Current Developments in Biotechnology and Bioengineering Series Editor: Professor Ashok Pandey Centre for Innovation and Translational Research CSIR-Indian Institute of Toxicology Research Lucknow, India & Sustainability Cluster School of Engineering University of Petroleum and Energy Studies Dehradun, India Current Developments in Biotechnology and Bioengineering Filamentous Fungi Biorefinery Edited by Mohammad J. Taherzadeh Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden Jorge A. Ferreira Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden Ashok Pandey Centre for Innovation and Translational Research, CSIR-Indian Institute of Toxicology Research, Lucknow, India & Sustainability Cluster, School of Engineering, University of Petroleum and Energy Studies, Dehradun, India Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States Copyright © 2023 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-323-91872-5 For information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals Publisher: Susan Dennis Editorial Project Manager: Helena Beauchamp Production Project Manager: Kiruthika Govindaraju Cover Designer: Matthew Limbert Typeset by STRAIVE, India Contents Contributors Preface xiii xvii 1. World of fungi and fungal ecosystems Gabriela Ángeles de Paz, Ulises Conejo Saucedo, Rafael León Morcillo, and Elisabet Aranda 1 1. Introduction 1 2. Fungal morphology 1 3. Lifestyles of fungi 4 4. Taxonomy of fungi 8 5. Fungal diversity 13 6. Fungal ecosystems 14 7. Applications of fungi in biorefineries 18 8. Conclusions and perspectives 19 References 19 2. Fungal biotechnology Mohammadtaghi Asadollahzadeh, Marzieh Mohammadi, and Patrik Roland Lennartsson 31 1. Introduction 31 2. Fungal cultivation and requirements 33 3. Fungal biorefineries 42 4. Fungal metabolites 50 5. Fungal biomass 54 6. Conclusions and perspectives 57 References 57 v vi Contents 3. Fungal biology Soumya Mukherjee and Shakuntala Ghorai 67 1. Introduction 67 2. The fungal world 69 3. Classification of fungi 78 4. Conclusions and perspectives 97 Acknowledgments 98 References 98 4. Mycotoxins Manikharda, Hanifah Nuryani Lioe, Rachma Wikandari, and Endang Sutriswati Rahayu 105 1. Introduction 105 2. Major groups of mycotoxins: chemistry and processing stability 108 3. Occurrence of mycotoxins in food 124 4. Toxigenic fungi and factors affecting mycotoxins production 130 5. Prevention and reduction of mycotoxins 131 6. Detection and determination of mycotoxins 136 7. Conclusions and perspectives 137 References 138 5. Sampling, preservation, and growth monitoring of filamentous fungi Sharareh Harirchi, Neda Rousta, Sunita Varjani, and Mohammad J. Taherzadeh 149 1. Introduction 149 2. Applications of filamentous fungi 150 3. Isolation, identification, and sampling of filamentous fungi 151 4. Preservation of filamentous fungi 160 Contents vii 5. Growth monitoring of filamentous fungi 168 6. Conclusions and perspectives 175 Acknowledgments 175 References 175 6. Industrial wastes as feedstock for filamentous fungi growth Pooja Sharma 181 1. Introduction 181 2. Microbiology of filamentous fungi 183 3. Diversity of filamentous fungi 184 4. Wastes, residuals, and wastewater as nutrients 185 5. Role of filamentous fungi in pollution reduction 187 6. Removal of microplastic using filamentous fungi 189 7. Conclusions and perspectives 190 References 191 Further reading 196 7. Filamentous fungal morphology in industrial aspects Anil Kumar Patel, Ruchi Agrawal, Cheng-Di Dong, Chiu-Wen Chen, Reeta Rani Singhania, and Ashok Pandey 197 1. Introduction 197 2. Industrial bioprocess with filamentous fungi 198 3. Important factors for industrialization of bioprocess with filamentous fungi 200 4. Applications of filamentous fungi in pulp and biofuel industries 204 5. Conclusions and perspectives 212 References 212 Further reading 217 viii Contents 8. Bioreactors and engineering of filamentous fungi cultivation 219 Daniel G. Gomes, Eduardo Coelho, Rui Silva, Lucı́lia Domingues,
A. Teixeira and Jose 1. Introduction 219 2. Engineering fundamentals in bioreactor design 220 3. Traditional bioreactor designs for filamentous fungi cultivation 224 4. Main reactor designs in solid-state fermentation processes 235 5. Filamentous fungi morphology and reactor design 236 6. Conclusions and perspectives 242 Acknowledgments 243 References 243 9. Filamentous fungi processing by solid-state fermentation Marta Cebrián and Jone Ibarruri 251 1. Introduction 251 2. Main filamentous species and substrates 253 3. Bioreactors and process control parameters: Critical aspects 258 4. Products and current industrial applications 270 5. Conclusions and perspectives 282 References 284 10. Production of industrial enzymes by filamentous fungi Zohresadat Shahryari and Seyyed Vahid Niknezhad 1. Introduction 2. Applications of industrial enzymes 3. Advantages of enzymes production by filamentous fungi 4. Filamentous fungi in enzymes production 5. Platforms for enzymes production by filamentous fungi 6. Challenges for the development of industrial enzymes production by filamentous fungi 293 293 294 294 295 299 303 Contents 7. Strategies to improve enzymes production by filamentous fungi 8. Heterologous protein expression in filamentous fungi 9. Economic aspects and filamentous fungal enzymes market ix 305 311 313 10. A roadmap toward future research on filamentous fungal enzymes production 313 11. Conclusions and perspectives 314 References 314 11. Production of bioactive pigmented compounds by filamentous fungi
Laurent Dufosse 325 1. Introduction 325 2. New non-mycotoxigenic fungal work horses for the production of polyketide pigments as food colorants 327 3. Focus on azaphilones 328 4. Fungal hydroxyanthraquinoid (HAQN) pigments as potential food colorants 331 5. Focus on anthraquinones 332 6. Fungi from marine ecological niches as novel sources of chemically diverse pigments 334 7. Conclusions and perspectives 338 Acknowledgments 339 References 339 12. Filamentous fungi for food Rachma Wikandari, Manikharda, Ratih Dewanti-Hariyadi, and Mohammad J. Taherzadeh 343 1. Introduction 343 2. Filamentous fungi as protein source 344 3. Filamentous fungi in food applications 350 4. Nutritional consideration 373 x Contents 5. Safety of fermented foods using filamentous fungi 376 6. Industrial production of filamentous fungi-based food 379 7. Conclusions and perspectives 385 Acknowledgment 385 References 385 13. Filamentous fungi as animal and fish feed ingredients 399 Sajjad Karimi, Jorge A. Ferreira, and Mohammad J. Taherzadeh 1. Introduction 399 2. Animal compound feed 400 3. Single-cell proteins (SCPs) 404 4. Fungi kingdom 405 5. Fungal biomass composition as animal feed nutrient 406 6. Animal feed 421 7. Applications of fungal biomass as feed 423 8. Economic and environmental aspects 424 9. Conclusions and perspectives 425 Acknowledgments 425 References 425 14. Production of alcohols by filamentous fungi Behzad Satari and Hamid Amiri 435 1. Introduction 435 2. Feedstocks for fermentative production of alcohols 437 3. Production of ethanol by filamentous fungi 438 4. Metabolic engineering of filamentous fungi for production of ethanol 443 5. Ethanol recovery and concentration 444 6. Fermentative production of butanol 445 7. Conclusions and perspectives 448 References 448 Contents 15. Biological production of organic acids by filamentous fungi Vivek Narisetty, G. Renuka, K. Amulya, Kamalpreet Kaur Brar, Sara Magdouli, Parameswaran Binod, Vinod Kumar, S. Venkata Mohan, Ashok Pandey, and Raveendran Sindhu xi 455 1. Introduction 455 2. Itaconic acid 456 3. Gluconic acid 460 4. Citric acid 462 5. Oxalic acid 466 6. Conclusions and perspectives 468 Acknowledgments 471 References 471 16. Production of antibiotics by filamentous fungi Parameswaran Binod, Raveendran Sindhu, and Ashok Pandey 477 1. Introduction 477 2. History 478 3. Filamentous fungi producing antibiotics 479 4. Production of antibiotics 480 5. Examples of fungal antibiotics 485 6. Conclusions and perspectives 492 Acknowledgment 492 References 492 17. Production of fungal biopolymers and their advanced applications
