to increase the flow in
that pathway; any bottleneck is removed by the increased down the pathway. This
strategy has been applied to obtain increased yield of L-tryptophan by
Corynebacterium glutamicum.
21.5.2
Strategies for Increasing Precursor Availability
A major aim of metabolic engineering for increased amino acid production is to channel
as much carbon as possible from sugar into the production of a desired amino acid. After
bottlenecks in the terminal pathway are removed, the main factor limiting increased
production is the shifting of intermediates to the central metabolic pathway. The
complete genetic sequence of Corynebacterium glumicum is available. One strategy is to
amplify the genes for the enzymes leading to the formation of aromatic amino acids
erythrose 4-P and to L histidine through 5-P (Fig. 21.9).
!'"
Modern Industrial Microbiology and Biotechnology
Fig. 21.9 Strategies for Increasing Precursor Availability for the Production of Aromatic
Amino Acid and L-Histidine in Corynebacterium glutamicum
21.5.3
Metabolic Engineering to Improve Transport of
Amino Acids Outside the Cell
The aim of strain improvement is to prevent feedback inhibition when the amino acid
accumulates intracellularly. One manner in which feedback inhibition can be avoided is
through increased efflux of the amino acid. A gene which codes for increased efflux has
been introduced into E.coli resulting in a vastly increased production of L-cysteine (Fig.
21.10).
21.6
21.6.1
FERMENTOR PRODUCTION OF AMINO ACID
Fermentor Procedure
Starting from shake flasks the inoculum culture is grown in shake flasks and transferred
to the first seed tank (1,000–2,000 liters) in size. After suitable growth the inoculum is
Fig. 21.10 Increased Efflux of Amino Acid in E. coli through Metabolic Engineering
Production of Amino Acids by Fermentation
!'#
transferred to the second seed tank (10,000–20,000 liters), which serves as inoculum for
the production tank (50,000–500,000 liters).
The fermentation is usually batch or fed-batch (Chapter 9). In batch cultivation all the
nutrients are added at once at the beginning of the fermentation, except for ammonia
which is added intermittently to help adjust the pH, and fermentation continues until
sugar is exhausted. In a fed-batch process, the fermentor is only partially filled with
medium and additional nutrients added either intermittently or continuously until an
optimum yield is obtained. The fed-batch appears preferable for the following reasons:
(a) Most amino acid production requires high sugar concentrations of up to 10%. If all
were added immediately, acid would be quickly produced which will inhibit the
growth of the microorganisms and hence reduce yield.
(b) Where auxotrophic mutants are used, excess supply of nutrients leads to reduced
production due to overgrowth of cells or feed back regulation by the nutrient.
(c) During the lag phase of growth, the oxgen demand of the organism may exceed
that of the organism leading to reduced growth.
21.6.2
Raw Materials
The main raw materials used are cane or beet molasses and starch hydrolysates from
corn or cassava as glucose. In the US, the preferred carbon source is corn syrup from corn,
whereas in Europe and South America it is beet molasses.
As nitrogen source, inorganic sources such as ammonia or ammonium sulfate is
generally used.
Phosphates, vitamins and other necessary supplements are usually provided with
corn steep liquour.
21.6.3
Production Strains
Apart from the glutamatic acid bacteria already discussed, E. coli and Bacillus subtilis are
also good amino acid producing organisms. The glutamic acid bacteria previously
classified as four different species are now regarded one species. The optimum
temperature of C glutamicum is 30°C, whereas that of E. coli is higher. Hence, E. coli may be
prefered for production in tropical countries.
Production strains for amino acids are generally classified as wild-type, capable of
producing amino acids under defined conditions, but generally low-yielding in quantity,
auxotrophic or regulatory mutants, in which feedback regulations are bypassed by
partially starving them of their requirements or by removal of metabolic controls through
mutation, and by gentically modifying the organism by amplifying genes coding for ratelimiting enzymes. The strains used belong to the last two categories and have been
developed using classical mutation methods or through genetic engineering (Chapter 7).
The selection of the strain is not only for high yields but also for those least producing
undesirable side products. For instance, when branched chain amino acids are
produced, it is essential that other branched chain amino acids do not occur as this
increases the cost of separation and extraction.
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Modern Industrial Microbiology and Biotechnology
21.6.4
Down Stream Processing
After fermentation, the cells may be filtered using a rotary vacuum filter (Chapter 10).
Sometimes filtration can be improved by using filteraids. These filteraids, usually
kiesselghur, which are based on diatomaceous earth, improve the porosity of a resulting
filter cake leading to a faster flow rate. Before filtration a thin layer is used as a precoat of
the filter (normally standard filters).
The extraction method of the amino acid from the filtrate, depends on the level of purity
desired in the product. However two methods are generally used: the chromatographic
(ion exchange) method or the concentration-crystallization method.
Crystallization is often used as a method to recover the amino acid. Due to the amphoteric
character (contains both acidic and basic groups) of amino acids, their solubility is
greatly influenced by the pH of the solution and usually show minima at the isoelectric
point (zero net charge). Since temperature also influences the solubility of amino acids
and their salts, lowering the temperature can be used in advance as a means of obtaining
the required product. Precipitation of amino acids with salts, like ammonium and
calcium salts, and with metals like zinc are also commonly used. This is followed by acid
(or alkali) treatment to obtain the free or acid form of the amino acid.
Ion exchange resins have been widely used for the extraction and purification of amino
acids from the fermentation broth. The adsorption of amino acids by ion exchange resins
is strongly affected by the pH of the solution and by the presence of contaminant ions.
There are two types of ion exchange resins; cation exchange resins and anion exchange
resins. Cation exchange resins bind positively charged amino acids (this is in the
situation where the pH of the solution is lower then the isoelectric point (IEP) of the amino
acid), whereas anion exchange resins bind negatively charged amino acids (pH of the
solution is higher than IEP). Elution of the bound amino acid(s) is done by introducing a
solution containing the counterion of the resin. Anion exchange resins are generally
lower in their exchange capacity and durability than cation exchange resins and are
seldom used for industrial separation. In general, ion exchange as a tool for separation is
only used when other steps fail, because of its tedious operation, small capacity and high
costs.
SUGGESTED READINGS
Araki, K. 2003. Amino Acids Kirk-Othmer Encyclopedia of Chemical Technology. 2, 554-618.
Currell, B.R.C., Mieras, V.D., Biotol Partners. 1997. Biotechnological Innovations in Chemical
Synthesis Elsevier.
Ikeda, M. 2003. Amino Acid Production Processes. Advances in Biochemical Engineering/
Biotechnology, 79, 1–35.
Kelle, R., Hermann, T., Bathe, B. 2005. L-Lysine. In: Handbook of Corynebacterium glutamicm.
L Eggelin, and M Bott, (eds). Taylor and Francis, Boca Raton FI, USA, pp. 465-488.
Kimura, E. 2003. Metabolic Enginering of Glutamate Production. Advances in Biochemical
Engineering/Biotechnology, 79, 37–57.
Production of Amino Acids by Fermentation
!'%
Mueller, U., Huebner, S. 2003. Economic Aspects of Amino Acid Production. Advances in
Biochemical Engineering/Biotechnology, 79, 137–170.
Pfefferle, W., Mockel, B., Bathe, B., Marx, A. 2003. Advances in Biochemical Engineering/
Biotechnology, 79, 59–112.
Sano, K. 1994. Host – Vector Systems for Amino Acid-Producing Coryneform Bacteria.
Improvement of Useful Enzymes by Protein Engineering. In: Recombinant Microbes for
Industrial and Agricultural Applications. Y Murooka, T. Imanaka, (eds). Marcel and Dekker,
New York, USA. pp. 485-507.
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Modern Industrial Microbiology and Biotechnology
+0)26-4
22
Biocatalysts: Immobilized
Enzymes and Immobilized
Cells
22.1 RATIONALE FOR USE OF ENZYMES FROM
MICROORGANISMS
Enzymes are organic compounds which catalyze all the chemical reactions of living
things – plants, animals and microorganisms. They contain mainly protein; some of them
however contain non-protein components, prosthetic groups. When excreted or
extracted from the producing organism they are capable of acting independently of their
source. It is this property of independent action which drew early attention to their
industrial use.
All enzymes have infrastructural backbones of protein. In some enzymes only proteins
exist, while in others, covalently attached carbohydrate groups may be present; often
these carbohydrate groups may play no part in the catalytic activity of the enzyme,
though they may contribute to the stability and solubility of the enzyme. Metal ions
known as co-factors and low molecular weight organic compounds, known as coenzymes may also be present. Co-factors and co-enzymes are important for the stability
and activity of the enzyme. They have a tendency to be detached and it is important to
provide conditions which ensure their retention.
Most industrial enzymes are obtainable from microorganisms. The advantages of
using microorganisms are numerous, in contrast with their production from plants (e.g.
malt diastase) and animals (e.g. pepsin) and are as follows:
(a) Plants and animals grow slowly in comparison with microorganisms;
(b) Enzymes form only small portions of the total plant or animal and large tracts of
land as well as huge numbers of animals would be necessary for substantial
productions. These limitations make plant and animal enzymes expensive.
Microbial enzymes on the other hand are not subject to the above constraints and
may be produced at will in any desired amount.
(c) By far the greatest attraction for the production of microbial enzymes, however, is
the great diversity of enzymes which reflects the diversity of microbial types in
Biocatalysts: Immobilized Enzymes and Immobilized Cells
!''
nature. Thus largely, though not entirely, because of the widely varying
environmental conditions in nature, microbial enzymes have been isolated which
operate under extreme environmental conditions. For example microorganisms
produce amylases functioning at temperatures as high as 110°C and proteases
operating at pH values as high as 11 or as low as 3.
(d) Finally, following from greater understanding of the genetic basis for the control of
physiological function in micro-organisms it is now possible to manipulate
microorganisms to produce virtually any desired metabolic product, including
enzymes.
22.2
CLASSIFICATION OF ENZYMES
Based on catalyzed reactions, the enzyme committee (EC) of the International Union of
Biochemistry and Molecular Biology (IUBMB) recommended the classification of
enzymes into six groups. The nomenclature of enzymes is based on the number assigned
to these six major groups, and the sub-groups found within the major groups. Enzymes
are also known by long-standing common names which are also widely used.
The IUBMB committee also defines subclasses and sub-subclasses. Each enzyme is
assigned an EC (Enzyme Commission) number. For example, the EC number of catalase
is EC 1.11.1.6. The first digit indicates that the enzyme belongs to oxidoreductase (class
1). Subsequent digits represent subclasses and sub-subclasses. Thus the enzyme rennet
used in cheese manufacture and also known as chymosin, has the number of EC 5.3.1.5.
The six major EC groups are as follows.
1. Oxidoreductases catalyze a variety of oxidation-reduction reactions. Common names
include dehydrogenase, oxidase, reductase and catalase.
2. Transferases catalyze transfers of groups (acetyl, methyl, phosphate, etc.). Common
names include acetyltransferase, methylase, protein kinase, and polymerase. The first
three subclasses play major roles in the regulation of cellular processes.
3. Hydrolases catalyze hydrolysis reactions where a molecule is split into two or more
smaller molecules by the addition of water. Some examples are:
Proteases: Proteases split protein molecules. They are further classified by their optimum
pH as acid, alkaline or neutral. They may also be classified on the basis of their active
centers into the following:
(i) Serine proteases: These have a residue in their active center and are specifically
inhibited by diisopropyl phosphofluoridate and other organophosphorus
derivates.
(ii) Thiol proteases: The activity of these depends on the presence of an intact-SH group
in their active center. They are specifically inhibited by thiol reagents such as
heavy metal ions and their derivatives, as well as alkylating and oxidizing agents.
(iii) Metal proteases: These depend on the presence of more of less tightly bound divalent
cations for their activity.
(iv) Acid proteases: Acid proteases contain one or more side chain carboxyl groups in
their active center.
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Modern Industrial Microbiology and Biotechnology
Nucleases split nucleic acids (DNA and RNA). Based on the substrate type, they are
divided into RNase and DNase. RNase catalyzes the hydrolysis of RNA and DNase acts
on DNA. They may also be divided into exonuclease and endonuclease. The
exonuclease progressively splits off single nucleotides from one end of DNA or RNA.
The endonuclease splits DNA or RNA at internal sites.
Phosphatase catalyzes dephosphorylation (removal of phosphate groups).
4. Lyases catalyze the cleavage of C-C, C-O, C-S and C-N bonds by means other than
hydrolysis or oxidation. Common names include decarboxylase and aldolase.
5. Isomerases catalyze atomic rearrangements within a molecule. Examples include
rotamase, protein disulfide isomerase (PDI), epimerase and racemase.
6. Ligases catalyze the reaction which joins two molecules. Examples include peptide
synthase, aminoacyl-tRNA synthetase, DNA ligase and RNA ligase.
22.3
USES OF ENZYMES IN INDUSTRY
Most of the enzymes used in industry are hydrolases (i.e., those which hydrolyze large
molecules). In particular amylases, proteases, pectinases, and to a lesser extents lipases
have been most commonly used. Enzymes are used in a wide range of industries and
some uses are discussed below.
(i) Production of nutritive sweeteners from starch: Enzymic hydrolysis has now almost
completely replaced the use of acid in starch hydrolysis (Chapter 4). The sweeteners
which have been produced from starch are high conversion (or high DE) syrup, high
maltose syrup, glucose syrup, dextrose crystals and high fructose syrup. These
sweeteners are often called corn syrups because they are produced from maize, although
starch from any source (e.g. cassava, sorghum, or potatoes) may be used. The processes of
production of sweeteners from corn consists of the gelatinization of starch production of
water-soluble dextrins with a-amylase, the subsequent application of a de-branching
enzyme (e.g. pullulanase) and, depending on the sugar sought, the application of a third
enzyme. a-Amylase from B. licheniformis is particularly suitable for dextrinization
because its optimum temperature is 110°C, a convenient temperature on account of the
need to boil starch to gelatinize it. If high maltose syrup is sought a-amylase is applied,
while gluco-amylase is applied if glucose syrup is sought (Fig. 22.1). Dextrose crystals are
usually produced by removing minerals with ion exchange resins and then crystallizing
the liquid after concentration.
Nowadays, most sweeteners produced from starch are in the form of high fructose
corn syrup (HFCS), whose production is discussed below. Glucose has a rather bland
taste and is not as sweet as sucrose. Fructose, on the other hand, is about 1.7 as sweet as
sucrose. In the confectionary industry therefore glucose resulting from the hydrolysis of
starch is converted to fructose by the enzyme glucose isomerase which rearranges the
glucose molecule to yield fructose and the syrup itself into a glucose/fructose syrup.
Glucose isomerase is completely specific for monomeric D-glucose. The maltose,
maltotriose and higher maltooligosaccharides present in glucose syrup are untouched
by the enzyme. An acceptable composition of high-fructose glucose syrups in commerce
is: fructose 42%, glucose, 50%, maltose, 6%, and maltotriose, 2%. These high fructose
Biocatalysts: Immobilized Enzymes and Immobilized Cells
"
STARCH
Heat “solution” of starch
GELATINIZATION
a-Amylase (sometimes with prior acid treatment)
DEXTRINIZATION
Fungal Amylase
Amyloglucosidase
pH ADJUSTMENT
PULLULANASE
b - AMYLASE
HIGH CONVERSION
SYRUP
HIGH MALTOSE
SYRUP
GLUCOSE SYRUP
De-ash with
ion exchange,
concentrate,
and crystallize
Ph ADJUSTMENT
CRYSTALLINE
GLUCOSE
Pass through column of immobilized
glucose isomerase
HIGH FRUCTOSE
SYRUP
Fig. 22.1
Hydrolysis of Starch for High Fructose Corn Syrup Production
mixtures are used in place of sucrose and invert sugar in the baking and beverages
industries. HFCS production indeed represents one of the major uses of enzymes.
Besides its role as a sweetener, fructose has other qualities which make it superior to
glucose. It is regarded as a low-calorie sweetener, since it is so much sweeter than
sucrose. Furthermore, fructose is favored for intravenous infusions or drip over glucose
because the body tolerates it better. Twice as much fructose as of glucose can therefore be
permitted in drips, representing a greater caloric intake in as much fluid. It is therefore the
preferred sugar for patients in a state of shock. Finally it is more quickly absorbed than
glucose and does not need the enzyme and hormonal system required for absorbing the
former.
"
Modern Industrial Microbiology and Biotechnology
Organisms which have served as sources for glucose isomerase include more than two
dozen strains of Streptomyces, several of Arthobacter, Nocardia, Micromonospora as well as
Lactobacillus brevis and Pseudomonas hydrophilla.
(ii) Proteolytic enzymes in the detergent industry: The detergent industry is at present one
of the greatest consumers of enzymes, and uses mostly proteases. Blood and pus stains
from hospital linen and other protein dirt precipitate and coagulate on clothes and are
ordinarily difficult to remove. The inclusion of proteolytic enzymes in a detergent or
washing soap greatly facilitates the removal of such stains. The proteolytic enzymes used
for this purpose should have a high pH optimum of 9-11, which is the pH of detergents,
and a high temperature optimum of 65-70°C since hot water facilitates laundering.
Furthermore, the enzyme should be able to cleave peptide bonds randomly and facilitate
the dissolution of the protein. Such proteolytic enzymes have been produced mainly by
alkalophilic and aerobic spore-formers such as strains of Bacillus licheniforms and Bacillus
amyloliquifaciencs. The latter has the advantage of producing a-amylases as well.
It is worth mentioning that the history of the use of enzymes in detergents has not
always been smooth. Soon after their introduction in the early 1960s, some factory
workers handling the enzymes suffered from allergic reactions. Strong public protests led
to the withdrawal of enzymes in detergents and, although a commission of inquiry
showed that there was no danger to the user, the use of enzymes in detergents suffered a
temporary setback. Subsequently, enzymes are added in dust-free encapsulated
preparations, to avoid inhalation by producers and users.
(iii) Microbial rennets: Rennin is an acid protease found in gastric juice of young
mammals where it helps to digest milk. It is used in the manufacture of cheese and
functions by hydrolyzing a polypeptide fragment from milk protein-kappa casein to
leave paracasein; this then forms an insoluble complex with cations to give a firm curd.
The commercial form of rennin known as rennets is obtained from the fourth stomachs of
young calves. It is therefore expensive and tedious to produce since it involves the
maturation, gestation and delivery of cows. Due to this, a search for substitutes ensued.
Strains of Mucor miehi, M. pusillus, Endothia parasitica, Bacillus polymyxa, B. subtilis and
Aspergilus are used to produce acid proteases which successfully substitute for rennin.
Indeed, microbial rennets constitute about the third largest use of microbial enzymes.
(iv) Lactase: Lactase hydrolyzes the disaccharide lactose into its component galactose
and glucose, both of which are sweeter than lactose and correspond to the addition of
0.9% sucrose. Thus, dairy products containing lactose, such as yoghurt, and ice cream,
can be sweeter and more acceptable to consumers without the extra expense of
extraneously added sugar. Galactose and glucose are also metabolized by a far wider
range or organisms than can attack lactose. The result is that lactase-hydrolyzed whey
can be used to produce alcohol or soft drinks. Furthermore, milk in which lactose is
hydrolyzed is preferred by individuals in some parts of the world where intestinal
lactase is low. Finally, when lactose occurs in high concentrations, such as is in ice
cream, it tends to crystallize out giving the impression during consumption that grains of
sand are present in the product. The addition of lactase prevents such crystallization.
Lactase is now produced commercially from Kluyveromyces fragilis, Saccharomyces lactis or
Aspergillus niger.
Biocatalysts: Immobilized Enzymes and Immobilized Cells
"!
(v) The textile industry: In the textile industry large amounts of starch, gelatin and their
derivatives such as glue are used to strengthen threads (yarns) of synthetic and natural
materials (e.g. cotton) to enable them to stand abrasion during weaving and also to polish
sewing thread. Starch and its derivatives are furthermore used to restrict dye stuffs and
prevent their diffusion to other portions of the fabric.
At the end of the manufacturing the starch is removed with thermostable a-amylases
from Bacillus licheniformis. The cloth is passed through hot water to remove inorganic
salts and to raise the temperature. It is then passed into an enzyme solution where it is
allowed to remain from 15 minutes to several hours depending on the enzyme
concentration and other factors.
Natural silk threads are proteinaceous in nature and consist of a highly resistant
protein known as fibroin, but they are held together by a semi-soluble protein gum known
as sericin. Sericin is removed with a neutral proteolytic enzyme; the silk threads are then
sized and woven into cloth after which they are de-sized in an amylase or protease
depending on the sizing material.
(vi) Pectinases for use in fruit juice and wine manufacture: Pectinases are enzymes which
attack pectic substances, a group of complex acidic polysaccarides. Pectic substances are
high molecular weight substances made up of poly – D – galacturonic acid. As the
carboxylic acid groups of the sugar units are partially esterified with methanol, they are
regarded as poly-uronides. They are the cementing material holding plant cells together.
The Agricultural and Food Chemistry section of the American Chemical Society has
proposed the following nomenclature for the various pectic substances:
Pectic Substances: a group designation for those complex carbohydrate derivatives which
occur in or are prepared from plants and contain a large proportion of
anhydrogalacturonic acid units. The carboxyl groups of polygalactoronic acids may be
partly esterified by methyl groups and partly or completely neutralized by one or more
bases.
Protepectin: The water-insoluble parent pectic substance which occurs in plants and
which, upon restricted hydrolysis, yields pectin or pectinic acids.
Pectinic acids: are colloidal polygalacturonic acids containing more than a negligible
proportion of methyl ester groups.
Pectic substances may be regarded as polygalacturonides composed of unbranched
a–1, 4 galacturonic acid residues (Fig. 22.2) but with other non-uronides bound to the
chain. However, pectic materials are not uniform because of the great variations which
have been observed by many authors in the molecular weights, degree of esterification
and acetylation, amount and type of neutral sugars and non-uronide residues found in
various preparations of pectic substances.
Pectinolytic enzymes or pectinases are widely distributed in plants and among
microorganisms. These enzymes vary greatly in their mechanisms of action, but may be
grouped into two: esterases which de-esterify pectin to pectic acid and depolymerases
which depolymerize pectin, pectin acid or short-chain galacturonic acid (ligo-Dgalacturonates) derived from pectin and pectic acids (Table 22.1). Aspergilli (A.niger, A.
oryzae, A. wentii, and A. flavus) and other fungi (Table 22.1) are used for industrial enzyme
production. The industrial enzymes themselves are a mixture of various pectinolytic
enzymes.
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Modern Industrial Microbiology and Biotechnology
COOCH3
H
H
H
OH
OH
COOCH3
OH
H
H
H
H
OH
H
H
H
H
H OH
H
H
COOCH3
H
OH
Fig. 22.2 Structure of Pectin
Table 22.1
Distribution of pectinolytic enzymes in some microorganisms
Source
PMGE
Bacillus sp.
Erwinia aroideae
Pseudomonas sp.
Ps. Marginalis
Xanthomonas campestris
Clostridium multifermentans
Aspergillus niger
Penicillium digitatum
Key:
PMGE
=
PG
=
PGL
PMG
=
=
PMGL
=
OG
=
OGL
=
PG
+
+
+
+
+
+
+
+
PGL
PMG
PMGL
+
+
+
+
+
+
OG
OGL
+
+
+
+
+
+
+
Polymethylgalacturonase esterase (de-esterify pectin to pectic acid by
removal of methoxy residues)
Polygalacturonases (hydrolyze pectic acids randomly, successive bonds or
alternate bonds)
Polygalactorunate lyase (hydrolyzes pectic acids by transelimination)
Polymethylgalacturonase (hydrolyzes pectin in random or
sequential fashion)
Polymethylgalacturanate lyase (cleaves pectic acid randomly or
sequentially)
Oligogalacturonase (hydrolyzes oligo-galactosiduronates i.e. breakdown
products of pectin and pectic acids)
Oligogalacturonate lyase (acts as OG but by transelimination)
Pectinolytic enzymes are used principally in the fruit juice, fruit processing and the
wine industries. In the fruit juice and fruit industry, pectinases are used to disintegrate
the fruits, and to clarify the resulting juices to give a clear sparkling liquid after the
filtration of the debris.
Another application of pectinases which does not involve the isolation of the enzymes
but which deserves mention is the retting of plants for flax (linen) from (Linum
usitatissimum) and hemp (Cannabis sativa) and of jute from Corchorus sp. Retting has not
been studied intensively probably because of the advent of man-made fibres. Pectinolytic
enzymes are produced anaerobically by Clostridium spp. when the plants are immersed
in water. When aerobic conditions prevail as in ‘dew-retting’ the organisms which have
been isolated include Bacillus comesii, and the fungi, Cladosporium, Aspergillus, and
Penicillium.
Biocatalysts: Immobilized Enzymes and Immobilized Cells
"#
(vii) Naringinase is used for removing the bitter tasting substance from citrus fruits,
especially grape fruits. Naringin is a flavonoid found in grapefruits, and gives grapefruit
its characteristic bitter flavor. Flavonoids are a group of polyphenolic secondary
metabolites secreted by plants and found widely among plants. They are present in many
plant-based foods such as tea and soybeans, and are generally believed to be beneficial to
health. Although naringin is supposed to have some beneficial effect such as stimulating
our perception of taste by stimulating the taste buds (for which reason some people eat
grape fruits before a meal), the bitter taste is undesirable in fruit juices. Therefore,
grapefruit processors attempt to select fruits with a low naringin content, or use
naralginase produced by strains of Aspergillus spp. to remove it.
Fig. 22.3 Structure of Naringin
(viii) Enzymes in the baking industry: Flour contains 72-75% starch, 11-13% protein, and
0.04-0.4% minerals. It also contains amylases and proteases derived from wheat. These
enzymes play major roles in the nature of the final baked product. When the flour is
deficient in amylases, unusually low amounts of sugar and intermediate products are
produced, giving rise to low volume, dry texture and other undesirable characteristics in
the finished bread. Similarly, while wheat brands rich in gluten (or ‘hard’ wheat) are
suitable for bread making because they are highly elastic, they are not suitable for cakes.
For the latter, ‘soft’ wheat with low gluten contents is required. ‘Hard’ wheat is rendered
‘soft’ however by the hydrolysis of some of the gluten using proteases. Bakery amylases
and proteases are derived from fungi.
(ix) Enzymes in the alcoholic beverages industry: Amylases derived from fungi or bacilli
may be employed in the distilled alcoholic beverages to hydrolyze starch to sugars prior
to fermentation by yeasts. The enzymes may also be used to hydrolyze unmalted barley
and other starchy adjuncts converting them to maltose and thus reducing the cost of beer
brewing. Although it is unusual, turbidities due to starch may arise in beer due to the
destruction of the amylases following the use of too high a temperature during malt
kilning. Amylases are used to remove turbidities due to starch. In beer, chill-hazes are due
mainly to protein-tannin (protein-polyphenol) precipitates. Chill-hazes may be removed
in several ways, one of which is the addition of proteases. Proteases from Aspergillus niger
are often used (Chapter 12).
(x) Leather baiting: After animal skin has been trimmed of flesh, it is de-haired with lime
or a proteolytic enzyme and it is then baited. The purpose of baiting, is to prepare the dehaired skins and hides for tanning. Baiting used to be done by keeping the skin in a warm
suspension of chicken and dog dung, which probably yielded proteolytic bacteria.
Nowadays proteolytic enzymes from Bacillus spp. and Aspergillus spp. are used. The
"$
Modern Industrial Microbiology and Biotechnology
effect of baiting is to make the fibrous protein collagen of which leather is composed more
amenable to the subsequent processes of leather manufacture.
(xi) Some medical uses of microbial enzymes: At present, the most successful medical
applications of enzymes are the use of proteolytic enzymes from Bacillus spp. and other
bacteria for the treatment of burns and skin cancers, and the treatment of life-threatening
disorders within the blood circulation using hemolytic enzymes produced from Bhemolytic streptococci. Other uses are given below:
(a) Fungal acid proteases may be used to treat alimentary dyspepsia, because of the
acid resistance of the enzyme. Fungal amylases may also be used to help digestion.
(b) Dextrans deposited on the teeth by Streptococcus mutans may be removed with the
use of fungal dextranase often introduced into the toothpaste, thus helping to fight
dental decay.
(c) L-asparaginase produced from E. coli and other gram-negative bacteria may be
used in the treatment of certain kinds of leukemia.
(d) Penicillinases produced by many organisms are sometimes used in emergency
cases of penicillin hypersensitivity.
–
SO2(e) Rhodanase which catalyses the reaction, S2O 32– + CN –
3 + SCN
has been used to combat cyanide poisoning. Rhodanase is produced by the
thermoacidophilic bacterium Sulfobacillus sibiricus.
The above are only a few of some of the uses to which microbial enzymes have been put
in the medical area.
22.4
PRODUCTION OF ENZYMES
22.4.1
Fermentation for Enzyme Production
Most enzyme production is carried out in deep submerged fermentation; a few are best
produced in semi-solid media.
22.4.1.1
Semi solid medium
This system, also known as the ‘Koji’ or ‘moldy bran’ method of ‘solid state’ fermentation
is still widely used in Japan. The medium consists of moist sterile wheat or rice bran
acidified with HCl; mineral salts including trace minerals are added. An inducer is also
usually added; 10% starch is used for amylase, and gelatin and pectin for protein and
pectinase production respectively. The organisms used are fungi, which appear
amenable to high enzyme production because of the low moisture condition and high
degree of aeration of the semi-soluble medium.
The moist bran, inoculated with spores of the appropriate fungi, is distributed either in
flat trays or placed in a revolving drum. Moisture (about 8%) is maintained by
occasionally spraying water on the trays and by circulating moist air over the
preparation. The temperature of the bran is kept at about 30°C by the circulating cool air.
The production period is usually 30-40 hours, but could be as long as seven days. The
optimum production is determined by withdrawing the growth from time to time and
assaying for enzyme. The material is dried with hot air at about 37°C–40°C and ground.
The enzyme is usually preserved in this manner. If it is desired, the enzyme can be
Biocatalysts: Immobilized Enzymes and Immobilized Cells
"%
extracted. Growth in a semi-solid medium seems sometimes to encourage an enzyme
range different from that produced in submerged growth. Thus, Aspergillus oryzae on
semi-solid medium will produce a large number of enzymes, primarily amylase,
glucoamylose, and protease. In submerged culture amylase production rises at the
expense of the other enzymes. Similarly, if Aspergillus oryzae producing takadiastase (a
commercial powder containing amylase and some protease) is grown in submerged
culture four protease components are formed whereas on semi-solid medium not only are
two proteases formed, but these are less heat resistant than those produced in submerged
fermentation.
22.4.1.2
Submerged production
Most enzyme production is in fact by submerged cultivation in a deep fermentor (Chapter
9). Submerged production has replaced semi-solid production wherever possible
because the latter is labor intensive and therefore expensive where labor is scarce, and
because of the risk of infection and the generally greater ease of controlling temperature,
pH and other environmental factors in a fermentor.
The medium must contain all the requirements for growth, including adequate
sources of carbon, nitrogen, various metals, trace elements, growth substances, etc.
However, a medium adequate for growth may not be satisfactory for enzyme production.
For the production of inducible enzymes, the inducers must be present. Thus, pectic
substances need to be in the medium when pectinolytoc enzymes are being sought.
Similarly, in the production of microbial rennets soy bean proteins are added into the
medium to induce protease production by most fungi. The inducer may not always be the
substrate but sometimes a breakdown or end-product may serve. For example, cellobiose
may stimulate cellulose production.
Sometimes some easily metabolizable components of the medium may repress enzyme
production by catabolite repression. Strong repression is often seen in media containing
glucose. Thus, a-amylase synthesis is repressed by glucose in Bacillus licheniformis and B.
subtilis. Fructose on the other hand represses the synthesis of the enzyme in B.
stearothermophilus. In many organisms protease synthesis is repressed by amino acids as
well as by glucose. It is therefore usual to replace glucose by more slowly metabolized
carbohydrates such as partly hydrolyzed starch. High enzyme yield may also be
obtained by adding constantly, low amounts of the inducer.
End-product inhibition has also been widely observed. Some specific amino acids
inhibit protease production in some organisms. Thus, isoleucine and proline are
involved in the case of B. megaterium while sulphur amino acids inhibit protease
formation in Aspergillus niger.
Temperature and pH requirements have to be worked out for each organism and each
desired product. The temperature and pH requirements for optimum growth, enzyme
production, and stability of the enzyme once it is produced are not necessarily the same
for all enzymes. The temperature adopted for the fermentation is usually a compromise
taking all three requirements into account.
The oxygen requirement is usually high as most of the organisms employed in enzyme
production are aerobic. Vigorous aeration and agitation are therefore done in the
submerged fermentations for enzyme production. Batch fermentation is usually
employed in commercial enzyme fermentation and lasts from one to seven days.
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Modern Industrial Microbiology and Biotechnology
Continuous fermentation, while successful experimentally, does not appear to have been
used in industry.
In a few cases the enzyme production is highest during the exponential phase of
growth. In most others, however, it occurs post-exponentially. Furthermore, different
enzymes are produced at different stages of the growth cycle. Thus Asp. niger produces
mostly a–amylases in the first 72 hours but mainly maltase thereafter.
22.4.2
Enzyme Extraction
The procedures for the extraction of fermentation products described in Chapter 10 are
applicable to enzyme extraction. Care is taken to avoid contamination. In order to limit
contamination and degradation of the enzyme the broth is cooled to about 20°C as soon
as the fermentation is over. Stabilizers such as calcium salts, proteins, sugar, and starch
hydrolysates may be added and destabilizing metals may be removed with EDTA. Antimicrobials if used at all are those that are normally allowed in food such as benzoates
and sorbate. Most industrial enzymes are extra-cellular in nature. In the case of cell
bound enzymes, the cells are disrupted before centrifugation and/or vacuum filtration.
The extent of the purification after the clarification depends on the purpose for which
the enzyme is to be used. Sometimes enzymes may be precipitated using a variety of
chemicals such as methanol, acetone, ethyl alcohol or ammonium sulfate. The precipitate
may be further purified by dialysis, chromatography, etc., before being dried in a drum
drier or a low temperature vacuum drier depending on the stability of the enzymes to
high temperature. Ultra-filtration separation technique based on molecular size may be
used.
22.4.3
Packaging and Finishing
The packing of enzymes has become extremely important since the experience of the
allergic effect of enzyme dust inhalation by detergent works. Nowadays, enzymes are
packaged preferably in liquid form but where solids are used, the enzyme is mixed with
a filler and it is now common practice to coat the particles with wax so that enzyme dusts
are not formed.
22.4.4
Toxicity Testing and Standardization
The enzyme preparation should be tested by animal feeding to show that it is not toxic.
This test not only assays the enzyme itself but any toxic side-product released by the
microorganisms. For a new product extensive testing should be undertaken, but only
spot checks need to done for a proven non-toxic enzyme in production. The potency of the
enzyme preparation, based on tests carried out with the substrate should be determined.
The shelf life and conditions of storage for optimal activity should also be determined.
22.5
IMMOBILIZED BIOCATALYSTS: ENZYMES AND CELLS
The major handicap in the traditional use of enzymes is that they are used but once. This
is mainly because the enzymes are unstable in the soluble form in which they are used
and because recovery would be expensive, even if it were possible. It is not surprising that
Biocatalysts: Immobilized Enzymes and Immobilized Cells
"'
influenced by the idea of the catalyst in the chemical industry ways should be sought to
re-use biological catalysts. The immobilization of enzymes and cells provides a basis for
the re-use of enzymes and cells. Interest in immobilized enzymes has grown since the
1960s and numerous conferences and papers have been held and given on them.
Immobilized cells have received a great deal but perhaps slightly less attention judging
from the literature, since their study came a little later than that of immobilized enzymes.
(i) Imobilized enzymes: An immobilized enzyme may be defined as an isolated or
purified enzyme confined or localized in a defined volume of space.
(ii) Immobilized cells: Immobilized cells, also referred to as controlled biological
catalysts, may be defined as a high density of cells physically confined on a solid
phase or in pellets or clumps and in which cell movement is restricted for the
period of their use as biological agents. This definition excludes cells in a
chemostat, or cells which are recovered by centrifugation in a batch culture and
returned to the fermentation. It is not a completely satisfactory definition as the
term ‘high density’ can be elastic. Cell immobilization has existed or been
exploited long before it became recognized as potentially valuable in industry.
Thus, microorganisms in natural habitats such as soil, marine, alimentary canal,
dental plaque or in the ‘Orleans’ process of vinegar production where cells are
immobilized on wood shavings, the activated and trickling filter treatment of
wastewater, may be seen as examples of immobilized cells.
22.5.1
Advantages of Immobilized Biocatalysts in General
The advantages of immobilized enzymes beside reuse are as follows:
(i) They can be easily separated from the reaction mixture containing any residual
reactants and reused in subsequent conservations.
(ii) Immobilized enzymes are more stable over broad ranges of pH and temperature.
(iii) Enzymes are absent in the waste-stream
(iv) Immobilized systems specially lend themselves to continuous processes.
(v) Reduced costs in industrial production.
(vi) Greater control of the catalytic effect.
(vii) Greater ease of new applications for industrial and medical purposes.