dric Delattre, Gustavo Cabrera-Barjas, Aparna Banerjee, Ce Saddys Rodriguez-Llamazares, Guillaume Pierre, Pascal Dubessay, Philippe Michaud, and Akram Zamani 497 1. Introduction 497 2. Fungal cell wall 499 xii Contents 3. Fungal cell wall polysaccharides 500 4. Effect of growing conditions on production of fungal polysaccharides 505 5. Extraction and purification of fungal biopolymers 508 6. Applications of fungal biopolymers 512 7. Conclusions and perspectives 523 References 523 18. Versatility of filamentous fungi in novel processes € lru Bulkan, Jorge A. Ferreira, Mohsen Parchami, Taner Sar, Gu and Mohammad J. Taherzadeh 1. Introduction 2. Brewery waste 3. Fruit industry waste 4. Bioethanol industry wastes 5. Fish processing industry waste 6. Oil processing industry waste 7. Potato processing industry waste 8. Sugar processing industry waste 9. Dairy processing industry 533 533 534 540 544 548 550 553 556 558 10. Conclusions and perspectives 560 Acknowledgments 561 References 561 Index 575 Contributors Ruchi Agrawal The Energy and Resources Institute, TERI Gram, Gwal Pahari, Haryana, India Hamid Amiri Department of Biotechnology, Faculty of Biological Science and Technology; Environmental Research Institute, University of Isfahan, Isfahan, Iran K. Amulya Bioengineering and Environmental Sciences, Department of Energy and Environmental Engineering, CSIR-Indian Institute of Chemical Technology, Hyderabad, India Elisabet Aranda Institute of Water Research; Department of Microbiology, University of Granada, Granada, Spain Mohammadtaghi Asadollahzadeh Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden Aparna Banerjee Centro de investigación en Estudios Avanzados del Maule (CIEAM), Vicerrectorı́a de Investigación Y Posgrado, Universidad Católica del Maule, Talca, Chile Parameswaran Binod Microbial Processes and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology (CSIR-NIIST), Thiruvananthapuram, Kerala, India Kamalpreet Kaur Brar Department of Civil Engineering, Lassonde School of Engineering, York University, Toronto, ON; Industrial Waste Technology Center, Abitibi Temiscamingue, QC, Canada € lru Bulkan Swedish Centre for Resource Recovery, University of Borås, Borås, Gu Sweden Gustavo Cabrera-Barjas Universidad de Concepción, Unidad de Desarrollo Tecnológico (UDT), Coronel, Chile Marta Cebrián AZTI, Food Research, Basque Research and Technology Alliance (BRTA), Parque Tecnológico de Bizkaia, Derio, Bizkaia, Spain Chiu-Wen Chen Department of Marine Environmental Engineering, National Kaohsiung University of Science and Technology, Kaohsiung City, Taiwan xiii xiv Contributors Eduardo Coelho CEB—Centre of Biological Engineering, University of Minho, Braga, Portugal Gabriela Ángeles de Paz Institute of Water Research, University of Granada, Granada, Spain dric Delattre Universite  Clermont Auvergne, Clermont Auvergne INP, CNRS, Institut Ce Pascal, Clermont-Ferrand; Institut Universitaire de France (IUF), Paris, France Ratih Dewanti-Hariyadi Department of Food Science and Technology, IPB University, Bogor, Indonesia Lucı́lia Domingues CEB—Centre of Biological Engineering, University of Minho, Braga, Portugal Cheng-Di Dong Department of Marine Environmental Engineering, National Kaohsiung University of Science and Technology, Kaohsiung City, Taiwan  Clermont Auvergne, Clermont Auvergne INP, CNRS, Institut Pascal Dubessay Universite Pascal, Clermont-Ferrand, France  Chemistry and Biotechnology of Natural Products (CHEMBIOPRO), Laurent Dufosse University of Reunion Island, ESIROI Food Science, Saint-Denis Cedex 9, Reunion Island, France Jorge A. Ferreira Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden Shakuntala Ghorai Department of Microbiology, Raidighi College, Raidighi, India Daniel G. Gomes CEB—Centre of Biological Engineering, University of Minho, Braga, Portugal Sharareh Harirchi Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden; Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran Jone Ibarruri AZTI, Food Research, Basque Research and Technology Alliance (BRTA), Parque Tecnológico de Bizkaia, Derio, Bizkaia, Spain Sajjad Karimi Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden Vinod Kumar School of Water, Energy, and Environment, Cranfield University, Cranfield, United Kingdom Patrik Roland Lennartsson Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden Hanifah Nuryani Lioe Department of Food Science and Technology, IPB University, Bogor, Indonesia Contributors xv Sara Magdouli Department of Civil Engineering, Lassonde School of Engineering, York University, Toronto, ON; Industrial Waste Technology Center, Abitibi Temiscamingue, QC, Canada Manikharda Department of Food and Agricultural Product Technology, Universitas Gadjah Mada, Yogyakarta, Indonesia  Clermont Auvergne, Clermont Auvergne INP, CNRS, Institut Philippe Michaud Universite Pascal, Clermont-Ferrand, France Marzieh Mohammadi Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden Rafael León Morcillo Institute of Water Research, University of Granada, Granada, Spain Soumya Mukherjee University of Toledo, Toledo, OH, United States Vivek Narisetty School of Water, Energy, and Environment, Cranfield University, Cranfield, United Kingdom Seyyed Vahid Niknezhad Burn and Wound Healing Research Center; Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran Ashok Pandey Centre for Innovation and Translational Research, CSIR-Indian Institute of Toxicology Research, Lucknow; Sustainability Cluster, School of Engineering, University of Petroleum and Energy Studies, Dehradun, India Mohsen Parchami Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden Anil Kumar Patel Department of Marine Environmental Engineering; Institute of Aquatic Science and Technology, National Kaohsiung University of Science and Technology, Kaohsiung City, Taiwan  Clermont Auvergne, Clermont Auvergne INP, CNRS, Institut Guillaume Pierre Universite Pascal, Clermont-Ferrand, France Endang Sutriswati Rahayu Department of Food and Agricultural Product Technology, Universitas Gadjah Mada, Yogyakarta, Indonesia G. Renuka Department of Microbiology, Pingle Government Degree College for Women, Warangal, India Saddys Rodriguez-Llamazares Centro de Investigación de Polı́meros Avanzados (CIPA), Edificio Laboratorio CIPA, Concepción, Chile Neda Rousta Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden xvi Contributors Taner Sar Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden Behzad Satari Department of Food Technology, College of Aburaihan, University of Tehran, Tehran, Iran Ulises Conejo Saucedo Institute of Water Research, University of Granada, Granada, Spain Zohresadat Shahryari Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden; Avidzyme Company, Shiraz, Iran Pooja Sharma Environmental Research Institute, National University of Singapore; Energy and Environmental Sustainability for Megacities (E2S2) Phase II, Campus for Research Excellence and Technological Enterprise (CREATE), Singapore, Singapore Rui Silva CEB—Centre of Biological Engineering, University of Minho, Braga, Portugal Raveendran Sindhu Microbial Processes and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology (CSIR-NIIST), Thiruvananthapuram, Kerala, India Reeta Rani Singhania Department of Marine Environmental Engineering, National Kaohsiung University of Science and Technology, Kaohsiung City, Taiwan Mohammad J. Taherzadeh Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden  A. Teixeira CEB—Centre of Biological Engineering, University of Minho, Braga, Jose Portugal Sunita Varjani Gujarat Pollution Control Board, Gandhinagar, Gujarat, India S. Venkata Mohan Bioengineering and Environmental Sciences, Department of Energy and Environmental Engineering, CSIR-Indian Institute of Chemical Technology, Hyderabad, India Rachma Wikandari Department of Food and Agricultural Product Technology, Universitas Gadjah Mada, Yogyakarta, Indonesia Akram Zamani Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden Preface Advances in Filamentous Fungi Biorefinery is a book in the Elsevier series on Current Developments in Biotechnology and Bioengineering (Editor-in-Chief: Ashok Pandey). This book explores various fundamental and industrial aspects of filamentous fungi for the manufacture of different products used in our society. Fungi are part of the ecosystem, and the world would look totally different without them. Most people know only mushrooms and their fruiting bodies as fungi. However, the main fungal biomass is their filaments, which can have many applications. Filamentous fungi can grow on a large variety of materials that contain carbohydrates, proteins, fats, etc., by degrading the macromolecules and then assimilating the monomers to grow and produce various enzymes and metabolites. This means that there are a large number of substrates on which to grow fungi, from agricultural and forest residuals to industrial residuals and products, to household wastes and wastewaters. Depending on the ecosystem and the environmental or cultivation conditions, fungi can grow in various morphologies, and many fungal strains are dimorphic, meaning that they can grow like yeast or short or long filaments. In addition, they can grow in various environmental conditions, such as aerobic or anaerobic. They adapt their enzyme machinery as required to these conditions, producing a variety of metabolites that are necessary for the fungi to grow. As fungi grow on a large number of substrates, they can produce various extracellular enzymes such as hydrolytic enzymes to degrade biopolymers. Therefore, fungi are an industrial source of enzyme production. Ultimately, we should not forget that the only goal of fungi is to grow. However, in certain conditions they can produce, for example, enzymes and/or various metabolites, which can be used as products. Bearing in mind the single goal of fungi, one of their major products is always fungal biomass or mycelium. This biomass normally contains protein, fat, and other biopolymers such as chitosan or beta-glucan in its cell wall, and a variety of bioactive compounds. As a result, the biomass of many filamentous fungi can be a good source for food and feed. Some of these fungi, particularly among the zygomycetes and ascomycetes, are edible and can be used for different food preparations such as tempeh, oncom, and koji. However, there is also an interest nowadays in developing new food and feed such as fish feed from fungi as an environmentally friendly and healthy alternative to meat, chicken, or even soy-based vegetarian products, for example. However, as some fungi produce mycotoxins, the fungal strain and the process conditions should be chosen carefully in order to avoid any risks to humans or animals. The aim of this book is to explore comprehensively the advances in using filamentous fungi as the core of industrial biorefinery. The book provides a thorough overview and xvii xviii Preface understanding of fungal biology, biotechnology, and ecosystems, and covers a variety of industrial products that can be developed from fungi. The contents of this book are organized in 18 chapters to cover: (a) the fungal ecosystem, biology, and biotechnology; (b) the fungal growth and process in solid state and submerged fermentation, particular aspects of bioreactors, and sampling, preservation, and process monitoring; (c) mycotoxins as an important aspect to choose the fungal strains for processes; (d) products of biorefinery that the fungi including food, feed, organic acids, alcohols, bioactive pigmented compounds, and antibiotics; and (e) fungi in novel processes. We are grateful to the authors for compiling the pertinent information in their chapters, which we believe will be a valuable resource for both the scientific community and readers in general. We are also grateful to the expert reviewers for their useful comments and scientific insights, which helped shape the book’s organization and which improved the scientific discussions and overall quality of the chapters. The Editors (Mohammad Taherzadeh and Jorge Ferreira) acknowledge support from the Swedish Agency for Economic €xtverket) through a European Regional Development Fund and Regional Growth (Tillva “Ways2Tastes.” Finally, our sincere thanks go to the staff at Elsevier, including Dr. Kostas Marinakis (former Senior Book Acquisition Editor), Dr. Katie Hammon (Senior Book Acquisition Editor), and Bernadine A. Miralles (Editorial Project Manager), and the entire Elsevier production team for their support in publishing this book. Editors Mohammad J. Taherzadeh Jorge A. Ferreira Ashok Pandey 1 World of fungi and fungal ecosystems Gabriela Ángeles de Paza, Ulises Conejo Saucedoa, Rafael León Morcilloa,#, and Elisabet Arandaa,b b a INSTITUTE OF WATER RESEARCH, UNIVERSITY O F GRANADA, GR ANADA, SPAIN DEPARTMENT OF MICROBIOLOGY, UNIVERSITY O F GRANADA, GR ANADA, SPAIN 1. Introduction Fungi, belonging to Eukarya, are highly diverse and less explored. They are cosmopolitan and play important ecological roles as saprotrophs, mutualists, symbionts, parasites, or hyperparasites. Advances in molecular phylogeny have allowed to clarify the complex relationships of anamorphic fungi (fungi imperfecti) and to place some of them outside the fungi. Exiting progress have been made in developing fungi for modern and postmodern biotechnology, such as obtaining enzymes, alcohols, organic acids, pharmaceuticals, or recombinant deoxyribonucleic acid (DNA). Filamentous fungi and yeasts are extensively used as efficient cell factories in the production of bioactive substances and metabolites or for native or heterologous protein expression. This is due to their metabolic diversity, secretion efficiency, high-production capacity, and capability of carrying out post-translational protein modifications. The commercial exploitation of fungi has been reported for multiple industrial sectors, such as those involved in the production of antibiotics, simple organic compounds (citric acids), fungicides or food and beverages. 2. Fungal morphology Fungi can colonize new places by growing as a system of branching tubes, known as hyphae, whose aggregates form the mycelium (filamentous fungi). Mycelium can be found in the substrates where the fungi growth or belowground and play an important role in obtaining nutrients for growth and development. The hyphae are characterized by the presence or absence of septa, cross-walls that are distinctive among different taxonomic groups. They are absent in Oomycota and Zygomycota, known as coenocytic hyphae (koinos ¼ shared, kytos ¼ a hollow vessel). The presence of septa is a common feature of Basidiomycota and Ascomycota, in which the exchange of cytoplasm or organelles is ensured by septal # Current affiliation: Institute for Mediterranean and Subtropical Horticulture “La Mayora” (IHSM), CSICUMA, Campus de Teatinos, Málaga, Spain. Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00010-7 Copyright © 2023 Elsevier Inc. All rights reserved. 1 2 Current Developments in Biotechnology and Bioengineering FIG. 1 Septum types in fungal hyphae. pores. These pores can be simple or dolipores, pores with a distinctive morphology that have a barrel-shaped swelling that surrounds the central pore (Fig. 1). Under senescence processes, differentiation or simply under mechanical breaking off, different organelles act as septal pore plugs, preventing the detrimental effect of trauma senescence or permitting differentiation processes. They include Woronin bodies, hexagonal crystals, elongated crystalline bodies, nuclei, mitochondria, or de novo deposition of plugging material (Markham, 1994). The septum represents a specialized structure for cell division. Not all fungi grow as hyphae; some occur as yeasts (yeast-like fungi). They usually grow on surfaces where penetration is not required (such as the digestive tract). Such fungi have attracted the attention of biotechnologists because they grow rapidly and can easily be manipulated. Other fungi can switch between yeast-like fungi and filamentous fungi; they are known as dimorphic fungi and include some pathogens such as the plant pathogen Ustilago maydis or the human pathogen Candida albicans. This attribute is highly important as a model of differentiation in eukaryotic organisms (Bossche et al., 1993). Dimorphism is a common treat in pathogenic fungi (animals and plants pathogen) and usually is regulated by different factors such as temperature, glucose, pH, nitrogen source, carbon dioxide levels, chelating agents, transition metals and inoculum size or initial cell density (Romano, 1966). However, a number of fungi with unknown pathogenic activity have important industrial applications such as the production of chitosan, chitin from Saccharomyces, bioremediation process by Yarrowia, ethanol or enzyme production (Doiphode et al., 2009). This fact is of special interest in industry, since (i) it is possible to overcome the operational problems generated during hyphal growth in bioreactors, (ii) morphology could be an indicator of biotechnological process (enzymes secretion or proliferation vs penetration), or (iii) can be an advantage for biocontrol formulations (Doiphode et al., 2009). From a point of view of biotechnology, the morphology of fungi has an important implication since the adaptation of the cultivation system must be optimized. Chapter 1 • World of fungi and fungal ecosystems 3 Filamentous fungi can growth as disperse mycelial or pellets, depending on the mechanical conditions of the cultivation. Industrial cultivation processes with fungi have been optimized over decades to increase productivity (Walker and White, 2017). The fungal cell wall is a dynamic and complex structure and usually based on glucans and chitin (Ruiz-Herrera, 1991). However, the chemical composition varies among different taxonomic groups, with implications for biotechnological processes since different enzymatic activities occur in the cell wall. The balance between wall synthesis and lysis could influence hyphal morphology and cell growth, with impacts on the retention of chemical compounds through bio-adsorption processes, enabling the successful industrial fermentation of filamentous fungi. In addition, some components of the cell wall, such as chitosan and chitin are considered high-value products for their use in biomedicine, agriculture, paper making, food industry, and textile industry that can be easily extracted using different technologies (Nwe et al., 2011; Table 1). 