(viii) Immobilized enzymes permit the use of enzymes from organisms which would not
normally be regarded as safe (i.e. non-GRAS).
22.5.2
Methods of Immobilizing Enzymes
Immobilized enzymes have been classified in a number of ways. The classification
method adopted here is the one published in 1995 by the International Union of Pure and
Applied Chemistry (IUPAC), and which divides methods of immobilized enzymes into
four broad groups, based on:
(a) covalent bonding of the enzyme to a derivatized water-insoluble matrix,
(b) intermolecular cross-linking of enzyme molecules using multifunctional reagents,
(c) adsorption of enzyme onto a water-insoluble matrix, and
"
Modern Industrial Microbiology and Biotechnology
Immobilized Enzyme
Bound (Chemical)
Covalently linked
Adsorbed
Entrapped (Physical containment)
Micro-encapsulation
Matrix-entrapped
Fig. 22.4 Methods of Enzyme Immobilization
(d) entrapment of the enzyme molecule inside a water-insoluble polymer lattice or
semi-permeable membrane.
The IUPAC groups can be divided into two basic groups, the chemical and the
physical methods as shown in Fig. 22.4.
The IUPAC emphasizes that in dealing with immobilized enzymes, the properties of
the free enzyme, the type of support used and the methods of support activation and
enzyme attachment must be specified.
22.5.2.1 Immobilization by covalent linkage
This is by far the most widely studied method. The covalent linkage is achieved between
a functional group on the enzyme not essential for catalytic activity and a reactive group
on a solid water-insoluble support. The functional groups available on enzymes for
linkage are amino and carboxyl groups, hydroxyl groups, imidazole groups, indole
groups, phenolic groups and sulphydryl groups. The nature of the enzyme’s functional
group through which immobilization is to be effected determines the reaction which will
be used to bind the enzyme to the support. Some of the reactions which have been used
are acylation, amination and arylation and alkylation.
Some supports which have been used for immobilization include agarose, celluose,
dextran, chitin, starch, polygalacturonic acid (pectin), polyacrylamide, polyvivyl
alcohol, polystyrene, polyprpylene, polyamino acids, polyamide, glass and metal aides
and bentonite. Many of these are organic, but recently the use has been advocated of
inorganic support on the grounds of reuse of inorganic materials, non-toxicity, good halflife of enzymes immobilized on inorganic supports, and the ease with which inorganic
materials can be fashioned to suit any particular enzyme system.
In the above description the reaction has been a direct one between functional groups
on the enzyme and reactive sites on the support. However, in some cases intermolecular
linkage may occur through the mediation of a crosslinking reagent, In such cases the
cross linking agent has a number of different active sites. Some of these react with the
support and others with the enzyme. One of the most commonly used cross-linking agent
is glutaraldehyde which has two cross-linking sites. Several others are available. The
advantage of the covalent bonding method of enzyme immobilization are:
(i) The coupling of the enzyme to the support is easy to conduct and consists of
allowing support and enzyme to interact and therefore facilitates centrifuging and
washing off any enzymes not bound.
Biocatalysts: Immobilized Enzymes and Immobilized Cells
"
(ii) The enzyme-support derivative is easy to manipulate and adapt because of the
great physical and chemical variation in the available support: they can be used in
a variety of reactors including stirred tank, fluidized bed-reactors and can also be
modified into flat sheets fiber.
(iii) Covalent coupling has been widely described and methods for carrying it out are
readily available in the literature.
(iv) The supports themselves are widely available commercially.
The disadvantages are that some preparations are tedious to make; the chemical
bonding may inactivate the enzyme in some cases; and finally covalently-bound waterinsoluble enzyme-substrate derivatives act poorly on high molecular weight substrates.
22.5.2.2
Immobilization by adsorption
This method is both simple and inexpensive and consists of bringing an enzyme solution
in contact with a water-insoluble solvent surface and washing off the unadsorbed
enzyme. The extent of the adsorption depends on a number of factors including the
nature of the support, pH, temperature, time, enzyme concentration. In principle, though
not always in practice, adsorption is reversible. Adsorbents which have been used
include alumina, bentonite, calcium carbonate, calcium phosphate, carbon, cellulose,
charcoal, clay, collagen, diatomaceous earth, glass, ion-exchange resins, sephadex, and
silica gel. Apart from the ease of the operation, the other advantage is that the enzymes are
unlikely to be inactivated because the system is mild. The disadvantage is that in cases of
weak binding the enzyme may be easily washed away.
22.5.2.3 Immobilization by micro-encapsulation
Micro-encapsulation consists in packaging the enzyme in tiny usually spherical
capsules ranging from 5-300 m in diameter in semi-permeable (permanent) or liquid (nonpermanent) membranes. The former are more commonly employed. To prepare microcapsules a high aqueous concentration of the enzyme is first prepared. The aqueous
enzymes solution is then emulsified in an organic solvent or solvents with a surfactant
which is soluble in the organic solvents. Two methods are then used to form microcapsules from this enzyme-surfactant emulsion.
In one method known as the interfacial polymerization technique, the enzyme
solution contains the enzyme as well as one component of the membrane that will form
round the micro-capsule. The emulsion is stirred vigorously and more of the organic
solvent(s) containing the rest of the capsule-forming reagent is added.
In the second method, the coacervation-dependent method, the added organic
solvents contain all the components of the polymer. In both cases the enzyme droplets are
formed during the vigorous stirring. The semi-permeable membrane is allowed to harden
around the micro-droplets; the micro-capsules are then washed and then transferred.
Semi-permeable membranes have been made of cellulose nitrate, polystyrene, etc.,
with the coacervation method.
Micro-capsule formation by the interfacial method has been produced from nylon and
widely investigated because of its application in medicine e.g. in urease immobilization
in artifical kidneys. The organic solvent usually used for the polyamide Nylon-6,10
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Modern Industrial Microbiology and Biotechnology
semi-permeable membrane from hexamethylenediamine and sabacoyl chloride is a
chloroform-cyclohezane mixture.
O
O
||
||
H2N – (CH2)6 – NH2 + Cl – C – (CH2) – C – Cl – HCI
Hexamethlenediamine
Sabacoyl chloride
O
O
O
O
||
||
||
||
– NH – (CH2)6 – NH – C – (CH2)8 – C (CH2)6 – NH G – (CH2)8 – C – NH–
Non-permanent liquid membranes are prepared by emulsifying the aqueous enzyme
solution in a surfactant to form the liquid membrane-encapsulated enzyme. It is not
commonly used.
The advantages of the permanent (semi-permeable) micro-capsulation method are:
(i) An extremely large surface is provided by the tiny bubbles of enzymes. A microcapsule with 20mm diameter would for example have a surface area of
2,500 cm2/ml.
(ii) The specificity of the micro-capsule is increased by the possibility of using a
membrane which will favor the diffusion of substrates of certain types.
The disadvantage is that a high concentration of enzyme is needed and only low
molecular weight substances pass through. With the non-permanent liquid membranes,
the same advantages accrue; the disadvantage however is leakage.
22.5.2.4
Immobilization by entrapment
In the entrapment of enzymes, no reaction occurs between support and the enzyme. A
cross-linked polymeric network is formed around the enzyme; alternatively the enzyme
is placed in a polymeric substance and the polymeric chains cross-linked.
Polyacrylamide gels have been widely used for this purpose, although enzymes do leak
through the network in some cases.
The advantages of the entrapping method are: (i) its simplicity, (ii) the small amount of
enzyme used, (iii) the unlikelihood of damage to the enzyme, (iv) applicability to water
insoluble enzymes.
The disadvantages include leakage of enzymes and some chemical and thermal
enzyme damage during gel formation.
22.5.3
Methods for the Immobilization of Cells
Three general methods are available for immobilizing microbial cells.
(i) Ionic binding to water-soluble ion-exchangers: Cells of E. coli and Azotobacter agile
bound to Dowex-l resin have been studied while mold spores have been bound to
ion-exchange cellulose derivatives. In both cases successful demonstration of
succinic acid oxidation and invertase activity were demonstrated. This method is
however not entirely satisfactory as enzymes may leak out following autolysis
during continuous enzyme reaction.
Biocatalysts: Immobilized Enzymes and Immobilized Cells
"!
(ii) Immobilization in cross-linked chemicals: Microbial cells have been immobilized by
cross-linking each other with bi-functional reagents such as glutaraldehyde. But
non-cross-linking agents are equally effective.
(iii) Entrapment in a polymer matrix: This appears to be the most widely used method of
immobilizing cells. In this method the cells are entrapped in a polymer matrix
where they are physically restrained. The following matrixes have been used:
polyacrylamide, collagen, cellulose triacetate, agar, alginate and polystyrene.
Methods for immobilization of cells and enzymes are given in Fig. 22.5.
22.5.3.1
Advantages of immobilized cells
Immobilized cells have the following advantages over conventional batch fermentation
as well as over immobilized enzymes.
(i) In batch fermentation, a significant proportion of the substrate is ‘wasted’ for the
growth of the microbial population and for producing other substances other than
enzymes required for the conversion at hand. Once the cells are immobilized
however, they need to be offered nutrients for growth.
(ii) When cells are immobilized the reactions are more homogeneous and can be
treated more like catalysts.
Fig. 22.5
Methods of Immobilizing Enzymes and Cells
""
Modern Industrial Microbiology and Biotechnology
(iii) The lag period which occurs in a conventional batch fermentation is eliminated for
the accumulation of products associated with non-growth phase of the cells.
(iv) It is more feasible to run immobilized cells continuously at high dilution rates
without the risk of washout which would occur in a conventional continuous
culture system.
(v) Higher and faster yield is possible because of the greater density of cells;
furthermore, toxic materials are continuously removed.
(vi) It is possible to recharge or resuscitate the cells by inducing growth and
reproduction among resting cells.
(vii) A high capital cost is involved in installing, and operating a fermentor; in systems
where comparison have been made, immobilized cells are cheaper than the
conventional batch production.
(viii) The use of immobilized cells eliminates the need for enzyme extraction and
purification. Furthermore, systems involving multi-enzyme reactions can occur
more easily in intact cells harboring these enzymes.
(ix) Immobilized cells are more suited to multiple step processes
(x) Cofactor regeneration is not a problem
Immobilized cells are particularly appropriate under the following conditions:
(i) When the enzymes are intracellular: the use of immobilized cells would eliminate
the need for breaking the cells for enzyme isolation.
(ii) When extracted enzymes are unstable during or after immobilization.
(iii) When the micro-organism does not produce enzymes which can cause
undesirable side reactions; or when such side-reaction producing enzymes can be
readily inactivated.
(iv) When the substrates and products are not high molecular weight substances.
22.5.3.2
Disadvantages of immobilized cells
Some of the disadvantages of the conventional system of cultivation of organisms spill
over to the immobilized cell thus:
(i) The cells may produce enzymes other than the one (s) sought.
(ii) Genetic changes, although with reduced likelihood in comparison with conventional fermentation, can also occur during immobilization.
(iii) Immobilization may result in the loss of a specific catalytic activity due to enzyme
inactivation, resulting from the immobilization process or to diffusional barriers
hindering substrate access, or product removal from the organisms.
(iv) Cells located in the center of a cell flow may be deprived of nutrients or be
inactivated by accumulating toxic wastes.
(v) Contamination by other microorganisms can occur.
22.6 BIOREACTORS DESIGNS FOR
USAGE IN BIOCATALYSIS
A variety of bioreactors are available for immobilizing enzymes and cells and these are
shown in Fig. 22.6.
Biocatalysts: Immobilized Enzymes and Immobilized Cells
a). batch stirred fermentor
b). continuous stirred tank
c). continuous packed-bed
i) downward flow
ii). upward flow
iii). Upward flow and re-cycle
d). continuous fluidized-bed
e). continuous ultrafiltration
Fig. 22.6 Various Designs of Bioreactors for Use in Biocatalysis.
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22.7
Modern Industrial Microbiology and Biotechnology
PRACTICAL APPLICATION OF IMMOBILIZED
BIOLOGICAL CATALYST SYSTEMS
Immobilized enzymes and cells have been intensively studied in the hope that they can
be used industrially. Only some of the expectations have been realized because of
economic reasons. Soluble ‘once only’ enzymes marketed in the form of powder or liquids
are available at low prices. Amylases and glucoamylases used in the starch industry are
so low-priced comparatively that immobilized forms can hardly compete. The only
immobilized enzyme currently used on a large scale is glucose isomerase used to produce
about 2 million tons of high-fructose syrups around the world, but especially in the USA,
Europe, and Japan. High fructose syrup competes successfully with sugar from beet or
cane.
Several immobilized enzymes or whole cell processes are being applied in the
Japanese pharmaceutical industry. These include L-amino acid from racemic acyl – D – L
– amino acids, L-aspartic acid from ammonium fumarate, L-citrulline from L-arginine,
and the production of 6-amino penicillanic acid (6-APA) for semi synthetic penicillin
production. In the United States and Europe 6-APA is produced with immobilized
enzymes. In Italy whole milk lactose hydrolysis is carried out by fiber entrapped lactase.
Many other applications are nearing the point in their development where they are
ready for commercialization: saccharification of starch by immobilized glucoamylase;
cheese whey lactose hydrolysis by bound >-galactosidase; beer chill-proofing, steroid
transformations, protein-hydrolysis to improve digestibility. Industrial processes do not
receive publicity rapidly and it is not unlikely that some of these may well have been
commercialized already.
Immobilized cells have been used industrially in Japan for the transformations
mentioned above except for L-amino acid isolation from racemic mixtures. The mostwidely employed use of immobilized cells however are glucose isomerization and the
hydrolysis of raffinose in beet sugar using mycelial pellets of the fungus Mortierrella sp.
Raffinose (in beet molasses) is hydrolyzed to sucrose and galactose by >–galactosidase
(mellibiase) produced by the fungus.
The potentials of immobilized enzymes and cells are yet far from realized. When
economic conditions permit them to become so, the whole process of fermentation as we
know it today may be revolutionized and fermentors may become largely for the growth
of cells for subsequent use in immobilized enzyme or cell production.
22.8 MANIPULATION OF MICROORGANISMS FOR
HIGHER YIELD OF ENZYMES
Until recently higher yields in enzyme production have been achieved, as has been the
case with most other industrial microbial products, by empirical means using selection
from natural variants, mutants obtained through treatment with various mutagens, and
improvement in the environmental conditions of the fermentor or semi-solid medium.
With these methods the rate of enzyme production has increased from two to fivehundred times.
In recent times more knowledge has accumulated about various aspects of enzyme
production including those of molecular and other dimensions, and the manipulation of
Biocatalysts: Immobilized Enzymes and Immobilized Cells
"%
industrial organisms in general. In this section some of these new developments and
their use or possible use in increasing enzyme yield will be discussed. Some of them
promise the possibility of producing virtually any enzyme extracelluarly and at will.
22.8.1
Some Aspects of the Biology of Extracellular
Enzyme Production
(i) The nature of extra-cellular enzymes secretion: Extracellular enzymes have been
defined as those which are secreted into the medium outside the cell without involving cell
lysis. This distinction is important because most extraccellular enzyme-producing
organisms are Gram-positive organisms. Gram-negative organisms, in general produce
enzyme in the medium only when the cell is lysed. Most Gram-negative organisms do not
therefore, according to this definition, produce ‘true’ extra-cellular enzymes. However, it
has been found in recent times that some Gram-negative organisms do in fact secrete
extracellular enzymes. Furthermore many Gram-negative organisms do in fact produce
and secrete enzymes across the cytoplasmic membrane. Such enzymes are however held
within the periplasmic zone (Fig. 22.7) of the Gram-negative cell wall and hence do not
find their way to the medium. Thirdly mutants of Gram-negative cells defective in the
ability to synthesize cell wall components continue to synthesize and secrete
polypeptides into the environment. On account of these observations extra-cellular
enzymes have been redefined as those which are secreted across the cell membrane. In
terms of industrial microbiology it is an apt definition as methods for deranging the
molecular arrangements of cell walls exist, which when successfully applied to Gramnegative bacteria secreting into the periplasmic space convert them to extra-cellular
secreters. Such methods include the formation of protoplasts by the prevention of cell
wall formation using suitable antibiotics, limited digestion by trypsin, solubilization of
the cell wall with a combination of a detergent (e.g. laurodeoxycholate and a chelating
agent).
(ii) Some biochemical properties of extra-cellular enzymes: Bacterial extracellular
enzymes vary in molecular weight from 12,000 to 500,000 but in the main they range from
20,000 to 40,000. Secondly, most but not all bacterial exoproteins lack cysteine. It has been
suggested, but not entirely accepted, that this absence of cysteine will confer the property
of malleability on extra-cellular enzymes thus facilitating their export.
(iii) Site of synthesis of extra-cellular proteins: Even in cells actively secreting extracellular proteins, an examination of the cytoplasm shows a complete absence or only a
trace amount of the enzymes being excreted. Early report for instance claiming that a–
amylase of >–amyloliquifaciens is first produced as a high molecular weight precursor
have been shown to be wrong on the basis of radio-isotope (labeling) experiments. Since
no evidence exists for the cytoplasmic synthesis of extracellular proteins it has been
suggested they are synthesized on ribosomes associated with the cells in much the same
way as in eucaryotic cells. In eucaryotic cells, membrance-bound polysomes are engaged
in secreting proteins for export whereas polysomes secrete non-exportable proteins.
According to the currently accepted model synthesis takes places on the cell
membrane and is secreted directly into pores in the cell membrane. Indeed synthesis and
secretion are one process, following the system in eucaryotic cells. Some evidence for this
are as follows. In many bacteria a considerable but variable fraction of the ribosomes is
"&
Modern Industrial Microbiology and Biotechnology
Left = Gram-negative wall; Right = Gram-positive wall; Dotted areas = hydrophobic
zones; cc=capsular carbohydrate; cp = capsular polysaccharide; ec = cytoplasmic
membrane enzymes in the cytoplasmic which synthesize cell wall macro-molecules; lp =
lipoprotein; p = structural and encymic proteins of the outer layers of the Gram-negative
wall; s = structural proteins of cytoplasmic membrane; sp = enzymes in the periplasmic
zone; ps = permeases.
Fig. 22.7
Generalized Structure of the Bacterial Cell Wall
associated with the cytoplasmic membrane. In exponential phase of Bacillus licheniformis
for example, 96% of the ribosomes are membrane bound. It is also know that exoenzyme
synthesis is more sensitive to antiobiotic inhibition than general protein synthesis. This
has been interpreted as being so because of the membrane bound ribosomes are more
accessible to the antibiotic.
(iv) Control of extra-cellular enzyme secretion by gene cloning: When the terminal portion
of the b-galactosidase gene of E. coli was replaced with a gene that codes for a protein of the
outer cell wall membrane of the bacteria, the b-galactosidase activity which is normally
intracellular was formed extracellularly depending on the size of the latter that attached
on the b-galactosidase gene. This and other similar experiments show that in due course it
may be possible to produce virtually any enzyme extracellularly by gene cloning.
(v) Some methods for increasing enzyme yields: The increased yields which have been
observed in enzyme production is based on strain selection, improved environmental
factors, regulatory controls and genetic manipulation.
(a) Strain selection: Strain selection from natural variants of the same species or even
entirely new species have resulted in the array of enzymes available in some
industries e.g. the starch hydrolyzing industries.
The natural strains may then be mutagenized for increased variation in the gene
pool. Strains have been selected in the above two manners for a wide variety of
properties including temperature-tolerant enzymes and resistance to feedback
regulation.
Biocatalysts: Immobilized Enzymes and Immobilized Cells
"'
(b) Environmental factors: Exoenzymes may be constitutive, but the majority are
inducible or partially so. Inducers are therefore important in increasing the yields
of many extracellular enzymes. Since many of the substrates are insoluble they
cannot enter the cell, and hence their analogues or gratuitious inducers (those that
induce the enzyme but are not substrates or breakdown products) have been used.
Inducers are usually cheap in order to bring down costs. Thus, corn cobs are
hydrolyzed to produce xylose which act as inducers for glucose isomerase.
Most extracellular enzymes are produced in the idiophase and maximum
production is usually in the late log and early stationary phase. This period
coincides with the period when the organism is released from catabolite
repression. Increased yields may therefore be achieved by feeding low levels of the
substrate or feeding them intermittently. Yields may also be increased by
increasing cell-wall permeability. Surfactants may be incorporated into the
medium for this purpose although how they affect the wall permeability is not fully
understood.
(c) Regulatory control: Control of regulation is through induction and catabolite and
feedback regulations. Mutants resistant to all three have been produced with
consequent boost in production. An example of inducer-resistance is in the case of
the glucose isomerase producing antinomycete Streptomyces phaechromogenes, the
wild strain of which will not germinate on L-lyxose, another form of D-xylose. It
will grow on lyxose only if germination is first obtained on xylose. Mutants were
selected which would germinate directly on lyxose, thus eliminating the need for
xylose.
The bypassing of catabolite repression has led to the production of large
amounts of enzymes. This has been achieved by using toxic analogues of the
substrates. Thus, 2-deoxy-glucose is used as a toxic analogue when seeking for
mutants able to over produce glucosidase.
Feedback regulation is not very applicable to enzyme synthesis, although some
examples are known.
(d) Genetic manipulation: As had been indicated earlier the gene specifying the extracellular secretion may be cloned on those controlling the synthesis of particular
enzymes, thus causing the enzyme to be secreted extracellularly.
The number of copies of specific genes may be increased by gene amplification
methods thus increasing enzyme yield several times. For example, plasmids specifying
particular extra-cellular enzymes may continue to replicate while the parent
chromosome is inhibited by, for example, chloramphenicol thereby permitting an
amplification of the genes – sometimes up to 2,000 copies or up to 40% of the cells total
DNA. The result is increased yield of the enzyme.
SUGGESTED READINGS
Anon. 1995. Classification and Chemical Characteristics of Immobilized Enzymes. (Technical
Report), International Union of Pure and Applied Chemistry. Pure and Applied Chemistry,
67, 597-600.
Butterfoss, G.L., Kuhlman, B. 2006. Computer-Based Design of Novel Protein Structures. Annual
Review of Biophysics and Biomolecular Structure, 35, 49–65.
"
Modern Industrial Microbiology and Biotechnology
Chaplin, M.F., Bucke, C. 1990. Enzyme Technology. Cambridge University Press. New York,
USA.
Cheetam, S.J. 2004. Bioprocesses for the Manufacture of Ingredients for Food and Cosmetics.
Advances in Biochemical Engineering/Biotechnology, 86, 83–158.
Desai, M.A. 2000. Downstream Processing of Proteins. Humana Press Totowa, New Jersey, USA.
Fogarty, M., Kelly, C.T. (eds) 1990. Microbial Enzymes and Biotechnology. 2nd ed. Elsevier
Applied Science London and New York.
Imanaka, T. 1994. Improvement of Useful Enzymes by Protein Engineering. In: Rombinant
Microbes for Industrial and Agricultural Applications. Y. Murooka, T. Imanaka, (eds). Marcel
and Dekker. New York, USA. pp. 449-465.
Kennedy, J.F. 1995. Principles of Immobilization of Enzymes. In: Handbook of Enzyme
Biotechnology. A Wiseman, (ed) 3rd ed. Ellis Horwood, London, UK. pp. 235–310.
Mitchel, D.A., Berovic, M., Krieger, N. 2000. Biochemical Engineering Aspects of Solid State
Bioprocessing. Advances in Biochemical Engineering/Biotechnology. 68, 61–132.
Pasechnik, V.A. 1995. Practical Aspects of Large-scale Protein Purification. In: Handbook of
Enzyme Boiotecnology. A Wiseman, (ed) 3rd ed. Ellis Horwood, London, UK. pp. 379–418.
Puri, M., Kaur, H., Kennedy, J.F. 2005. Covalent immobilization of naringinase for the
transformation of a flavonoid. Journal of Chemical Technology & Biotechnology, 80, 1160 1165.
Puri, M., Banerjee, U.T. 2002. Production, purification, and characterization of the debittering
enzyme naringinase. Biotechnology Advances, 18, 207-217.
Roy, I., Sharma, S., Gupta, M.N. 2004. Smar Biocatalysts: Design and Application. Advances in
Biochemical Engineering/Biotechnology, 86, 159–190.
Schugerl, K. 2000. Recovery of Proteins and Microorganisms from Cultivativation Media by
Foam Flotation. Advances in Biochemical Engineering/Biotechnology. 68, 191-233.
Tanaka, A, Tosa, T, Kobayashi, T, (1993). Industrial Applications of Immobilized Biocatalysts
New York: Dekker.
Mining Microbiology: Ore Leaching (Bioleaching) by Microorganisms
+0)26-4
"
23
Mining Microbiology: Ore
Leaching (Bioleaching) by
Microorganisms
23.1
BIOLEACHING
The term bioleaching refers to the conversion of an insoluble metal (generally a metal
sulfide, e.g., CuS, NiS, ZnS) into a soluble form (usually the metal sulfate, e.g., CuSO4,
NiSO4, ZnSO4). When this happens, the metal is extracted into water; this process is
called bioleaching. As these processes are oxidations, this process may also be termed
bio-oxidation. However, the term bio-oxidation is usually used to refer to processes in
which the recovery of a metal is enhanced by microbial decomposition of the mineral, but
the metal being recovered is not solubilized. An example is the recovery of gold from
arsenopyrite ores where the gold remains in the mineral after bio-oxidation and is
extracted by cyanide in a subsequent step. The term bioleaching is clearly inappropriate
when referring to gold recovery (although arsenic, iron, and sulfur are bioleached from
the mineral). Biomining is a general term that may be used to refer to both processes.
Bacterial leaching of metals is a process in which the ores of the metals, usually their
sulphides are solubilized by bacterial action. Basically the process is a chemical
oxidation following the equation
Microorganisms
MS + 2O2
MSO4
where M is a bivalent metal. The solution collected after the solubilization is processed to
recover the metal. In the case of insoluble sulfides such as is the case with lead sulfide, the
fact of insolubility may be used to separate it from the dissolved metals.
Bacterial leaching has been practiced by man over many centuries without any
understanding of the microbiological basis of the process. It was used for mining copper
by the Romans in Wales, in Rio Tinto, Spain in the 18th century and in the USA in this
century. Indeed about 12% of copper produced in the USA is obtained by bacterial
leaching of low grade ores.
"
Modern Industrial Microbiology and Biotechnology
23.2
COMMERCIAL LEACHING METHODS
There are two types of processes for commercial microbially-assisited metal recovery: the
irrigation-type and the stirred tank type.
23.2.1
Irrigation-Type Processes
The irrigation-type processes can be grouped into three: the dump, the heap and the insitu methods. The most widely used methods are dump and heap leaching. The metal
most commonly bioleached metal in the irrigation methods is copper.
23.2.1.1
Dump leaching
In this method large quantities of low-grade ore are placed in valleys with impermeable
grounds. The dumps are shaped by bull-dozers into cones which may be as high as
600 ft with 600 ft diameter at the base. Acid solutions usually H2SO4, known as leach
solutions, are then sprayed into, or flooded over, the dumps or injected into it through
steel pipes. The acid provides the low pH required by the microorganisms whose
activities are responsible for dissolving the ores. Liquid collected at the bottom of the
dump contains dissolved metal which is recovered after processing.
In some processes the dumps are subjected to preconditioning, irrigation, rest, and
conditioning, each of which may extend for a year. The irrigation is done with an ironand sulfate-rich recycyled waste-water from which the copper has been removed.
Microorganisms growing in the dump bring about the reactions which cause the
insoluble copper sulfides to become soluble copper sulfate. The copper sulfate is collected
from the bottom of the dump and the copper is recovered by solvent extraction and
electrolysis. One of the best-known dump leaching sites is the Kennecot Copper mine in
Bingham Canyon, Utah, USA; another is Bala Ley plant in Coledo, Chile.
23.2.1.2
Heap leaching
Heap leaching is very similar to dump leaching, except that steps are taken to make the
process more efficient through ores of smaller particles and a smaller scale of operation.
The ores are crushed to make them finer, and then staked in much smaller heaps of about
6 to 50 ft high. Aeration pipes may be included to permit forced aeration so as to speed up
the process. To further speed up the reaction inorganic ammonium salts and phosphates
may be added. The heaps are placed in mounds on drainage pads or in concrete tanks
with false bottoms through which liquid collects in a dump. Tanks have capacities of
about 12,000 tons. On account of the steps taken to accelerate the process the operation is
complete in months rather than in years. An example of a large modern heap plant is to be
found in Quabrada Blanca in northern Chile.
Heap bioleaching may also be used to mine gold in low grade gold ores. In this case the
ore is initially flooded with acid-ferric iron. Thereafter, it is treated with recycled heap
effluent. After the ore is sufficiently broken down, the ore is mixed with lime and the gold
extracted with cyanide.
23.2.1.3
In-situ Mining
In in-situ irrigation bioleaching the ore is not brought to the surface and the ore is
extracted in situ. In some instances such as in the copper mine at San Manuel, Arizona,
Mining Microbiology: Ore Leaching (Bioleaching) by Microorganisms
" !
injection wells are used to introduce acidified leaching fluids into the mineral deposit. It
percolates and collects in disused mines or a specially prepared catchment location. The
geology of the location must have a suitable impermeable layer. In spite of this leaching,
fluid is lost in this process.
The mining of uranium is generally done in situ in the mine. The uranium ore (uranite)
is not attacked by acid producing bacteria. However it is solubilized when it is oxidized
from the tetravalent form which is insoluble, to the soluble hexavalent form by chemical
oxidants such as ferric iron, MnO2, H2O2 chlorates or nitric acid. In uranium leaching
mediated by bacteria, the role of the bacteria is to produce ferric iron which then oxidizes
the uranite to make it soluble.
23.2.2
Stirred Tank Processes
Stirred tank bioreactors are highly aerated tanks much like the fermentors already
discussed except that the sterility maintained in fermentors used, for example for
antibiotics, is not necessary in these bioreactors. They are expensive to construct and
maintain and hence they are used only for high value minerals such as gold.
They are usually arranged in series and function as continuous fermentors. The tanks
in the first stage are usually arranged in parallel so that the fermentation broth is retained
long enough for the cell numbers to reach a steady state without being washed out. The
mineral ore is suspended in water to which fertilizer grade (NH4)2SO4 and KH2PO4 are
added. The pH is adjusted 1.5–2.0. The bioreactor is vigorously aerated using air spargers
and baffles. The released heat is removed by a jacket of cold water. Systems using aerated
bioreactors use pretreatments for the recovery of gold, especially where the gold is finely
divided in a mixture of pyrite and arsenopyrite (i.e. iron ores mixed with arsenic). Such
ores are known as recalcitrant ores. Normally gold is recovered from rich ores by
treatment with cyanide. However, because gold forms only a small proportion of
recalcitrant aesenopyrite ores, the ores are pretreated in the aerated bioreactors. During
the pretreatment which lasts for about four days microbes oxidize the arsenopyrite which
is decomposed into iron and sulfate releasing the gold, and is then treated with cyanide.
Stirred bioreactors are used to pretreat gold ores in many countries around the world
including Australia, Brazil, Ghana, and Peru. The Ghana plant in Sensu is perhaps the
largest fermentation in the world with 24 tanks each with a capacity of 1 million liters.
Aerated bioreactors are used for other minerals in France and Uganda (cobalt) and
South Africa (nickel).
23.3
MICROBIOLOGY OF THE LEACHING PROCESS
The primary biomining microorganisms involved in ore leaching have several properties
in common:
(i) They are Gram-negative specialized chemolitho-authotrophic able to use ferrous
iron and reduced inorganic sulphur compounds or both as electron donors;
(ii) They are able to fix carbon with energy derived from the oxidation of inorganic
compounds such as ferrous iron, sulfur and sulfides according to the following
equations:
" "
Modern Industrial Microbiology and Biotechnology
4FeSO4 + 02 + 2H2SO4
(S8 + 1202 + 8H2)
H2S + 202
®
®
®
2Fe2 (SO4) + 2H2O
8H2SO4
H2SO4
(1)
(2)
(3)
(iii) They are acidophilic and will thrive under very acid conditions at pH ranges of 1.5
– 2.0.
(iv) Although they can use electron acceptors other than oxygen they grow best in
highly aerated solutions.
(v) Some are able to fix atmospheric nitrogen, when the oxygen supply is limited
(vi) Some are obligately autotrophic, while others are facultative autotrophs and able
to grow in the presence of organic matter.
(vii) The organisms are usually found naturally in waters in contact with exposed
sulphides or in mines.
The organisms involved are the following:
(a) Acidothiobacillus: Organisms belonging to this genus were previously classified as
Thiobacillus. Following 16S rRNA analysis they have been reclassified as
Acidothiobacillus to accommodate the very acidophilic members of the genus. These
include Acidothiobacillus ferrooxidans (formerly Thiobacillus ferooxidans) which is
the most intensively studied and until recently was thought to be the sole
microorganism involved. It is an obligate autotroph and obtains its energy for the
fixation of CO2 from the oxidation of ferrous irons, sulfides, and other sulfur
compounds such as thiosulfate.
(b) Leptospirillum ferrooxidans: This grows only in soluble ferrous iron and not on
sulfur or mineral sulfides.
(c) Acidothiobacillus thiooxidans (formerly Thiobacillus thiooxidan): It cannot oxidize
iron, but grows on elemental sulfur and soluble compounds including those
generated in the leaching systems of T. ferrooxidans.
(d) Other bacteria: Thiobacillus organosporus is facultatively chemolithotrophic (i.e.
while it is autotrophic it will also grow when organic compounds are available to
it). It oxidizes sulfur, but not iron or sulfides. Mixed with other organisms e.g.
Leptospirriullum ferrooxidans it will degrade iron sulfides which neither can do
alone.
Unlike other industrial microbiology processes there is no conscious attempt to use
pure cultures. The highly acidophilic organisms create conditions which are unsuitable
for other organisms.
23.4
LEACHING OF SOME METAL SULFIDES
(i) Copper sulfides: One of the world’s main source of copper is the iron-copper sulfide,
chalcopyrite (CuFe2S2). Others are chalcocite (Cu2S) and covellite (CuS). The
reactions in which these minerals are leached by bacterial action are complex, but
the end equations may be given as follows:
Mining Microbiology: Ore Leaching (Bioleaching) by Microorganisms
" #
Chalcopyrite
Bacteria
60CuFeS2 + 25SO2 + 90H2O
+ 20H Fe (SO4) 2. 2Fe (OH) 3
+ 20H2 SO4
60CuSO4
(4)
Chalcocite
Bacteria
10 Cu2S + 10H2 SO4
20CuSO4 + 10H2O
(5)
(ii) Uranium extraction: Bacteria especially T. ferrooxidans, oxidize ferrous sulphate to
ferric sulphate (Equation I). The latter then reacts with uranium ores thus:
Uranite
UO2 + Fe2 (SO4)3
UO2 SO4 + FeSO4
(6)
The tetravalent form in the ore is insoluble in the leach solution while the
oxidized hexavalent form is. There is thus a two-stage action which seems
especially appropriate as the uranyl ion is toxic to most strains of T. ferrooxidans.
(iii) Cobalt and nickel sulfides leaching: The major nickel bearing sulfide ore is usually
pentlandite which contains about 1% of nickel. The reaction for leaching with T.
ferrooxidans is given as follows:
Bacteria
(Ni, Fe) 9 S8 + 175/8 O2 + 3¼ H2SO4
4½ NiSO4
+ ¼ Fe2 (SO4) 3 + 3¼ H20
(7)
(iv) Zinc and lead sulfide leaching: Zinc and lead sulfides respectively may be oxidized
by T. ferrooxidans according to the equations:
Bacteria
ZnS + 202
Zn SO4
(8)
Pb SO4
(9)
Bacteria
PbS + 202
23.5 ENVIRONMENTAL CONDITIONS AFFECTING
BACTERIAL LEACHING
The following factors affect the efficiency of leaching via bacterial action. Much of this
has been studied using T. ferrooxidans.
(i) Temperature: The optimum temperature for the bacterial solubilization of metals
lies between 25°C and 45°C for different strains of T. ferrooxidans. Above 55°C the
activity is mainly chemical. No minimum temperature has been established, but it
is believed that action stops at freezing.
(ii) pH: T. Ferrooxidans is acidophilic and has been studied at pH values ranging from
1 to 5. The optimum pH values for acting on many minerals lie between pH 2.3 and
2.5. Other organisms outside T. ferrooxidans appear to have about the same pH
requirements.
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Modern Industrial Microbiology and Biotechnology
(iii) Nutrient status of the leaching medium: Like any other bacterium, the iron-oxidizing
thiobacilli must have the appropriate nutrients in the leaching medium. The
energy source is, as has been stated, ferrous sulfate, but minerals containing iron or
sulfur may be utilized. The carbon is obtained from CO2 of the air. It also needs
nitrogen, potassium, magnesium, phosphate, and calcium. Most of these are
available from the surrounding rocks; where the deficiency of one or more of them
is established, it must be replaced in the leaching solution for optimum
productivity. The thiobacilli are strict aerobes and oxygen deficiency leads to
limitations in leaching productivity.
(iv) Particle size: In general the finer the particle size of the ore the greater the extraction
of the leaching solution. Below a certain particle size however, especially in the
case of low grade ores, the ratio of the mineral to unwanted part of the ore increases
and productivity falls.