2.1 General aspects of reproduction Fungi can reproduce sexually or asexually (vegetative reproduction). These two reproduction modes differ according to the fungal morphology and taxonomic group (yeast, filamentous, or dimorphic fungi) (Hawker, 2016). In yeasts, the most frequent mode of vegetative reproduction is budding, which has been studied in detail for Saccharomyces cerevisiae. This mode can be multilateral, bipolar, unipolar, or monopolar budding. However, fission by forming a septum can also occur in yeasts such as Schizosaccharomyces pombe (host for heterologous expression). This fission can be binary or a bud fission, in which a cross-wall at the base of the bud separates both cells. Ballistoconidiogenesis is a specific vegetative reproduction in species such as Bullera (β-galactosidase) or Sporobolomyces (carotenoids and fatty acids). Pseudomycelia are typical in dimorphic species in which a single filament is produced when cells fail to separate after budding or fission (Walker and White, 2017). In filamentous fungi, such as basidiomycetes, this vegetative reproduction can occur by fragmentation of the hyphae; in some ascomycetes, the formation of mitotic spores has been observed. Sexual reproduction is a complex mechanism and differs according to the taxonomic groups; it involves the formation of a meiotic spore by planogametic copulation, gametangial contact, gametangial copulation, spermatization, and somatogamia. Table 1 Percentages of dry weight of the total cell wall fraction of the main components (chitin, cellulose, glucans, protein, and lipids) in different groups of fungi. Group Chitin Cellulose Glucans Protein Lipids Oomycota Chytridiomycota Zygomycota Ascomycota Basidiomycota 0 58 9 1–39 5–33 25 0 0 0 0 65 16 44 29–60 50–81 4 10 6 7–13 2–10 2 n/a 8 6–8 n/a n/a (Data not available). Data adapted from Ruiz-Herrera, J., Ortiz-Castellanos, L., 2019. Cell wall glucans of fungi. A review. Cell Surf. 5, 100022. 4 Current Developments in Biotechnology and Bioengineering 2.2 Fungal nutrition Filamentous fungi and yeasts are chemo-organotroph microorganisms with relatively simple nutritional needs. Fungi are heterotrophic organisms since they lack photosynthetic pigments. Most fungi are aerobes, but we can find representatives of obligate anaerobes (Neocallimastix) or facultative anaerobes (Blastocladia). In aerobic respiration, the terminal electron acceptor is oxygen; however, based on oxygen availability, we can find obligate fermentative or facultative fermentative fungi, including Crabtree-positive (Saccharomyces cerevisiae), Crabtree-negative (Candida utilis), non-fermentative (Phycomyces, Rhodotorula rubra) or obligate aerobes (most fungi) (Walker and White, 2017). 2.2.1 Nutrient uptake Filamentous fungi and few yeast species obtain their nutrients via extracellular enzymes; they absorb smaller molecules produced after extracellular digestion. These enzymes can be wall-bound-enzymes or may diffuse externally into the environment, depending on the lifestyle of the fungus (Section 3). Nutrient distribution through the hyphae might occur by passive (diffusion-driven) or active translocation (metabolically driven) through the protoplasm (Olsson and Gray, 1998; Persson et al., 2000). In both cases, nutrient translocation allows filamentous fungi to growth in habitats where the spatial distribution of nutrients and minerals is irregular and variable, including environments with low nutrient concentrations or polluted areas, by exploiting the resources available in other parts of the mycelium (Boswell et al., 2002). The enzymatic system in fungi depends on the taxonomic group. Some ecophysiological artificial groups have been established based on the capability to produce enzymes. These groups include the former ligninolytic fungi because of their ability to secrete a set of enzymes involved in the degradation of lignin. These enzymes play a significant role in biotechnology since they can be used in biorefineries. They include lipases produced by some yeasts (Candida, Yarrowia lipolytica), hydrolytic enzymes such as glycoside hydrolases (GHs), polysaccharide lyases (PLs), glycosyltransferases (GTFs), carbohydrate esterases (CEs), lytic polysaccharide monooxygenases (LPMOs), non-catalytic carbohydrate-binding modules (CBMs) and enzymes with “auxiliary activities,” including all enzymes involved in lignocellulosic conversion such as laccases, peroxidases, manganese peroxidase, lignin peroxidases, versatile peroxidases, DyP-type peroxidases (Levasseur et al., 2013). These enzymes are classified in the CAZymes database, which describes the families of structurally related catalytic and CBMs of enzymes that degrade, modify, or create glycosidic bonds (http://www.cazy. org/). These enzymes are important in biotechnology, particularly in biorefineries, for the deconstruction of plant biomass into simple sugars to obtain fermentation-based products and for the recovery of value-added compounds (Contesini et al., 2021). 3. Lifestyles of fungi Fungi, as heterotrophic eukaryotic microorganisms and efficient producers of enzymes, can live in different habitats and on different organic substrates. In general terms, fungi Chapter 1 • World of fungi and fungal ecosystems 5 are either saprophytic—they feed on nutrients from organic, non-living matter in the surrounding environment-, symbiotic—they share a mutually beneficial relationship with another organism-, parasitic—they feed off a living host that may survive (biotrophs) or die (necrotrophs)- or hyperparasitic—they live at the expense of another parasites. Saprotrophic fungi are important for the recycling of nutrients, especially phosphate minerals and carbon incorporated in wood and other plant tissues. Their role as decomposers of organic matter is fundamental, since together with bacteria, they prevent the accumulation of organic matter, ensure the distribution of nutrients and play a crucial role in the global carbon cycle in terrestrial and aquatic ecosystems (Kjøller and Struwe, 2002; Cebrian, 2004; Moore et al., 2004). Besides, filamentous fungi play other significant roles in natural ecosystems. For instance, in terrestrial systems, fungi maintain the soil structure due to their filamentous branching growth and participate in the transformation of rocks and minerals (Gadd, 2008), incorporating new elements into the ecosystem that may be used by other organisms. In addition to the important role in natural processes, and as stated before, the decomposition of organic matter by fungi represents an important trait for biotechnological purposes due to the potential use of individual microbial strains or enzymes for the use of renewable resources, such as plant biomass. In general terms, saprotrophic basidiomycetes can degrade plant litter and wood more rapidly than other fungi because of their high capacity to decompose lignin and other plant polymers, allowing them to spread rapidly in the environment (Osono and Takeda, 2002; Martı́nez et al., 2005; Baldrian, 2008). Nevertheless, litter and wood decomposition is a successive process, of which basidiomycetes and ascomycetes govern different phases (Osono, 2007; Vo