SUGGESTED READINGS
Kelly, D.P., Norris, P.R., Brierley, C.L. 1979: In: Microbial Technology: Current State, Future
Prospects. A.T., Bull, D.C. Ellwood, C. Ratledge, (eds). Cambridge University Press,
Cambridge. UK. pp. 263-308.
Lundgren, D.G. 1980. Ore Leaching by bacteria Ann. Rev. Microbiol. 24, 263-283.
Rawlings, D.E. 2002. Heavy Metal Mining Using Microbes Annual Review of Microbiology, 56,
65-91.
Zajic, J.E. 1969. Microbial Biogeo-chemistry Academic Press, New York, USA.
Section
/
Production of Commodities
of Medical Importance
Modern Industrial
Microbiology and Biotechnology
+0)26-4
24
Production of Antibiotics
and Anti-Tumor Agents
As currently defined, antibiotics are chemicals produced by microorganisms and which
in low concentrations are capable of inhibiting the growth of, or killing, other
microorganisms. Anti-microbial substances are also produced by higher plants and
animals. Such substances are however excluded by this definition. Bacteriocins although
produced by microorganisms are also not included in this definition because they are not
only larger in molecular size than the usual antibiotics, but they are mainly protein in
nature; furthermore they affect mainly organisms related to the producing organism. In
comparison with bacteriocins, conventional antibiotics however are for more diverse in
their chemical nature and attack organisms distantly related to themselves. Most
importantly, while the information specifying the formation of ‘regular’ antibiotics is
carried on several genes, that needed for bacteriocins being single proteins need single
genes. It will be seen later that in the last few years this definition has been somewhat
broadened by some authors to include materials produced by living things – plants,
animals or microorganisms – which inhibit any cell activity.
Antibiotics may be wholly produced by fermentation. Nowadays, however, they are
increasingly produced by semi-synthetic processes, in which a product obtained by
fermentation is modified by the chemical introduction of side chains. Some wholly
chemically synthesized compounds are also used for the chemotherapy of infectious
diseases e.g. sulfonamides and quinolones. But these will not be considered since they
are not produced wholly or partially by fermentation. Some antibiotics e.g.
chloramphenicol were originally produced by fermentation, but are now more cheaply
produced by chemical means.
Thousands of antibiotics are known; and every year dozens are discovered. However,
only a small proportion of known antibiotics is used clinically, because the rest are too
toxic.
24.1
Classification and Nomenclature of Antibiotics
Several methods of antibiotic classification have been adopted by various authors. The
mode of action has been used, e.g. whether they act on the cell wall, or are protein
inhibitors, etc. Several mechanisms of action may operate simultaneously making such a
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Modern Industrial Microbiology and Biotechnology
method of classification difficult to sustain. In some cases they have been classified on the
basis of the producing organisms, but the same organism may produce several
antibiotics, e.g. the production of penicillin N and cephalosporin by a Streptomyces sp.
The same antibiotics may also be produced by different organisms. Antibiotics have been
classified by routes of biosynthesis; however, several different biosynthetic routes often
have large areas of similarity. The spectra of organisms attacked have also been used, e.g.
those affecting bacteria, fungi, protozoa, etc. Some antibiotics belonging to a well known
group e.g. aminoglycosides may have a different spectrum from the others. The
classification to be adopted here therefore is based on the chemical structure of the
antibiotics and classifies antibiotics into 13 groups. This enables the accommodation of
new groups as they are discovered (Table 24.1).
Table 24.1 Grouping of antibiotics based on their chemical structures
Chemical Group
Example
Aminoglycosides
Ansamacrolides
Beta-lactams
Chloramphenicol and analogues
Linocosaminides
Macrolides
Nucleosides
Puromycin
Peptides
Phenazines
Polyenes
Polyethers
Tetracyclines
Streptomycin
Rifamycin
Penicillin
Chloramphenicol
Linocomycin
Erythromycin
Puromycin
Curamycin
Neomycin
Myxin
Amphothericin B
Nigericin
Tetracycline
One well-known example of each group has been given to facilitate recognition of the
groups.
The nomenclature of antibiotics is also highly confusing as the same antibiotic may
have as many as 13 different trade names depending on the manufacturers. Antibiotics
are therefore identified by at least three names: the chemical name, which prove long and
is rarely used except in scientific or medical literature; the second is the group, generic, or
common name, usually a shorter from of the chemical name or the one given by the
discoverer; the third is the trade or brand name given by the manufacturer to distinguish
it from the product of other companies.
The production of antibiotics is a very wide subject and because of space limitations
only beta-lactam antibiotics will be discussed. Even among them, only penicillin and
cephalosprin will be discussed in any detail.
24.2
BETA-LACTAM ANTIBIOTICS
The Beta-lactam antibiotics are so-called because they have in their structure the fourmembered lactam ring. Figure 24.1 shows the structures of the various Beta-lactam
antibiotics.
Production of Antibiotics and Anti-Tumor Agents
Fig. 24.1
"!
The Beta-lactam Antibiotics
The Beta-lactam structure is not very common in nature and besides the antibiotic
groups to be discussed it is only found in some alkaloids and some anti-metabolite toxins
including pachystermines from the higher plant, Pachystradra terminalis, wild-fire toxin
from Pseudomonas tabici and the anti-tumor antibiotics, phleomycins and bleomycins
from Streptomyces verticillus.
The Beta-lactam antibiotics include the well-established and clinically important
penicillins and cephalosphorins as well as some relatively newer members:
cephamycins, nocardicins, thienamycins, and clavulanic acid. Except in the case of
nocardicins these antibiotics are derivatives of bicyclic ring systems in which the lactam
ring is fused through a nitrogen atom and a carbon atom to ring compound. This ring
compound is five-membered in penicillins (thiazolidine), thienamycins (pyrroline) and
clavulanic acid (oxazolidine); it is six-membered (dihydrothiazolidine) in
cephalosporins and cephamycins (Fig. 24.1).
The Beta-lactam antibiotics inhibit the formation of the structure-conferring
petidoglycan of the bacterial cell wall. As this component is absent in mammalian cells,
Beta-lactam antibiotics have very low toxicity towards mammals.
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Modern Industrial Microbiology and Biotechnology
24.2.1
24.2.1.1
Penicillins
Strain of organism used in penicillin fermentation
In the early days of penicillin production, when the surface culture method was used, a
variant of the original culture of Penicillium notatum discovered by Sir Alexander Fleming
was employed. When however the production shifted to submerged cultivation, a strain
of Penicillium chrysogenum designated NRRL 1951 (after Northern Regional Research
Laboratory of the United States Department of Agriculture) discovered in 1943, was
introduced. In submerged culture it gave a penicillin yield of up to 250 Oxford Units (1
Oxford Unit = 0.5988 of sodium benzyl penicillin) which was two to three times more
than given by Penicillium notatum. A ‘super strain’ was produced from a variant of NRRL
1951 and designated X 1612. By ultraviolet irradiation of X-1612, a strain resulted and
was named WISQ 176 after the University of Wisconsin where much of the stain
development work was done. On further ultra violet irradiation of WISQ 176, BL3-D10
was produced, which produced only 75% as much penicillin as WISQ 176, but whose
product lacked the yellow pigment the removal of which had been difficult. Present-day
penicillin producing P. chrysogenum strains are far more highly productive than their
parents. They were produced through natural selection, and mutation using ultra violet
irradiation, x-irradiation or nitrogen mustard treatment. It was soon recognized that
there were several naturally occurring penicillins, viz. Penicillins G, X, F, and K (Fig.
24.2).
Penicillin G (benzyl penicillin) was selected because it was markedly more effective
against pyogenic cocci. Furthermore, higher yields were achieved by supplementing the
medium with phenylacetic acid, analogues (phenylalanine and phenethylaninie) of
which are present in corn steep liquor used to grow penicillin in the United States.
Present day penicillin-producing strains are highly unstable, as with most industrial
organisms, and tend to revert to low-yielding strains especially on repeated agar
cultivation. They are therefore commonly stored in liquid nitrogen at – 196° or the spores
may be lyophilized.
Penicillin has since been shown to be produced by a wide range of organisms including the fungi Aspergillus, Malbranchea, Cephalosporium, Emericellopsis, Paecilomyces,
Trichophyton, Anixiopsis, Epidermophyton, Scopulariopsis, Spiroidium
and the
actionomycete, Streptomyces. The only type of penicillin produced by actinomycetes however is Penicillin N (with the chemical structure D-a (@- aminoadipyl) penicillin usually
accompanied by cephamycins and/or deacetyl – 3 – 0-carbamoylcephalosporin C.
24.2.1.2
Fermentation for penicillin production
The inoculum is usually built up from lyophilized spores or a frozen culture and
developed through vessels of increasing size to a final 5-10% of the fermentation tank. As
the antibiotic concentration in the fermentation beer is usually dilute the tanks are
generally large for penicillin and most other antibiotic production. The fermentors vary
from 38,000 to 380,000 liters in capacity and in modern establishments are worked by
computerized automation, which monitor various parameters including oxygen content,
Beta-lactam content, pH, etc.
The medium for penicillin production now usually has as carbohydrate source
glucose, beet molasses or lactose. The nitrogen is supplied by corn steep liquor. Cotton
Production of Antibiotics and Anti-Tumor Agents
"!!
Fig. 24.2 Natural and Biosynthetic Penicillins
seed, peanut, linseed or soybean meals have been used as alternate nitrogen sources. The
nitrogen source is sometimes exhausted towards the end of the fermentation and it must
then therefore be replenished. Calcium carbonate or phosphates may be added as a
buffer. Sulfur compounds are sometimes added for additional yields since penicillin
contains sulfur. The practice nowadays is to add the carbohydrate source intermittently,
i.e. using fed-batch fermentation. Lactose is more slowly utilized and need not be added
intermittently. Glucose suppresses secondary metabolism and excess of it therefore limits
penicillin production. The pH is maintained at between 6.8 and 7.4 by the automatic
addition of H2SO4 or NaOH as necessary.
Precursors of the appropriate side-chain are added to the fermentation. Thus if benzyl
penicillin is desired, phenylacetic acid is added. Phenyl acetic acid is nowadays added
continuously as too high an amount inhibits the development of the fungus. High
yielding strains of P. chrysogenum resistant to the precursors have therefore been
developed.
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Modern Industrial Microbiology and Biotechnology
Penicillin production is stimulated by the addition of surfactants in a yet unexplained
mechanism. The temperature is maintained at about 25°C, but in recent times it has been
found that yields were higher if adjusted according to the growth phase. Thus, 30-32°C
was found suitable for the trophophase and 24°C for the idiophase. Aeration and
agitation are vigorous in order to keep the components of the medium in suspension and
to maintain yield in the highly aerobic fungus.
Penicillin fermentation can be divided into three phases. The first phase (trophophase)
during which rapid growth occurs, lasts for about 30 hours during which mycelia are
produced. The second phase (idiophase) lasts for five to seven days; growth is reduced
and penicillin is produced. In the third phase, carbon and nitrogen sources are depleted,
antibiotic production ceases, the mycelia lyse releasing ammonia and the pH rises.
24.2.1.3
Extraction of penicillin after fermentation
At the end of the fermentation the broth is transferred to a settling tank. Penicillin is
highly reactive and is easily destroyed by alkali conditions (pH 7.5-8.0) or by enzymes. It
is therefore cooled rapidly to 5-10°C. A reduction of the pH to 6 with mineral acids
sometimes accompanied by cooling helps also to preserve the antibiotic. The
fermentation broth contains a large number of other materials and the method used for
the separation of penicillin from them is based on the solubility, adsorption and ionic
properties of penicillin. Since penicillins are monobasic carboxylic acids they are easily
separated by solvent extraction as described below.
The fermentation beer or broth is filtered with a rotary vacuum filter to remove mycelia
and other solids and the resulting broth is adjusted to about pH 2 using a mineral acid. It
is then extracted with a smaller volume of an organic solvent such as amyl acetate or
butyl acetate, keeping it at this very low pH for as short a time as possible. The aqueous
phase is separated from the organic solvent usually by centrifugation using Podbielniak
centrifugal countercurrent separator (Chapter 9).
The organic solvent containing the penicillin is then typically passed through
charcoal to remove impurities, after which it is back extracted with a 2% phosphate buffer
at pH 7.5. The buffer solution containing the penicillin is then acidified once again with
mineral acid (phosphoric acid) and the penicillin is again extracted into an organic
solvent (e.g. amyl acetate). The product is transferred into smaller and smaller volumes of
the organic solvent with each successive extraction process and in this way, the
penicillin becomes concentrated several times over, up to 80-100 times. When it is
sufficiently concentrated the penicillin may be converted to a stable salt form in one of
several ways which employ the fact that penicillin is an acid: (a) it can be reacted with a
calcium carbonate slurry to give the calcium salt which may be filtered, lyophilized or
spray dried. (b) it may be reacted with sodium or potassium buffers to give the salts of
these metals which can also be freeze or spray dried; (c) it may be precipitated with an
organic base such as triethylamine.
When benzyl penicillin is administered intramuscularly it is given either as the
sodium (or potassium) salt or as procaine penicillin. The former gives high blood levels
but it quickly excreted. Procaine penicillin gives lower blood levels, but it lasts longer in
the body because it is only slowly removed from the blood. It is produced by dissolving
sodium or penicillin in procaine hydrochloride.
Production of Antibiotics and Anti-Tumor Agents
24.2.1.4
"!#
Production of semi-synthetic penicillins
In the late 1940s it was shown by labeling experiments that penylacetamide derivatives
were directly incorporated into the benzyl penicillin molecule. The possibility was
recognized of inducing the mold to produce new antibiotics antibiotic by the
introduction of various precursors. Phenoxymethyl penicillin (penicillin V) which had
greater acid stability than penicillin G, allythiomethyl penicillin (Penicillin O) which
was less likely to induce allergic reactions and butylthiomethyl penicillin (Penicillin S)
were thus produced. The natural penicillins (formed in unsupplemented media) and the
biosynthetic (produced by the addition of specific side-chain precursors) are indicated in
Fig 24.2 The high expectations of making new penicillins by the introduction of sidechains during fermentation, did not however, result in many new pencillins.
In 1959 6-amino penicillanic acid (6-APA) was isolated from precursor-starved
P. chrysogenum fermentations and this ushered in the era of semi-synthetic penicillins
and indeed other semi-synthetic antibiotics. Today the only ‘natural’ penicillins used are
benzyl penicillin (Penicillin G) and phenoxymethyl penicillin (Penicillin V). All others
are semi-synthetic.
In preparing semi-synthetic penicillins, 6-APA is not produced by starving P.
chrysogenum of precursors, because yields are low. It is prepared by cleaving from
penicillin G or penicillin V, the 6-acyl group by chemical means or with enzymes
(acylases) produced by a wide range of microorganisms including bacteria, yeasts, and
molds and even mammals (hog kidney acylase). The various acylases have different
substrates. Actinomycete and mold acylases usually attack penicillins with aliphatic
side-chains, e.g. penicillin V or phonoxymetyl penicillin (Penicillin); penicillin G is
attacked more slowly. On the other hand, bacterial acylases attack penicillin G rapidly.
Immobilized enzymes and cells are being used in these processes.
The introduction of the acyl side chain is done by reacting 6-APA with a suitable
derivative of a carboxylic acid, usually a chloride, in organic solvents under anhydrous
or aqueous conditions. In the latter system it is done in acetone-water mixtures in the
presence of sodium bicarbonate. The resulting penicillins can be extracted by solvent
extraction as already described, followed by charcoal treatment.
Semi-synthetic penicillins were developed to meet some of the short-comings of benzyl
penicillin. Some of the desired properties were greater intrinsic activity against Grampositive bacteria, increased antibacterial spectrum including Gram-negative organisms,
gastric acid stability and oral absorbability and resistance to beta-lactamases, (i.e.
enzymes which open the Beta-lactam ring). All penicillins to some extent bind to the
serum albumin and therefore reduce the quantity of the antibiotic available to attack
microorganisms. The less binding therefore, for pencillins of otherwise equal activity, the
more bactericidal. The final desirable property is reduced ability to induce
hypersensitivity, a phenomenon which occurs in 9-10% of the population.
24.2.2
Cephalosporins
Cephalosporins in general have a broader spectrum than penicillins and are less likely to
induce allergic reactions among those who react to penicillins. The first cephalosporin
was discovered in 1948 and was produced by Cephalosporium acremonium. It was later
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Modern Industrial Microbiology and Biotechnology
shown that the organism in fact produced five antibiotics. Five of these were steroids (and
therefore hydrophobic) and were active only against Gram-positive bacteria. A
hydrophilic component active against both Gram-positive and Gram-negative bacterial
was also found and named cephalosporin N. While purifying cephalosporin N, another
compound, cephalosporin C was discovered. It was latter found that cephalosporin C
was stable to acid and to pencillianse; most importantly it was active against Gramnegative bacteria although it had about one-tenth the activity of cephalosporin N against
Gram-positives. With further study Cephalosporin N was found to be a penicillin and
renamed Penicillin N. Cephalosporin is now known to be produced by as wide a range of
micro-organisms as produce penicillins and these include fungi and actinomycetes. The
natural cephalosporins and cephamycins are given in Fig 24.3.
24.2.2.1
Production of cephalosporin
While two of the clinically important penicillins (Penicillin G & V) are produced entirely
by fermentation the rest being semi-synthetic, all of the cephalosporins in use on the other
hand are semi-synthetic. They however have as their starting point Cephalosporin C
produced by fermentation, otherwise the production of both antibiotics is similar.
24.2.2.2
Strain of organism used
The original strain of Cephalosporium acremonium (C Ml 49, 137 – Common Wealth
Mycological Institute, Kew Gardens, London) produced by violet mutagenesis, C.
acremonium 8650. This latter organism is the parent of most of the various commercially
used C. acremonium. In passing, Cephamycins, (Fig. 24.3) are produced by Streptomyces
lipmanni and S. clavuligenis.
24.2.2.3
Fermentation
The medium used for cephalosporin fermentation is same as used for penicillin N.
Paraffins have however been used to produce several cephalosporins. Methionine,
arginine, ornithine, spermine, cadaverine, and lysine have been shown to increase
cephalosporin production.
24.2.2.4
Extraction of cephalosporin after fermentation
While penicillins are carboxylic acids, cephalosporin C is amphotheric having both
alkaline and acidic properties. For this reason it cannot be extracted directly into organic
solvents. Cephalosporin C is more commonly isolated by ion exchange and precipitation.
The broth is acidified to a low pH after filter-aid filtration in a rotary vacuum filter. The
broth usually contains penicillin N and deactylacephalosporin. The low pH destroys
penicillin N and converts the deactylcelphalosporin to cephalosporin G.
While 6-APA can be made during fermentation by starving P. chrysogenum of the sidechain precursor, 7-amino cephalosoporanic acid (7-ACA) cannot be produced by
fermentation even if precursors are not added; neither can it be produced by the
enzymatic side-chain cleavage as with 6-APA. The production of 6-amino
cephalosporanic acid is therefore by chemical means. 7-ACA is obtained by the chemical
removal of the Alpha-amino adipic side chain of cephalosporin when preparing
Production of Antibiotics and Anti-Tumor Agents
Fig. 24.3 Natural Cephalosporins and Cephamycins
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Modern Industrial Microbiology and Biotechnology
cephalosporins which have Alpha-3 acetoxymethyl group or a derivative from it.
However, cephalosporins with a 3-methyl substituent (deacetoxy-cephalosporanic
acids) are derived from penicillins. The more complex nature of cephalosporins in
comparison with penicillins offers greater opportunities for the production of semi
synthetics and besides 6-ACA, 6-ADCA (6 amino decephalosporanic acid) is also used
as a basis for the production of semi synthetic cephalosporins. The methods of their
preparation are similar to those described for the semi-synthetic penicillins.
24.2.2.5
Use of cephalosporins
Among cephalosporins, cephalothin occupies the same position as benzyl-penicillin,
which continues to be widely used despite some of its deficiencies and the presence of
newer products. Cephalothin is broad-spectrum although ineffective against some
Gram-negative organisms such as Proteus and Pseudomonas. It is administered preferably
intravenously because it is poorly absorbed and because intra-muscular pain is high in
some individuals. New cephalosporins have been produced which are effective against
Gram-negative organisms e.g. cefazolin and cefenandole. Oral cephalosporins include
cefatrizine and cefachlor.
24.2.3
24.2.3.1
Other Beta-Lactam Antibiotics
Cephamycins, (7-Methoxycephalosporins)
Three cepham antibiotics produced by Streptomyces were discovered in 1971. A cepham
with a methoxy group at position C-7 was produced by Streptomyces lipmanni while Streptomyces clavuligenis produced cephalosporin (A-16886-A) and 7-methoxycephalosporin
(A-16886-B) with carbamoxy-loxymethyl function at C-3 position. Penicillin N was produced simultaneously in both strains. Soon afterwards several species of Streptomyces
able to produce 7-methoxycephalosporins were found (Fig. 24.1).
As with other cephams and penicillin N, the production of cephamycins is stimulated
by methionine. They are generally broad-spectrum though the extent to which they affect
Gram-positive and Gram-negative organisms vary among different compounds in the
group. Cephamycin C is for instance specially active against Proteus and E. coli. They are
resistant to hydrolysis by cephalosporinases produced by cephalosporin-resistant
bacteria. They appear most important for the present as starting points for the synthesis
of new semi-synthetic cephams.
24.2.3.2
Nocardicin
The nocardicins were first isolated in 1976 using a super sensitive mutant strain of E. coli,
strain ES 11, whose minimum inhibitory concentrations on penicillin G (100)
Cephalosporin (400) and Nocardium A were 0.8, 0.4, and 0.4 respectively. They are
produced by Nocardia uniformis subspecies suyamanensis. This antibiotic is novel among
the Beta-lactams (Fig. 24.1) in that it is monocyclic (i.e., no ring is fused to the Beta-lactam
ring). In comparison with the cepham, cephazolin, it is highly effective against the Gramnegative Proteus and Shigella but has limited activity against Pseudomonas. An enhanced
activity occurs however when Pseudomonas is treated in vivo. Against Gram-positive
bacteria and yeasts and molds it is completely ineffective. Norcadicin A and B differ
slightly in their structures and are very non-toxic to mammals.
Production of Antibiotics and Anti-Tumor Agents
"!'
24.2.2.3 Clavulanic acid
This antibiotic was first described in 1976 using another novel method of isolation. In
this method, agar plates containing 10 mcg/ml of benzylpenicillin are seeded with Betalactamase – producing Klebsiella aerogenes. Test samples which did not inhibit the
organisms without the introduced penicillin give a zone of inhibition on the test plate
when a diffusible Beta-lactamase inhibitor was present. Clavulanic acid, cephalosporins
and penicillin N are produced by strains of Streptomyces clavuligerus using the above
method. Clavulanic acid was not however discovered with the classical method. It is a
weak antibiotic but has broad-spectrum activity against bacteria. However, it has an
obvious potential value if it can be used along with penicillinase-susceptible antibiotics
of greater potency than itself. Structurally it resembles cephalosporins.
24.2.3.4
Thienamycins
Thienamycin was first described in 1976. Like the cephalosporins and the cephamicins it
was discovered as a fermentation product with broad spectrum activities and was
produced by Streptomyces cattleya. Thienamycins are reported to be broad spectrum even
at lower concentrations while being as non-toxic as the known natural and semisynthetic Beta-lactams.
24.3
THE SEARCH FOR NEW ANTIBIOTICS
In the 1970s the view was that the fight against communicable diseases was about to be
won. The rates of bacterial disease were falling through vaccination and the effectiveness
of the available antibiotics. Many pharmaceutical companies decided to focus attention
away from anti-microbial drugs production as there seemed to be little need for new
compounds. The situation has changed and there now appears an urgent need for new
antimicrobial drugs. This section will discuss the need for new antibiotics, the classical
methods for searching new antibiotics, and some of the newer methods for prospecting
them.
24.3.1
24.3.1.1
The Need for New Antibiotics
The problem of multiple resistance to existing antibiotics
Microorganisms have developed multiple resistance to many of the antibiotics currently
in common use. This is due to several factors some inherent in the nature of
microorganisms, others relating to use (or misuse) of antibiotics by humans. Some of the
factors pertaining to the human use of antibiotics include the wide spread and sometimes
unnecessary use of antibiotics, the prophylactic use of antibiotics, and the use of low
doses of antibiotics for encouraging the growth of farm animals. In the case of bacteria,
their sheer numbers means that there is a potentially large number of genotypes waiting
to be selected and their short generation time fuels the rapidity of this selection. Finally
their ability to horizontally transfer genetic materials through plasmids, transposons,
and by conjugation and transformation set the stage for the (almost) inevitability of the
development of resistance among microorganisms, especially bacteria.
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Modern Industrial Microbiology and Biotechnology
24.3.1.2
The development of previously non-pathogenic
microorganisms into pathogens
As clinical practice now use more invasive methods and people live longer, more and
more people now depend on adequate antimicrobial coverage. Especially in patients
who are immunocompromised, microorganisms which were previously non-pathogenic
or ordinary commensals have become pathogens due to the widespread use of
antibiotics. Thus, Proteus sp., Acinetobacter sp. and yeasts have all gained new status as
pathogens especially in intensive care units.
24.3.1.3
Need to develop anti-fungal antibiotics
Currently there are few satisfactory systemic anti-fungal and anti-viral antibiotics. In the
case of anti-fungal agents, currently few satisfactory systemic antifungal antibiotics
outside Amphothericin B seem to exist; even Amphothericin B is not always efficacious.
There is a similar dearth in the anti-viral antibiotics.
24.3.1.4
Need to develop antibiotics specifically for
agricultural purposes
The growing needs for antibiotics used in agriculture for combating plant diseases, in
animal feeds and in veterinary practice dictate that antibiotics be found specially for food
production, but not for human medicine because of the problem of resistance.
24.3.1.5
Need for anti-tumor and anti-parasitic drugs
Although microbial metabolites for combating tumors, helminthes and parasites cannot
be strictly described as antibiotics in the conventional sense, their production by the
fermentation of microorganisms may allow the loose use of the term anti-tumor
antibiotics and anti-helminthes antibiotics. Antibiotics need to be produced from
microorganisms for these purposes.
24.3.2
The classical method for searching for antibiotics:
random search in the soil
The classical method for the search for new antibiotics is by random search in the soil.
This method will be described briefly below as a setting for more recent methods.
Although the first important commercially produced antibiotic was discovered by
chance, most present day antibiotics were discovered by systematic search. The soil is a
vast repository of microorganisms and it is to the soil that search is turned when
antibiotics are being sought. The stages to be discussed below are not necessarily rigidly
followed; they are merely meant to indicate in a general manner some of the activities
involved in the development of antibiotics. The most important are:
(i) The primary screening: Several methods have been employed in primary screening.
(a) The crowded plate: This method is used to isolate soil organisms able to produce
antibiotics against other soil organisms. A heavy aqueous suspension (1:10; 1:100)
of soil is plated on agar in such a way as to ensure as much as possible the
development of confluent growth. Organisms showing clear zones around
Production of Antibiotics and Anti-Tumor Agents
""
themselves are isolated for further study. Different groups of organisms could be
encouraged to develop by altering the media used.
This method has the disadvantage that slow-growing antibiotic-producing
organisms such as actinomycetes are usually over grown and are therefore hardly
isolated. Furthermore, the test organisms used in this method are soil organisms.
The susceptibility of soil organisms to the antibiotics produced in the test, may
therefore be unrelated to the susceptibility of clinically important organisms.
(b) The direct-soil-inoculation method: This method is used when the aim is to isolate
antibiotics against a known organism or organisms. Pour plates containing the
test organisms are prepared. Soil crumbs or soil dilutions are then placed on the
plates. Antibiotic producing organisms develop which then inhibit the growth of
the organisms in the plate. They are recognized by the cleared zone which they
produce around themselves and they may then be picked out.
(c) The cross-streak method: This method is used for testing individual isolates,
especially actinomycetes which may be obtained from soil without any previous
knowledge of their antibiotic-producing potential. The organism may come from
one of the two methods already indicated above.
The purified isolate is streaked across the upper third of plate containing a
medium which supports its growth as well as that of the test organisms. A variety
of media may be used for streaking the antibiotic producer. It is allowed to grow for
up to seven days, in which time any antibiotic produced would have diffused a
considerable distance from the streak. Test organisms are streaked at right angles
to the original isolates and the extent of the inhibition of the various test organisms
observed (Fig. 24.4).
(d) The agar plug method: This method is particularly useful when the test organism
grows poorly in the medium of the growth of the isolate such as fungi. Plugs about
0.5 cm in diameter are made with a sterile cork borer at progressive distances from
the fungus. These plugs are then placed on plates with pure cultures of different
organisms. The diameters of zones of clearing are used as a measure of antibiotic
production of the isolate. The method may be used with actinomycetes.
Fig. 24.4 The Cross Streak Method for the Primary Search of Antibiotic Producing Organisms
""
Modern Industrial Microbiology and Biotechnology
(e) The replica plating method: If a large number of organisms are to undergo primary
screening, one rapid method is the use of replica plating. This is a well-known
method used in microbial genetics. It was discussed in Chapter 5 dealing with the
production of mutants. The method consists of placing a sterile velvet pad on the
colonies formed in the crowded plate or soil inoculation plate, or on series of
discrete colonies to be tested for antibiotic properties. The pad is thereafter
carefully touched on four or five plates seeded with the test organisms. As a
landmark is placed on the pad as well as on the plates it is possible to tell which
colonies are causing the cleared zones on the tested plates (Fig. 24.5).
(ii) Secondary screening: Organisms showing suitably wide zones of clearing against
selected target organisms are cultivated in broth culture in shake flasks using
components of the solid medium in which the isolate grew best. Crude methods of
isolating the active antibiotic are developed by extracting the broth using a wide range of
extractive methods. With each extraction the resultant material is assessed for activity
against the target organisms at various dilutions. The extract is either spotted on filter
paper discs placed on agar seeded with the test organism or introduced into wells dug
out from the seeded agar with sterile cork borers. In this manner the most efficient
extractive methods and the spectrum of activity of the organisms are determined.
Colonies 1 - 7 are transferred by a velvet pad to plates seeded with E. coli, Bacillus sp., and Proteus sp.
respectively. Note that colonies 1,5,7 produce dear zones in E. coli, Bacillus sp., and Proteus sp.
respectively.
Fig. 24.5 Replica Plating Method of Testing Antibiotic Producing Colonies
Production of Antibiotics and Anti-Tumor Agents
""!
Secondary screening is aimed at eliminating at an early stage any antibiotic which
does not appear promising either by virtue of low activity, other undesirable properties or
because it has been discovered previously.
Antibiotic spectrum: The minimal inhibitory concentration (MIC) is a means of
determining the activity of the isolated antibiotic and comparing this activity with those
of existing antibiotics. Tests involving agar diffusion such as filter paper discs or agar
wells described above are rapid and very useful for initial screening. However, they
involve not only the intrinsic anti-microbial potency of the antibiotic produced but also
its ability to diffuse through agar. The MIC has the advantage that it is performed in broth
thereby eliminating the disadvantage of large-molecule slower-diffusing antibiotics.
(iii) Other properties: The other qualities of the antibiotic outside anti-microbial activity
depend on its intended use. For antibiotics meant for clinical use, information on a
number of the following may be sought at this stage:
(a) Toxicity to mammals, determined by intra peritoneal injection into animals;
(b) Haemolysis is tested by observing the effect on blood agar;
(c) Serum binding is tested by adding serum to the broth before testing against
susceptible organisms;
(d) The inactivation of the antibiotic by several enzymes from various organs is tested
by exposing the antibiotic to them;
(e) Acid stability is tested if the antibiotic is meant for oral fermentation
(f) Tetragonicity tests, which determine the effect on the unborn are carried out on
laboratory animals;
(g) For plant antibiotics, phytotoxicity as shown by damage to leaves in the laboratory
and in the green house, is determined;
(h) For feed antibiotics, low absorbability and low toxicity are desirable and are tested.
Several other tests designed to certify the safety of administering the antibiotic may be
carried out at this stage or performed later.
As part of the secondary screening, initial studies on the chemical nature of the crude
antibiotics is determined using paper chromatography, ultraviolet absorption, solubility
in acid and alkali, optical rotation, infra-red absorption, nmr, etc. The aim of this study is
to see if it is an already known antibiotic. If it is, then further work on it may be
abandoned. It is important to make this decision early. As has been shown previously,
the same antibiotic is often produced by a large number of different organisms. This
method known as ‘finger printing’ is more fully discussed when anti-tumor antibiotics
are examined.
(iv) Further laboratory evaluation: If and after all the above, the antibiotics is promising,
then further experimentation is done in shake flasks as a preparation for pilot
production. The optimal conditions of growth are determined; the most suitable medium,
optimal pH, temperature, length of fermentation etc. are all determined.
(v) Pilot plant production: The results obtained in previous experimentation are fed into
the pilot plant. The material produced is subjected to further safety tests and chemical
analysis. Enough materials are made available for the structure to be determined.
"""
Modern Industrial Microbiology and Biotechnology
Methods for the isolation are improved and perfected. Clinical tests on a limited scale
may be carried out at this stage. The Journal of Antibiotics regularly carries several articles
describing the procedures for the chemical analysis of many new antibiotics. Unless the
organism is already known, it is also described and may be named. The mode of action,
the route of biosynthesis and strain improvement are undertaken, but the production of a
good antibiotic does not await their successful completion.
(vi) Plant production: The production plant utilizes all the information obtained in the
pilot experimentation.
(vii) Certification: A government agency must approve the antibiotic before it becomes
available for general use. In the US, it is the Food and Drug Administration. In the UK and
EU countries, it is the European Medicines Agency (EMEA) which was established in
1993 and is based in London. The process for the certification of drugs by the FDA is
discussed in greater detail later in Chapter 28.
(viii) Marketing and financing: The marketing and financing of the business are of
paramount importance since the aim of the producing firm is profit maximization.
24.4 COMBATING RESISTANCE AND EXPANDING THE
EFFECTIVENESS OF EXISTING ANTIBIOTICS
Many ways have been devised to combat microbial resistance to existing antibiotics or
expand the effectiveness and to discover new ones. These include modifying the
procedures for the classical search in soil, searching for antibiotics in novel
environments, and chemically manipulating the antibiotics directly or through using
mutant microorganisms.
24.4.1
Refinements in the Procedures for the Random
Search for New Antibiotics in the Soil
To increase the chances of finding new antibiotic - producing organisms, some
refinements of the classical methods of searching for antibiotic producers were
introduced as shown below.
24.4.1.1
The use of super-sensitive mutants
By using super-sensitivity strains of test organisms, organisms producing only small
amounts of an antibiotic may be detected. Antibiotics so produced are useful because
they may have a wider spectrum than those of the same class already in existence.
Furthermore they may provide better substrates for semi or muta-synthesis. This method
has led to the discovery of novel Beta-lactam antibiotics, thienamycin, olivanic acid,
nocardicins, clavulanic acid etc. It is remarkable that several natural clavulanic acid type
compounds (e.g. 2-hydroxymethylclavam) have significant antifungal properties. The
use of super-sensitive mutants has shown that Beta-lactam antibiotics are produced by a
wider spectrum of organisms – ascomycetes, fungi imperfecti and actinomycetes – than
was previously thought.
Production of Antibiotics and Anti-Tumor Agents
24.4.1.2
""#
The application of criteria other than death or inhibition
Reactions such as irregular growth of the fungal mycelium, inhibition of sporulation, or
some readily ascertainable deficiency rather than death may be used to follow the
antibiotic effect. When anti-fungal antibiotics are sought, the clear zone principle is
employed using yeasts or fungal spore in the test plate. With this method the existing
antifungal antibiotics, namely polyenes as well as cycloheximide and actimycins were
found. If criteria less drastic than death e.g. abnormal growth of hyphae, or inhibition of
zygosphore formation, then a wide range of antibiotics may be found. Thus some new
antibiotics have been found in actinomycetes including boromycin, venturicidin and
mikkomycin, with this method.
24.4.1.3 Search for antibiotics effective in
conjunction with other antibiotics
Some antibiotics while being effective are not permeable through the wall of the test
organism or the pathogen. Such antibiotics should be sought in nature by using testorganisms with deficient cell walls, protoplasts, or by the incorporation of detergents or
EDTA which enable the permeation of antibiotics into the organism. When they are
discovered they can be made permeable by using them in conjunction with wallinhibiting antibiotics or compounds, or coupling them to compounds which bacteria
ingest by active transport.
24.4.1.4 Use of organisms of recent clinical
importance as test organisms
The classical search used routine organisms such as E. coli and Bacillus. The result as has
been shown has been that fewer and fewer new antibiotics have been discovered. In
recent times new organisms previously of little importance in clinical practice have
emerged, due to the widespread use of antibiotics as important medical organisms. These
include anaerobes, Gram-negatives e.g. Proteus, Beta-lactamase producing gonococci,
facultatives, haemolysis, etc. These should be used as the test organisms in place of the
previous ones.
24.4.2
Newer Approaches to Searching for
Antibiotics
In spite of the above modifications and refinements in the classical methods for searching
for new antibiotics, antibiotics with new structures were not discovered and crossresistance among the available antibiotics continue to occur. In recent times some newer
approaches to discovering new antibiotics have been adopted.