rı́šková and Baldrian, 2013). This capacity has been widely used in at industrial scale in various applications because of the oxidation of phenolic and non-phenolic lignin-derived compounds. Few examples are fungal laccases, ligninolytic enzymes which degrade complex recalcitrant lignin polymers and are widely used in the food industry (Minussi et al., 2002; MayoloDeloisa et al., 2020), in cosmetic, pharmaceutical, and medical applications (Golz-Berner et al., 2004; Niedermeyer et al., 2005; Hu et al., 2011; Ueda et al., 2012; Sun et al., 2014), in the paper and textile industry (Bourbonnais et al., 1995; Ozyurt and Atacag, 2003; Rodrı́guez Couto and Toca Herrera, 2006; Virk et al., 2012) or in the nano-biotechnology (Li et al., 2017; Kumari et al., 2018). Apart from industrial applications, the potential capacity of saprophytic fungal intra- and extracellular enzymes to degrade/transform complex polymers, such as lignin, is used in the biodegradation of organic xenobiotic pollutants. Among them, oxidoreductases represent the most important group of enzymes used in xenobiotic bioremediation transformations, including peroxidases, laccases, and oxygenases, and can catalyze oxidative coupling reactions using oxidizing agents to support the reactions (Sharma et al., 2018; Baker et al., 2019). These enzymes are produced by a wide diversity of fungi, which are some of the most extensively fungi used to detoxify xenobiotic compounds; they belong to the basidiomycetes group called “white rot fungi” and include the genera Trametes, Pleurotus, and Phanerochaete spp. (Aust, 1995; Pointing, 2001; Baldrian, 2003; Asif et al., 2017), but also ascomycetes genera such as Aspergillus or Penicillium (Aranda, 2016; Aranda et al., 2017). 6 Current Developments in Biotechnology and Bioengineering Pathogenic and parasitic fungi virtually attack all groups of organisms, including bacteria, other fungi, plants, and animals, including humans. According to their nutritional relationship with the host, parasitic fungi can be divided into biotrophic parasites, which obtain their sustenance directly from living cells, and necrotrophic parasites, which first destroy the parasitized cell and then absorb its nutrients. Besides, fungi might be facultative parasites, which are capable of growing and developing on dead organic matter and artificial culture media, or obligate parasites, which can only obtain food from living protoplasm and, therefore, cannot be cultured in non-living media (Brian, 1967; Lewis, 1973) Fungi possess the broadest host range spectrum of any group of pathogens. For instance, the filamentous ascomycetous fungus Fusarium oxysporum causes vascular wilt on many different plant species (Pietro et al., 2003), but it is also responsible for causing life-threatening disseminated infections in immunocompromised humans (Boutati and Anaissie, 1997). Nonetheless, there are many examples of fungal pathogens that infect only one host (Shivas and Hyde, 1997; Zhou and Hyde, 2001), highlighting the high host specificity of diseases produced by certain fungal infections. To explain this dual aspect of fungal infection specificity, the pathogenic strains of parasitic fungi are divided into formae speciales, defining the existence of different subgroups within species based on their host specificity (Armstrong and Armstrong, 1981; Anikster, 1984). In terms of agriculture, the estimated crop losses due to fungal diseases would be sufficient to feed approximately 600 million people a year (Fisher et al., 2012). To colonize plants, fungi secrete hydrolytic enzymes, including cutinases, cellulases, pectinases and proteases that degrade these polymers and permit fungal entrance through the external plant structural barriers. These enzymes are also required for the saprophytic lifestyle of fungi. As mentioned above, some fungi are facultative parasites and may attack plant roots from a saprophytic base in the soil through the mycelium, progressively causing the death of the host and thereafter living as saprophytes (Zhou and Hyde, 2001). Some fungi have developed other mechanisms to colonize plant hosts, such as via specialized penetration organs, called appressoria, or via penetrating through wounds or natural openings, such as stomata (Knogge, 1996). Fungal pathogens are responsible for numerous diseases in humans and for the extinction of amphibian and mammal populations (Brown et al., 2012; Fisher et al., 2012). For example, Batrachochytrium dendrobatidis, an aquatic chytrid fungus that attacks the skin of over 500 species of amphibians, and Geomyces destructans, a ascomycete fungus that attacks numerous bat species, seriously threaten the survival of these animals and might lead to the decline in the populations of other species (Colón-Gaud et al., 2009; Fisher et al., 2009; Lorch et al., 2011). For humans, fungal infections are rarely life-threatening; however, superficial fungal infections of the skin, hair and nails are common worldwide and affect approximately one-quarter of the human population (Schwartz, 2004). Airborne pathogenic fungi can also cause different respiratory diseases and also be lethal in immunocompromised patients (Mendell et al., 2011). The infection process is, generally, similar to that in plants. However, and contrary to plant infections, appressoria have not been described for Chapter 1 • World of fungi and fungal ecosystems 7 animal-pathogenic fungi, except for a few similarly shaped structures formed by Candida albicans (Kriznik et al., 2005). Instead to appressoria, in animals, fungal pathogens use other mechanisms, such as the binding of specific receptors that facilitate the endocytosis € rnberger of host cells and, therefore, their entrance into the living tissue (Woods, 2003; Nu et al., 2004). Nonetheless, and similar to fungal infections in plants and other fungal lifestyles such as saprophytes, this process may also be mediated by lytic enzymes, such as proteases, that degrade the surface of the host cells and permit fungal penetration into the € rnberger et al., 2004; Schaller et al., 2005). living host (Nu A special type of parasitic fungi is represented by hyperparasites, fungi that live at the expense of another parasite, which is highly common among fungi ( Jeffries and Young, 1994). Hyperparasitism is often used in agriculture for plant protection as an alternative to chemical treatments (Brožová, 2004). A classic example is Trichoderma harzianum, a fungus extensively used as biological agent against a wide range of fungal parasites. Nonetheless, it is estimated that 90% of all fungi used in plant protection products belong to the genus Trichoderma (Benı́tez et al., 2004). Fungi may also live as mutualistic symbionts, associating with other organisms with benefits for both parties. Remarkable examples of these symbioses are mycorrhizae and lichens. Mycorrhizae are the symbiotic association of soil fungi with the roots of vascular plants. Generally, fungi colonize plant roots and provide nutrients and water, which are captured from the soil through the external hyphal network, whereas plants supply organic molecules derived from photosynthesis, such as sugars or fatty acids, to the obligate biotrophic fungi (Harrison, 1999; Keymer et al., 2017). This represents a universal symbiosis, not only because almost all plant species are susceptible to form the symbiosis, but also because such symbioses can be established in the majority of terrestrial ecosystems, even under highly adverse conditions (Mosse et al., 1981). Moreover, this symbiosis contributes to global carbon cycles as plant hosts divert up to 20% of photosynthates to the host fungi (Smith and Read, 2010). There are three different types of mycorrhizae: endomycorrhizae, ectomycorrhizae, and ectendomycorrhizae. Endomycorrhizae are characterized by the presence of hyphae inside the cells of the root cortex. It is estimated that at least 90% of all known vascular plants, (about 300,000 species), form this type of mycorrhizae. On the other hand, in the ectomycorrhizae symbiosis, the hyphae of the fungus do not penetrate the cells of the cortex of the roots and form a dense hyphal sheath, known as the mantle, surrounding the root surface. It is believed that at least 3% of vascular plants develop this type of mycorrhizae, including almost all species of the most important forest tree genera. Finally, the ectendomycorrhizae present characteristics of both endo- and ectomycorrhizae, namely a mantle surrounding the plant roots and fungal hyphae that penetrate the root cells (Smith and Read, 2010). Mycorrhizal symbiosis plays an essential role in the establishment and functioning of terrestrial ecosystems, being involved in natural processes such as nutrient cycling and, in part, in the structure and dynamics of populations and plant communities (Newman, 1988; Klironomos et al., 2011). In terms of agriculture, mycorrhizae improve the 8 Current Developments in Biotechnology and Bioengineering productive capacity of poor soils, such as those affected by desertification, salinization, and wind erosion, because of the fungal capacity to obtain and translocate nutrients and water to the host plants (George, 2000). In addition, the symbiosis enhances soil aggregation and structure (Miller and Jastrow, 2000) and contributes to defence against diverse plant pathogens (Elsen et al., 2001; Azcón-Aguilar et al., 2002; Garcı́a-Garrido and Ocampo, 2002) and abiotic stresses, such as drought or salinity (Ruiz-Lozano et al., 1996; Ruiz-Lozano, 2003). Another form of symbiotic association of fungi is represented