24.4.2.1
Search in novel environments
Systematic search for antibiotics is usually from soil. Other natural bodies exist which
can provide novel organisms. Two of such habitats will be discussed in this section,
namely the sea and white blood cells.
""$
Modern Industrial Microbiology and Biotechnology
24.4.2.1.1 The sea as a habit for prospecting for micro-organisms
producing antibiotics (and other drugs)
The seas and oceans occupy 70% of the earth surface. Until recently they were not
exploited as sources of antibiotic-producing organisms. Although they would present
new difficulties such as the need for a boat, they are a unique habitat. They are not only
twice the land area of the earth, they contain large amounts of salt and other mineral
nutrients, have fairly constant temperature, and have a higher hydrostatic pressure and
less sunlight in the deeper regions. The coastal area is constantly changing with tides
and such areas should be expected to have a wide variety of organisms, peculiar to the
littoral environment.
Although deep ocean exploration is still in its infancy, many scientists now believe
that the deep sea harbors some of the most diverse ecosystems on Earth. This diversity
holds tremendous potential for human benefit. More than 15,000 natural products have
been discovered from marine microbes, algae, and invertebrates, and this number
continues to grow. The uses of marine-derived compounds are varied, but the most
exciting potential uses lie in the medical realm. More than 28 marine natural products are
currently being tested in human clinical trials, with many more in various stages of
preclinical development. To date, most marketed marine products have come from
shallow and often tropical marine organisms, due mainly to the ease of collecting them.
But increasing scientific interest is now being focused on the potential medical uses of
organisms found in the deep sea, much of which lies in international waters. These
organisms have developed unique adaptations that enable them to survive in dark, cold,
and highly pressurized environments. Their novel biology offers a wealth of
opportunities for pharmaceutical and medical research and a growing body of scientific
evidence (Table 24.2) suggests that deep sea biodiversity holds major promise. The
medicines in the Table do not include antibiotics, but it could be because search for them
was not conducted in this particular study. Nevertheless the search for antibiotics in the
sea has indeed led to the discovery of new and unique antibiotics. These include
antibiotic SS-228R from Chainia sp. effective against Gram-positive bacteria and tumors,
bromopyrrole from a marine Pseudomonas, and leptosphaenin from marine fungi.
24.4.2.1.2
Antibiotic sources other than microorganisms: bactericidal/
permeability increasing protein (BPI)
Antibiotics are produced also by higher organisms – plants and animals – and they also
should be screened by the regular method of antibiotic screening. However because of
established practice, antibiotics from such higher organisms have been usually screened
for anti-tumor and anti-viral activity. Nothing intrinsic in materials from higher
organisms should stop them from acting against microorganisms in suitable cases. In
this section Bactericidal/permeability increasing protein (BPI) will be discussed as an
example of a novel antimicrobial agent derived from a living thing higher than
microorganisms.
BPI, is a protein and has been studied for several decades. It is derived from the white
blood cells, polymorphonuclear leucocytes, the primary phagocytic white blood cells
responsible for part of the body’s innate immune response. BPI has great affinity for the
lipopolysaccharide layer (LPS) of Gram-negative bacteria. It immediately arrests the
Production of Antibiotics and Anti-Tumor Agents
""%
Table 24.2 Deep sea compounds in development for medical use (July, 2005)
Name
Application
Source
Depth/Location Status
E7389
Cancer:
non-small
cell lung
and other
types
Cancer:
solid tumors
Sponge:
Lissodendoryx
sp.
330 ft (100 m) Phase I
New Zealand clinical trials
Sponge:
Discondermia
Dissolute
460 ft (140 m) Phase l trials
Bahamas
(completed)
Cancer
Sponge: Order
Lithistida,
Family
Corallistadae
Coral:
Sarcodictyon
roseum
1,460 ft (442 m) Preclinical
Jamaica
Development
330 ft (100 m) Preclinical
Mediterranean Development
Toxicity
similar to
Taxol ®
Microbe:
Selinospora
More than
Preclinical
3,300 ft (1,000 Development
m) North
Pacific Ocean
Will enter
clinical trails
in 2005;
potency 35x
omuralide
Discodemorlide
Diclyostatin -1
Sarcodictyin/ Cancer
Eleutherobin
(related
compounds)
SalinosporConcer:
amide A
melanoma,
colon, breast,
non-small
cell lung
Topsentin
Antiinflammatory:
arthritis, skin
irritations
Cancer: colon
(preventive)
Alzheimer’s
Orthopedic
Bone grafting
implants
Sponge:
1980 ft
Spongosporites (1,000 m)
ruetzleri
Bahamas
Preclinical
Development
Coral: Family
Isididae
Preclinical
Development
More than
3,300 ft
(1,000 m)
North Pacific
Ocean
Comments
Toxicity
similar to
Taxol ®;
works on
multi-drug
resistant
tumors
Toxicity
similar to
Taxol ®
Resources
risk of
mammalian
disease
growth of Gram-negative bacteria, increases the permeability of the outer and inner
membranes of the Gram-negative bacterial wall and eventually kills the organism. It is
attractive as a possible antibiotic for several reasons. First, it is highly potent and specific
against Gram-negative bacteria, which include many important human pathogens; it is
at least 10 times more potent than any known mammalian anti-microbial protein or
peptide. Second, it is non-toxic to mammalian cells. Third, it maintains its anti-microbial
""&
Modern Industrial Microbiology and Biotechnology
activity in the complex environment of body fluids, unlike many mammalian antimicrobial agents. Finally it is not only a potent antimicrobial agent, but it also reduces the
effect of the inflammatory response known as ‘septic shock’ which the
lipopolysaccharide of the Gram-negative cell wall induces in patients. None of the other
proteins able to bind to the Gram-negative lipopolysaccharide is able to neutralize the
effect of toxic shock. It is currently at the stage of clinical trials.
24.4.3
Chemically Modifying Existing Antibiotic:
The Production of Semi-synthetic Antibiotics
The production of a semi-synthetic antibiotic involves the use of a fermentation-derived
antibiotic, which is then modified by the addition of side chain to give rise to an antibiotic
with new properties. As has been discussed, a well-known example is the modification of
penicillin G to 6-APA and the subsequent use of chemical reaction to produce semisynthetic penicillins. Another example achieved in a different manner is the chemical
alteration of specific sites in an antibiotic in order to render the antibiotic immune to an
enzyme which destroys it, as is done with streptomycin.
24.4.4
Modifying an Existing Antibiotic Through
Synthesis by Mutant Organisms: Mutasynthesis
This method is one in which a mutant of an antibiotic producing organism is fed different
precursors leading to the production of new antibiotics. Since the original production of
hybridicins from the neomycin synthesizing Strep. fridiae, this method has been used in
the production of paromomycin by Strep. rimogus forma paromonycins, ribostamycin by
Strep. ribosidificus and butrosin by Bacillus circulans.
24.5
24.5.1
ANTI-TUMOR ANTIBIOTICS
Nature of Tumors
Each cell in the animal (and human) body has a definite function which it carries out in
cooperation with other cells. Thus the brain, the skin and the intestines are composed of
specialized cells which cooperate to carry the functions of these organs. Sometimes
however a cell in any part of the body may no longer cooperate with others with which it
normally functions in an organ. Such cells divide indiscriminately and independently of
the others to form a structure called a tumor or a neoplasm. Sometimes the body restricts the
growth of tumors by forming a capsule round them. Under these conditions they do not
spread: they are known as benign tumors. Other tumors however grow rapidly and are
not restricted by a capsule. Such tumors are malignant. The cells in malignant tumors
often break off and are carried via blood vessels and lymphatic vessels to other parts of
the body where they initiate new tumors. When such secondary growth occurs away
from the primary tumor the situation is known as metastasis.
Tumors are further classified according to the type of tissue they attack. Some of these
will be mentioned: a malignant tumor composed of epithelial cells is called a cancer or a
carcinoma. Adenocarcinomas are tumors formed around the mucous membranes such as in
the alimentary canals. Sarcomas are connective tissue tumors. The term ‘hard’ tumor is
Production of Antibiotics and Anti-Tumor Agents
""'
sometimes used to distinguish neoplasms formed in the solid parts of the body such as
the gut, bones, brain etc. from those of blood such as leukemia which is a neoplasm of the
white blood cells. Neoplasms are treated by one or more of three methods: (1) by surgery
to remove the cancer; (2) by radiation, which aims at selectively destroying the cancer
cells and (3) by chemotherapy or the use of chemicals which affect the tumor cells without
damaging the normal cells. When such chemicals are produced by microorganisms they
are called anti-tumor antibiotics. Chemotherapy is particularly useful when the disease
has metastasized to several sites in the body so that it becomes practically impossible to
achieve any success by surgery or by radiation. It is also used after treatment by surgery
or radiation to attack those cancerous cells missed by the other two treatments. Many of
the chemotherapeutic agents used in cancer treatment are secondary metabolites
produced by microorganisms, especially of the genus Streptomyces. This chapter is
concerned with these metabolites known as anti-tumor antibiotics.
24.5.2
Mode of Action of Anti-tumor Antibiotics
The anti-tumor antibiotics are heterogeneous in their chemical natures. Some of the best
known groups used in clinical practice include anthracyclines, actinomycins and
bleomycins (Fig. 24.7). In terms of their modes of action their common characteristic
seems to be interaction in some form with DNA. Daunomycin and adriamycin which are
anthracyclines link up base pairs and thus inhibit RNA and DNA synthesis.
Mithramycin and chromomycin A3 which are actinomycins inhibit DNA – dependent
RNA synthesis. On the other hand bleomycins which are peptides react with DNA and
cause it to break. Other anti-tumor antibiotics operate through alkylation e.g.
streptonigrin, mitomycin C, and profiromycin. Still others interfere with membrane
functions or interact with the micro-tubules in the cell.
The basis of all chemotherapy whether with anti-bacterial or with anti-tumor drugs is
the ability of the drugs to selectively attack the pathogen or the errant tumor cell. In the
well-known case of penicillin for example, the absence of mucopeptides in animal cell
wall is the key to the operation of the drug. Several mechanisms have been suggested
which allow the selective attack of anti-tumor drugs on tumor cells. These include
inability of the tumor cell to repair damage by the anti-tumor drug, higher distribution of
the drug in tumor than in normal cells, greater ability to inactivate tumor cells. These are
based on structural and bio-chemical differences between normal and tumor cells.
Unfortunately anti-tumor antibiotics as well as other anti-tumor drugs do not always
discriminate successfully between tumor and normal cells. Varying degrees of toxicity
therefore usually accompany the use of anti-tumor antibiotics. The most severe of these is
damage to the bone marrow which is involved in synthesis of blood components a
damage that may be fatal. Toxicity is in many cases being successfully handled clinically
by various means including reduced dosage, change of route of administration, etc. Less
toxic antibiotics are also being produced by semi-synthesis or by the modification of the
antibiotic molecule.
24.5.3
Search for New Anti-tumor Antibiotics
The search for anti-tumor antibiotics is more difficult than that of anti-bacterial or antifungal agents in terms of methodology and interpretation. In the search for the latter
"#
Modern Industrial Microbiology and Biotechnology
R3
R4
R5
X
Y
Adriamycin
Daunomycin
OCH3
OCH3
CH2OH
CH3
H
H
H
H
H
H
O
O
H
H
Carminomycin
OH
CH3
H
H
H
O
H
Fig. 24.7
General Formula of Anthracyclines
agents it is usually possible to isolate the pathogen from the diseased animal and test the
isolate against a wide range of possible antibiotics in vitro and in vivo in experimental
animals. In general an antibiotic successfully tested in vivo in experimental animals will
be expected to be reasonably efficacious in treating human disease. In the case of antitumor agents the relationship is not so straight-forward.
As is the case with anti-microbial antibiotic no order of procedure can be prescribed.
The description that follows is built up from published materials from several groups,
especially at the National Cancer Institute, and the Cancer Research Laboratories,
Kalamazoo, both in the USA. The stages involved in the search and initial development of
anti-tumor antibiotics may be enumerated as follows.
24.5.3.1
Screening of potential antibiotic producing organism
A wide variety of microorganisms is obtained from all over the world and their
fermentation broth is evaluated for the presence of anti-tumor drugs. Cultures whose
identities are established as well as those still to be established are obtained from culture
collections and individual scientists around the world. Fresh isolations are also made
from natural habitats including soil, aquatic and other environments.
Production of Antibiotics and Anti-Tumor Agents
"#
For the isolation (and often the maintenance of the organisms, a wide variety of carbon
sources is used. Especially in well-studied groups, such as actinomycetes, novel carbon
sources are used in the hope that fermentation broth may contain some anti-tumor
antibiotics, as a result of the blockage of certain pathways or the enhancement of others.
Such carbon sources include monosaccharides, glycosides, substituted sugars,
polyhydric alcohols, oligosaccharides, terpens, and hydrocarbons.
Isolations of microorganisms are done by sprinkling soil on plates containing the
above carbon sources. Perfusion technique in which soil is bathed constantly with a
solution containing the chosen carbon source may also be used. Isolates are grown in
shake flasks using the various carbon sources and a variety of environmental conditions
including pH, minerals, temperature, nitrogen sources, and aeration.
24.5.3.1.1 In vitro prescreening
Since large numbers of samples are generated, it would be extremely expensive to test
them directly in tumor-bearing animals. Prescreens are therefore used. Such prescreens
should ideally select the broths successfully containing potentially in vivo active
components, should be relatively inexpensive in terms of money and time and should
require only small quantities of the test broth. In vitro screens are also used to follow the
course of fermentation. In vitro methods are particularly essential because the frequency
of occurrence of active anti-tumor components in fermentation beers is low, and when
present at all, the concentration especially initially is also low. Some of the in vitro
screening methods which are currently in use are as follows:
(i) Use of anti-microbial activity as prescreens: As most anti-tumor agents will also inhibit
micro-organisms, the latter came to be used during the early stages of the search for these
drugs. Indeed a number of drugs possessing carcinostatic properties were first isolated
as anti-microbial agents. These include actinobolin, cycloheximide, and actinomycin.
Azaserine was the first anti-tumor drug isolated following activity against a bacterium,
which in this case was E. coli. A wide range of microorganisms can and indeed have been
used in prescreens. However, many groups favor using a set containing a few
microorganisms to facilitate the identification by ‘finger printing’ of already discovered
antibiotics. Finger-printing will be discussed more fully below. A set of microorganisms
which has been used include Bacillus subtilis ATCC 6633, Sarcina lutea ATCC 9341,
Torulopsis albida NRRL Y1400 and Escherichia coli M 1262.
The beer to be tested is spotted on filter paper disks placed on freshly made pour plates
of these organisms or in agar wells dug from such plates. The extent of the inhibition is
determined by the diameter of the zone of clearing.
(ii) Anti-metabolite activity: The use of anti-metabolites as a prescreen was based on the
observation that some drugs or broths which did not inhibit microorganisms in complex
media such as nutrient agar did so on synthetic minimal media. It was soon shown that
by adding various compounds to the minimal medium it was possible to determine the
missing metabolite. Broth samples are incorporated into rich agar and minimal agar
respectively. Those broths which are more active against susceptible microorganisms in
synthetic minimal medium are regarded as potential leads. This methods led to the
discovery of 5-Azacytidine and its principal advantage is that it gives an idea at an early
stage of the mode of action of the active component of the broth.
"#
Modern Industrial Microbiology and Biotechnology
(iii) Inhibition of tumor cells in culture: The ability of the broth to inhibit animal tumor cells
in tissue culture is tested. Among cells which have been used are L1240 (mouse leukemia
cells), KB (human carcinoma cells of the nasopharynx) and p388 mouse leukemia cells.
L1240 appears to be most widely used. The tumor cells are grown in liquid culture with
and without the broth over a period of about three days. They are then counted in a
coulter counter. The potency of the same is given as ID50 or ID90 or the dilution that will
cause 50% or 90% inhibition of growth as compared with control.
(iv) Nuclear cytotoxicity: Instead of using cell cultures, nuclei from tumor cells can be
isolated and the broth tested against these. Isolation may be achieved using citric acid,
detergents, organic solvents, and glycerol. The limitation of the system is that it can be
used to detect only those agents which in some way affect nuclear synthesis. It however
lends some insight into the mode of action of the agent.
24.5.3.2
Finger printing of anti-tumor antibiotics in culture
Broths showing some activity by any of the above prescreening methods are subjected to
‘finger-printing’ or ‘dereplication’ in order to avoid the isolation of already known
antibiotics. The data used include the following:
(i) The anti-microbial spectrum of the active components of the broth is determined
using a set of microorganisms. Besides bacteria and yeasts some workers use
protozoa and algae.
(ii) The characteristics of the culture used in the fermentation.
(iii) Chromatographic analyses of the broth using paper, thin layer, and high
performance liquid chromatography.
(iv) Ultraviolet absorption.
24.5.3.3
In vivo assessment
If the results of the finger-printing indicate that the active components are new then in
vitro testing is done. The one common characteristic of tumors or neoplasms is the
uncontrolled growth of cells. Outside this property they are in fact biologically
heterogeneous in terms of site of origin, cell type involved, the course of the disease, or
response to curative procedures. In assessing active components in vivo therefore this
diversity is acknowledged by testing various types of artificially induced tumors usually
in mice. These include mouse tumors of the colon, breast, lungs, and white blood cells
(leukemia). Human cancer from the colon, breast and lungs, are also grafted on these
regions of mice whose thymus glands have been removed to avoid rejection of the grafts.
Often it is necessary to have an in vivo prescreen before subjecting the broth to the
above tests. Some workers have found that mouse leukemia P388 is more sensitive as an
in vivo prescreen than L1240.
The parameters for in vivo tests are increased lifespan of the animal or tumor growth
inhibition as measured by tumor weight inhibition over the control.
The in vivo test is expensive in time and money. It takes three to four weeks to perform
whereas cell or microbial cultures take about three days or less.
Production of Antibiotics and Anti-Tumor Agents
24.5.3.4
"#!
Extraction and manipulation of the pure drug
Since the active component may often be present in very low concentration it is necessary
to obtain the active component in the broth in a reasonably pure form. Subsequent to this
it is characterized chemically and then subjected to further animal tests before being
assessed clinically. Since many anti-tumor antibiotics have serious side effects,
analogues of the drugs are produced and modifications to the molecule are then carried
out. Other procedures, e.g. improvement in the environmental conditions of the broth,
development of optimum isolation procedures, scale up, sales, etc. are carried out as for
anti-microbial antibiotics.
24.5.3.5
Towards a new definition of ‘antibiotic’
The current definition of the term ‘antibiotic’ which restricts them to chemicals produced
by microorganisms is credited to Waksman who had won the Nobel Prize for discovering
streptomycin. However, when screenings have been done outside microorganisms, the
higher organisms so screened have been shown to produce anti-microbial substances.
Such substances are low molecular weight secondary metabolites in the same way as
regular antibiotics are. Due to this, there is now a tendency to extend the term antibiotic to
all secondary metabolites, irrespective of their origin, which are able to inhibit various
growth processes at low concentration. Not only that, even wholly synthetic
antimicrobials such as ciprofloxacin are now legitimately termed antibiotics. It is not an
altogether unreasonable redefinition. After all, the word antibiotic derives from two
origins, anti (against) and bios (life). Nothing in the word itself restricts antibiotics both
in origin or in use to microbial life
24.6
NEWER METHODS FOR SEARCHING FOR
ANTIBIOTIC AND ANTI-TUMOR DRUGS
In recent times newer method have been developed for searching for new antibiotics, antitumor agents and other drugs. These methods include computer-aided drug designing,
synthesis of new drugs by combinatorial chemistry and genome-based methods. These
are further discussed in Chapter 28, where drug discovery is examined.
SUGGESTED READINGS
Allsop, A., Illingworth, R. 2002. The impact of genomics and related technologies on the search
for new antibiotics. Journal of Applied Microbiology, 92, 7-12.
Anon, 1993. Congress of the United States, Office of Technology Assessment. Pharmaceutical
R&D: Costs, Risks and Rewards: 1993; pp. 4-5.Washington, DC, USA.
Anon, 1999. From Test Tube to Patient: Improving Health Through Human Drugs. Special
Report, Center Drug Evaluation and Research. Food and Drug Administration. Rockville,
MD, USA.
Austin, C. 2004. The Impact of the Completed Human Genome Sequence on the Development of
Novel Therapeutics for Human Disease. Annual Review of Medicine, 55, 1-13.
Bansal, A.K. 2005. Bioinformatics in the microbial biotechnology – a mini review. Microbial Cell
Factories, 4, 4-19.
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Modern Industrial Microbiology and Biotechnology
Beamer, L. 2002. Human BPI: One protein’s journey from laboratory to clinical trials. ASM News.
68, 543-548.
Behal, V. 2000. Bioactive Products from Streptomyces. Advances in Applied Microbiology. 47, 113156.
Bull, A.T., Ward, A.C., Goodfellow, M. 2000. Search and Discovery Strategies For Biotechnology:.
The Paradigm Shift. Microbiology and Molecular Biology Reviews, 64, 573-548.
Dale, E., Wierenga, D.E., Eaton, C.R. 2001. Processes of Product Develpoment. http://
www.allpcom/drug-dev.htm. Accessed on September 28, 2005 at 12.05 pm GMT.
Debouck, C., Metcalf, B. 2000. The Impact of Genomics on Drug Discovery. Annual Review of
Pharmacology and Toxicology, 40, 193–208.
Fan, F., McDevitt, D. 2002. Microbial Genomics for Antibiotic Target Discovery. In: Methods in
Microbiology. Vol 33, Academic Press. Amsterdam: The Netherlands. pp. 272–288.
Feling, R.H., Buchanan, G.O., Mincer, T.J., Kauffman, C.A., Jensen, P.R., Fenical, W. 2003.
Salinosporamide A: a highly cytotoxic proteasome inhibitor from a novel microbial source, a
marine bacterium of the new genus Salinospora. Angewandte Chemie International Edition,
42, 355-357.
Fraser, C.M., Rappuoli, R. 2005. application of microbial Genomic Science To Advanced
Therapeutics. Annual Review of Medicine, 56, 459–74.
Handelsman, J., Rondon, M.R., Brady, S.F., Clardy, J., Goodman. R.M. 1998. Molecular biological
access to the chemistry of unknown soil microbes: a new frontier for natural products.
Chemistry and Biology, 5, 245-249.
Hill, D.C., Wrigley, S.K., Nisbet, L.J. 1998. Novel screen methodologies for identification of new
microbial metabolites with pharmacological activity. Advances in Biochemical Engineering
and Biotechnology, 59, 75–124.
Maxwell, S., Ehrlich, H., Speer, L., Chandler, W. 2005. Medicines from the Deep Sea. Washington,
DC, USA.
Rosamond, J., Allsop, A. 2000. Harnessing the Power of the Genome in the Search for New
Antibiotics. Science, 287, 1972-1976.
Wlodawer, A., Vondrasek, J. 1998. Inhibitors of HIV-1 Protease: A Major Success Of StructureAssisted Drug Design. Annual Review of Biophysics and Biomoecular Structurrs, 27, 249–84.
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25
Production of
Ergot Alkaloids
25.1
NATURE OF ERGOT ALKALOIDS
Alkaloids are laevorotatory basic naturally occurring hetereocyclic organic nitrogen
containing compounds which are biosynthesized from amino acids by plants,
microorganisms and some animals. Many of them are pharmacologically active and are
consequently used as drugs. The main precursors for alkaloid biosynthesis are ornithine,
lysine, aspartic acid, phenylalanine, tyrosine and tryptophan. For example the alkaloid
in tobacco, nicotine, is derived from ornithine while phenylalanine and tyrosine give rise
to simple alkaloids such as ephedrine or more complex ones such as morphine.
The name alkaloid (literally alkali like) derives from their basic nature, because of
which they readily form salts with acids present in the natural sources from which they
are derived. Their chemical classification is based on the carbon-nitrogen skeletons.
Ergot alkaloids, the subject of this chapter are of the indole type and are derived from
tryptophan.
The ergot alkaloids are so called because they were originally derived from ergot, a
sclerotium (twisted mat of fungal hyphae) formed as a disease on the grain of rye (Secale
cereale L.) a temperate cereal. The dried ergot is known among pharmacists as secale
cornutum. The cause of the rye disease is a fungus, an ascomycete, Claviceps purpurea. As
will be seen later, ergot contains several (more than 40) highly potent alkaloids and the
unwitting consumption of grain attacked by fungi producing ergot alkaloid has led to
‘ergotism’, (previously known as ‘Holy fire’ or ‘St. Anthony’s fire’ when it was not
understood) a disease characterized by among other symptoms, convulsions. . Ingestion
of contaminated grain, most often after the grain has been made into bread, causes
ergotism, also known as the ‘Devil’s curse’ or ‘St. Anthony’s fire,’ and has been a problem
for centuries. It has been noted in writings from China as early as 1100 B.C. and in
Assyria in 600 B.C., and Julius Caesar’s legions suffered an epidemic of ergotism during
one of their campaigns in Gaul (France). In 994 A.D., an epidemic in France killed
between 20,000 and 50,000 people, and in 1926, at least 11,000 cases of ergotism occurred
in Russia.
Ergotism can cause convulsions, nausea, and diarrhea in mild forms, and there is
some thought that an outbreak of ergotism may have been the cause of the ‘bewitchings’
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Modern Industrial Microbiology and Biotechnology
which led to the Salem witch trials in the United States in 1691. Ergotism may also have
caused some of the extreme destruction associated with the French Revolution. In the
Middle Ages, ergotism was described as causing victims to die “miserably, their limbs
eaten up by the holy fire that blacked like charcoal.” People turned to the church for help,
assuming that the disease was retribution for their sins. In particular, they prayed to St.
Anthony for deliverance, giving rise to the name for the disease. Ergotism takes two
forms, gangrenous ergotism, in which tingling effects were felt in fingers and toes
followed in many cases by dry gangrene of the limbs and finally loss of the limbs, and
convulsive ergotism, in which the tingling was followed by hallucinations and delerium
and epileptic-type seizures. In both cases, death was slow and painful. Ergotism has now
been recognized as a result of infection by a mycotoxin, and the ergotism plagues have
been eliminated.
About 50 ergot alkaloids are known today. While most of these alkaloids are derived
from the Claviceps sclerotium formed on the rye grain, hundreds of other cereals and
grasses can serve as hosts for the fungus. About 50 species of Claviceps itself are known.
In addition, in recent times the alkaloids have been produced by other fungi including
Aspergillus, Penicillium, and Rhizopus. Some recent fungi shown to produce alkaloids are
Balansia epichloe, B. henningsiana, B. strangulans, Myriogenospore atrementose and Epichre
typhine. Furthermore, ergot alkaloids have recently been found in the seeds of some
higher plants, Ipomea, Rivea, Agyreis which belong to Convulvulaceae the family to which
the flower morning glory belongs.
The life cycle of Claviceps is given in Fig. 25.1. The sclerotium forms in the size and
shape of the grain which it replaces. These sclerotia fall to the ground at the end of the
growing season and remain dormant till the beginning of the next growing season, when
they germinate and form ascocarps (perithecia). The ascospores are distributed by wind
to the newly formed flowers of grains. The germinated ascospores yield hyphae which
produce masses of conidia supported in a sugary liquid which attracts insects. These
insects further help distribute the conidia to other plants.
The ergot alkaloids are classified as indole alkaloids, which are derived from
tyrptophan. With one exception, chanoclavine, the ergot alkaloids possess the basic
tetracyclic (four-ringed) structure known as ergoline (Fig. 25.2).
The naturally occurring ergot alkaloids can be divided into two groups. (a) lysergic
acid derivaties (Fig. 25.2) and (b) clavine alkaloids. Although the clavine alkaloids were
the first to be prepared in fermentation broths, and were used for biosynthetic studies,
they are much less known than the lysergic (Fig. 25.3) acid ones in terms of their
fermentation and even in terms of pharmacological activity. Therefore only the lysergic
alkaloids will be handled in this discussion.
The lysergic acid derivates can be further divided into two depending on the nature of
the amide substituents. First are the simple amide substituents and second are the
peptide alkaloids in which a cyclic peptide is attached to lysergic acid. The greatest
interest appears to center on the peptide alkaloids. In this group a modified tripeptide
containing proline and an a–hydroxy - a-amino acid which has undergone cyclic
foundation with the carbonyl atom of proline substitutions at C-2 and C5 of the cyclol
peptide moeity creates variability as shown in Fig. 25.4. This group includes ergotamine.
Production of Ergot Alkaloids
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Fig. 25.1 Life cycle of the Ergot Fungus
Ergoline
Fig. 25.2 Structure of Ergoline
25.2
Fig. 25.3
Lysergic Acid
USES OF ERGOT ALKALOIDS AND THEIR DERIVATES
Ergot alkaloids and their derivates are powerful drugs and may be used as such or may
be the basis of semi-synthetic preparations. Almost all of them have one important
pharmacological effect or another, depending on the nature of the substituents and on the
tissue of the body concerned. Thus ergotamine will cause vessels to constrict while
ergometrine has a minimal effect on blood vessels but will cause the uterus (womb) to
constrict; LSD on the other hand will excite the brain cells in a manner not achieved by the
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Modern Industrial Microbiology and Biotechnology
Ergonovine (uterine contraction; treating post-partum hemorrhages). Methysergide (cranial
vasodilator; treatment of migrane headaches). Ergotamine (treatment of severe migrane head aches). 2Bromo-=-ergokryptine (semi-synthetic, reduction of lactation in women). Ergometrine (used for
treating post-partum hemorrhages). Lysergic Acid Diethylamide (for treating psychiatric disordrers).
Fig. 25.4 Some Therapeutically Useful Ergot Alkaloids
Production of Ergot Alkaloids
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other three. These various effects are exploited in using ergot alkaloids as drugs in the
manner shown below:
(i) Ergot alkaloids (e.g. ergometrine) have been used in mid-wifery to induce labor for
centuries.
(ii) Ergonovine, (the 2-aminopropanolamide derivative) is used to stop bleeding after
birth due to its stimulatory effect on the sympathetic nervous system.
(iii) Ergotamine blocks the sympathetic system and is used for treating strong
headaches such as migraines.
(iv) The diethylamide derivate of lysergic acid (known as LSD) is a powerful
hallucinogenic drug and is often utilized illegally for this purpose in many
countries, as well as for experimental psychotherapy.
(v) Most of the clavine alkaloids do not possess strong pharmaceological properties.
However a few of the didydro derivates have found use as strong stimulants of
oxytoxic (milk secreting) activity or of uterine contractions.
In recent times newer uses have been found for ergot alkaloids especially semisynthetic ones. These include:
(vi) ‘Nigericoline’, a derivate of lysergic acid which is a receptor-blocking agent and is
used for treating peripheral and cerebral circulation disorder.
(vii) ‘Lysenyl’ a diethyl derivate of isolysergic acid is used to treat hypertension and
migraine, since it is a serotonin antagonist.
(viii) In recent times it has been found that some ergot alkaloids affect functions
controlled by the hypothalamic pituitary system particularly the release of
prolactin (which deals with milk secretion) from the pituitary gland. Since the
prolactin level seems to play a part in the growth and development of certain breast
cancers they are used for therapy.
(ix) Some of newer ergot alkaloids have also been implicated as potential therapeutic
agents in the treatment of diseases such as Parkinson’s disease, lack of milk
production after child birth, and cancer of the prostrate.
(x) The alkaloids have been used as models for the synthesis of several potent drugs.
25.3
PRODUCTION OF ERGOT ALKALOIDS
A large market based mainly in Europe derives from ergot drugs; this market seems to be
expanding as more and more pharmacological properties are discovered in the ergot
drugs.
The methods for producing these drugs are three.
(a) Isolation from field cultivated ergot.
(b) Fermentation of the ergot fungus
(c) Partial or total chemical synthesis.
Until now the bulk of the production seems to be the extraction of field inoculated
ergot. The pattern seems however to be changing. Fermentation is increasing and as with
the antibiotics wholly new drugs are being produced by semi-synthesis with substrates
derived from fermentation.
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Modern Industrial Microbiology and Biotechnology
(a) Isolation from field cultivated ergot (or parasitic production): This method is widely used in
Europe. Inoculation of the rye plants with conidiospores of Claviceps and other fungi
takes place two to three weeks before flowering begins and may continue during
flowering. Harvesting of ergotized ears of rye takes place about two months later. This
method has numerous disadvantages. Firstly, only one crop a year can be obtained.
Secondly, the yield of alkaloid in terms of quality (type) and quantity is highly
unpredictable. Thirdly, the vicissitudes of the weather attendant on all field operations
make the operation highly unstandardizable.
(b) Fermentation production: Due to the above problems, efforts have been put in over the
years to devise fermentation methods. A good measure of success has been achieved
especially for the clavine alkaloids and simple lysergic acid derivates. Ergot alkaloids
can be produced in submerged fermentation by Claviceps or Penicillium species, which are
used for their industrial production. Initial work in Japan showed that submerged
cultures did not produce the typical alkaloids associated with the sclerotium but instead
produced a series of new non-peptide bases (clavines) which did not possess any
significant pharmacological action. Attempts were made by many workers to influence
alkaloid production by modification of the culture medium and the fungus strain. The
first pure ergot alkaloid, ergotamine, was obtained by Stoll in 1920. Subsequently, others
reported the discovery of the “water soluble uterotonic principle of ergot” which was
subsequently determined to be ergonovine (also called ergometrine). As a result of further
successful experiments the commercial manufacture of simple lysergic acid derivatives
by fermentative growth of a strain of Claviceps paspali became feasible
(i) Production of clavine alkaloids: Different species of Claviceps parasitizing a variety of
grasses have been isolated and grown in liquid medium and the different alkaloids
assayed. Clavine alkaloids were obtained from Cl. litoralis, Cl. microcephals. One
strain Cl. purpurea produced a clavine and the other a pepetide alkaloid. The
medium used contained mannitol (5%) ammonium citrate (0.7%), KH2PO4 (0.1%)
and MgSO4 (0.03%). The pH was 5.2. The use of 10% sucrose instead of mannitol
gave higher yields.
The fermentation lasted from 30 days to 40 days and by classical strain
improvement methods yields of up to 1.0-1.5 gm/liter were obtained.
Improvements have since been obtained by other workers who found that a
mixture of mannitol (6.5%) and glucose (1%) gave up to 1 gm/liter yield in about
14 days. A high carbon to nitrogen in the medium increased yield.
(ii) Production of simple lysergic acid derivates: The production of simple lysergic acid
derivates was achieved in 1961 using Claviceps paspali isolated from an infected
Paspalum digitatum.
Higher yields have since been attained by slight modifications of the original
mannitol-succinic acid-mineral salts medium. Fumaric acid gave higher yields
when it was used in place of succinic acid. The fermentation lasted nine days. The
addition of hydrophilic non-ionic surfactants had the greatest effects on yield.
Aeration was vigorous.
(iii) Peptide alkaloids: The physiology of ergotamine formation has been studied using
Claviceps purpurea, C. litoralis, Elymus mollis. Improved ergotoxine yields were
Production of Ergot Alkaloids
"$
obtained by the mutation and selection of a strain of Claviceps purpurea. This strain
on a mannitol-ammonium-succinate medium produced up to 40 gm/liter.
25.4
PHYSIOLOGY OF ALKALOID PRODUCTION
(i) Induction: Tryptophan is the central precursor of all ergot alkaloids and it is
therefore required in the medium. In addition to being a direct precursor of ergot
alkaloids, tryptophan is also a factor in the induction and derepression of enzymes
necessary for alkaloid synthesis. If the amino acid is not added to the medium the
organism manufactures it. When it is added the organism accumulates it in its
hyphae.
(ii) Feedback regulation: Feedback regulation studies have been hampered by the cell
wall as studies using protoplasts of Claviceps sp. have unambiguously
demonstrated. It was shown that the addition of elymoclavine inhibited (not
repressed) the first enzyme in the synthesis of the alkaloid, namely dimethylollyl
tryptophane synthase (DMAT synthase). This was demonstrated by supplying to
washed stationary phase cultures of the producing organism, basal culture
medium or basal culture medium supplemented with elymoclavine. Cultures with
fresh medium synthesized the alkaloid at a slower rate than when supplemented.
Both of them however reached the same final alkaloid concentration.
(iii) Phosphate repression: Like many secondary metabolites ergot alkaloid formation is
inhibited by increasing the level of phosphate. In this case the unfavorable limit
was 1.1. gm/liter. The addition of tryptophan helped to nullify the effect of
phosphate, showing that phosphate inhibition was mediated through tryptophan
probably by preventing its accumulation from exceeding the amount required for
alkaloid synthesis induction. In support of this explanation, it is noted that 5methyltryptophan also overcomes the inhibition of alkaloid synthesis by
tryptophan. Phosphate inhibited culture had very low levels of the first enzyme in
the synthetic chain, namely DMAT synthase; tryptophan caused more than 10fold increase in the activity of the enzyme.
(iv) Catabolite regulation: High levels of glucose (5%) greatly inhibited enzyme
production. The addition of a small amount of camp restored alkaloid synthesis to
a little extent.
(v) Alkaloid formation and morphological structures: It is interesting to note that alkaloids
seem to form in one structure and accumulated in another. Thus, in the plant genus
Ipomea, alkaloids are formed in the leaves and accumulated in the seeds, which do
not produce alkaloids at all. In ergotoxine alkaloid fermentation, alkaloids are not
elaborated until specific morphological structures, pellets, are formed in the
medium. The medium composition appears to influence the formation of these
pellets. Sucrose appears to encourage their formation while they are poorly formed
in malt. The peptide alkaloids are found in these structures, while clavines and
simpler derivates of lysergic acid are found in the medium.
(vi) Biosynthesis: Work on biosynthesis of alkaloids has been greatly facilitated by the
use of protoplasts. Being secondary products, alkaloids are produced by pathways
different from those of general metabolism. Furthermore, synthesis initiates with
carbon limitation. The ergot skeleton is derived from tryptophan, mevalonic acid,
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Modern Industrial Microbiology and Biotechnology
and methionine. The central precursor of the ergoline skeleton is tryptophan,
which is initially dealkylated. The five-carbon unit is obtained from mevalonic
acid. Although a component of the ergoline unit, it is neither limiting nor does it
participate in the regulation of alkaloid synthesis induction. Mevalonic acid may
be formed from the malonyl COA pathway with biotin and decarboxylation.
Clavines are simpler in terms of biosynthesis than lysergic acid derivates.
Lysergic acid itself is a key substrate in ergot alkaloid synthesis and the simpler
amide as well as the peptide derivates come from it (see Fig. 25.5).
Surprisingly not many enzymes appear to be involved in the synthesis of
alkaloids. Two of them have so far been isolated: Dimethyl tryptophan synthase
(DMTPS) and chanoclavine synthase. DMTPS catalyses the first specific reaction
of the formation of 8-ergoline and introduces the isoprene residue to the C4 position
of tryptophan. Analogues of tryptophan increase the activity of the enzyme. The
compound produced, dimethyl tryptophan (4-isoprenyltryptophan) is 5-10 times
more effective as a precursor of the clavines than tryptophan.
Chanoclavine cyclase catalyses the cyclization of chanoclavine – 1 to the fourring 8-ergoline i.e. to elgmoclavine and agroclavine, both individually and
simultaneously. Some biogenetic relationships among alkaloids are shown in Fig.
25.5
(vii) Extraction of the alkaloid: Alkaloids are readily isolated at an alkaline pH by various
organic solvents such as ether, chloroform, and ethyl acetate.
Fig. 25.5
Synthetic Routes for Ergot Alkaloids
Production of Ergot Alkaloids
"$!
The earliest procedures used for extraction were designed to obtain alkaloids
from sclerotia. In general the sclerotia would be dried, powdered, alkalinized, and
extracted with tartaric acid. For further purification, the tartaric acid solution was
made alkaline and extracted with chloroform. This chloroform layer would then
contain all the alkaloids. This has led to the terms water-soluble alkaloids which
include the simple amide lysergic acid derivatives and the clavines, whereas the
water-insoluble alkaloids now include the peptide-type. Nowadays the method
adopted for both water-soluble and water-insoluble peptides is to extract the
powdered ergot with chloroform to which a small amount of methanolic ammonia
has been added. The chloroform extract is concentrated to a small volume, diluted
with ether and extrated with concentrated H2SO4. On neutralization with
ammonia the water-soluble alkaloids can be extracted with water and the waterinsoluble ones with carbon-tetrachloride.
SUGGESTED READINGS
Boichenko, L.V., Boichenko, D.M., Vinokurova, N.G., Reshetilova, T.A., Arinbasarov, M.U. 2001.
Screening for Ergot Alkaloid Producers among Microscopic Fungi by Means of the
Polymerase Chain Reaction, Microbiology, 70: 306-307.
Dongen, van P.W.J., de Groot, A.N.J.A. 1995. History of ergot alkaloids from ergotism to
ergometrine, European Journal of Obstetrics & Gynaecology and Reproductive Biology, 60:
109-116.
Evans, W.C. 1996. Pharmacognosy. 14th ed, W B Saunders Company Ltd, London, UK.
Groot, N.J.A. de Akosua, van Dongen, Pieter W.J, Vree, Tom, B., Hekster, Yechiel A., van
Roosmalen, Jos. 1998. Ergot Alkaloids - Current Status and Review of Clinical Pharmacology
and Therapeutic Use Compared with Other Oxytoxics in Obstretrics and Gynaecology, Drugs,
56: 525-8.
Hardman, J.G., Limbird, L.E. (eds) 1996. The Pharmacological Basis of Therapeutics, 9th ed,
McGraw-Hill
Kobel, H., Kobel, J. 1986. Ergot alkaloids. In: Biotechnology, H.J., Rehm, G. Reed, (eds) Vol 4,. 2nd
Ed. VCH, Weinheim, Germany, pp. 569-609.
Komarova, E.L., Tolkachev, O.N. 2001. The Chemistry of Peptide Ergot Alkaloids,
Pharmaceutical Chemistry Journal, 35: 504-506.
Lange, Klaus W. 1998. Clinical Pharmacology of Dopamine Agonists in Parkinson’s Disease,
Drugs & Aging, 13: 385-386.
Langley, D. 1998. Exploiting the Fungi: Novel Leads to New Medicines, Mycologist, 11: 165-166.
Menge, J.M.M. 2000. Progress and Prospects of Ergot Alkaloid Research. Advances in
Biochemical Engineering/Biotechnology. 68, 1-20.
Thompson, F., Muir, A., Stirton, J., Macphee, G., Hudson, S. 2001. Parkinson’s Disease, The
Pharmaceutical Journal, 267: 600-612.
Votruba, V., Flieger, M. 2000. Separation of Ergot Alkaloids by Adsorption on Silicates,
Biotechnology Letters, 22: 1281-1282.
Rehacek, Z., Sajdl, P. 1990. Ergot Alkaloids: Chemistry, Biological Effects, Biotechnology.
Academia Praha: Czech Republic.
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26
Microbial Transformation of
Steroids and Sterols
26.1
NATURE AND USE OF STEROIDS AND STEROLS
Steroids are a large group of organic compounds with the perhydro- 1, 2-cyclopentano –
phenanthrene nucleus, which consists of four fused rings (Fig. 26.1).
Sterols are hydroxylated steroids – that is, they are alcohols derived from steroids. The
hydroxyl (OH) group of sterols is usually substituted at position C3. Unsaturation is
usually at C5 and often as C7 and C22. The term sterol comes from the Greek (Steros = solid)
because the earliest members studied were solid alcohols resulting from the
unsaponifiable (i.e. could not be broken down by NaOH) fractions of fats of plants and
animals. As the variety of known structures increased the general term steroid came into
use about 1935. In higher animals the principal sterol is cholesterol but a wider variety
exists in lower animals and in plants (Fig. 26.1).
Steroids and sterols are widely distributed in nature and are present in bile salts,
adrenal-cortical and sex hormones, insect molting hormones, sapogenins, alkaloids and
some antibiotics.
Steroids and sterols differ from each other in two ways: (a) the number, type, and
position of the substituents; (b) the number and position of the double bonds in the ring.
Steroid molecules are usually flat. However, the substituents at each of the junctions of
Rings A and B, Rings B and C, and Rings C and D may be either above or below the plane
of the ring. When the substituent group lies above the plane (denoted by a solid line) of the
molecule the substituent is denoted by >; when it is below (denoted by a broken line) it is
denoted by =. When as is the case in many steroid hormones a double bond exists
between C4 and C5 the situation is denoted ,4. The individual compounds are named
systematically as derivatives of steroidal hydrocarbons the more important of which are
gonane, estrane, androstane, pregnane, cholane and cholestane. Thus cortisone which is
a derivative of pregnane is ,4 - pregnene – 17=, 24 – diol – 3 11, 20 – trione.
The steroid hormones of the mammalian body have profound effects on the body
function and even the behavior of the animal. Thus, the male hormones, androgens
secreted by the testes are responsible for the development of the male reproductive organs
and the secondary sexual characters such as hairiness among several other functions.
Microbial Transformation of Steroids and Sterols
Fig. 26.1
"$#
Structures of some Steroids and Sterols
The female sexual hormones include estrogens and progesterone. Estrogens are
produced by the ovary – they stimulate the development of the female reproductive
organs and secondary sexual characteristics such as enlarged breasts, etc.
Progesterone is produced by the corpus luteum, a body formed by the mature egg in the
female ovary. In association with the oestrogens, progesterone prepares the uterus for the
implanation of the fertilized egg in the uterine wall. Corticosteroid hormones are
produced by the cortex surrounding the adrenal glands, which are themselves located
just above the kidneys. The main steroid hormones produced by the glands are
corticosterone, cortisol, and aldosterone. Aldosterone is involved with mineral
metabolism, mainly of sodium ions and hence indirectly of the blood pressure. Cortisol
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Modern Industrial Microbiology and Biotechnology
and corticosterone help the body handle physiological stress including extreme cold. It is
important to underscore, even at the risk of sweeping generalization, the significance of
hormones as a background towards appreciating the impetus for the transformation of
steroids.
Among insects steroid hormones are also very important in post-embryonic
development: juvenile hormones control larval growth; ecdysone controls
metarmorphosis of larval-larval larval-pupal and pupal-adult moulting processes; a
third hormone affects the brain and controls the production and release of the moulting
hormone. These hormones and their laboratory synthesized analogues (pheromones) are
used for controlling insects. Bile salts, sterols, oestrogens, progesterone, androgens
cortisone and cortisol and other steroids from animal and plants were isolated and
studied from 1903.
26.2
USES OF STEROIDS AND STEROLS
The world sale of steroids runs into billions of dollars (see Table 26.1)
26.2.1
Sex Hormones
As will be seen below, many steroids and sterols are manufactured through microbial
action. The largest economic impact of synthetic estrogen and progestin production has
been for use as contraceptive agents and for treatment and prevention of osteoporosis.
Contraceptive steroid mixtures have also been used to treat a variety of related abnormal
states including endometriosis, dysmenorrhea, hirsutism, polycystic ovarian disease,
dysfunctional uterine bleeding, benign breast disease, and ovarian cyst suppression.
Estrogens are routinely prescribed to post-menopausal women to prevent the
development and exacerbation of osteoporosis because it can increase bone density and
reduce fractures.
Table 26.1
Total worldwide sales of systemic sex hormones and corticosteroids
Sales, $x 106
Steroid class
Systemic sex hormones
Corticosteroids
Topical
Systemic
Respiratory
Nasal
Inhalants, systemic
Steroids for sensory organs
Total
1990
1994
3,582
5,436
1,558
903
988
382
606
396
7,427
1,891
1,181
2,170
665
1,505
507
11,185
Testosterone, alkylated testosterone, or testosterone esters are the primary anabolic–
androgenic steroid drugs. Most of these synthetic testosterone derivatives were in failed
attempts to separate the hormones’ masculinizing (androgenic) and skeletal musclebuilding (anabolic) effects. The medicinal uses for these drugs include treatment of
Microbial Transformation of Steroids and Sterols
"$%
certain types of anemias, hereditary angioedema, certain gynecological conditions,
protein anabolism, certain allergic reactions, and use in replacement therapy in gonadal
failure states.
Anabolic–androgenic steroids are best known for their nonmedical, and illegal, use to
aid in body-building or to increase skeletal muscle size, strength, and endurance by
athletes.
26.2.2
Corticosteroids
The greatest portion of steroid drug production is aimed at the synthesis of
glucocorticoids which are highly effective agents for the treatment of chronic
inflammation. Glucocorticoids exert their effects by binding to the cytoplasmic
glucocorticoid receptor within the target cell and thus either increase or decrease
transcription of a number of genes involved in the inflammatory process. Specifically,
glucocorticoids down-regulate potential mediators of inflammation such as cytokines
(Chapter 28). Typical oral glucocorticoids used to treat rheumatoid arthritis are
prednisone and 6 =-methylprednisolone. Corticosteroids are the most efficacious
treatment available for the long-term treatment of asthma, and inhaled corticosteroids are
considered to be a first-line therapy for asthma. They are also used to treat rhinitis, or
nasal congestion and inflammations of the skin.
26.2.3
Saponins
These are used for their hypocholesterolemic (cholesterol lowering) activity. Synthetic
steroids that are structurally related to saponins have been shown to lower plasma
cholesterol in a variety of different species
26.2.4
Heterocyclic Steroids
Dihydrogentesterone (DHT) is a more potent androgen than testerone. Elevated levels of
DHT lead to enlarged prostate (benign hyperplasia), sometimes prostate cancer, and
male baldness and the enzyme antagonistic to DHT steroid 5 =-reductase is being
developed as treatment for these ailments.
26.3
MANUFACTURE OF STEROIDS
In 1937, the first microbial transformation of steroids was carried out. Testerone was
produced from dehydroepiandrosterone using Corynebacterium sp. Subsequently,
cholesterol was produced from 4-dehydroeticholanic acid and 7-hydroxycholesterol
using Nocardia spp. These developments were virtually unexploited until 1949, when the
dramatic curative effect of cortisone on rheumatoid arthritis, a disease in which painful
swellings occur at the joints of the body, was announced. The cortisone used in this work
had been prepared by complex and tedious chemical synthesis beginning with
deoxycholic acid, a bile acid. So tedious was this that it took 32 chemical steps and two
years to produce only 11 gm of cortisone acetate. Additionally, it was difficult to find
enough of the starting materials. To meet the demand for cortisone to treat the large
number of people suffering from rheumatoid arthritis, a great burst of activity along the
four following lines ensured:
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Modern Industrial Microbiology and Biotechnology
(i) The improvements of the original chemical method along lines suitable for
commercial production;
(ii) The development of methods of chemical synthesis using other more available and
more abundant starting materials including steroids and steroid-containing
compounds of plant origin. One of such compounds was diosgenin (a glycoside
formed from a steroid and glucose) obtained from a species of yams, Dioscorea
composita and the South African ‘elephant foot’ Testudinaria sylvatica. The other
was a plant sterol, stigmasterol obtained from soy bean, Glycine max.
(iii) Total chemical synthesis of cortisone and cortisol from relatively simple materials;
(iv) The use of biological agents to transform readily available steroids by introducing
oxygen at carbon C11, a process which took 12 steps in chemical synthesis. The use
of biological agents originally consisted of the use of ground or homogenized
adrenal tissues and fungi.
The first of the two methods mentioned above had moderate successes and for some
time provided steroids for clinical use. The third method remained an academic exercise.
The fourth method however gave dramatic results of which microbial transformations
eventually became more important. Two of the earliest such microbial transformations
were the conversion of progesterone to 11 – a hydroxy progesterone by the introduction of
– OH at the position 11 using Rhizopus nigricans and the conversion of cortisol to
prednisolone by Corynebacterium simplex (Fig. 26.2).
Fig. 26.2
Some Steroid Transformations Brought about by Microorganisms
Microbial Transformation of Steroids and Sterols
"$'
This latter transformation was notable because the new product was more active than
the starting one. Since then a large number of steroid analogues have since been
produced using a wide variety of microorganisms. Indeed virtually every steroid is
transformable in some way by some microorganism or the other.
From the beginning of 1960, intensive research interest shifted from rheumatoid
steroids to the area of sex hormones, especially the progresterone-based drug principally
used for birth control pills. This shift of interest was as a result of concern for rising world
population. The disclosure about 1965 of the steroidal nature of insect hormones
stimulated interest in them as a means of controlling insect pests of agriculture and food
and vectors of disease.
A large number of steroids have since been produced and tested for a variety of
purposes. Several of them have been found useful as anti-inflammatory, anti-tumor and
anti-allergy drugs; as birth control pills; for treatment in heart disease and a vast array of
medical and veterinary uses.
The use of microorganisms to transform steroids revolutionized the steroid
transformation industry. For example the price of cortisone, a widely used antiinflammatory drug, fell from US $200 per gm in 1949 to less then US $1.0 per gm in 1979
as a result of this development.
The microbial transformation of steroids differs from the ‘traditional’ fermentations
such as that of penicillin thus:
(i) In many cases steroid transformations are one-step-processes which bring about
relatively minor structural changes in the substrate, i.e. the steroid molecule. This
differs from the synthesis of penicillin and many other fermentation products in
which the product is synthesized entirely from the substrate offered in the medium.
(ii) Whereas in many industrial fermentations, the process of production is completed
in the fermentor, in the case of steroid transformations, readily available steroids
are micro-biologically transformed into important intermediates which are then
converted chemically to the final product. Alternately, the chemical syntheses are
first performed and the products transformed microbiologically later.
26.3.1
Types of Microbial Transformations in
Steroids and Sterols
Transformations by microorganisms affecting various positions in a wide range of
steroids and sterols have been carried out. Although steroid hormones have been most
widely studied, the transformation of bile acids, plant and animal sterols, steroid
alkaloids have also occurred. The transformation reaction include: hydroxylation,
dehydrogenation, reduction, side chain degradation, lactone formation, aromatization,
isomerization, epoxidation, hydrolysis, esterification, halogenation, and cleavage of the
steroid skeleton.
All of these have been carried out on steroid hormones, but only some of them have
been done on the other natural steroids and sterols. Two examples of these reactions have
already been described: Progesterone is converted to 11= - hydroxyl progesterone by
hydroxylation or the introduction of an OH group at position 11. Similarly, cortisol is
converted to prednisolone by dehydrogenation at position 1. Some other examples are
given in Fig. 26.2.
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Modern Industrial Microbiology and Biotechnology
A major transformation in which interest has grown sharply in recent times is the
cleavage of the C17 side chain of sterols. An important source of steroids for the synthesis
and production of pharmacologically active steroids used in contraceptives,
corticosteroids, geriatic drugs etc. is diosgenin (Fig. 26.1) from Dioscorea spp. Due to the
shortage of diosgenin, interest has shifted to more abundant sterols from phytosterols (i.e.
sterols from plants) and cholesterols from animals. The phytosterols include soy bean
sterols mainly >-sitosterol and stigmasterol and tall oil sterols mainly sitosterol and
campesterol. For these to be used as starting materials for the production of progesterone
and other drugs, the C17 side chain must be cleaved hence the interest. The microbial
removal of the side chain offers more promise than chemical means. Unfortunately microorganisms which cleave off the side-chain will also attack the D ring to which the chain
is attached. Three methods have therefore been evolved to solve the problem of inhibiting
ring degradation, while cleaving the chain.
(i) The substrate may be modified structurally by chemical means so that the ring is
stable while the side-chain is cleaved. Thus while cholesterol rings are degraded
when the side-chain is cleaved by Nocardia sp. 3-Acetoxy-9- hydroxy-5-cholestene
is not. This later compound can be prepared from cholesterol by three chemical
steps. The cleavage of the side-chain of cholesterol yields esterone which can then
be used for further transformations.
(ii) The enzymes which open the D nucleus may be selectively inhibited. The key stage
in the opening of the ring is at the ninth position and since the enzyme for this
hydroxylation contains metals, the enzyme and its process may be inhibiting by
using chelating agents which remove metals from them.
(iii) Finally, mutants have been developed which will degrade only the side chain. One
of the best known is a mutant of Mycobacterium sp.
26.3.2
Fermentation Conditions Used in Steroid Transformation
The media used are highly variable, but in the main are not very complex. They are
basically mineral salts media containing some carbon source such as glucose, dextrin or
glycerol. Nitrogen sources may be ammonium salts, corn steep liquor, soybean, or a
protein digest. In some cases yeast extract is added.
Steroid and sterols are lipids; they are not water soluble and therefore must be
dissolved in a water-miscible lipid-solvent. Acetone, ethanol, propylene glycol, and
methanol are suitable because they dissolve a reasonable amount of the steroid while
being relatively non-inhibitory to the enzymes; dimethyl formamide dissolves a
reasonable amount of the steroids but has only a minimum of toxicity. Sometimes the
steroid is added in small amounts at a time. In this way, any toxic effect of the solvent is
minimized.
The level of steroid added is variable and depends both on the transforming ability of
the organisms as well as its susceptibility to the toxic effects of the steroid. Normally 200800 mg/litre are added but much higher amounts are sometimes used. To solve the
problem of the insolubility of steroids in water, non-ionic surface-acting agents which
reduce surface tension e.g. Tween 80 are often added to the medium. Some polysaccharides in the medium e.g. yeast cell wall mannan, bind to the steroids and cause
them to be more available to the organism.
Microbial Transformation of Steroids and Sterols
"%
A wide range of microorganisms, mainly fungi and bacteria, are used in the
transformation of steroids. Some of these include the fungi Rhizopus nigricans, Curvularia
lunata, Fusarium spp. Cylindrocarpon radicicola as well as the bacteria Mycobacterium spp.,
Corynedbacterium simplex, and Streptomyces spp. As has been mentioned, there are
organisms to perform just about any conceivable transformation of the steroid molecule.
The transformation may occur at different stages of the growth and the steroid may be
added to the growing cultures either simultaneously with the inoculation of the culture
or the resting or stationary stage of the organism. Fungal spores may sometimes be
inoculated as the steroid is introduced into the medium. In recent times immobilized cells
have been employed in the transformations of steroids.
Steroid transformations require vigorous aeration and a temperature of about 28°C is
usually employed. The fermentation is usually complete in four to five days.
26.4
SCREENING FOR MICROORGANISMS
The screening for microorganisms capable of transforming steroids to yield products of
useful pharmacological properties is a continuing one. The processes which are followed
in the screening are as follows:
(i) The microorganism is isolated from soil or some suitable source and grown in a
suitable medium for 24-28 hours.
(ii) The steroid in a suitable carrier is added to the fermentation and the growth
continues for a further period which could be as long as one week.
(iii) The transformation products are extracted with solvents such as methyl acetate
and purified by chromatography etc.
(iv) The product is tested for pharmacological properties.
(v) Finally, the structure is elucidated by classical methods of organic chemistry.
SUGGESTED READINGS
Flickinger, Michael C., Drew and Stephen W. 1999. Encyclopedia of Bioprocess Technology Fermentation, Biocatalysis, and Bioseparation Wiley. Electronic ISBN: 1-59124-457-9.
Martin, C.K.A. 1984. Sterols. In: Biotechnology. Kiesich (ed) Vol 6A Biotransformations Verlag
Chemie. Weinheim: Germany. pp. 79–96.
Morgan, B.P., Moynihan, M.S. 1997. Steroids. Kirk-Othmer Encyclopedia of Chemical
Technology, 2, 71-113.
Smith, L.L. 1984. Steroids. In: Biotechnology. Kiesich (ed) Vol 6A Biotransformations Verlag
Chemie. Weinheim Germany: pp. 31-78.
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+0)26-4
27
Vaccines
27.1
NATURE AND IMPORTANCE OF VACCINES
Vaccines are materials which when introduced into the human body help protect the
vaccinated person against specified communicable diseases. Communicable diseases
are diseases caused by microorganisms, including viruses. Vaccines are preparations of
dead or weakened pathogens, or their products, that when introduced into the body,
stimulate the production of protective antibodies or T cells without causing the disease.
Vaccination is also called active immunization because the immune system of the
body is stimulated to actively develop its own immunity against the pathogen. Passive
immunity, in contrast, results from the injection of antibodies formed by another animal
(e.g., horse, human) which provide immediate, but temporary, protection for the recipient.
The name ‘vaccine’ comes from the Latin vacca (for cow). This is because the earliest
vaccination was done using the cow pox virus (which causes the disease in cow) as a
vaccine against small pox in humans. The English physician, Edward Jenner carried out
the above vaccination in the late 18th century and published his paper in 1798.
Over the past 200 or so years vaccination has contributed greatly to reducing
morbidity and mortality from communicable diseases. The greatest triumph of
vaccination is the eradication of smallpox from the earth; no naturally-occurring cases
has been reported since 1977. A program to try to eliminate another virus disease,
poliomyelitis (polio for short), from the world has been on for some time and the
indications are that the number of cases has drastically dropped. Except for the few cases
caused by oral polio vaccine (OPV) (see below), in which the live virus reverts, the disease
has now been eliminated from the Western hemisphere. Outbreaks of polio still occur in
Africa, the Indian subcontinent, and parts of the Near East. Due to the success of
vaccination near 100% reduction has been obtained in the cases of many diseases which
were previously sources of great mortality and morbidity. These include diphtheria,
measles, mumps, pertusis, rubella and tetanus. Table 27.1 gives a list of the most
commonly used vaccines today.
27.2
BODY DEFENSES AGAINST
COMMUNICABLE DISEASES
In order to better understand the nature of vaccines and their design and production, it is
important that the defenses of the human body against communicable diseases be
Vaccines
"%!
Table 27.1 Vaccines most commonly used in the world
Disease
Preparation
Notes
Diphtheria
Toxoid
Tetanus
Toxoid
Often given to children in a
single preparation (DTP; the
‘triple vaccine’) or the nowpreferred DTaP using acellular
pertussis
Pertussis
Killed bacteria (‘P’) or their
purified components
(acellular pertussis = ‘aP’)
Inactivated virus previously
grown on monkey or
human diploid cells
Attenuated virus
inactivated virus
previously grown on
monkey or human
diploid cells
Protein (HBsAg) from
the surface of the virus
uses acellular pertussis and
IPV (Salk)
Polio
Hepatitis B
Diphtheria, tetanus,
pertussis, polio, and
hepatitis B
Measles
Mumps
Rubella
Chickenpox
(Varicella)
Influenza
Pneumococcal
infections
Staphylococcal
infections
Attenuated virus
1 Attenuated virus
2 Vaccine: Live in duck cells
Attenuated virus
Pig, chick embryo or
canine tissue-culture grown
Attenuated virus
Egg-grown virus, formalin
inactivated, highly purified
by zonal ultracentrifugation
Hemagglutinins
Capsular polysaccharides
7 capsular polysaccharides
conjugated to protein
2 capsular polysaccharides
conjugated to protein
Inactivated polio vaccine: IPV
(Salk)
Oral polio vaccine; OPV (Sabin)
Both vaccines trivalent (types 1,
2, and 3)
Made by genetic engineering
Pediarix®; combination
vaccine given in 3 doses to
infants
Often given as a mixture
(MMR) Does not increase the
risk of autism. (Nor do any
vaccines containing thimerosal
as a preservative.)
Caused by the varicella-zoster
virus (VZV)
Contains hemagglutinins
from the type A and type B
viruses recently in circulation
A mixture of the capsular
polysaccharides of 23 common
types. Works poorly in infants.
Mobilizes helper T cells; works
well in infants.
To prevent infection by Staph.
aureus in patients hospitalized
and/or receiving dialysis
Contd.
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Modern Industrial Microbiology and Biotechnology
Table 27.1
Contd.
Disease
Preparation
Notes
Meningococcal disease
Polysaccharides
Used chiefly to prevent
outbreaks among the military
Prevents ear infections in
children
Available in single shot with
HBsAg (Twinrix®)
Vaccine prepared from human
diploid cell cultures (HDCV)
has replaced the duck vaccine
(DEV)
Hemophilus influenzae, Capsular polysaccharide
type b (Hib)
conjugated to protein
Hepatitis A
Inactivated virus
Rabies
Smallpox
Inactivated virus
Active:
(1) >-propiolactoneinactivated virus grown in
embryonated duck eggs
(2) phenol-inactivated virus
grown in rabbit brain
Passive: equine hyperimmune serum
Attenuated live virus:
attenuated by passing
through calves
Anthrax
Extract of attenuated bacteria
Typhoid
Three available:
1. killed bacteria
2. live, attenuated
bacteria (oral)
3. polysaccharide
conjugated to protein
Live attenuated virus
Prepared in chick embryo:
Dakar strain or 17D strain
Live attenuated mycobacterium BCG (Bacille calmette
Guerin) strain (BCG)
Yellow fever
Tuberculosis
Despite the global eradication
of smallpox, is used to protect
against a possible bioterrorist
attack
Primarily for veterinarians and
military personnel
Rarely used in the US
discussed briefly. Ordinarily the human body is surrounded by microorganisms: in the
air it breathes, the water it drinks, in the soil around it and on the clothes he wears. Most
of these are not normally pathogenic. But even the pathogenic ones do not always cause
disease when they come in contact with the human body because the body has evolved
ways of dealing with microorganisms and preventing them from causing disease,
collectively known as the immune system. The immune system is a complex network of
cells and organs which work together to protect the body from communicable diseases. It
has two components: the innate or non-specific immunity and the acquired or specific
methods. While the innate immunity eliminates the organism no matter the type,
acquired or specific immunity specifically recognizes and selectively eliminates the
microorganism or foreign molecule.
Vaccines
27.2.1
"%#
Innate or Non-specific Immunity
The innate or non-specific defense mechanisms are the first line defense against invading
microorganisms. They will act irrespective of the type of microorganism. Briefly they
consist of the following:
(a) Anatomic barriers: these include mechanical barriers such as skin, which physically
keeps out microorganisms and mucous membranes of the alimentary canal
respiratory and urinogenital tracts which entrap microorganisms. In addition the
mucous membranes harbor a normal set or flora of microorganisms which keep out
foreign organisms.
(b) Physiologic barriers: The physiology of the human body keeps out some pathogens.
Thus the high temperature of the human body, including the fever response keeps
out some microorganisms as does the acidic nature of the stomach. Chemical
mediators such as lysozyme found in tears breakdown bacterial cell walls.
(c) Phagocysis and endocytosis: white blood cells kill and digest whole microorganisms, while specialized cells engulf and breakdown foreign particles.
(d) Inflammatory responses: Tissue damage and infection induce leakage of vascular
fluid serum protein with antibacterial fluid and influx of white blood cells leading
to pus formation.
27.2.1.1
Acquired or Specific immunity
Acquired or specific immunity has three important properties among others, which are
crucial in understanding vaccines and how they function.
(i) Antigenic specificity: An antigen is a material usually a protein which binds
specifically to an antibody or to T-cell receptor (see below). Great specifity occurs in
the anatigen-antibody or antigen-T-cell receptor relations. Often a small difference
of a single amino acid can decide whether or not binding to antibody or T-cell will
take place.
(ii) Immunologic memory: Once the acquired immune system has recognized and
responded to an antigen, it exhibits immunologic memory: a second encounter
with the same antigen induces an increased response.
(iii) Self/non-self recognition: Specific immunity recognizes foreign bodies in
contrast to those of the body and seeks to destroy the intruders. In rare cases the
system of recognition breaks down and the system fails to recognize body cells and
proceeds to destroy them, giving rise to auto-immune diseases.
Specific immunity has two components, humoral and cell-mediated immunity which
are mediated by white blood cells known as lymphocytes, and as will be seen below both
sectors are linked. Humoral immunity is mediated by B Lymphocytes, while cellmediated immunity is brought about by T Lymphocytes. White blood cells including
lymphocytes and like red blood cells are produced from stem cells in the bone marrow.
The B lymphocytes, remain in the bone marrow to mature, while T lymphocytes mature in
the thymus, a small organ located above the heart.
27.2.2.2
Specific immunity: humoral immunity
Humoral immunity is also known as antibody immunity. Antibodies are soluble proteins
in the blood which bind to foreign agents and mark them for destruction, or neutralize
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Modern Industrial Microbiology and Biotechnology
toxins produced by microorganisms. Also known as immunoglobulins, antibodies are
glycoproteins by nature (i.e. proteins to which carbohydrates are conjugated). (see Fig.
27.1)
-S-S-, Disulphide bonds; CHO, carbohydrate molecule attached to the constant region of the heavy chain;
CH2, CH3, Constant regions of the heavy chain in the biological activity end of the antigen molecule; C L,
Constant region of the light chain; VL, Variable region of the light chain; CH1, Constant region of the heavy
chain in the constant part antigen binding end; VH, Variable region of the light chain (see text)
Fig. 27.1 General Structure of an Antibody Molecule
When B lymphocytes mature each has one unique antigen-binding molecule, an
antibody attached to its membrane; up to 10 different antibody molecules may be carried
on the B lymphocytes. When such a mature B lymphocyte which has not encountered any
antigen, known as a naïve lymphocyte, encounters an antigen for which its membrane
bound antibody is specific, it begins to divide rapidly and differentiates into two types of
cells: memory cells and plasma cells. The memory cells have a longer span of life and
continue to express membrane-bound antibody just like the original parent cell. The
plasma cells, on the other hand live for four to five days and do not have cell-membrane
bound antibodies; instead they produce antibody in a form in which it can be secreted,
often in huge amounts, sometimes reaching 2,000 molecules per second. The memory
cells are the source of the long-term protection which vaccines confer.
Antibodies (immunoglobulins) are Y- shaped and consist of two identical light chains
and two identical heavy chains (Fig. 27.1). The upper end of the Y of the light and heavy
chains of the antibody molecule is the variable region. The amino acids in this region vary
greatly among different antibodies and this variability confers on antibodies the vast
specificity for which they are known. The lower ends of the light and heavy chains are the
‘constant’ regions and do not show the variability found at the tips of the Y. The
variability in protein composition in the ‘constant’ region of the heavy chains (Fig. 27. 1)
Vaccines
"%%
leads to antibodies being divided into five major classes, each with a different and
distinct property: IgG, IgA, IgD, IgM, and IgE. IgG is the most abundant (80%) of all Igs,
and it is the only one able to cross the placenta, helping to confer maternal immunity on
the newborn.
An antibody recognizes an antigen in a specific manner and the immune system
acquires memory towards it. The first encounter with an antigen is known as the primary
response. Re-encounter with the same antigen causes a secondary response that is more
rapid and powerful. This is the basis on which vaccines function; they induce the
memory lymphocytes to proliferate and the resulting plasma cells to produce soluble
antibodies (Fig. 27.2).
Antibodies are proteins known as immumoglogulins (Ig). The are five different kinds of immunoglobulins
IgA, IgD, IgE, IgG, and IgM. The animal body produces antibodies when challemged with materials to which
the body can react by producing antibodies (known as antigens). When the animal body is challenged with
the same antigen a second time the production of antibodies is not only produced in a shorter time, but the
antibody production is more pronounced as shown in the figure above. In the figure above IgM is produced
in the first challenge and IgM in the second. (see text).
Fig. 27.2 Antibody Response of the Animal Body to a Second Challenge of an Antigen
27.2.2.3
Specific immunity: cell-mediated immunity
While B lymphocytes mediate antibody or humoral immunity, T lymphocytes are
responsible for cell-mediated immunity. T lymphocytes do not have cell membrane
bound antibodies, nor do they secrete antibodies. Instead they have T-cell receptors
(TCRs). Unlike antibodies which can recognize antigens directly, T-cell receptors can
recognize an antigen only if the antigen is associated with cell membrane proteins
known as major histocompatibility compatibility (MHC) molecules, of which two classes
exist: MHC I and MHC II.
When a naïve T cell encounters an antigen associated with an MHC on a cell, the T cell
proliferates and differentiates into memory T cells, T helper cells (TH), and T cytotoxic
cells (TC). TH and TC cellscarry on their membranes different glycoproteins. TH cells carry
glycoprotein CD4, while TC cells carry CD8.
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Modern Industrial Microbiology and Biotechnology
When a TH cells interacts with an antigen linked to an MHC II compound, it is
activated to produce cytokins which also activate B cells to produce antibodies. Cytokins
also activate TC cells when they interact with an antigen linked to an MHC I compound,
to differentiate into cytoxic T lymphocytes (CTLs). CTLs do not secrete cytokins; instead
they monitor the body cells and eliminate any cells which are foreign or contain foreign
bodies such as cancer cells or cells containing viruses. To ensure that self cells are not
attacked by CTLs, they attack only cells displaying foreign foreigns complexed to an
MHC molecule on the surface of cells called antigen presenting cells (APCs). Antigen
presenting cells adsorb foreign antigens such as viruses, digest them and display the
peptides from them on their surfaces. CTLs identify such cells and destroy them. APCS
are specialized white blood cells. The relationships between B lymphocytes, T
lymphocytes, cytokins, Tc cells and CTLs are depicted in Fig. 27.3. The cell-mediated
immune response is important in cases where the pathogen is intracellular as in viruses
Fig. 27.3 Scheme Showing Immune System in Man
27.2.2.3
Antigens and Epitopes
Antigens are macromolecules that elicit an immune response in the body. Antigens can
be proteins, polysaccharides or conjugates of lipids with proteins (lipoproteins) and
polysaccharides (glycolipids). Antigens are generally very large and complex and the
lymphocytes may not recognize all the sites of a particular antigen. Rather both B and T
lymphocytes recognize discreet sites on an antigen known as epitopes or antigenic
determinants. The aim in vaccine production is to ensure that epitopes exist on the vaccine
which will elicit humoral or cell-mediated response.
Vaccines
27.3
"%'
TRADITIONAL AND MODERN METHODS OF
VACCINE PRODUCTION
Traditionally three types of vaccines have been used: attenuated live vaccines, killed
vaccines and bacterial toxoids. Recent advances in molecular biology and genomic
science have spilled over into vaccine production. This chapter discusses the traditional
vaccines, but will also discuss the newer approaches which have been influenced by
advances in molecular biology and genomic science.
27.3.1
27.3.1.1
Traditional Vaccines
Live attenuated organisms
In live attenuated vaccines, the organism has been cultured so as to reduce its pathogenicity,
but still retains some of the antigens of the virulent form. They consist of the living pathogens whose virulence has been reduced (attenuated) by passaging them through hosts
different from the usual. Alternatively, non-virulent strains of the pathogen may be used.