by lichens, which are composite organisms composed of algae or cyanobacteria, called photobionts, living among filaments of a fungus, the so-called mycobiont. The algae, as an autotrophic organism, provides the fungus with organic compounds and oxygen derived from its photosynthetic activity, whereas the fungus, as a heterotroph, supplies the algae with carbon dioxide, minerals and water, since contrary to plants, lichens lack vascular organs to directly control their water homeostasis (Proctor and Tuba, 2002; Lutzoni and Miadlikowska, 2009). Most of the lichenized fungal species (98% approximately) belong to the phylum Ascomycota, whereas only few orders are in the phylum Basidiomycota and mitosporic fungi (Hawksworth et al., 1996). Although some lichens inhabit partially shaded areas and forests (Neitlich and McCune, 1997), most lichens often live in highly exposed places under intense light intensities, such as deserts or arctic and alpine ecosystems. For this reason, the mycobiont normally produces secondary colored compounds, called lichen compounds, that strongly absorb UV-B radiation and prevent damage to the algae’s photosynthetic apparatus (Fahselt, 1994). These compounds are a source of structurally diverse groups of natural products, with a wide range of biological activities including antibiotic, analgesic, and antipyretic activities (Yousuf et al., 2014) and have traditionally been used in the cosmetic and dye industry as well as in food and natural remedies (Oksanen, 2006). In nature, lichens are important as early-stage primary succession organisms. For instance, they are the pioneers in the colonization of rocky habitats and, after dying, their organic matter might be used by other organisms (Lutzoni and Miadlikowska, 2009; Muggia et al., 2016). As poikilohydric organisms, their water status passively follows the atmospheric humidity (Nash, 1996), and they can tolerate irregular and extended periods of severe desiccation. This allows them to colonize habits that cannot be colonized by most plants. Despite this, many lichens also grow as epiphytes on plants, mainly on the trunks and branches of trees. However, they are not parasites or pathogens since they do not consume or infect the holding plant (Ellis, 2012). In addition, lichens adsorb and are sensitive to heavy metals and pollutants (Garty, 2001), making them perfect environmental indicators. 4. Taxonomy of fungi Understanding how fungi have adapted to so many ecosystems, the way in which they have evolved, but above all, taxonomic classification, has not been an easy task. Fungi, after plants and animals, are one of the most diverse and dominant groups in almost Chapter 1 • World of fungi and fungal ecosystems 9 all ecosystems. It is estimated that there are between 1.5 and 5.1 million species. However, a new estimation of the number of fungi ranges between 500,000 and almost 10 million € cking, 2017), although only almost 10% have been identified so far (Hawksworth and Lu (Blackwell, 2011; Hibbett et al., 2016) In the middle of the 18th century, the scientist Carl von Linnaeus implemented a binomial system to classify living beings (Systema naturae). This system is based on the classification of organisms according to their morphological characteristics and phenotypic traits. Fungi were considered as part of the plant kingdom (Linnaeus, 1767). Some years after, Whittaker classified the fungi as an independent group, which he called “true fungi” (Eumycota) (Whittaker, 1969). Then, different taxonomic classifications continued until the middle of the 19th century. Advances in the classification of fungi have always gone hand in hand with the development of new technologies, such as electron microscopy, new biochemical, and physiological analysis methods, the study of secondary metabolites, cell wall composition and fatty acid composition, as well as molecular technologies, among others (Guarro et al., 1999). In the last two decades, the development of PCR techniques and, later, genome sequencing, has significantly contributed to the advance in fungal taxonomy. This promoted rapid changes, and therefore, different proposals for the reclassification of fungi have been made, tripling the number of phyla from 4 to more than 12. However, less than 5% of the identified species have been taxonomically classified ( James et al., 2020). Nextgeneration sequencing tools have allowed fungal genome sequencing, transcriptomes, and mitochondrial genomes that provided relevant information for phylogenetic studies in fungi. Additionally, specific regions of ribosomal RNA (rRNA), such as internal transcribed spacers (ITSs), large subunit (LSU), small subunit (SSU) and intergenic spacer of rDNA, as well as various markers including translation elongation factor 1 (TEF1), glycerol-3-phosphate dehydrogenase (GAPDH), histones (H3, H4), calmodulin gene, RNA polymerase II largest subunit (RPB1) genes and mitochondrial genes (cytochrome c oxidase I and ATPase subunit 6), have played an important role in the development of the fungal taxonomy (Zhang et al., 2017). The organization of such information (genomes) by the scientific community has required different efforts. On the one hand, the Y1000 + project aims to sequence the genomes of 1000 yeast species (https://y1000plus.wei.wisc.edu), and on the other hand, the 1000 Fungal Genomes project (http://1000.fungalgenomes.org/home) has the objective of sequencing 1000 fungal genomes. The database UNITE is a recent database that concentrates the sequences of the ITS ribosomal region of fungi included in the International Nucleotide Sequence Database (http://www.insdc.org/). This database resulted from the collaboration of researchers and taxonomic specialists who collected and deposited fungal sequences, specifically with the purpose of building a database that registers, analyses, and shares this information with the scientific community (Kõljalg et al., 2020). In the last 14 years, the taxonomy of fungi has been under major changes. The kingdom of fungi, proposed by Hibbett et al. (2007), includes one subkingdom (Dikaria) and seven 10 Current Developments in Biotechnology and Bioengineering FIG. 2 Fungal classification proposed by Hibbett et al. (2007). phyla: Blastocladiomycota, Glomeromycota, Chytridiomycota, Neocallimastigomycota, Microsporidia, Ascomycota, and Basidiomycota; four subphyla, namely Entomophthoromycotina, Kickxellomycotina, Mucoromycotina, Zoopagomycotina, and a total of 31 classes (Hibbett et al., 2007)(Fig. 2). Over the last few years, different approaches, reclassifications, and updates on fungal taxonomy have been made (Gryganskyi et al., 2012; Hyde et al., 2013; Slippers et al., 2013; Phookamsak et al., 2014; Ariyawansa et al., blová et al., 2015; Li et al., 2016; Spatafora et al., 2016; Marin-Felix et al., 2017, 2019; Re 2018; Voglmayr et al., 2019; Mitchell et al., 2021). Tedersoo et al. (2018) described and proposed an updated classification for the fungal kingdom based on divergence time and phylogenies of particular taxa. Under this point of view, nine subkingdoms have been proposed (1Rozellomyceta, 2Aphelidiomyceta, 3Blastocladiomyceta, 4Chytridiomyceta, 5Olpidiomyceta, 6Basidiobolomyceta, 7Zoopagomyceta, 8Mucoromyceta and 9Dikarya); each subkingdoms divides into one or more phyla (18 phyla-1Rozellomycota, 2Aphelidiomycota, 3Blastocladiomycota, 4Chytridiomycota, 4 Monoblepharomycota, 4Neocallimastigomycota, 5Olpidiomycota, 6Basidiobolomycota, 7 Entomophthoromycota, 7Kickxellomycota, 7Zoopagomycota, 8Mucoromycota, 8Mortierellomycota, 8Calcarisporiellomycota, 8Glomeromycota, 9Entorrhizomycota, 9Basidiomycota, and 9Ascomycota). Additionally, each phylum divides into one or more subphyla Chapter 1 • World of fungi and fungal ecosystems 11 FIG. 3 Fungal classification proposed by Tedersoo et al. (2018). (20 subphyla—1Rozellomycotina, 2Aphelidiomycotina, 3Blastocladiomycotina, 4Chytridiomycotina, 4Monoblepharomycotina, 4Neocallimastigomycotina, 5Olpidiomycotina, 6Basidiobolomycotina, 7Entomophthoromycotina, 7Kickxellomycotina, 7Zoopagomycotina, 8 Mucoromycotina, 8Mortierellomycotina, 8Calcarisporiellomycotina, 8Glomeromycotina, 9 Entorrhizomycotina, 9Agaricomycotina, 9Pucciniomycotina, 9Ustilaginomycotina, 9Wallemiomycotina, 9Pezizomycotina, 9Taphrinomycotina, 9Saccharomycotina), and 76 classes are included (Fig. 3). An alternative classification was proposed by Naranjo-Ortiz and Gabaldón (2019), based on nine main lines: Opisthosporidia, Neocallimastigomycota, Blastocladiomycota, Chytridiomycota, Mucoromycota, Glomeromycota, Zoopagomycota, Ascomycota, and Basidiomycota. The main differences with respect to other proposals can