Live agents may be used for one or more of the following reasons:
(i) When the protection-inducing substance is produced as a diffusible product of
metabolizing organisms e.g. Bacillus anthracis.
(ii) When it is not feasible to produce sufficient amounts of nonviable agents and a
small concentration of the living agent can propagate within the vaccinated
subject to overcome the deficiency.
(iii) When immunity is induced by the modification of parasitized cells.
Live vaccines in use include those against polio (Sabin oral polio vaccine - OPV), foot
and mouth disease of farm animals, mumps, measles, rubella (German measles),
tuberculosis, rabies and yellow fever. For tuberculosis the vaccine is derived the Bacillus
Calmette-Guérin (BCG) strain of Mycobacterium tuberculosis, a weakened version of the
bacterium that causes tuberculosis in cows. BCG is used as a vaccine against
tuberculosis in many European countries; it is however not commonly used in the U. S.
The OPV has advantages and disadvantages when compared with the (inactivated)
Salk polio vaccine (IPV). OPV can be given by mouth rather than by injection, and it can
spread to the other members of the vaccinee’s family thus immunizing them as well. Its
disadvantage is that on rare occasions, the virus regains full virulence and cause the
disease. On account of this, the Salk vaccine has gained prominence over the Sabin
vaccine in some countries.
27.3.1.2
Killed vaccines
These consist of suspensions of fully virulent organisms (bacteria or viruses) killed as
mildly as possible in order not to destroy the antigenic determinants on the organism.
Killing can be achieved by heat, (usually about 60°C for 1 hour) chemicals (phenol,
alcohol, formalin, >-propiolactone) or ultraviolet irradiation. Killed vaccines do not
provide as prolonged antigenic stimuli as living vaccines and two, three or more subcutaneous injections are required to give adequate protection. Examples of killed
vaccines include TAB vaccine against typhoid fever and which consists of heat-killed
phenol-preserved suspension of Salmonella typhii and Salm. Paratyphii A & B, whooping
cough, cholera, and the Salk IPV.
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27.3.1.3
Bacterial toxoids
Toxoids are inactivated bacterial exotoxins. The toxins from Clostridium botulinum,
Clostridium tetani and Corynebacterium diphtheriae are inactivated by treatment in
formalin. Toxoids induce antibody production when injected into the body, although
they are themselves harmless. In some diseases, of which diphtheria and tetanus are
good examples, the bacterial metabolite, a protein toxin which they liberate, is the cause
of the disease and not the bacteria themselves. Exposing the toxin with formaldehyde,
denatures the protein. However, some epitopes on the protein molecule are retained and
they elicit antibody production.
27.3.2
Newer Approaches in Vaccinology
The advent of genomics, proteomics, and biotechnology, as well as the increased
understanding of pathogenesis and immune responses to various pathogens have led to
the development of safer, more effective and cheaper vaccines. Some of these are
described below.
27.3.2.1
Sub-unit or surface molecule vaccines
Subunit vaccines contain antigens or epitopes that induce protection rather than the
whole organism. The materials usually come from the surface of the organism and hence
they are also known as surface molecule vaccines. The potential advantages of using
subunits as vaccines are the increased safety, less antigenic competition, since only a few
components are included in the vaccine. One of the disadvantages of subunit vaccines is
that they generally require strong adjuvants and these adjuvants often induce tissue
reactions. (Adjuvants are compounds administered with vaccines so as to increase the
immunogenicity of the vaccines.) Second, the duration of immunity is generally shorter
than with live vaccines. Sometimes peptide epitopes may be used. Apart from requiring
adjuvants, a pathogen can escape immune responses to a single epitope; hence several
peptides linked together are used to broaden the immune response to different epitopes.
Subunit vaccines are currently available for typhoid and whooping cough. Several
vaccines employ purified surface molecules. One of them, the influenza vaccine contains
purified hemagglutinins from the viruses currently in circulation around the world.
Another example is vaccine against hepatitis B virus. The gene encoding a protein
expressed on the surface of the virus, the B surface antigen or HBsAg, can now be
expressed in E. coli cells and provides the material for an effective vaccine; hepatitis B
infection is strongly associated with the development of liver cancer. For the vaccine
against Streptococcus pneumoniae which causes pneumonia in humans about 80 different
strains of the organism are used. They differ in the chemistry of their polysaccharide
capsules which surround them and the current vaccine consists of purified capsular
polysaccharides of the 23 most common strains.
27.3.2.2
Conjugate vaccines
These are similar to subunit vaccines in the sense that only a part of the organism is used
in making the vaccine. Some bacteria which are encapsulated cause important childhood
diseases such as septicemia, pneumonia and meningitis. The bacteria are Hemophilus
influenzae type B (HiB), Neissseria meningitides and Streptococuus pneumoniae. The capsules
Vaccines
"&
of these bacteria are made of carbohydrates which the immune system of adults recognize
as foreign, but which that of infants do not and hence cannot make antibodies against
them. To solve the problem protein from diphtheria or tetanus toxoids is linked or
conjugated to the carbohydrate to make a vaccine. This enables a baby’s immune system
to respond to the combined vaccine and produce antibodies, initiating an immune
response against the disease-causing organism. The licensed conjugate vaccines against
Haemophilus influenzae type b (Hib), previously the major cause of bacterial meningitis in
babies and young children, have virtually eliminated the disease in the United States.
27.3.2.3
Other (Experimental) vaccines
(i) Polynucleotide (DNA) Vaccines
A recent development in vaccinology is immunization with polynucleotides. This has
been referred to as genetic immunization or DNA immunization. The rationale for this is
that cells can take-up DNA and express the genes within the transfected cells. Thus, the
animal body itself produces the vaccine. This makes the vaccine relatively inexpensive to
produce. Some of the advantages of polynucleotide immunization are that it is extremely
safe, induces a broad range of immune responses (cell-mediated and humoral
responses), long-lived immunity, and, most importantly, can induce immune responses
in the presence of maternal antibodies. Most recently, it has also been used for
immunizing animal fetuses. Thus, animals are born immune to the pathogens and at no
time in the animal’s life are they susceptible to these infectious agents. Although an
attractive development, there is a great need to develop better delivery systems to improve
the in vivo efficiency.
(ii) Edible Vaccines
Edible vaccines can safely and effectively trigger an immune response against the
Escherichia coli bacterium and the Norwalk virus. Attempts are being made to genetically
engineer potatoes, bananas, and tomatoes that, when eaten, will initiate an immune
response against harmful intestinal bacteria and viruses.
27.3.2.4
Reverse vaccinology
The name reverse vaccinology mimics the established term reverse genetics - meaning the
process of identifying a protein or enzyme through its gene product. Despite the
numerous successful vaccines produced over the last 200 years of vaccinology and the
advances made in vaccine discovery techniques, there are still problems with existing
approaches. Traditionally, the initial point in preparing a vaccine is to grow the
pathogen, which could often be very fastidious and difficult to grow, then the antigens
had to be identified one by one. Sometimes many years might be spent just working one
antigen, and traditionally only the most abundant and the easiest to purify and identify
(and not necessarily the best) have been studied.. To begin with however, not all
pathogens will successfully grow in vitro as is required for conventional methodologies,
with Hepatitis B and C viruses being prime examples of such organisms. With reverse
vaccinology the organism need not even be seen; the genomic constitution of the
organism already in databases need only to be accessed in silico. Reverse vaccinology
utilizes the wealth of information provided by genome sequencing to identify and
characterize a whole host of new vaccine targets.
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Modern Industrial Microbiology and Biotechnology
The technology has two major facets, in silico and in vitro/vivo. The in silico aspect is the
identification, annotation and then localization of ORFs and their products. Identified
targets can then be used for laboratory study (in vitro/in vivo) where they are expressed,
purified and tested for immunogenicity. Genome-based vaccine discovery was applied
for the first time to serogroup B meningococcus, a bacterium which is a major cause of
sepsis and meningitis, that had defied all traditional approaches to vaccine development.
The in silico sequence of the genome predicted 600 potential antigens. Of them 350 were
expressed in Escherichia coli, purified and used to immunize mice. Twenty nine were
found to induce bactericidal antibodies, which will lead to protection. A subgroup of the
genome-derived antigens is now being tested in clinical trials. Reverse vaccinology is
now a standard technology. Vaccines projects are not now undertaken without
knowledge of the the sequence of the pathogen. Successful examples of genome-based
vaccine discovery are pneumococcus, group B streptococcus, Staphylococcus aureus, and a
variety of viruses.
27.3.2.5
Some definitions of terms used in dealing with vaccines
(i) Active natural immunity is immunity arising from a natural disease. Small pox for
example, is suffered once in a life-time because of the immunity conferred on the
sufferer by the disease, due to antibodies produced during the illness.
(ii) Active artificial immunity is due to the use of vaccines, toxoids, etc., which stimulate
the individual to produce his own antibodies.
(iii) Passive natural immunity is most easily exemplified by maternal immunity in which
new-born animals are immune from certain disease for a short period early in their
lives due to the crossing of the placenta by certain antibodies (immunoglobulins).
In some animals including man maternal immunity is also acquired by the
young’s consumption of colostrum (thick cream-colored milk produced during the
first few days after childbirth.
(iv) Passive artificial immunity occurs when ready-made antibodies are introduced into
the body. An example is the use of anti-tetanus serum in which serum from a horse
which has been immunized against tetanus is used to protect an individual
against tetanus.
27.4
PRODUCTION OF VACCINES
27.4.1
Production of Virus Vaccines
Viruses multiply only in living cells. Viruses to be used for vaccine production must
therefore be grown in such cells. In practice they are grown for vaccine purposes in tissue
cultures which will first be described briefly below.
27.4.1.1
Tissue cultures and their cultivation
The growth of animal cells in vitro in monolayers is known as tissue or cell cultures.
Tissue cultures will be discussed briefly here because they are used in an area of
industrial microbiology which deals with the manufacture of biological materials used
in pharmacy, human medicine and veterinary practice. Such biologicals include
vaccines, interferon, hormones, immunological reagents and cellular biochemical such
as insulin, enzymes, plasminogen, and plasminogen activators.
Vaccines
"&!
(i) Cells used: The cells used in tissue cultures for the production of biolgocials are
derived from three sources:
(a) Primary cells are obtained by treating certain tissues derived from healthy animals
with disaggregating enzymes such trypsin and transferred for the first time into an
in vitro growth environment. Such tissues include decapitated avian embryo,
kidneys from virus-free green monkeys widely use for many biologicals (including
polio vaccines), rabbit kidney (for rubella and vccinia), calf kidney (for measles in
Japan).
When embryo of chicks or ducks are used, the avian embryos are harvested from
eggs, obtained from special isolated flocks, and then minced and treated with
disaggregating enzymes such as trypsin, collagenase, hyaluronidase, and
pronase. The fibroblast cells released by this process are attachment dependent,
requiring solid surfaces for growth. The successful commercial manufacture of
viral vaccines in attachment-dependent cell systems rely on the establishment and
maintenance of healthy cell monolayers. The appropriate growth and
maintenance media must be carefully selected, and careful attention must be paid
to nutrient depletion, waste accumulation, and changes in pH over time.
To produce measles and mumps vaccines, those viruses are grown and
attenuated by passage through cultures of chicken embryo cells. For foot-andmouth disease which affects farm animals a wide range of primary cells are used:
bovine kidney, goat heart, and lung, skin and kidney of camels.
(b) Diploid cells are obtained from well-defined human cell lines. Serially passaged
diploid strains of cultured human cells were first described in the 1960s and at the
concept of using human diploid cell lines for vaccine preparation was hotly
debated for fear that cancer-causing DNA or human viral agents might be
unknowingly co-administered with the vaccine. Extensive karyological (i.e.,
chromosomal) characterization and thorough searches for viral contaminants
were necessary to ensure that cultures were free of exogenous infectious agents
before the first diploid cell product, poliomyelitis vaccine, was licensed in the
United States. Such diploid cell lines must have shown a karyology or
chromosome characteristics identical with the parent tissue, be free from bacterial,
viral or fungal contaminants. ). Human diploid cell lines (WI-38 or MRC-5) have
since been used to produce a number of licensed vaccine products against
poliovirus, adenovirus types 4 and 7, rubella (German measles) virus, rubeola
(measles) virus, rabies, hepatitis A, and varicella virus (chickenpox).
(c) Established cell lines include those which are capable of growth for an indefinite
number of passages. They are used for veterinary rather than human vaccines. A
good example is the baby hamster kidney cells. A distinguishing feature of the
human diploid cells such as WI-38 or MRC-5 is that they have a finite life span,
reaching senescence after 40 to 60 population doublings. In contrast, continuous
cell lines exhibit no such constraints and divide indefinitely. An example of a
nonhuman continuous cell line that has been used successfully for the
manufacturing of vaccines such as polio, rabies, and influenza is Vero. Vero cells
are a continuous monkey kidney cell line. Serially cultured or continuous cell lines
are advantageous in that each new production batch is derived from a uniform
master cell bank, characterized to be free of contaminating infectious agents,
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Modern Industrial Microbiology and Biotechnology
contaminating proteins, or nucleic acids. Proper maintenance of a master cell bank
is critically important for the consistency of cell culture products.
(ii) Medium for tissue culture: Various media are available for the cultivation of cells.
They consist essentially of inorganic salts, amino acids, vitamins, nucleotides, and low
molecular growth factors such as hormones, steroids, and fatty acids. Another important
component is serum and this is often used in conjunction with peptones, tryptic digests
and albumin hydrolysates. A mixture of antibiotics is also often added to remove
contaminants.
(iii) Cell Culture fermentors: Conventionally, animal cells grow on the surface of the
glass containers in which they are cultured. Cylindrically shaped roller bottles have been
used successfully to establish monolayers of chick fibroblasts on their inner surfaces,
which can be monitored microscopically through the clear plastic. The cell sheets are
continuously bathed by a growth medium contained within the bottle during slow axial
rotation on special roller racks. After the cell sheets are established, they are infected by
introduction of the specific virus. After incubation, the cells and virus may be harvested
(e.g., varicella). In the case of rubella, viral fluids may be harvested at approximately twoto three-day intervals, through 10 to 12 harvest cycles. Although millions of doses have
been successfully manufactured using roller bottles, capacity is limited by the space that
a large number of roller bottles requires and by the time-consuming and often labor
intensive manipulations for harvesting and pooling the viral fluids from them. Multidisk
reactors offer an alternative to roller bottles for attachment-dependent cell lines. They
consist of 10-L stainless steel reaction vessels containing approximately 100 parallel
titanium disks that slowly rotate through growth media and provide solid surfaces for
cell attachment and monolayer formation. For the production of vaccines and other
biologicals many thousands of bottles are stacked together. The tendency in recent times
is to develop large units of up to 1,000 liters in many small units.
In summary, because cells still require surfaces for growth even on such a large scale,
various arrangements are used. In some fermentors, plates or discs made of plastics, glass
or metal are supported with a central frame, and which are bathed with a sterile tissue
culture solution. The other consist of packed beds of plastic or glass materials over which
the medium flows; cells adhere to the surfaces of the support material. In yet others a bank
of roller tubes through which medium is circulated support growth on the tubes’ internal
surfaces.
(iv) Cell harvest: Cells may be harvested by the use of trypsin (or other proteolytic
enzymes such as papain or pronase), by the use of chelating agents e.g. EDTA, by
physical scraping off or a combination of one or more of these methods.
27.4.1.2
Production of salk polio vcaccine
The production of salk polio vaccine will be discussed as an example of the production of
a virus vaccine. The cells of the kidneys of rhesus monkeys are caused to separate into
individual members by treatment with trypsin. The suspension of cells is then
distributed in shallow containers, and covered with a suitable medium. The cells have a
tendency to adhere to glass and incubation of the cultures at 37°C for four to six days
permits a confluent growth of a monolayer of cells. The culture fluid is removed and is
replaced by maintenance medium, which contains no protein as subjects using the
vaccine may react adversely to protein if this is present.
Vaccines
"&#
Live virus are inoculated into the tissue culture medium and incubated at 37°C for four
days. The viruses lyse the kidney cells in a manner characteristic of the particular virus
and which is described as the cytopathic effect. The viruses are harvested by centrifuging
to remove cell debris. They are then inactivated by treatment with formalin. The success of
inactivation is checked by injection into embryonated chick egg or in experimental
animals. The inactivating agent is removed before the virus is stored at 4°C sometimes
with addition of glycerol.
27.4.2
Production of Bacterial Toxoids
Many clostridia (Gram-positive spore-forming anaerobic rods) cause disease in man and
animals by the production of exotoxins. Some examples include:
Cl. tetani
tetanus
Cl. botulinum
botulism
Cl. welchii
gas gangrene
Aerobes may also cause disease by the production of exotoxins e.g. Corynebacte-rium. It
is possible to collect the toxins so produced in cell-free extracts from in vitro cultivation.
The toxin can then be inactivated by treatment with formaldehyde. Such inactivated
toxin known as a toxoid is antigenic and is able to cause the body to produce antibodies
to the original toxin. Toxoids are non-toxic, and are used to artificially induce active
specific immunity.
In industrial practice toxoids to clostridial toxins are prepared by inactivating toxins
produced from the clostridia grown in large fermentors under anaerobic conditions.
The media used usually consist of hydrolysates of proteins from horse meat, and are
sterilized at 15 p.s.i. Nowadays synthetic media containing inorganic components are
preferred as toxins therein are easier to isolate from such fermentations. Since the
fermentation is anaerobic, satisfactory growth and toxin production are easily obtained
in deep fermentors. The only agitation required is to provide uniform temperature.
Nitrogen is also blown through to flush away oxygen from the system.
At the end of the incubation, the bulk of the bacterial cells is harvested by centrifuging.
The supernatant is further filtered through bacterial filters, before the toxin present
therein is converted to toxoid. This is done by incubating the filtrate at 37°C in contact
with formalin. The inactivation is tested from time to time by injection into animals.
Protein from the medium is removed by precipitation with ammonium sulphate at 4°C.
Excess (NH4)2SO4 is removed by dialysis, the product is filter-sterilized and diluted to
final strength with buffered saline.
27.4.3
Production of Killed Bacterial Vaccines
A suspension of the organism (usually produced by scrapping from surface cultures on
agar) is washed thoroughly with centrifugation. It is then killed usually with heat. For
non-spore-forming bacteria, treatment at 60°C for half hour is usually enough. The
efficiency of the killing is tested by streaking the killed cells on agar. The density of cells
needed to immunize laboratory animals is worked out in experimental animals and that
needed for man is obtained by proportion by relating number to weight in both man and
animals.
"&$
27.5
Modern Industrial Microbiology and Biotechnology
CONTROL OF VACCINES
(i) Stringency of standards: Vaccines produced for man’s use must conform to standards
laid down by different countries, and manufactures must conform to them. The World
Health Organization is also interested in ensuring high standards and has setup its own.
The controls are stringent and designed to ensure (a) that the material is potent; (b) it is
safe, (c) it will not give rise to unpleasant or undesirable side effects. The control of
vaccines is stringent, time-consuming and very expensive because of the expertise
involved. Indeed a good deal of the cost of the vaccine is due to the expenses incurred in
the tests. No vaccine can be considered ready for use for the health of the general public
until it has been extensively tested and conform to the standards of the WHO.
Live vaccines, whether of bacteria or viruses are, understandably more generally
stringently tested than killed vaccines or toxoids. Thus, while the killed vaccines/toxins
for the bacterial diseases dipheria, tetanus, cholera, typhoid fever and pertussis are
merely tested for sterility, toxicity and potency, the live vaccines for tuberculosis, BCG, are
tested for contaminations, virulent organisms, identity, skin reactivity, viable counts and
stability.
One reason for the stringency of the testing of virus vaccines is that when polio virus
was grown on monkey tissue kidney, it was soon found that these monkeys themselves
harbored a large number of viruses. Laboratory-grown animals were therefore resorted
to, including ducks, chicken, rabbits, or dogs. Even though these contained fewer viruses,
the tests had to be gone through because any viruses in the substrate could find its way
into man.
(ii) Potency and field potency testing: These are based on the ability of the vaccine or
toxoid to immunize animals against a lethal, paralytic, skin-test or intracerebral (in the
case of pertusis) challenge. In potency testing a set of test animals are protected with the
vaccine while the control is not. In a potent vaccine the disease should appear only in the
control when the animals are inoculated. A large effort in vaccine production is devoted
to potency testing. Field trials must be carried out on the vaccine, but this is usually carried
out by government regulatory agencies rather than the manufacturer, although he is
usually consulted during the tests. The manufacturer usually recommends any
adjuvants which may be found necessary. Adjuvants are immunological enhancers and
have the following qualities:
(i) A smaller quantity of antigen can be used in single and combined vaccines.
(ii) Owing to the reduced antigen quantity or its slower release, there are fewer local
systemic reactions.
(iii) A better immune response is obtained, an important situation in disease, where a
high antibody level is required for protection.
(iv) Many antigens may be included in a single vaccine.
(v) A reduced number of inoculations may be given.
Some adjuvants used are aluminium compounds, groundnut oil and calcium phosphate.
(iii) Quantity of antigen used in vaccine: This is to be determined by experimentation by
titrating the quantity of antibodies produced against the level of antibody produced. In
some cases large quantities of vaccine may be necessary for immunization. It should be
noted however that in some cases too high a dose of antigen may paralyze the immune
system.
Vaccines
"&%
(iv) The crowding-out effect: Vaccines may be inactivated when multiple vaccines are
administered.
(v) Choice of test animal: The test animal in evaluating a vaccine is of paramount importance. The vaccine must be able to induce antibody production in the animal being used.
(vi) The route of administration: This may be important in determining the efficiency of
the response.
27.6
VACCINE PRODUCTION VERSUS OTHER ASPECTS
OF INDUSTRIAL MICROBIOLOGY
It is important that some differences between vaccine production and routine industrial
fermentations be discussed in order to emphasize its uniqueness.
(i) The cells used in vaccine manufacture are usually pathogenic and therefore complete
sterility must be maintained. Furthermore, while contaminants may merely hinder
production in other industrial fermentations, in vaccine production, contaminants
may mean the introduction of an undesirable organism into the human body.
(ii) The fermentation is usually small (25-1,000 liters) compared to, say, antibiotic
fermentation (500,000 liters). This is because the amount required per person is
usually small.
(iii) Potency cannot be determined during cultivation, hence reproducibility during
production is viewer the less very essential.
SUGGESTED READINGS
Anon, 1979. Microbial Processes: Promising Technologies for the Developing countries. National
Academy of Science, Washington, DC, USA.
Dove, A. 2004. Making Prevention Pay Nature Biotechnology, 72, 387-391.
Fraser, C.M., Rappuoli, R. 2005. Application of Microbial Genomic Science To Advanced
Therapeutics. Annual Review of Medicine, 56, 459-474.
Gurunathan, S., Klinman, D.M., Seder, R.A. 2000. DNA VACCINES: Immunology, Application,
and Optimization. Annual Review of Immunolology, 18, 927–974.
Kuby, J. 1997. Immunology 3rd Ed W H Freeman and Co. New York, USA.
Meinginer, B., Mongeot, H., Favre, H. 1980. Development in Biological Standardization, 46, 249256.
Mora, M, Veggi, D., Santini, L., Pizza, M., Rappuoli, R. 2003. Reverse Vaccinology Drug Discovery
Today 8, 459-464.
Rappuoli, R. 2001. Reverse Vaccinology, a Genome-based Approach to Vaccine Development.
Vaccine, 19, 2688-91.
Robertson, B.H., Nicholson, J.K.A. 2005. New microbiology Tools For Public Health And Their
Implications. Annual Review of Public Health. 26, 281–302.
Spier, R.E. 1980. Adv. Biochem. Eng. 14, 141-162.
Stoughton, R.B. 2005. Applications of DNAmicroarrays in Biology. Annual Reviews of
Biochemistry 74, 53–82.
Telford, J.L., Pizza, M., Grandi, G., Rappuoli, R. 2002. Reverse Vaccinology: From Genome to
Vaccine In: Methods in Microbiology. Vol 33, Academic Press. Amsterdam the Netherlands:
pp. 258–269.
Yewdell, J.W., Haeryfar, S.M.M. 2005. Understanding Presentation of Viral Antigens To Cd8+ T
Cells In Vivo: The Key to Rational Vaccine Design. Annual Review of Immunology 23, 651–82.
Zinkernagel, R.M. 2003. On Natural And Artificial Vaccinations. Annual Review of
Immunolology, 21, 515–46.
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28
Drug Discovery in Microbial
Metabolites: The Search for
Microbial Products with
Bioactive Properties
Microorganisms produce a wide array of chemically diverse secondary metabolites
which are not necessarily anti-microbial in nature. Many of them have turned out to be
very important to the pharmaceutical industry, while some are of importance in the
agricultural industry. In Chapter 24 we discussed the production of anti-microbial and
anti-tumor agents by microorganisms; in this chapter we will look at other products with
pharmaceutical relevance outside antibiotics.
Organized search for microbial metabolites of pharmaceutical and clinical
importance began in the late 1960s when methods were developed for the isolation of
enzyme inhibitors of microbial origin. This led to the discovery of many drugs of clinical
importance. One such enzyme inhibitor is an beta-lactamase inhibitor which is
administered with Beta-lactam antibiotics; the other is an inhibitor of cholesterol
accumulation, while a third is the immunosupressant, cyclosporin A. This section will
discuss the conventional methods for assaying microbial metabolites as a means of
discovering those with positive bioactive activities with the potential of resulting in new
drugs. Some of the newer methods which have come into being following the recent
successes with the human genome project and such developments as the involvement of
the computer in biotechnology, or bioinformatics, will also be touched upon. The
examples given will illustrate how knowledge of a disease helps us look for drugs
against it from among the metabolites of microorganisms.
Prior to the assay for possible drug activities the microbial metabolite must be studied
using various chemical methods including solvent extraction, precipitation,
chromatography, spectroscopic methods; spectral libraries should be searched to
eliminate known compounds. The assays may be cell-based, receptor-binding or enzyme
assays. Examples from each type of assay are given below to illustrate the immense
diversity of microbial metabolites. Many assays are available and the examples given are
a small selection, and designed to expose the student to the general procedure for drug
Drug Discovery in Microbial Metabolites
"&'
discovery in microbial metabolites. Finally the processes of drug approval by regulatory
agencies will be discussed using those of the Food and Drug Administration (FDA) as
illustration.
It will be seen that the processes of drug discovery are beyond the ordinary capabilities
of the microbiologist working single-handedly, and thus illustrate the team-work nature
of industrial microbiology and biotechnology discussed earlier in this book. The human
physiologist, the biochemist, the clinician and many others may be involved in the
process of drug discovery.
28.1 CONVENTIONAL PROCESSES OF DRUG DISCOVERY
28.1.1
Cell-based Assays
Cell-based tests are used to screen for novel microbial metabolites which inhibit a cellular
function, but where a specific molecular target has not been identified. The active
compound(s) may interact with the cell at a number of points and the cell may even be
killed. Compounds with activity at the cell level need to be further screened to identify the
exact mechanism by which they affect the cell. A number of cell reactions are used to
determine whether or not microbial metabolites are bioactive and hence their protential to
become new drugs.
28.1.1.1
Inflammatory reactions
When a foreign protein such as a bacterium enters the body, the body reacts by
developing a non-specific inflammatory reaction in which white blood cells rush to the
site; it may also develop antibodies to the foreign protein. The ability to enhance or initiate
or to suppress immunologic reaction is used to assess bioactivity in microbial
metabolites. One test used is the mixed lymphocyte reaction (MLR) test. This test is an in
vitro assay of TH –cell proliferation in a cell-mediated response. In an MLR test cytotoxic
lymphocytes (CTLs) are generated by co-culturing spleen cells from two different species,
the rat and the mouse (Chapter 27). The T lymphocytes undergo extensive transformation
and cell proliferation. The degree of cell proliferation is assessed by adding labeled [3H]
thymidine to the culture medium and monitoring the uptake of the label into DNA during
cell divisions. Both components proliferate unless one population is rendered
unresponsive with an inhibitory antibiotic such as mitomycin C or has been killed by
irradiation.
28.1.1.1.1 Immunomodulation
Microbial metabolites with the ability to enhance immunologic reactions are those able to
enhance the incorporation of [3H] thymidine into spleen cells and T-cells in the MLR test.
Similarly the ability of a microbial metabolite to restore antibody production to mice
unable to produce antibodies is an indication of bioactivity. Kifunesine a compound
produced by the actinomycete, Kitasatosporia kifunense has been found to have
immunomodulatory activity.
28.1.1.1.2 Immunosupression
The enhancement of the body’s immune system so as to protect it from disease by foreign
organisms such as bacteria is normally desirable. However under some conditions it is
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Modern Industrial Microbiology and Biotechnology
necessary to suppress the body’s immune system. One such situation is when
immunosupression is desirable during tissue or organ transplantation when a person
receives body parts from another. The immunologic apparatus of the recipient individual
would normally reject the donated part because it is foreign. To avoid the rejection
immunosuppressive drugs are given to the recipient person. The presence of
immunosuppressive metabolites is tested by a modification of the MLR test, known as the
one-way MLR. In this test one component of the mixture, the stimulator cells, is treated
with mitomycin C to inactivate them. Within 24-48 hours the untreated cells, the
responder cells begin to divide and incorporate [3H] thymidine as well as express
antigens foreign to the responder T cells. The presence of an immunosuppressive
metabolite is indicated by its blockage of the expression of the foreign antigens in the
responder cells. An example of an immunosuppressive microbial metabolite is an
analogue of the antibiotic cyclosporin, identified as FR901459 which is produced by the
fungus Stachybotris chartarum.
28.1.1.1.3
Macrophage activation
Macrophages are white blood cells which migrate into tissues through out the body
(histocytes in connective tissue, alveolar macrophages in lung, microglial in the central
nervous system, mesangial in kidney, Kupffer cells in liver, and osteoclasts in bone). They
engulf and digest foreign matter and are active in antigen processing and presentation
and the phagocytic effectors of cell-mediated immunity and hypersensitivity. They not
only destroy bacteria invading the body, but they also destroy cancer cells. Activation of
macrophage cells is detected by the spreading of the macrophages as observed under the
scanning electron microscope. TAN-999 is an alkaloid produced by a Streptomyces sp.
and shown to activate macrophages.
28.1.1.2
Cardiovascular disease: Inhibition of
platelet aggregation
Platelets are cell fragments from megakaryocytes. Some megakaryocytes give rise to red
blood cells, while others fragment to give platelets. Blood contains 150,000 to
350,000 platelets per ml. They are important in blood clotting. When the wall of a blood
vessel is damaged by disease or by a trauma such as a cut, thrombin is formed and this
acts on the soluble fibrinogen present in the blood to form insoluble fibrin strands. The
fibrin strands trap platelets to form blood blots. Blot clots forming within the blood
vessels are dangerous, and if large enough could block blood vessels leading to the heart
causing a heart attack or in blood vessels leading to the brain, a stroke. Platelet
aggregation inhibitors are useful in preventing cardiovascular disorders through
preventing clots. Platelet aggregation inhibition is measured against a control by a
turbidometric assay of the platelets to which the metabolite and thrombin are mixed.
28.1.1.3
Cardiovascular disease: Angiogenesis inhibitors
Angiogenesis is the process of new blood vessel formation and is essential for the
formation of solid tumors. Metabolites are tested for anti-angiogenesis effects by placing
pellets of the metabolites dried on small cores of sterile filter paper on a five-day chick
embryo. Metabolites containing anti-angiogenesis compounds prevent blood vessel
formation where the paper was placed.
Drug Discovery in Microbial Metabolites
"'
28.1.2 Receptor Binding Assays
Receptor binding assays have been important in drug discovery. Preparations from
animal tissues have been used as membrane receptors in a wide variety of targets. The
bound ligand is then separated by centrifugation or filtration and a percentage inhibition
calculated on the basis of controls. Ligand receptor interactions are used to search for
microbial metabolites which have potential in drug discovery for dealing with
inflammatory (immunity) diseases, cancers, cardiovascular disease, and central nervous
system diseases.
28.1.2.1
Receptor binding in inflammatory disease
28.1.2.1.1 Leukotriene B4 (LTB4) binding inhibitors
Leukotrienes are produced by a variety of white blood cells. Among them, LTB4 is a
powerful mediator of inflammatory reactions, causing the loss of granules in granulecontaining white blood cells leading to allergic reactions. LTB4 antagonists may
therefore be useful in treating inflammatory diseases. In the search for LTB4
anatagonists, labeled LTB4 is incubated with a suspension of membrane from white
blood cells and the test microbial metabolite. After the incubation, the bound labeled
LTB4 is separated from the free ligand by filtration and centrifugation.
28.1.2.1.2 CD4 binding inhibitors
CD4 is a glycoprotein present on the surface of mature helper/inducer T white blood cells
(lymphocytes) (see ch. 27). It binds to class II MHC (major Histocompatibility Complex II)
and this‘ stabilizes the T cell receptor and its attachment, the antigen-MHCII complex.
Inhibition of this interaction can have important suppressive effects on immune
responses and thus it is an attractive target in the search for immunosuppressants.
Additionally the CD4 molecule is an important anti-viral target because it is the cellular
receptor for the HIV virus. An assay which has been used to search for anti-CD4
compounds was based on the interaction between soluble recombinant CD4 and a
monoclonal antibody. The assay enabled the discovery of new anti-CD4 compounds of
fungal origin.
28.1.3
Enzyme Assays
Enzymes are work horses of living things; all activities of living things are mediated
through enzymes. It is therefore not surprising that enzymes are widely targeted in the
search for pharmacologically active compounds. Only a few examples will be given in
this section.
28.1.3.1
Inflammatory diseases
28.1.3.1.1 Cell surface sugar metabolism inhibitors
Sugars conjugated with various compounds are important in mammalian cell surfaces.
They are important in cell adhesion, which is important in inflammatory disease as well
as in cancer. Numerous enzymes are involved in the metabolism of sugars presented at
cell surfaces. An enzyme which has been targeted is a-D-mannosidase. Broth cultures of
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Modern Industrial Microbiology and Biotechnology
Streptoverticullum verticullus var. quantum was found to contain antagonists to the
enzyme, designated mannostatins A and B.
28.1.3.1.2
Human leukocyte elastase inhibitors
Human leukocyte elastase is one of the most destructive enzymes known. It hydrolyzes
several compounds found in connective tissue including elastin, proteoglycan and
collagen. The enzyme is released by certain white blood cells and it may be involved with
the destructive processes associated with chronic inflammatory diseases. A peptide
metabolite of Streptomyces resistomycificus was found to antagonize the enzyme.
28.1.3.1.3
Cardiovascular disease: Inhibition of cholesterol metabolism
Cholesterol is important in cardiovascular disease because it is deposited on the walls of
blood vessels, thereby decreasing the blood vessel diameter leading to high blood
pressure and in some cases to occlusion of the blood vessels. This may lead to heart
attacks or strokes depending on whether the occlusions occurs in a blood vessel leading
to the heart or one leading to the brain. Acyl co-enzyme A cholesterol transferase (ACAT)
plays an important part in atherogenesis and cholesterol adsorption from the intestine.
ACAT inhibitors may therefore be useful in treating arteriosclerosis and
hypercholesteremia (high cholesterol). Many new ACAT inhibitors have been identified
in recent times, including some from Fungi.
28.2
28.2.1
NEWER METHODS OF DRUG DISCOVERY
Computer Aided Drug Design
The search for anti-microbial compounds and other drugs can nowadays start at the
computer ie in silico. New drugs can be created at the computer and their efficacy
determined through assessing whether or not they will bind to proteins on ‘pathogenic
micro-organisms’ or ‘disease tissues’. Drug discovery and development is immensely
expensive and time-consuming. The success rate of new chemical entities selected for
clinical development is approximately 20% with most failures attributed to unacceptable
pharmacokinetic properties Undesirable properties, such as poor absorption, low and
variable bioavailability, drug interactions may be predicted from in vitro and in silico
data, thus facilitating selection of the most appropriate lead compound. The in silico
approach is not only rapid, but it is also cost-effective. The successful in silico antibiotic or
drug must then be tested in the wet laboratory using in vitro and in vivo methods as
required by regulatory agencies discussed later in this chapter. Perhaps the best example
of in silico drug development is the development of inhibitors of HIV-1 protease by
computer-aided drug design. HIV-1 genome encodes an aspartic protease (HIV-1 PR).
Inactivation of HIV-1 PR by either mutation or chemical inhibition leads to the
production of immature, noninfectious viral particles thus the function of this enzyme
was shown to be essential for proper virion assembly and maturation. It is not surprising,
then, that HIV-1 PR was identified over a decade ago as the prime target for structure- or
computer-assisted (sometimes called ‘rational’) drug design. The structure-assisted drug
design and discovery process utilizes structural biochemical methods, such as protein
crystallography, nuclear magnetic resonance (NMR), and computational biochemistry,
Drug Discovery in Microbial Metabolites
"'!
to guide the synthesis of potential drugs. This information can, in turn, be used to help
explain the basis of their activity and to improve the potency and specificity of new lead
compounds. Put in another language once the structure of a target is known, the structure
of a compound which will attack it can be computer-designed and then synthesized.