be found in the incorporation of Opisthosporidia. This group incorporates three lineages: Rozellidea, Aphelidea, and Microsporidia. In turn, the group of Chytridiomycota was divided into three classes: Monoblepharidomycetes, Hyaloraphidiomycetes, and Chytridiomycetes, whereas Mucoromycota includes the two subphyla Mucoromycotina and Mortierellomycotina. Additionally, Basidiomycota comprised Pucciniomycotina, Ustilagomycotina, Agaricomycotina, Wallemiomycotina, and Bartheletiomycetes. Finally, Ascomycota contains three main clades: Taphrinomycotina, Saccharomycotina, and Pezizomycotina. Most recently, the classification of fungi proposed by Wijayawardene et al. (2020) has coincided, for many clades, with the proposal of Tedersoo et al. (2018). The subkingdom taxonomic rank was removed, and 16 phyla were recognized: Rozellomycota, 12 Current Developments in Biotechnology and Bioengineering Blastocladiomycota, Aphelidiomycota, Monoblepharomycota, Neocallimastigomycota, Chytridiomycota, Caulochytriomycota, Basidiobolomycota, Olpidiomycota, Entomophthoromycota, Glomeromycota, Zoopagomycota, Mortierellomycota, Mucoromycota, Calcarisporiellomycota, and three higher fungi (Dikarya-Entorrhizomycota, Basidiomycota, Ascomycota). In this study, only four subphyla were proposed (Mucoromycota Mortierellomycota, Entomophthoromycota, and Calcarisporiellomycota); in the case of Dycaria (Ascomycota and Basidiomycota), seven subphyla are described (Fig. 4). Some changes and additions have also been described, including the creation of the phylum Rozellomycota which contains the classes Rudimicrosporea and Microsporidea. The order Metchnikovellida was moved to the class Rudimicrosporea, and the most significant changes occurred in the phyla Ascomycota and Basidiomycota; the class Bartheletiomycetes, which was included in the phylum Basidiomycota, was changed to the subphylum Agaricomycotina. Moreover, Agaricomycetes, Dacrymycetes, Tremellomycetes and Bartheletiomycetes were grouped together. From the class Collemopsidiomycetes, the subphylum Pezizomycotina was eliminated, and new classes were included (Candelariomycetes and Xylobotryomycetes). The order Collemopsidiales was moved to the class Dothideomycetes. In the class Geminibasidiomycetes, the subphylum Wallemiomycotina was excluded, and only the class Wallemiomycetes remained. FIG. 4 Fungal classification proposed by Wijayawardene et al. (2020). Chapter 1 • World of fungi and fungal ecosystems 13 Despite these efforts, there are large numbers of genera, orders and families that have not yet been classified. Only in the phyla Ascomycota and Basidiomycota, remain not assigned families for 876 genera (Wijayawardene et al., 2018). According to a compilation from the Royal Botanic Gardens, in the last 10 years, 350 new families have been described, including Pucciniaceae with 5000 species, Mycosphaerellaceae with 6400 species, Cortinariaceae and Agaricaceae consisting of 3000 species. On the other hand, about 30% of the new incorporations are basidiomycetes, whereas 68% are ascomycetes. Until now, the continuous contribution of different groups of researchers, which has resulted in the growth of databases, and the development of new technologies and molecular tools have helped to establish a universal classification for fungi. The organization of this enormous amount of information through taxonomy allows to correlate the different styles of life as well as the structural, genetic, and metabolic characteristics, which, as mentioned here, are used to classify fungi. This taxonomic panorama allows us to show the great diversity of species that exist on earth, study their evolution and, in some cases, take advantage of certain metabolic functions that can be applied in different industrial processes; most of the fungi used belong to the phyla Ascomycota and Basidiomycota. These fungi play an important role in the production of various products or intermediates in the generation of bioethanol (fungi from the subphyla Agaricomycotina, Pezizomycotina, and Mucoromycotina), biodiesel (fungi from the subphyla Pezizomycotina, Ustilaginomycotina, and Saccharomycotina), biogas (fungi from the subphyla Mucoromycotina, Agaricomycotina, Pucciniomycotina, Pezizomycotina, Saccharomycotina), the pre-treatment of lignocellulosic biomass (fungi from the subphyla Basidiomycotina and class Agaricomycetes), applications in the pulp and paper industry (Agaricomycotina, Pezizomycotina), xylitol production (Saccharomycotina), and lactic acid production (Mucoromycotina, Saccharomycotina), among others (Kumari et al., 2018). 5. Fungal diversity An increasing number of studies are addressing fungal diversity. The total richness and diversity of fungal taxa across the studies published mainly involve Ascomycota (56.8% of the taxa) and Basidiomycota (36.7% of the taxa), with a total fungal diversity of around 6.28 million taxa. These studios represent a conservative estimate of global fungal species richness (Baldrian et al., 2021). In the largest study of fungal diversity, around 45,000 operational taxonomic units (OTUs) were recovered from 365 sites worldwide, using 1.4 million ITS sequences (Tedersoo et al., 2014). One-third of the total OTUs showed 97% of similarity compared with others reported in public databases; thereby, 30,000 new and different OTUs have been detected. These results greatly contributed to the discovery of new fungal species. The subkingdom Dikarya (Ascomycetes and Basidiomycetes) contains most of the fungal diversity on earth in terms of described species, but is small compared to the size of the total fungal kingdom. Recently, more taxa have been described, such as Cryptomycota 14 Current Developments in Biotechnology and Bioengineering (Jones et al., 2011; Lara et al., 2010), a chytrid group, Archaeorhizomycetes and other soil ascomycete groups (Porter et al., 2008; Rosling et al., 2003; Schadt and Rosling, 2015). Furthermore, around 150 genera have been estimated as being a part of the fungal group called Microsporidia, with 1200–1300 species (Lee et al., 2009); the actual figures are presumably higher than the figures for the host diversity. However, molecular diversity studies have not been detailed enough to elucidate this information (Krebes et al., 2010; McClymont, et al., 2005). Those approximations have been made possible due to DNA sequencing and the widespread use of the formal fungal barcode Nuclear ribosomal Internal Transcribed Spacer ITS 1–2. It is recognized as the official molecular marker of choice for the exploration of fungal diversity in environmental samples (Kõljalg et al., 2013) and counts with a vast and up-to-date database, which is necessary for data analysis (Schoch et al., 2012). Nevertheless, it cannot differentiate between all groups and cryptic species. Therefore, identification and diversity analysis of fungi is still greatly challenging (De Filippis et al., 2017). Moreover, the relationships between fungal diversity and their environments have not been completely described, which is also the case for the processes and mechanisms involved (Branco, 2019). As a consequence, two disciplines have been recognized to understand the relationship between fungal diversity and their environment, community ecology, and population genetics (Branco, 2019). Community ecology studies focus on the species level, addressing both ecological and biological questions, with a high level of accuracy and reliability. In contrast, population genetics studies have determined species assemblies and ranges, comprehending fungal intra-specific variation, dispersion, and establishment and including the identification of key traits influencing fitness (Mittelbach and Schemske, 2015). Cryptic species are biological entities that have already been named and described; however, they are morphologically different, and molecular studies are needed to elucidate, detect and enumerate these differences at the alpha diversity level (Bickford et al., e and Giovannoni, 2003; Sogin et al., 2006). These 2007; Horton and Bruns, 2001; Rapp studies highlight their diversity potential, proposing the measurement by genetic distances to know how hyper-diverse they are and to determine their species-level differences in many multicellular groups. Although novel molecular tools and new methods of identification have been used over the years, fungal diversity is barely known. This is mainly due the species with different morphological and ecological features (Hawksworth, 2004). 