An aspect of in silico drug discovery would appear to be a process known as tethering.
To facilitate the drug discovery process, many researchers are turning to fragment-based
approaches to find lead molecules more efficiently. One such method, tethering, allows
for the identification of small-molecule fragments that bind to specific regions of a protein
target. These fragments can then be elaborated, combined with other molecules, or
combined with one another to provide high-affinity drug leads.
28.2.2
Combinatorial Chemistry
The essence of combinatorial chemistry or techniques involving ‘molecular diversity’ is
to generate enormous populations of molecules and to exploit appropriate screening
techniques to isolate active components contained in these libraries. This idea has been
the focus of research both in academia, but more especially in the pharmaceutical or
biotechnology industry. Its developments go hand in hand with an exploding number of
potential drug targets emerging from genomics and proteomics research.
Fig. 28.1 Illustration of Combinatorial Chemistry
Synthesis of molecules in a combinatorial fashion can quickly lead to large numbers of
molecules. For example, a molecule with three points of diversity (R1, R2, and R3) can
generate NR1 ´ NR2 ´ NR3 possible structures, where NR1, NR2, and NR3 are the number
of different substituents utilized.
In this a technique a large number of structurally distinct molecules are synthesized at
a time and submitted for pharmacological assay. The key of combinatorial chemistry is
that a large range of analogues is synthesized using the same reaction conditions, the
same reaction vessels. In this way, the chemist can synthesize many hundreds or
thousands of compounds in one time instead of preparing only a few by simple
methodology.
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Modern Industrial Microbiology and Biotechnology
In the past, chemists have traditionally made one compound at a time. For example
compound A would have been reacted with compound B to give product AB, which
would have been isolated after reaction work up and purification through
crystallization, distillation, or chromatography. In contrast to this approach,
combinatorial chemistry offers the potential to make every combination of compound A1
to An with compound B1 to Bn (Fig. 28.1). Combinatorial chemistry has been helped by
developments in automation or robotics and miniaturization of processes. These enable
many hundreds of compounds to be synthesized and screened. The starting points of the
compounds to be ‘amplified’ by combinatorial chemistry could be from plants, animals
or micro-organisms. Combinatorial chemistry methods are used for discovering other
drugs besides antimicrobial agents.
28.2.3
Genomic Methods in the Search for New Drugs,
Including Antibiotics
The traditional approach to the development of novel antibiotics has relied on random
screening (mainly of the soil) for new active molecules, using simple antibiotic activity
especially death of the test organisms for primary selection. The disadvantages of using
death as the main criterion for selecting anti-microbial agents are shown in Table 28.1.
The result is that very few new antibiotics have been discovered over many years; on
account of this progress has been achieved by modification of existing antibiotics. As a
result, resistance cross-reaction across available antibiotic groups has become common.
More imaginative approaches were limited by knowledge and technology. The
completion of the Human Genome Project and the availability of genome sequence data
for many micro-organisms including pathogenic ones, has stimulated research on the
identification of novel targets for antimicrobial compounds by providing a complete
catalogue of genes which can be compared at various levels.
Recent genomic advances have enabled ‘target-based’ initiatives in the search for
antimicrobials. Traditionally screening has been done against whole cells such as
microbial cells. Such an approach has a number of disadvantages. First they are
inherently insensitive and often lead to the isolation of toxic compounds. Second, the
screens will only identify targets that are lethal to the bacteria under the conditions of
growth in the laboratory. Third since the exact nature of the target inhibited by any new
compound is unknown, rational modification of the molecule to enhance its activity and
moderate toxicity is not possible. This approach has been replaced by attempts to identify
effective drug targets and to design antagonists to disrupt the activity of the target.
Comparative analysis of microbial genome sequence data has revealed that: (i) the
genome of each organism contains a large number of open reading frames- ie sequences
which code for proteins– 20% - 40% for which the proteins are unknown and about 10%
of these unknown proteins are unique to the organisms (Chapter 3); (ii) the microbial
genome is a dynamic entity shaped by multiple forces including gene duplication and
gene loss, genome rearrangements, and acquisition of new genes through lateral
transfers; (iii) each of these mechanisms has been shown to play a major role in the
evolution of pathogens and has important implications in epidemeologic studies and the
spread of antibiotic resistance and pathogenicity; (iv) differences between the pathogen
and non-pathogen are best explained not by the presence or absence of a gene but by
Drug Discovery in Microbial Metabolites
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Table 28.1 Comparison of the screening strategies for novel antimicrobial compounds
Whole-cell screening
Target-based screening
(Looking directly for compounds
which kill microorganisms)
(Looking for biochemical inhibitors)
Advantages
1
Selection for compounds
which penetrate cells
1
2
Antimicrobial properties
established
Highly reproducible has been
used successfully historically
2
3
More sensitive (can detect weak or
poorly penetrating compounds
suitable for chemical optimization)
Easy screening
3
Different approach can target new
areas of biology facilities rational
drug design
Disadvantages
1
Insensitive
1
Need to turn an in vitro inhibitor
into an antibacterial drug
(complicated by penetration issues)
2
3
Most active compounds are toxic
No rational basis for compound
optimization (target unknown)
2
Genetic validation of targets (by
gene knockout or reduced
expression) can be misleading
4
Mixed mechanisms of action in
recent years has failed to deliver
subtle single nucleotide changes, and virulence genes have been shown to be inactivated
by such single-nucleotide changes.
The ideal antimicrobial genomic target should be (i) different from the existing targets;
(ii) essential for the viability of the pathogen; (iii) absent or substantially different in the
human host, a parameter much easier to assess now with the availability of the complete
human genome; (iv) conserved across the appropriate range of organisms; (v) easy to
assay, especially in high throughput processes; (vi) easy to identify the target’s inhibitors
and (vii) suitable for rapid structural analysis. .
Genomics has enabled the identification of targets through (a) large-scale
identification of novel potential targets through in silico comparison of pathogenic and
non-pathogenic strains; (b) examining existing metabolic information on the organisms
present in databases; (c) identifying genes necessary for bacterial growth and survival by
experimental means, including transposon mutagenesis, targeted mutagenesis of
conserved genes, and the expression of anti-sense RNA. These studies suggest that
depending on the organism, the number of essential genes range from 150 to 500.
The potential new targets identified include aminoacyl tRNA synthetases,
polypeptide deformylase, fatty acid biosynthesis, protein secretion, and cell signaling.
The next step is to identify small molecular inhibitors of these proteins, often by
exploiting the diversity of chemical compounds to be found in combinatorial libraries.
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Modern Industrial Microbiology and Biotechnology
The target approach is however not without its shortcomings. These include the
possibility that the antibacterial drug may not penetrate the cell (Table 28.1).
28.2.4
Search for Drugs Among Unculturable
Microorganisms
Natural products, primarily of microbial origin, have accounted for one-third of the more
than $100 billion of sales in the US and in excess of $250 billion worldwide
pharmaceutical market and are also an important source of specialty chemical,
agrochemical, and food or industrial processing products. Although the pharmaceutical
industry appears to be spending more money in the search for new drugs, the results do
not match the increased expenditure.
About $20 billion is spent currently on search for new drugs in the US, or about 20
times the figure in the 1970s. Yet only about 40 new drugs (new chemical entities, NCEs)
were introduced in the mid-1990s compared to 60-70 in the 1970s. One reason for this is
the diminishing returns from existing sources of search. Many of the sources of
pharmaceuticals are bacteria. It is now known that culturable bacteria represented only
about 5% of all bacteria. To combat the problem of diminishing returns from searching
among culturable organisms, Oceanix Biosciences Corporation has developed and
patented a biotechnology-based for the production of new pharmaceutical from
unculturable bacteria. The procedure of the company named Combinatorial Genomics
TM
for which US Patent no 5,773,221 was granted by the US Patent and Trademark Office
consists essentially isolating nonculturable micro-organisms or their high molecular
weight DNA directly from environmental samples followed by the integration and
expression of that genetic material in well characterized microbial host species. As to be
expected, the results are unpredictable since it is based on a random and
phenomenological genetic survey of unknown genetic materials.
The environmental DNA may be isolated either in a ‘naked’ form and subsequently
encapsulated in liposomes prior to use, or may be contained in non-culturable microbial
cells which are converted into spheroplasts or protoplasts prior to use. Liposomes,
spheroplasts, or protoplasts containing environmental DNA are then fused, employing
standard cell fusion techniques such as polyethylene glycol (PEG) mediated fusion or
electrofusion, with spheroplasts or protoplasts (Chapter 9) of well characterized and
easily cultured host microorganisms. Well characterized host microbes can be employed
as recipient organisms including Gram-positive and Gram-negative bacteria as well as
certain fungal and archaebacterial host species. Following a fusion event between a host
microbe protoplast or spheroplast (auxotrophic) cell and a prepared environmental
DNA sample containing liposomes, protoplasts, or spheroplasts the viable, colonyforming cells will be those in which the delivered environmental DNA is expressed.
Protoplast and liposome fusion are a versatile and well explored technique to induce
genetic recombination in a variety of prokaryotic and eukaryotic microorganisms. In the
presence of a fusogenic agent, such as Polyethylene glycol (PEG), or by treatment in
electrofusion chambers, protoplasts and liposomes are induced to fuse and form hybrid
cells. During the hybrid state, the genomes reassort and extensive genetic recombination
can occur. The final, crucial step is the regeneration of viable cells from the fused
protoplasts, without which no viable recombinants can be obtained. The patentees
Drug Discovery in Microbial Metabolites
"'%
named the process Combinatorial Genomics in mimic of combinatorial chemistry where
numerous compounds are prepared, in the same manner as numerous recombinations
may occur between the host organisms and the unknown DNA isolated from the
environment.
Growth is on agar plates and the colonies selected are tested for i) their further
characterization in additional antibiotic tests employing a wider range of indicator
microbial species, ii) their anti-cancer activity in a battery of malignant cell lines, and iii)
their agonist or antagonist activity in a relevant central nervous system (CNS). Those
bioactive agents which display promising activity as antibiotic agents, as anti-cancer
agents or as pharmacologically relevant materials are then purified and subjected to
chemical structural analysis. Novel bioactive agents are examined for their safety and
toxicology properties.
28.4
APPROVAL OF NEW ANTIBIOTIC AND OTHER
DRUGS BY THE REGULATING AGENCY
Discovering and developing safe and effective new medicines is a long, difficult and
expensive process. The US system of approval for new drugs is perhaps the most rigorous
in the world. In the US the regulatory agency for certifying new medicines as safe and
effective is the Food and Drug Administration (FDA). In EU countries drugs are regulated
by the European Medicines Agency (EMEA) which was established in 1993.
In the US which is a major producer of new drugs, it takes 12 years on average for an
experimental drug to travel from the laboratory to the medicine chest. It is also an
expensive process and takes on the average about $360 million to get a new medicine
from the lab oratory to the medicine cupboard. Where a new medicine is a life saving one
for which few or no equivalent exists it can be put on fast track and may be approved in
six months.
Prior to submission for pre-clinical trial by the FDA, the firm itself would have carried
out tests. Subsequently the firm submits its product for testing by the FDA. Only five in
5,000 compounds that enter preclinical testing make it to human testing. As shown in the
table below, one of these five tested in people is approved. The processes of drug progress
are as follows beginning with the work of the firm. (see Table 28.2)
28.4.1
28.4.1.1
Pre-Submission Work by the Pharmaceutical Firm
Synthesis and extraction
The process of identifying new molecules with the potential to produce a desired change in
a biological system (e.g., to inhibit or stimulate an important enzyme, to alter a metabolic
pathway, or to change cellular structure).
The process may require: 1) research on the fundamental mechanisms of disease or
biological processes; 2) research on the action of known therapeutic agents; or 3) random
selection and broad biological screening. New molecules can be produced through
artificial synthesis or extracted from natural sources (plant, mineral, or animal). The
number of compounds that can be produced based on the same general chemical
structure runs into the hundreds of millions.
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Modern Industrial Microbiology and Biotechnology
Table 28.2
Table showing flow chart of approval for a new drug (including antibiotics) to travel
from the laboratory to the patient in the US
Lab
studies/
Preclinical
Testing
Clinical Trials
Phase I
Phase II
Phase III
2
3
Years
3.5
1
Test
Population
Lab and
animal
studies
Assess
safety
and
biological
activity
20 to 80
healthy
volunteers
Purpose
Success
Rate
28.4.1.2
5,000
compounds
evaluated
File
IND
at
FDA
FDA
100 to 300
patient
volunteers
Evaluate
Determine effectiveness,
effectiveness,
monitor
safety and
look for
dosage
side
effects
1000 to 3000
patient
volunteers
Verify
effectiveness,
adverse
reactions
from
long-term
use
2.5
File
NDA
at
FDA
5 enter trials
Clinical
Trials
Phase IV
12
Total
Review
process/
Approval
Additional
Post
marketing
testing
required by
FDA
(sometimes)
1
approved
Biological screening and pharmacological testing
Studies to explore the pharmacological activity and therapeutic potential of compounds.
These tests involve the use of animals, isolated cell cultures and tissues, enzymes and
cloned receptor sites as well as computer models. If the results of the tests suggest
potential beneficial activity, related compounds each a unique structural modification of
the original are tested to see which version of the molecule produces the highest level of
pharmacological activity and demonstrates the most therapeutic promise, with the
smallest number of potentially harmful biological properties.
28.4.1.3 Pharmaceutical dosage formulation and
stability testing
The process of turning an active compound into a form and strength suitable for human use.
A pharmaceutical product can take any one of a number of dosage forms (e.g., liquid,
tablets, capsules, ointments, sprays, patches) and dosage strengths (e.g., 50, 100, 250, 500
mg) The final formulation will include substances other than the active ingredient, called
excipients. Excipients are added to improve the taste of an oral product, to allow the
active ingredient to be compounded into stable tablets, to delay the drug’s absorption into
the body, or to prevent bacterial growth in liquid or cream preparations. The impact of
each on the human body must be tested.
28.4.1.4
Toxicology and safety testing
Tests to determine the potential risk a compound poses to man and the environment. .
These studies involve the use of animals, tissue cultures, and other test systems to
examine the relationship between factors such as dose level, frequency of administration,
Drug Discovery in Microbial Metabolites
"''
and duration of exposure to both the short- and long-term survival of living organisms.
Tests provide information on the dose-response pattern of the compound and its toxic
effects. Most toxicology and safety testing is conducted on new molecular entities prior to
their human introduction, but companies can choose to delay long-term toxicity testing
until after the therapeutic potential of the product is established.
All the above tests can take up to three and half years. If the results are promising, the
firm then submits the compound and all the tests and results obtained to the FDA as an
investigational new drug (IND).
28.4.2
Submission of the New Drug to the FDA
28.4.2.1 Regulatory review: Investigational new drug (IND)
application
An application filed with the U.S. FDA prior to human testing.
After completing its laboratory studies, the company files an IND with FDA to begin to
test the drug in people. The IND shows results of previous experiments, how, where and
by whom the new studies will be conducted; the chemical structure of the compound;
how it is thought to work in the body; any toxic effects found in the animal studies; and
how the compound is manufactured. In addition, the IND must be reviewed and
approved by the Institutional Review Board where the studies will be conducted, and
progress reports on clinical trials must be submitted at least annually to FDA. The IND
application is a compilation of all known information about the compound. It also
includes a description of the clinical research plan for the product and the specific
protocol for phase I study. Unless the FDA says no, the IND is automatically approved
after 30 days and clinical tests can begin.
28.4.2.2
Clinical trials
28.4.2.2.1 Phase I Clinical Evaluation
The first testing of a new compound in human subjects, for the purpose of establishing the
tolerance of healthy human subjects at different doses, defining its pharmacologic effects at
anticipated therapeutic levels, and studying its absorption, distribution, metabolism, and
excretion patterns in humans.
About 20 -80 healthy volunteers are used for this trail.
28.4.2.2.2 Phase II clinical evaluation
Controlled clinical trials of a compound’s potential usefulness and short term risks.
A relatively small number of patients, usually no more than several hundred subjects
(100 – 300), enrolled in phase II studies.
28.4.2.2.3 Phase III clinical evaluation
Controlled and uncontrolled clinical trials of a drug’s safety and effectiveness in hospital
and outpatient settings.
Phase III studies gather precise information on the drug’s effectiveness for specific
indications, determine whether the drug produces a broader range of adverse effects than
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Modern Industrial Microbiology and Biotechnology
those exhibited in the small study populations of phase I and II studies, and identify the
best way of administering and using the drug for the purpose intended. If the drug is
approved, this information forms the basis for deciding the content of the product label.
Phase III studies can involve several hundred to several thousand subjects (1,000 – 3,000).
28.4.2.3 Process development for manufacturing and
quality control
The firm’s manufacturing capability is assessed.
Engineering and manufacturing design activities to establish a company’s capacity to
produce a product in large volume and development of procedures to ensure chemical
stability, batch-to-batch uniformity, and overall product quality.
28.4.2.4
Bioavailability studies
The use of healthy volunteers to document the rate of absorption and excretion from the
body of a compound’s active ingredients.
Companies conduct bioavailability studies both at the beginning of human testing
and just prior to marketing to show that the formulation used to demonstrate safety and
efficacy in clinical trials is equivalent to the product that will be distributed for sale.
Companies also conduct bioavailability studies on marketed products whenever they
change the method used to administer the drug (e.g., from injection or oral dose form), the
composition of the drug, the concentration of the active ingredient, or the manufacturing
process used to produce the drug.
28.4.2.5
Regulatory review: New drug application (NDA)
The firm puts in an application for a new drug, New Drug Application (NDA)
An NDA is an application to the FDA for approval to market a new drug. All
information about the drug gathered during the drug discovery and development
process is assembled in the NDA Following the completion of all three phases of clinical
trials, the company analyzes all of the data and files an NDA with FDA if the data
successfully demonstrate safety and effectiveness. The NDA must contain all of the
scientific information that the company has gathered. NDAs typically run 100,000 pages
or more. By law, FDA is allowed six months to review an NDA. In almost all cases, the
period between the first submission of an NDA and final FDA approval exceeds that
limit; the average NDA review time for new molecular entities approved in 1992 was 29.9
months.
28.4.3
Approval
Once FDA approves the NDA, the new medicine becomes available for physicians to
prescribe. The company must continue to submit periodic reports to FDA, including any
cases of adverse reactions and appropriate quality-control records. For some medicines,
FDA requires additional studies (Phase IV) to evaluate long-term effects.
Drug Discovery in Microbial Metabolites
28.4.4
#
Post Approval Research
Experimental studies and surveillance activities undertaken after a drug is approved for
marketing.
Clinical trials conducted after a drug is marketed (referred to as phase IV studies in the
United States) are an important source of information on as yet undetected adverse
outcomes, especially in populations that may not have been involved the premarketing
trials (e.g., children, the elderly, pregnant women) and the drug’s long-term morbidity
and mortality profile. Regulatory authorities can require companies to conduct Phase IV
studies as a condition of market approval. Companies often conduct post-marketing
studies even in the absence of a necessity to do so.
SUGGESTED READINGS
Allsop, A., Illingworth, R. 2002. The impact of genomics and related technologies on the search
for new antibiotics. Journal of Applied Microbiology, 92, 7-12.
Anon, 1993. Congress of the United States, Office of Technology Assessment. Pharmaceutical
R&D: Costs, Risks and Rewards: 1993; Washington, DC, USA. pp. 4-5.
Anon, 1999. From Test Tube to Patient: Improving Health Through Human Drugs Special
Report, Center Drug Evaluation and Research. Food and Drug Administration. Rockville,
MD, USA.
Austin, C. 2004. The Impact of the Completed Human Genome Sequence on the Development of
Novel Therapeutics for Human Disease. Annual Review of Medicine, 55, 1–13.
Bansal, A.K. 2005. Bioinformatics in the microbial biotechnology – a mini review Microbial Cell
Factories, 4, 4–19.
Beamer, L. 2002. Human BPI: One protein’s journey from laboratory to clinical trials. ASM News.
68, 543-548.
Behal, V. 2000. Bioactive Products from Streptomyces. Advances in Applied Microbiology. 47, 113–
156.
Bull, A.T., Ward, A.C., Goodfellow, M. 2000. Search and Discovery Strategies For Biotechnology:.
The Paradigm Shift. Microbiology and Molecular Biology Reviews, 64, 573-548.
Dale, E., Wierenga, D.E., Eaton, C.R. 2001. Processes of Product Develpoment. http://
www.allpcom/drug-dev.htm. Accessed on September 28, 2005 at 12.05 pm GMT.
Debouck, C., Metcalf, B. 2000. The Impact of Genomics on Drug Discovery. Annual Review of
Pharmacology and Toxicology, 40, 193–208.
Erlanson, D.A., Wells, J.A., Braisted, A.C. 2004. Tethering: Fragment-Based Drug Discovery.
Annual Reviews of Biophysical and Biomolecular Structure, 33, 199–223.
Fan, F., McDevitt, D. 2002. Microbial Genomics for Antibiotic Target Discovery. In : Methods in
Microbiology. Vol 33, Academic Press. Amsterdam the Netherlands, pp. 272–288.
Feling, R.H., Buchanan, G.O., Mincer, T.J., Kauffman, C.A., Jensen, P.R., Fenical, W. 2003.
Salinosporamide A: a highly cytotoxic proteasome inhibitor from a novel microbial source, a
marine bacterium of the new genus Salinospora. Angewandte Chemie International Edition 42,
355-357.
Manyak, D.M., Carlson, P.S. 1999. Combinatorial GenomicsTM: New tools to access microbial
chemical diversity In : Microbial Biosystems: New Frontiers. C.R., Bell, M. Brylinsky, P.
Johnson-Green, (eds). Proceedings of the 8th International Symposium on Microbial Ecology
Atlantic Canada Society for Microbial Ecology, Halifax, Canada, 1999.
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Section
Waste Disposal
0
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Microbiology and Biotechnology
+0)26-4
29
Treatment of
Wastes in Industry
Wastes, unwanted materials, result inevitably from industrial activities in the same way
as they also do in domestic ones. If allowed to accumulate on the ground, or if dumped
indiscriminately into rivers and other bodies of water, unacceptable environmental
problems would result. Governments the world over usually institute legislation which
regulates the handling of wastes, including those resulting from industry. In the US the
Environmental Protection Agency (EPA) is the regulating agency. The EPA works to
develop and enforce regulations that implement environmental laws enacted by
Congress. EPA is responsible for researching and setting national standards for a variety
of environmental programs, and delegates to states the responsibility for issuing permits
and for monitoring and enforcing compliance.
The activities of industrial microorganisms usually occur in large volumes of water;
the resulting wastes are therefore transported in aqueous medium. This chapter will
examine briefly the treatment of waste water. The subject is of interest, not only from the
intrinsic need to dispose of wastes in industry, but especially because the basis for
ultimate waste disposal is microbial.
Waste carried in water, whether from industry or from domestic activity is known as
sewage. Waste water disposal constitutes a peculiar branch of industrial microbiology.
The methods to be discussed were evolved originally to handle domestic sewage, but they
have been extended for use in those industries, such as the food and fermentation industries, which yield wastes degradable by microorganisms. Sewage emanating from some
chemical industries especially those dealing with manmade chemicals are not only less
degradable but are sometimes toxic to microorganisms and man. The processes of biological waste-water treatment to be discussed here are really an aspect of industrial
microbiology within the definition of the subject adopted in this book, because they involve micro-organisms on a large scale, although there is no direct expectation of profit.
29.1 METHODS FOR THE DETERMINATION OF ORGANIC
MATTER CONTENT IN WASTE WATERS
Waste waters are sampled and analyzed in order to determine the efficiency of the treatment system in use. This is particularly important at the point of the discharge of the
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treated waste water into rivers, streams and other natural bodies of water. If waste water
discharged into a natural water is rich in degradable organic matter, large numbers of
aerobic microorganisms will develop to break down the organic matter. They will use up
the available oxygen and as a consequence fish and other aquatic life will die. Furthermore, anaerobic bacteria will develop following the exhaustion of oxygen; the activities of
the latter will result in foul odors. Some of the methods for analyzing the organic matter
content of waste waters are given below.
29.1.1
Dissolved Oxygen
Dissolved oxygen is one of the most important, though indirect, means of determining the
organic matter content of waters. The heavier the amount of degradable material present
in water, the greater the growth of aerobic organisms and hence the less the oxygen
content. The Winkler method is widely used for determining the oxygen in water. In this
method, dissolved oxygen reacts with manganous oxide to form manganic oxide. On
acidification in an iodide solution, iodine is released in an amount equivalent to the
oxygen reacting to form the manganic oxide. The iodine may then be titrated using
thiosulphate. Membrane electrodes are now available for the same purpose. In these
electrodes oxygen diffuses through the electrode and reacts with a metal to produce a
current proportional to the amount of oxygen reacting with the metal.
29.1.2
The Biological or Biochemical Oxygen
Demand (BOD) Tests
Due to the complexity of the organic materials introduced into water and the key role
played by oxygen in supporting the aerobic bacteria which break down this organic
matter, the method of the Biochemical Oxygen Demand (BOD) was developed. It is a
measure of the oxygen required to stabilize or decompose the organic matter in a body of
water over a five-day period at 20°C. In carrying out the test, two 250-300 ml bottles are
filled with water whose BOD is to be determined. The oxygen content of one is determined
immediately by the Winkler method and in the other at the end of five days incubation at
20°C. The difference between the two is the BOD.
Although it has been severely criticized, the BOD test is still widely used. Some of the
criticisms are that it takes too long to obtain results and that it may infact relate only
loosely to the actual organic matter content of water since it represents the overall value of
the respiration of the organisms present therein. Furthermore, many industrial wastes
contain materials which are either difficult to degrade or which may even be toxic to the
organisms. In such cases an inoculum capable of degrading the materials must be
developed by enrichment and introduced into the bottles.
29.1.3
Permanganate Value (PV) Test
This PV method determines the amount of oxygen used up by a sample in four hours from
a solution of potassium permanganate in dilute H2SO4 in a stoppered bottle at 27°C. It
gives an idea of the oxidizable materials present in water, although the actual oxidation
is only 30-50% of the theoretical value. The method records the oxidation of organic
Treatment of Wastes in Industry
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materials such as phenol and aniline as well as those of sulfide, thiosulfate, and
thiocyanate and would be useful in some industries. However because oxidation is
incomplete it is not favored by some workers.
29.1.4
Chemical Oxygen Demand (COD)
The chemical oxygen demand is the total oxygen consumed by the chemical oxidation of
that portion of organic materials in water which can be oxidized by a strong chemical
oxidant. The oxidant used is a mixture of potassium dichromate and sulfuric acid and is
refluxed with the sample of water being studied. The excess dichromate is titrated with
ferrous ammonium sulfate. The amount of oxidizable material measured in oxygen
equivalent is proportional to the dichromate used up. It is a more rapid test than BOD and
since the oxidizing agents are stronger than those used in the PV test, the method can be
used for a wider variety of wastes. Furthermore, when materials toxic to bacteria are
present it is perhaps the best method available. Its major disadvantage is that bulky
equipment and hot concentrated sulfuric acid are used.
29.1.5
Total Organic Carbon (TOC)
Total organic carbon provides a speedy and convenient way of determining the degree of
organic contamination. A carbon analyzer using an infrared detection system is used to
measure total organic carbon. Organic carbon is oxidized to carbon dioxide.
The CO2 produced is carried by a ‘carrier gas’ into an infrared analyzer that measures
the absorption wavelength of CO2. The instrument utilizes a microprocessor that will
calculate the concentration of carbon based on the absorption of light in the CO2. The
amount of carbon will be expressed in mg/L. TOC provides a more direct expression of
the organic chemical content of water than BOD or COD.
29.1.6
Total Suspended Solids (TSS)
The term ‘total solids’ refers to matter suspended or dissolved in water or wastewater,
and is related to both specific conductance and turbidity. Total solids (also referred to as
total residue) is the term used for material left in a container after evaporation and drying
of a water sample. Total Solids include both total suspended solids, the portion of total
solids retained by a filter and total dissolved solids, the portion that passes through a
filter. Total solids can be measured by evaporating a water sample in a weighed dish, and
then drying the residue in an oven at 103 to 105°C. The increase in weight of the dish
represents the total solids. Instead of total solids, laboratories often measure total
suspended solids and/or total dissolved solids. To measure total suspended solids
(TSS), the water sample is filtered through a preweighed filter. The residue retained on the
filter is dried in an oven at 103 to 105°C until the weight of the filter no longer changes.
The increase in weight of the filter represents the total suspended solids. TSS can also be
measured by analyzing for total solids and subtracting total dissolved solids.
29.1.7
Volatile Suspended Solids (VSS)
Volatile suspended solids (VSS) are those solids (mg/liter) which can be oxidized to gas
at 550°C. Most organic compounds are oxidized to CO2 and H2O at that temperature;
inorganic compounds remain as ash.
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29.2
Modern Industrial Microbiology and Biotechnology
WASTES FROM MAJOR INDUSTRIES
The composition of industrial wastes depend on the industry. Wastes from three key
industries in the US are given in Table 29.1 for illustration: the oil, the pulp and paper and
the food industries.
Table 29.1
Typical wastes from three industries
A
Typical Components of an Oil Refinery Waste Water
Handling of oil crude
Crude oil distillation
Thermal cracking
Alkylation, polymerization,
isomerization processes
Refining
Purification and extraction
Sweetening, stripping, fltration
Oil, sludge oil emulsions, sulfur- and nitrogen
corrosion inhibitors
Hydrocarbons, organic and inorganic acids,
phenols and sulfur
Phenols, triphenols, cyanides, hydrogen sulfide
cid sludge, spent acid, mineral acids (sulfuric,
hydrochloric), catalyst support
Hydrogen sulfide, ammonium sulfide, gums.
catalyst support
Phenols, glycols, amines, spent caustic
Sulfur and nitrogen compounds, copper chloride,
suspended matter
B
Typical Effluent Loads from Pulp and Paper Manufacture
Effluent
Pulps: unbleached sulfite
Pulps: bleached sulfite
Fine paper
Tissue paper
Kg/1,000 kg of product
Suspended solids
5-day BOD
10 - 20
200 - 300
12 - 30
220 - 400
25 - 30
7 -20
15 – 20
10 - 15
C
Typical Effluent Loads from Food industries
Effluent
Apple canning
Cherries canning
Mushrooms
Slaughter house
Parking house
Processing plant
Plant waste
Kg/1,000 kg of product
Suspended solids
5-day BOD
Cannery wastes
300 - 600
1680 - 5530
200 - 600
700 - 2100
50 - 240
76 - 850
Meat Packing Industry
3000 - 930
2200 - 650
2000 - 230
3000 - 400
800 - 200
800 - 200
Poultry
100 - 1500
150 - 2400
Treatment of Wastes in Industry
29.3
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SYSTEMS FOR THE TREATMENT OF WASTES
The basic microbiological phenomenon in the treatment of wastes in aqueous
environments is as follows:
(i) The degradable organic compounds in the waste water (carbohydrates, proteins,
fats, etc.) are broken down by aerobic micro-organisms mainly bacteria and to some
extent, fungi. The result is an effluent with a drastically reduced organic matter
content.
(ii) The materials difficult to digest form a sludge which must be removed from time to
time and which is also treated separately.
The discussion will therefore be under two headings: aerobic breakdown of raw
waste-water and anaerobic breakdown of sludge.
29.3.1
Aerobic Breakdown of Raw Waste Waters
The two methods which are usually employed include the activated sludge and the
trickling filter.
29.3.1.1
The activated sludge system
The activated sludge method is the most widely used method for treating waste waters.
Its main features are as follows:
(a) It uses a complex population of microorganisms of bacteria and protozoa;
(b) This community of microorganisms has to cope with an uncontrollably diverse
range of organic and inorganic compounds some of which may be toxic to the
organisms.
(c) The microorganisms occur in discreet aggregates known as flocs which are
maintained in suspension in the aeration tank by mechanical agitation or during
aeration or by the mixing action of bubbles from submerged aeration systems. Flocs
consist of bacterial cells, extracellular polymeric substances, adsorbed organic
matter, and inorganic matter. Flocs are highly variable in morphology, typically 40
to 400 mm and not easy to break apart (Fig 29.1).
Fig. 29.1 Diagram of a Floc
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Modern Industrial Microbiology and Biotechnology
(d) The flocs must have good settling properties so that separation of the biomass of
microorganisms and liquid phases can occur efficiently and rapidly in the
clarifier. Sometimes proper separation is not achieved giving rise to problems of
bulking and foaming.
(e) Some of the settled biomass is recycled as ‘returned activated sludge’ or RAS to
inoculate the incoming raw sewage because it contains a community of organisms
adapted to the incoming sewage.
(e) The solid undigested sludge may be further treated into economically valuable
products.
The advantages of the activated sludge system over the other methods to be discussed
are its efficiency, economy of space and versatility. The flow diagrams of the conventional
set-up and various modifications thereof are given in Fig. 29.2; others are shown in
Figs 29.3, 29.4 and 29.5.
Modifications of the Activated Sludge System
(i) The conventional activated sludge set-up: The basic components of the conventional
system are an aeration tank and a sedimentation tank. Before raw waste water enters the
aeration tank it is mixed with a portion of the sludge from the sedimentation tank. The
contents of the raw water are therefore broken down by organisms already adapted to the
environment of the aeration tank. The incoming organisms from the sludge exist in small
flocs which are maintained in suspension by the vigor of mixing in the aeration tank. It is
the introduction of already adapted flocs of organisms that gave rise to the name
activated sludge. Usually 25-50% of the flow through the plant is drawn off the
sedimentation tank. Other modifications of the activated sludge system are given below.
(ii) Tapered aeration: This system takes cognizance of the heavier concentration of
organic matter and hence of oxygen usage at the point where the mixture of raw sewage
and the returned sludge enters the aeration tank. For this reason the aeration is heaviest
at the point of entry of waste waters and diminishes towards the distal end. The
diminishing aeration may be made directly into the main aeration tank (Fig. 29.2b and c)
or a series of tanks with diminishing aeration may set up.
(iii) Step aeration: In step aeration the feed is introduced at several equally spaced points
along with length of the tank thus creating a more uniform demand in the tank. As with
tapered aeration the aeration may be done in a series of tanks.
(iv) Contact stabilization: This is used when the waste water has a high proportion of
colloidal material. The colloid-rich waste waster is allowed contact with sludge for a
short period of 1 - 1½ hours, in a contact basin which is aerated. After settlement in a
sludge separation tank, part of the sludge is removed and part is recycled into an aeration
tank from where it is mixed with the in-coming waste-water.
(v) The Pasveer ditch: This consists of a stadium-shaped shallow (about 3 ft) ditch in
which continuous flow and oxygenation are provided by mechanical devices. It is
essentially the conventional activated sludge system in which materials are circulated in
ditch rather than in pipes (Fig. 29.3).
(vi) The deep shaft process: The deep shaft system for waste water treatment was
developed by Agricultural Division of Imperial Chemical Industries (ICI) in the UK, from
Treatment of Wastes in Industry
a = Conventional aeration; b = Tapered aeration with direct introduction of
raw sewage; c = Tapered aeration with tank introduction of raw sewage; d
= Step aeration; e = Contact stabilization.
Fig. 29.2 Schematic Representation of Various Modifications of the Activated Sludge Set-up
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Modern Industrial Microbiology and Biotechnology
Fig. 29.3
The Pasveer Ditch: A Modification of the Activated Sludge Scheme in Which the Aeration
is Done in a Basin about ft Deep in Which the Sewage Circulates
their air-lift fermentor used for the production single cell protein from methanol. It
consists of an outer steel-lined concrete shaft measuring 300 ft or more installed into the
ground. Waste water, and sludge recycle are injected down an inner steel tube.
Compressed air is injected at a position along the center shaft deep enough to ensure that
the hydrostatic weight of the water above the point of injection is high enough to force air
bubbles downwards and prevent them coming upwards. The air dissolves lower down
the shaft providing oxygen for the aerobic breakdown of the wastes. The water rises in the
outer section of the shaft (Fig. 29.4). The system has the advantage of great rapidity in
reducing the BOD and about 50% reduction in the sludge. Space is also saved.
(vii) Enclosed tank systems and other compact systems: Since the breakdown of waste
in aerobic biological treatment is brought about by aerobic organisms, efficiency is
sometimes increased by the use of oxygen or oxygen enriched air. Enclosed tanks, in
which the waste water is completely mixed with the help of agitators, are used for
aeration of this type. Sludge from a sedimentation tank is returned to the enclosed tank
along with raw water as in the case with other systems. The advantage of the system is
the absence, (or greatly reduced) obnoxious smell from the exhaust gases, and increased
efficiency of waste stabilization. This system is widely used in industries the world over.
Compact activated sludge systems do not have a separate sedimentation tank. Instead
sludge separation and aerobic breakdown occur in a single tank. The great advantage of
such systems is the economy of space (Fig. 29.5).
29.3.1.1.1 Organisms involved in the activated sludge process
The organisms involved are bacteria and ciliates (protozoa). It was once thought that the
formation of flocs which are essential for sludge formation was brought about by the
Treatment of Wastes in Industry
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In this system of activated sludge, the sewage is pump underground and air is injected. Because of the
depth the pressure of the air is increased causing greater dissolution of oxygen. The advantage of this
method is the saving in space use.
Fig. 29.4 The Deep Shaft Aeration System
slime-forming organism, Zooglea ramigera. It is now known that a wide range of bacteria
are involved, including Pseudomonas, Achromobacter, Flavobacterium to name a few.
29.3.1.1.2 Efficiency of activated sludge treatments
The efficiency of any system is usually determined by a reduction in the BOD of the waste
water before and after treatment. Efficiency depends on the amount of aeration, and the
contact time between the sludge and the raw waste water. Thus in conventional activated
sludge plants the contact time is about 10 hours, after which 90-95% of the BOD is
removed. When the contact time is less (in the high-rate treatment) BOD removal is 60-
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Modern Industrial Microbiology and Biotechnology
Fig. 29.5 Compact Activated Sludge System
70% and the sludge produced is more. With longer contact time, say several days, BOD
reduction is over 95% and sludge extremely low.
With systems where oxygen is introduced as in the closed tank system or where there
is great oxygen solubility as in the deep shaft system, contact time could be as short as 1
hour but with up to 90% BOD reduction along with substantially reduced sludge.
29.3.1.2
The trickling filter
In the trickling filter no sludge is returned to the incoming waste water. Rather the waste
water is sprayed uniformly by a rotating distributor on a bed of rocks 6-10 ft deep. The
rotation may be powered by an electric motor or a hydraulic impulse. The water
percolates over the rocks within the bed which are 1-4 in diameter and is collected in an
under-drain. The liquid is then collected from the under drain and allowed in a
sedimentation tank which is an integral component of the trickling filter. The sludge from
the sedimentation tank is removed from time to time. Various modifications of this basic
system exist. In one modification the water may be pre-sedimented before introduction to
the filter. Two filters may be placed in series and the effluent may be recycled (Fig. 29.6
and 29.7).
Microbiology of the trickling filter: A coating of microorganisms form on the stones as the
waste water trickles down the filter and these organisms break-down the waste. Fungi,
algae, protozoa and bacteria form on the rocks. As the filter ages the aerobic bacteria
which are responsible for the breakdown of the organic matter become impeded, the
system becomes inefficient and flies and obnoxious smells may result (Fig. 29.7). The
microbial coating sloughs off from time to time.
Treatment of Wastes in Industry
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Fig. 29.6 Section through Trickling Filter Bed
Sludge
Removed
Raw
sewage
Effluent
Sedimentation
tank
Filter
Convential
Pump
Sludge
Removed
Sedimentation
tank
Raw
sewage
Single Stage
Primary
sedimendation
Filter
Secondary
sedimentation
Fig. 29.7 Scheme Illustrating Two Arrangements of Trickling Filter: Conventional and Single Stage
29.3.1.3
Rotating discs
Also known as rotating biological contactors, these consist of closely packed discs about
10 ft in diameter and 1 inch apart. Discs made of plastic or metal may number up to 50 or
more and are mounted on a horizontal shaft which rotates slowly, at a rate of about 0.5-
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Modern Industrial Microbiology and Biotechnology
Left: Transverse section
Right: Side view
Fig. 29.8 Structure of Rotating Discs (Rotating Biological Contactor)
15 revolutions per min. During the rotation, 40-50% of the area of the discs is immersed in
liquid at a time. A slime of micro-organisms, which decompose the wastes in the water,
builds up on the discs. When the slime is too heavy, it sloughs off and is separated from
the liquid in a clarifier. It has a short contact time and produces little sludge. The rotating
disc system can be seen as a modification of the tricking filter in which the waste water is
spread on rotating discs rather than on a bed of rocks.
29.4 TREATMENT OF THE SLUDGE: ANAEROBIC
BREAKDOWN OF SLUDGE
As has been seen above, sludge always accompanies the aerobic breakdown of wastes in
water. Its disposal is a major problem of waste treatment. Sludge consists of microorganisms and those materials which are not readily degradable particularly cellulose.
The solids in sludge form only a small percentage by weight and generally do not exceed
5%.
The goals of sludge treatment are to stabilize the sludge and reduce odors, remove
some of the water and reduce volume, decompose some of the organic matter and reduce
volume, kill disease causing organisms and disinfect the sludge. Untreated sludges are
about 97% water. Settling the sludge and decanting off the separated liquid removes
some of the water and reduces the sludge volume. Settling can result in a sludge with
about 96 to 92% water. More water can be removed from sludge by using sand drying
beds, vacuum filters, filter presses, and centrifuges resulting in sludges with between 80
to 50% water. This dried sludge is called a sludge cake. Anaerobic digestion is used to
decompose organic matter to reduce its volume. Digestion also stabilizes the sludge to
reduce odors. Caustic chemicals can be added to sludge or it may be heat treated to kill
disease-causing organisms. Following treatment, liquid and cake sludges are usually
spread on fields, returning organic matter and nutrients to the soil.
The commonest method of treating sludge however is by anaerobic digestion and this
will be discussed below.
Anaerobic digestion consists of allowing the sludge to decompose in digesters under
controlled conditions for several weeks. Digesters themselves are closed tanks with
provision for mild agitation, and the introduction of sludge and release of gases. About
50% of the organic matter is broken down to gas, mostly methane. Amino acids, sugars
alcohols are also produced. The broken-down sludge may then be de-watered and
Treatment of Wastes in Industry
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disposed of by any of the methods described above. Sludge so treated is less offensive and
consequently easier to handle. Organisms responsible for sludge breakdown are
sensitive to pH values outside 7-8, heavy metals, and detergents and these should not be
introduced into digesters. Methane gas is also produced and this may sometimes be
collected and used as a source of energy. Fig. 29.9 shows some anaerobic sludge digester
designs.
29.5
WASTE WATER DISPOSAL IN THE
PHARMACEUTICAL INDUSTRY
The treatment of wastes from a pharmaceutical industry is chosen to illustrate industrial
waste treatment because the wastes are representative of a broad range of materials and
include easily degradable organic materials, as well as sometimes some inorganic and
even toxic compounds. Which of the various methods of disposal is used by a particular
firm will depend on a number of factors foremost among which are: (a) the cost of the
disposal method; (b) the location of the industry; (c) the nature of the industry and hence
of its waste materials, and (d) the governmental regulations operating in the locality.
The above factors are all inter-related. For example, in siting the industry in the first
place, space for, and the type of method of, waste disposal would have been considered.
The cost of the disposal will be influenced not only by the nature and quantity of the
waste and consequently the method adopted to handle it, but also what distance needs to
be covered to have it disposed of. EPA regulations may for example dictate that the BOD
of the wastes be reduced to a certain level before being discharged into a stream; any BOD
reduction ultimately involves the expenditure of funds.
Nature of Wastes: The wastes from pharmaceutical firms may include easily degradable
materials such as emulsion syrup, malt and tablet preparations. These contain
considerable amounts of carbohydrates and hence yield wastes with high BOD.
Acids including the organic acids, acetic, formic and sulfanilic acids as well as the
inorganic HCl and H2SO4 may be added to wastes. They have to be neutralized before
being allowed into the treatment system.
Dissolved salts added in their own right or resulting from neutralization may also
enter the system. Many drugs, some toxic or inhibitory to bacteria, may also be added.
Pre-treatment: Before treatment acid (or alkali) is neutralized, dissolved salts are
removed usually by precipitation as calcium salts through lime addition, which also
neutralizes acidity. Chloride and sulfate may be removed by ion exchange or rendered
innocuous by dilution with water. Volatile compounds are stripped by pre-aeration.
Treatment: Before a routine is used within a treatment method, laboratory experiments
would have been carried out to determine how much of the wastes may be efficiently
handled within a given period. It may often be necessary to segregate the wastes, treating
the more easily biodegradable organic forms separately from those wastes rich in
inorganic materials. This is because the latter may require ‘seeding’ or the development of
microorganisms specifically able to grow in and degrade them. Seeding is achieved by
shaking a sample of the waste with a soil sample long enough for a special flora to
develop.
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Modern Industrial Microbiology and Biotechnology
Fig. 29.9 Anaerobic Digestion Systems
Treatment of Wastes in Industry
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SUGGESTED READINGS
Andrew, W. 1996. Biotechnology for Waste and Wastewater Treatment. Noyes Publications
Westwood, N.J., USA.
Eckenfelder, W.W. 2000. Industrial water pollution control. McGraw-Hill Boston, USA.
Kosric, N., Blaszczyk, R. 1992. Industrial Effluent Processing. Encyclopedia of Microbiology. Vol
2, Academic Press. San Diego, USA. pp. 473-491.
Lindera, K.C. 2002. Activated Sludge – the Process. Encyclopedia of Environmental Microbiology
Vol 1 Wiley-Interscience Publication. New York, USA. pp. 74–81.
Nielsen, P.H. 2002. Activated Sludge – the Floc. Encyclopedia of Environmental Microbiology Vol
1 Wiley-Interscience Publication. New York, USA. pp. 54–61.
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Glossary
Anabolism The biochemical processes involved in the synthesis of cell constituents from simpler
molecules usually requiring energy.
Anaerobic respiration When the final electron is an inorganic compound other than O2.
Anotation The process by which useful information is added to a raw genomic DNA sequence,
producing a frame work of understanding and enhancing its utility for downstream users.
Anticodon A sequence of three bases in transfer RNA which base-pairs with a codon in the
messenger RNA during protein synthesis.
Antiparallel This refers to nucleic acids, where one strand runs 5' ~ 3', the other 3' ~ 5'.
Antisense mRNA, an mRNA transcript that is complementary to endogenous mRNA; it is a
noncoding strand and complementary to the coding sequence of the endogenous mRNA.
Introducing a transgene coding for antisense mRNA is a strategy used to block expression of a
gene of interest.
Antisense RNA This is produced from a gene sequence inserted in the opposite orientation, so
that the transcript is complimentary to the normal mRNA and can therefore bind to it and
prevent translation.
Artificial chromosomes Cloning vectors which can carry very large inserts of foreign DNA and
exist in the cell very much like a cellular chromosome. The most widely used are bacterial artificial
chromosomes (BACs) and yeast artificial chromosomes (YACs).
ATP Adenosine triphosphate, the major energy carrier of the cell.
Autoradiography Detection of radioactivity in a sample labeled with a radioactive material, by
placing it in contact with a photographic film; the radioactive portions will imprint on the film
Autotroph An organism able to use CO2 as a sole source of carbon, for example plants and bluegreen algae (Cyanobacteria).
Auxotroph An organism that has developed a nutritional requirement through mutation.
Wthout the addition of the required material, it will not grow. The opposite is a Prototroph or wild
type.
B lymphocyte (B cell) A lymphocyte that has immunoglobulin surface receptors, produces
immunoglobulin, and may present antigens to T cells.
Bergey’s Manual A compendium of an approved list of bacteria, first published in 1923. The
second edition is currently being published in 5 volumes beginning in 2001 and is expected to be
completed in 2007. Bergey’s Manual of Systematic Bacteriology gets its name from Dr David H
Bergey first Chairman of the Editorial Board of the Manual published by the then Society of
American Bacteriologists (now called the American Society for Microbiology).
Bioinformatics The revolution in computer technology and memory storage capability has
made it possible to model grand challenge problems such as large scale sequencing of genomes
Glossary
#
and management of large integrated databases over the Internet. This vastly improved
computational capability integrated with large-scale miniaturization of biochemical techniques
such as PCR, BAC, gel electrophoresis and microarray chips has delivered enormous amount of
genomic and proteomic data. This integration of computation with biotechnology is
Bioinformatics.
cDNA, (complementary DNA) is single-stranded DNA made in the laboratory from a
messenger RNA template using the enzyme reverse transcriptase. This form of DNA is often
used as a probe in the physical mapping of a chromosome; it is also used when it is desired to
express a eukaryotic gene in a prokaryotic cell; for cloning into prokaryotic cell, the introns in a
eukaryotic mRNA are spliced off and the intron-free mRNA converted to cDNA with reverse
transcriptase.
cDNA library, A cDNA library is a collection of DNA sequences generated from mRNA
sequences. This type of library contains only DNA that codes for proteins and does not include
any non-coding DNA. The complete cDNA library of an organism gives an indication of the total
amount of the proteins it can possibly express. The cDNA sequence also gives the genetic
relationship between organisms through the similarity of their cDNA.
C’hemolithotroph An organism obtaining its energy from the oxidation of inorganic molecules.
Chemoorganotroph An organism obtaining its energy from the oxidation of organic.
Cistron A sequence of bases in DNA that specifies one polypeptide.
Clone A population of cells all descended from a single cell. Also, a number of copies of a DNA
fragment obtained by allowing an inserted DNA fragment to be replicated by a phage or plasmid
compounds.
Genetic Code The triplet codons that determine the types of amino acids inserted into a
polypeptide chain during translation. There are 61 codons for 20 amino acids and three stop
codons.
Genetic map The arrangement of genes on a chromosome.
Genome All the genes present in an organism.
Genomics The field of science that studies the entire DNA sequence of an organism’s genome.
The goal is to find all the genes within each genome and to use that information to develop
improved medicines as well as answer scientific questions.
Histone proteins These are present in eucaryotic chromosomes; histones and DNA give
structure to chromosomes in eucaryotes.
Introns Non-coding sequences within genes.
Kilo basepair 1,000 basepairs, a unit of DNA length abbreviated KB.
Knock out mice A transgenic mouse in which a gene function has been disrupted or knocked out.
It is used to produce animal models for the study of human disease.
Mass spectrometry is an analytical technique used to measure the mass-to-charge ratio of ions. It
is most generally used to find the composition of a physical sample by generating a mass
spectrum representing the masses of sample components. The technique has several
applications, including:
1. identifying unknown compounds by the mass of the compound and/or fragments
thereof.
2. determining the isotopic composition of one or more elements in a compound.
3. determining the structure of compounds by observing the fragmentation of the
compound.
4. quantitating the amount of a compound in a sample using carefully designed methods
(mass spectrometry is not inherently quantitative).
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Modern Industrial Microbiology and Biotechnology
5. studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals
in vacuum).
6. determining other physical, chemical or even biological properties of compounds with a
variety of other approaches.
Methanogenesis The biological production of methane.
Nuclear Magnetic Resonance (NMR) is a physical phenomenon based upon the magnetic
property of an atom’s nucleus. NMR spectroscopy is one of the principal techniques used to
obtain physical, chemical, electronic and structural information about a molecule. It is the only
technique that can provide detailed information on the exact three-dimensional structure of
biological molecules in solution. Also, nuclear magnetic resonance is one of the techniques that
has been used to build elementary quantum computers.
Operons Typically present in prokaryotes, these are clusters of genes controlled by a single
operator; an operator itself is a region of an operon, close to the promoter to which a receptor
protein binds.
Promoter A DNA sequence lying upstream of from the gene to which RNA polymerase binds.
Proteome The totality of the proteins present in a cell.
Proteomics The study of the study of the structure, function and regulation of the proteins in an
organism.
Sense mRNA, endogenous mRNA molecules which encode functional proteins; it is a 5' to 3'
mRNA molecule.
Shine-Dalgarno sequence A conserved sequence in prokaryotic mRNAs that is complementary
to a sequence near the 5´ terminus of the 16S ribosomal RNA and is involved in the initiation of
translation.
Site-directed mutagenesis A technique for construction a mutation in a gene in vitro by altering
a base or bases in the gene.
TATA box Also called Hogness Box, an AT-rich region of the DNA with the sequence TATAT/
AAT/A located before the initiation site.
Transcription factor Transcription factor is a protein that binds DNA at a specific promoter or
enhancer region or site, where it regulates transcription.
Transfer RNA (tRNA) A small RNA of 75 – 85 bases that carries the anticodon and the amino acid
residue required for protein synthesis.
Index
# !
Index
Acetic acid bacteria 23, 41, 84, 259, 264266, 270, 280, 283, 284, 349,
Acetobacter 23, 41, 84, 253, 280, 288
Acidothiobacillus ferrooxidans 424
Acquired immunity 475
Acridine 79, 131
Actinobacteria 22, 24, 26
Actinomycetes 27, 29, 30, 57, 60, 80, 83,
92, 93, 95, 131, 135, 136, 148, 175-177,
186, 203, 204, 230, 300, 432, 436, 441,
444, 445, 451,
Activated sludge system 509, 510, 512,
514
Adjuncts 65, 238, 239, 239, 244, 248, 281,
405
Agrobacterium tumefaciens 149
Air lift fermentors 202
Akamu 335, 349
Akpu 335
Alcohol 23, 56, 60, 62, 89, 137, 238, 251,
260, 263, 265, 285, 309, 311, 313, 314,
370, 373, 374, 377
properties 373
Alcoholic beverages from Africa 272
Alkanes 88, 89, 204, 294, 304
American Type Culture Collection
(ATCC) 9, 172
Amino acids
manufacture 384
production by mutants 390
production by Metabolic engineered
organisms 391
uses 380
Amino acids, metabolites from 96
synthetic routes 95
Aminoglycosides 430
Amphothericin B 430, 440
Amylases 70, 72, 239, 242, 244, 245, 258,
278, 299, 340, 341, 399, 402, 405, 416
Amylopectin 67, 70, 72, 162, 164
Amylose 67, 69, 162, 245
Anabolism 77, 78, 467, 520
Anaerobic digestion 516, 518
Ansamacrolides 430
Antibiotics 29, 34, 57, 80-83, 92, 97, 122,
132, 136, 137, 143, 147, 178, 202, 212,
219, 222, 228, 234, 286, 331, 345, 346,
380, 389, 417, 423, 429-431, 435, 436,
438-441, 443-446, 448-453, 459, 464,
484, 488, 494, 498
classification and nomenclature 429
new definition 453
newer search methods 445
search for 439
Anti-foams (antifoams) 190, 229
Antigens 34, 120, 318, 477, 478-482, 486,
490, 520
Anti-tumor antibiotics
search for 449
Ascomycetes 29, 319, 444
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Modern Industrial Microbiology and Biotechnology
Asepsis 222, 232
Auxotrophic mutants 113, 132, 390, 395
Axis of symmetry 139
Bacillus thuringiensis 8, 160, 316, 318, 319,
321, 323-326
delta-endotoxin 322
formulation 324
Bacillus thuringinensis var. israelensis 318
Bacterial artificial chromosomes (BACs)
46, 520
Bacterial phyla 22
Bacteriocins 141, 429
Bacteriophages 33, 131, 141, 146, 176,
221, 232, 329, 345
groups and morphology 233
Baker’s yeasts 294, 306, 308, 310, 312, 314
Barley grain 239, 243, 244
malt 57, 72, 238, 244, 281
Basidiomycetes 29, 31, 319
Bdellovibrio 33, 221
Beer brewing 65, 198, 237, 255, 307, 314,
405
bottom- and top-fermented 238
defects 240, 253, 255
Beer, types 237
Bergey’s Manuals 21
Beta alba 60
Beta-lactams 82, 430, 438, 439
Biochemical Oxygen Demand (BOD) 506
Bioinformatics 50, 51, 52, 488, 520
Bioleaching 421, 422
Biological control 316-319, 321
Biotechnology 3, 5, 19, 20, 22, 34, 36, 41,
51, 122, 126, 143, 152, 157, 171, 480,
488, 493
Blood meal 65, 385
>-Oxidation of fats 90
>-propiolactone 228, 230, 474, 479
Brandy 275-278
Bread 10, 31, 273, 308, 334-343, 405, 455
systems of Bread-making 339
Brewer’s yeasts 241
Carbon Decolorization 217
Carl R Woese 18
Cassava 63, 64, 65, 69, 239, 259, 299, 334,
335, 350, 352, 375, 395, 400
Catabolism 10, 77, 83, 84, 88, 95, 105, 117
Catabolite regulation 100, 103, 105, 115117, 461
Cavitator 288
CD binding inhibitors 491
Cell disruption 212
Cell-mediated immunity 475, 477, 490
Cellulose 10, 17, 23, 54, 62, 67, 73-75, 188,
201, 223, 299, 324, 407, 411, 413, 516
Cell-wall 73, 115, 389, 419
Cephalosporins 431, 435, 436, 438, 439
Champagne 266-269
Cheese 10, 153, 202, 281, 303, 313, 334,
344-347, 366, 416
manufacture 345
Chemical insecticides 315
mutagens 128
Chemical Oxygen Demand (COD) 507
Chemostat 199, 200, 409
Chemosterilants 227
Chloramphenicol 21, 92, 93, 117, 119,
143, 234, 429
and analogues 430
Chromatography 209, 214, 217, 263, 349,
369, 408, 443, 452, 488, 494
Citric 23, 79, 114, 132, 136, 185, 206, 251,
288, 365-368, 452
acid 23, 61, 79, 89, 132, 185, 206, 365,
368, 389, 452
Claviceps 29, 81, 115, 455, 460
Clinical trials 446-448, 482, 498, 499, 501
Cocoa 164, 335, 354, 355
Codons 38, 39, 46, 47, 130, 521
Coffee 186, 299, 335, 354, 355
Combinatorial chemistry 453, 493, 497
Conjugation 80, 127, 135, 136, 141, 144,
148, 321, 439
Contaminants, basis of loss 221
Contaminations 174, 198, 221, 222, 229,
275, 486
Index
Continuous brewing 202, 255, 257
fermentations 196, 198, 201
Corn grits 239
steep liquor 56, 57, 59, 60, 120, 309,
313, 322, 389, 432, 470
Corticosteroids 466, 467, 470
Corynebacterium manihot 351
Crystallization 60, 209, 217, 218, 365,
396, 402, 494
Culture collections 9, 122, 171, 172, 173,
450,
Dawa dawa 335
Decoction method 245-247
Deep sea 446, 447
Deuteromycetes 31
Disruption of cells 209
Distillers soluble 60
DNA chimera 137, 138
chips 42
modification and restriction 138
sequencing 34, 44, 45, 50
vaccines 481
Domains 18, 20, 21
properties 21
Drug discovery 43, 50, 51, 453, 489, 491,
492, 493, 500
approval procedures for new drugs
497
cell-based 489
enzyme assays 402
newer methods 492
receptor-binding 491
Drum driers 219, 311
Dryers 219
Dump leaching 422
Earth summit 3
Edible vaccines 155-157, 481
Electromagnetic spectrum 226
Embden-Meyerhof-Parnas (EMP
Pathway) 84
Enrichment methods 124
# #
Entner-Duodoroff Pathway (ED) 84
Environmental Protection Agency (EPA)
505
Enzymes 3, 8, 35, 55, 64, 70, 72, 79, 83,
101, 105, 107, 135, 153, 212, 242, 257,
398, 405-408, 435, 483
classification 399
industrial uses 400
manipulating organisms for higher
yields 416
production 406
Enzymes, use in brewing 257
Ergot alkaloids 29, 81, 96, 116, 455-462
production 459
Ethylene oxide 223, 227, 228
Extraction 10, 50, 125, 208, 213, 297, 323,
368, 372, 408, 434, 436, 453, 462, 497,
508
Feedback regulation 100, 105-109, 114,
117, 418, 419, 461
Fermentation 5, 9, 10, 25, 28, 31, 32, 55,
57, 60, 84, 86, 88, 110, 115, 125, 137,
157, 172, 183-199, 201-206
Fermented foods 10, 334, 343, 348, 350,
355, 356, 359, 360
advantages 334
Fermentor 10, 33, 183, 184, 185, 192, 196,
198, 203, 225, 229, 249, 255, 286, 288,
309, 313, 331, 394, 407, 512
aerated stirred tank batch bioreactor
184
aeration and agitation 186
anaerobic 195
construction materials 185
fermentor configurations 197
gas phase barriers 187
submerged 183, 184
surface or tray 206
Fermentors 4, 149, 167, 168, 183-185, 188,
195, 196, 200, 206, 225, 229, 293, 297,
309, 331, 416, 423, 432, 484
Filtration 10, 209-212, 217, 222, 224, 230,
244, 247, 288, 310, 323, 396, 408, 491
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Modern Industrial Microbiology and Biotechnology
Firmicutes 22-24, 26
Fish meal 66
sauce 335, 360
Flocs 210, 509, 512
Foam extraction (fractionation) 211
Foaming 188-190, 205, 286, 383, 510
Foams 188-190, 229
Fodder yeasts 306
Food yeasts 307, 311-313
Foo-foo 335, 350, 352
Formaldehyde 223, 228, 342, 369, 480,
485
Frings acetator 286-288
Fungi 18, 29, 30, 31, 57, 74, 83, 84, 92-96,
131, 135, 153, 167, 172, 176, 204, 206,
227, 257, 259, 278, 300, 315, 322, 323,
342, 347, 357, 403, 404, 430, 436, 444,
460, 468, 471, 492, 509
G+C content 26
Garri 334, 350-352
Gaseous sterilization 226, 229
Gasohol 5, 63, 374
Gene identification 47
Gene pool 171, 418
Gene transfer into
animals 151
bacteria 146
plants 148
Genetic code 38, 521
applications 152-170
engineering 3, 7, 8, 41, 126, 127, 135,
137, 138, 140, 142, 144, 151, 154, 158,
160, 161, 164, 166, 167, 377, 395, 473
Genetic improvement 122
macromolecules 35, 50
Genomics 34, 42, 46, 50, 480, 493, 495,
497, 521
Geotrichum candidum 351
Grape wines
classification 266
defects 265
fortified 269
yeasts used 263
Grapes (Vitis vinfera) 262
Growth factors 31, 54, 55, 60, 62, 63, 134,
372, 484
Hemicelluloses 62, 67, 73, 154, 244
Heterokaryosis 127
Hops 237-240, 248, 307
Hordeum vulgare 239
Hydrocarbons 21, 54, 88, 201, 294, 295,
300, 312, 451, 508
Idiophase 57, 58, 81, 83, 115, 116, 117,
120, 202, 419, 434
Idli 335, 359
Immobilized biocatalysts 408, 409
advantages 409
bioreactors for 415
cells 385, 409, 413, 416, 471
enzymes 409, 416, 435
Immune system 155, 164, 472, 477, 486,
490
Industrial alcohol 60, 222, 276, 307, 373
microbiology 3-5, 7, 9, 11, 19, 22, 29,
31, 41, 43, 50, 54, 62, 64, 66, 126, 129,
143, 152, 171, 230, 417, 482, 487
Industrial products
primary metabolites 78
secondary metabolites 79
Industrial vs Medical Microbiology 4
Inflammatory reactions 489, 491
Inoculum preparation 205
Intellectual property 5 (see also Patents)
Ion exchange 209, 214, 216, 217, 241, 366,
396, 411, 412, 517
Ionizing radiations 127, 226
Isolation de novo 123
Isoprene 94, 462
Itaconic acid 365
Jerusalem artichoke 65
Kilning 243, 258, 277, 405
Kokonte 335, 350, 352
Index
Lactic acid 24-27, 59, 79, 89, 196, 245,
259, 264, 273, 313, 345, 355, 369-372
acid bacteria 24-27, 29, 59, 221, 253,
259, 265, 270, 307, 334, 341, 345, 346,
348, 349, 351, 352, 353, 355, 358, 371
properties and uses 348
Lactobacillus helveticus 26, 273
Lactobacillus bulgaricus 29, 345
Lactobacillus mesenteroides 259
Lactobacillus plantarum 259, 341, 349, 353,
370
Lafun 335, 352
Lagering 237, 242, 243, 251, 255, 259
Leptospirillum ferrooxidans 424
Ligase 141, 400
Lignin 62, 73, 75, 298
Linamarin 350-352
Linocosaminides 430
Liquid extraction 212-214
Literature search 122
Lotaustralin 350, 352
Lymphocytes 475-478, 491
Lyophilization 175, 177
Macrolides 123, 430
Mahewu 349
Maize 35, 59, 60, 63, 67, 97, 239, 273, 334,
349, 400
Malting 64, 238, 239, 242, 258, 262
Mashing 238, 242, 244, 245, 258, 259
methods 245
Metabolic engineering 127, 136, 391, 394
pathways 77, 90, 120, 387
Metabolism 10, 23, 34, 76-84, 89, 99, 115,
174, 297, 461, 491, 499
Metagenomics 48, 49
Methylbromide 228
Microarrays 42, 43, 50
Microbial insecticides 326
metabolites 44, 122-125, 440, 488, 489,
491
metabolites, testing 124
# %
Microorganisms, advantages
in biotechnology and industrial
production 20
Microorganisms,
characteristics for use in biotechnology
31
Milk, composition 343
Millets 64, 258
Mining microbiology 423
Miso 335, 356, 358
Molasses 56, 57, 60, 61, 189, 201, 206,
275, 277, 278, 281, 294, 299, 307, 309,
310, 312, 322, 325, 368, 377, 395, 432
Mutagens practical isolation 131
Mutation 32, 101, 110, 113, 126-132, 136,
172, 241, 309, 346, 391, 432, 492
Naringin 405
Neomycin 116, 430, 448
Nephila clavipes 167
Non-specific immunity 474, 475
Nucleic acid biotechnology 3
Nucleoside antibiotics 92
Nucleosides 92, 304, 430
Ogi 334, 349
Ogili 335, 360
Oncom 335, 358
Open reading frame (ORF) 46, 47
Orsellenic acid 93, 94
Overproduction
derangement of regulatory
mechanisms, primary metabolites 110
derangement of regulatory
mechanisms, secondary metabolites
116
regulatory mechanisms 101
Oxygen 10, 25, 33, 89, 183-188, 191, 193196, 203, 249, 253, 264, 269, 286, 298,
331, 341, 424, 468, 506, 507, 514
Palindromes 139
Palm wine 259, 270-272, 281, 294
# &
Modern Industrial Microbiology and Biotechnology
Pasteurization 224, 234, 265, 270, 345
Patenting in microbiology and
biotechnology 7- 9
Patents 5-7, 122, 295, 322
Patents and trademarks office 6
Pectinases 352, 400, 403, 404
Pediococcus damnosus 254, 273
Pediococus streptococcus damnosus 221
Penicillin 3, 10, 30, 56, 81, 115, 214, 389,
406, 416, 430, 432, 434, 435, 469
natural and biosynthetic 433
semi-synthetic penicillins 435
Penicillium chrysogenum 81, 82, 116, 132,
136, 432
Pentose Phosphate Pathway (PP) 84, 92,
103
Peptides 59, 80, 101, 160, 190, 244, 342,
417, 430, 449, 463, 478, 480
Permeability 100, 108, 109, 114, 126, 213,
389, 419, 446, 447
Permeabilization 148, 213
Pharmamedia 59, 322
Phenazines 92, 430
Phenyl acetic acid 433
Phosphoketolase Pathway (PP) 84, 88
Phycomycetes 29, 319
Pickled cucumbers 335
Pilot fermentor 10, 205
Plasmids 7, 21, 135, 141, 143, 144, 146,
321, 419, 439
Polyenes 430, 445
Polyethers 191, 430
Polyketide 93
Polymerase Chain Reaction (PCR) 34, 39
applications 41
Precursors 57, 82, 120, 240, 254, 433-436,
448, 455
Preservation of microorganisms
by dehydration 176
by lowering the growth temperature
174
by reduction of nutrients 178
Primary and secondary metabolites 4, 83,
91, 137
Probes 192, 193, 229
Procaryotic and eukaryotic cells 17
Production fermentor 10, 205, 310
Production of vaccines 202, 482, 484
Promoters 38, 51, 80, 122
Protein denaturation 39
Protein folding 39
Protein synthesis 18, 19, 34, 36, 37, 38,
101, 102, 103, 107, 143, 147, 418, 520
negative control of 101
positive control of 102
Proteomics 50, 480, 493, 522
Protoplast fusion 127, 136
Purification 61, 208, 209, 217, 218, 368,
396, 408, 414, 463, 494, 508
Puromycin 116, 430
Pyruvate 84, 86, 108
Radiations 127, 226
Raw materials 10, 25, 55-59, 62, 66, 153,
201, 238, 278, 334, 383, 385, 388, 395
Raw materials, criteria for use 56
Restriction endonucleases 138-141, 145
Reverse vaccinology 481, 482
Rhizobium 327-333
inoculants 328
Ribosome sub-units 18
Ribosomes 18, 19, 35, 37, 38, 417
Rice 5, 56, 63, 65, 164, 166, 206, 239, 281,
356, 359, 368, 406
Rifamycin 80, 430
16S RNA 18, 22
18S RNA 19
Rotary vacuum filter 209, 210, 310, 396,
434, 436
Rum 4, 60, 64-66, 123, 159, 169, 192, 219,
226, 241, 272, 277, 307, 311, 370, 377,
392, 408, 435, 443, 490, 507
Saccharification 66, 72, 73, 246, 259, 277,
416
Index
Saccharomyces cerevisiae 178, 238, 249, 272,
278, 294, 307, 309, 314, 336, 375, 377
Saccharomyces uvarum 237, 314, 377
Saccharum officinarum 60
Sauerkraut 335, 353
Secondary metabolites 29, 57, 78-84, 91,
95, 97, 100, 115, 117, 122, 137, 170, 202,
405, 488
methods for deranging 120
Secondary metabolites, physiology 82
Shikimate-Chorismate 92, 93
Single Cell Protein (SCP) 293
Single cell protein
production substrates 294
safety 303
Site-directed mutation 127, 136
Solvent extraction 209, 213, 214, 215, 297,
372, 422, 488
Sorbitol 23, 191,
Sorghum 64, 65, 69, 239, 258-260, 273,
294, 348, 375, 400
beers 258
Soy sauce 335, 356-358
Spirit beverages
measuring alcoholic strength 274
production 275
Spirits 275, 278, 307, 314, 373
Starch 10, 26, 54, 59, 63, 67, 69, 70, 72, 75,
99, 116, 124, 153, 162, 164, 193, 201,
230, 238, 239, 242, 244-246, 258, 259,
275, 299, 310, 322, 336, 340, 343, 351,
395, 400, 403, 408, 416
Steam 10, 65, 74, 185, 223, 224, 225, 229,
276, 310, 372, 375
Sterility 10, 185, 186, 188, 221, 222, 383,
423, 486, 487
Sterility, methods for achieving 222
Steroids 93, 436, 464, 466-471, 484
Steroids and Sterols
microbial screening 471
microbial transformations 470
Sterols 464, 466, 470
Stout 57, 238, 243, 249, 252
# '
Strain improvement 14, 125, 126, 134,
394, 444, 460
Strains 26, 33, 80, 122, 125, 126, 136, 148,
171, 237, 238, 241, 249, 259, 275, 283,
286, 300, 308, 309, 311, 333, 346, 357,
368, 375, 377, 391, 395, 402, 405, 425,
432, 439, 479, 480
Streptococcus thermophilus 345
Streptomyces 29, 80, 93, 117, 120, 129,
135, 221, 402, 419, 430, 436, 449, 471,
490, 492
Streptomycin 21, 80, 117, 212, 216, 430,
448, 453
Sufu 335, 356
Sulfur dioxide 59, 67, 73, 228, 265, 268
Svedberg units 18, 37
Sweet potatoes 63, 64
Tea 186, 335, 354, 405
Terpenes and steroids 93
Tetracyclines 58, 93, 132, 430
Thiobacillus ferooxidans 424
Thiobacillus organosporus 424
Toxoids 479-482, 485, 486
Traditional biotechnology 3
vaccines 479
Transcription 21, 36, 38, 80, 101, 104, 105,
128, 144, 159, 162, 467, 522
Transduction 127, 134, 135, 151
Transfer RNAs (tRNAs) 39
Transformation 80, 115, 127, 135, 141,
146, 147, 439, 466, 467, 469-471, 489
Translation 36, 37, 38, 46, 47, 162, 205,
521, 522
Tricarboxylic acid cycle 84, 89, 114, 367
Trickling filter 285, 287, 409, 509, 514,
515
Triketide 94
Trophophase 57, 81, 115, 117, 120, 202,
434
Turbidostat 199, 200
Tyndallization 224
Ugba 335, 360
#!
Modern Industrial Microbiology and Biotechnology
UK patent law 6
Ultra violet light 226, 318, 319, 325
Ultraviolet 127, 128, 132, 227, 325, 432,
443, 452, 479
Uncultured microorganisms 49
Upstream/downstream processes 11
Use in biological classification 19
Vaccines 7, 34, 50, 148, 155, 169, 202, 228,
472, 479-487
Vectors 8, 141, 143, 144, 147, 151, 315,
319, 325, 469
Vegetables 26, 170, 280, 335, 353
Vinegar 23, 79, 196, 280-288
manufacture 283
processing 288
types 281
uses 280
Vitamin B 12 57, 63, 314
Volatile Suspended Solids (VSS) 507
Wastes 10, 56, 202, 294, 298-301, 505,
508, 509, 517
Water, importance in brewing 240
Wheat 63, 65, 69, 238, 271, 277, 302, 332,
335, 357, 405, 406
Whiskey 60
Wine making processes 262
Working stock 172
Yams 64, 65, 468
Yeast production 54, 198, 299, 306, 309,
312
Yeasts 3, 29, 31, 56, 63, 79, 84, 99, 108, 115,
126, 136, 172, 176, 185, 202, 209, 212,
228, 241, 249, 259, 263, 265, 272, 294,
297, 305, 307, 308, 311, 313, 331, 334,
337, 341, 349, 351, 356, 368, 375, 405,
452
Yeasts, factory production 309
Yoghurt 3, 10, 26, 202, 347, 402