6. Fungal ecosystems As mentioned above, fungi are highly diverse and constitute a major portion of various ecosystems in terms of biomass, genetic diversification, and total biosphere DNA (Bajpai et al., 2019). Their distribution is extraordinarily diverse and shows biogeographical patterns depending upon local and global factors, such as climate, latitude, dispersal limitation, and evolutionary relationships (Bajpai et al., 2019). In terms of diversity, the Chapter 1 • World of fungi and fungal ecosystems 15 highest alpha diversity of fungi has been found in soils and terrestrial environments, mainly in plant shoots, plant roots, and deadwood (Baldrian et al., 2021). These associations with plants lead us to infer that fungi play a dominant role in terrestrial environments. 6.1 Terrestrial ecosystems Fungi in terrestrial habitats exhibit different preferences related to the edaphic condition, with a higher diversity in tropical ecosystems. Fungal endemicity is especially strong in such regions. Nevertheless, this distribution depends on the group of the fungi and their features; for instance, ectomycorrhizal fungi and other classes are most diverse in temperate or boreal ecosystems. In general, several taxa show a cosmopolitan distribution throughout habitats (Tedersoo et al., 2014). Sequencing studies have been performed in different terrestrial environments, revealing several numbers of novel sequences clustered conservatively. For example, in forest soil, fungal diversity showed around 830 OTUs that were not matched to any fungal taxon previously described when blasted against NCBI (http://www.ncbi.nlm.nih.gov/) or e et al. (2009). This analysis resulted in an estimated diverUNITE (http://unite.ut.ee/) Bue sity of 2240 (71.5%) by using Chao1, a non-parametric richness tool. The authors also compared the sequences with a curated database of robustly identified sequences and found that 11% of the total sequences, excluding all “uncultured fungi,” remained unclase et al., 2009). sified and a further 20% belonged to the unclassified Dikarya (Bue Fungal diversity in forest soil is highly associated with plants. Their symbiosis plays an important role in vegetation dynamics. Moreover, strong relation and similarities in fungal alpha and beta diversity studies (Hooper et al., 2000; Wardle et al., 2004; Gilbert and Webb, 2007) have been reported since Hawksworth and Mound (1991) estimated around 1.5 M of fungal species only in this habitat. Mycorrhizal and saprotrophic fungi are usually the primary regulators of plant-soil feedbacks across a range of temperate grassland plant species; the most abundant families are Paraglomeraceae, Glomeraceae, and Acaulosporaceae, whereas the most abundant genera of saprotrophic fungi are Mortierella and Clavaria (Semchenko et al., 2018). 6.2 Aquatic ecosystems Aquatic ecosystems comprise the largest portion of the biosphere and include both freshwater and marine ecosystems. Numerous studies in different aquatic habitats have indicated that fungi are abundant eukaryotes in aquatic ecosystems (Grossart et al., 2019; Money, 2016). They can reach relative abundances of >50% in freshwater and about >1% in saline habitats (Comeau et al., 2016; Monchy et al., 2011). However, these results can be extremely variable and depend on the respective habitat and its environmental settings. The predominant fungi in aquatic habitats are mostly determined by cultivation methods. This, coupled with temporal dynamics, spatial connectivity, and vectors such 16 Current Developments in Biotechnology and Bioengineering as migration, can represent a huge limitation when studying aquatic fungal diversity, interactions, and distribution (Grossart et al., 2019). Nevertheless, advances in sequencing technologies have revealed novel fungal biodiversity and new approaches for the understanding of fungal phylogeny, lineages, and evolution (Hyde et al., 2021; Richards et al., 2015; Wurzbacher et al., 2019). Despite those limitations, many different aquatic fungi have been reported, such as species of the genera Aspergillus, Penicillium, Cladosporium, Aureobasidium, Cryptococcus, Malassezia, Candida, and Rhodotorula (López-Garcı́a et al., 2007; Edgcomb et al., 2011; Jones et al., 2014). In marine habitats, 1901 species have been reported (2012). The dominant species on the surface belong to the groups Ascomycetes and Basidiomycetes, whereas yeasts and some other filamentous forms have been found in the deepest regions (Bass et al., 2007; Tisthammer et al., 2016). As mentioned before, fungi play multiple roles, engaging with all members of the aquatic community. Their interactions highlight them as important drivers of many ecosystem functions (Deveau et al., 2018). Marine fungi are especially adept at living on or inside other living organisms such as algae, corals, sponges, and even other fungi. Regarding such interactions, we can find multiple fungal groups such as Basidiomycota, Ascomycota, Zigomycota, Chytridiomycota, and Cryptomycota (Corsaro et al., 2018). The most studied groups are Chrytridiomycota and Cryptomycota, early diverging lineages that may not have a terrestrial ancestor, unlike the majority of marine fungi (Grossart et al., 2019). However, there is still a huge gap in terms of ecological and interaction studies of fungi and higher organisms, requiring further analyses of these roles. Numerous studies have already been carried out in freshwater habitats. In such environments, we can find different fungi belonging to the phyla Aphelidiomycota, Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Monoblepharomycota, Mortierellomycota, and Rozellomycota (Wijayawardene et al., 2020). Different species of Phycomycetes, Hiphomycetes and some zoosporic fungi belong to the genus Chytridium, Tetracladium, Cercospora, and Ophioceras were also reported (Chauvet et al., 2016; Wijayawardene et al., 2020). Mangrove fungi are strongly related to the geochemical cycles in the water. Mangrove forests share several kinds of fungi with other aquatic environments, and recent studies have reported about 850 taxa, including yeasts and lower mangrove fungi found exclusively in mangrove environments (Devadatha et al., 2021). 6.3 Extremophile environments New and hardly explored habitats are rich in fungal diversity. Those extremophile fungi have developed several mechanisms, involving enzyme production, that allow them to grow and reproduce in such harsh conditions. These enzyme systems are potential candidates for the isolation of new bioactive compounds with unusual but highly useful structures (Chávez et al., 2015). This has resulted in an increase in studies on extremophile fungi. The phyla Ascomycota, Basidiomycota, and a few traditional Zygomycota are the major fungi in Antarctic habitats. However, the endemism in Antarctic fungi can largely Chapter 1 • World of fungi and fungal ecosystems 17 be ascribed to the classes Eurotiomycetes and Dothideomycetes. These fungi have developed several mechanisms, involving the production of rock-mineralizing enzymes. As a consequence, they can adapt to and colonize these extreme environments, showing a high tolerance to UV radiation, extreme temperatures, and drought. Moreover, they also show competitive advantages over other fungal groups (Treseder and Lennon, 2015). In this sense, Antarctic fungi appear to possess novel or unusual metabolic pathways, with a potential for biotechnological applications. Habitats with high temperatures have been extensively studied, and most of the resident fungi are limited primarily by water stress, carbon limitation, high salinity, and UV irradiation. However, some fungi display specific adaptations that enable them to survive in spite of these conditions ( Jones et al., 2018). This fact is of main concern for biotechnological applications since thermotolerant enzymes are desirable for various industrial processes. Although sampling in such regions is difficult, numerous studies have been performed, greatly contributing to a deeper knowledge about extremophile fungi under extremely arid and saline conditions (Fuentes et al., 2020). Yungay halities, for instance, showed a high diversity and number of species of Penicillium and Aspergillus. Also, in the Atacama Desert, numerous melanized fungi and Neucatenulostroma species have been reported, isolated from the hyper-arid core in this region, approximately 45 km south of Yungay and the Pacific coast (Culka et al., 2017). The yeasts Rhodosporidium toruloides, Exophiala sp., Cryptococcus friedmannii, and Holtermanniella watticus are great examples of the versatility of fungi and can survive and thrive in volcanoes. They are resistant to UVC, UVB, and UVC radiation, high NaCl concentrations, and extremely high temperature (Pulschen et al., 2015), making them promising model organisms for astrobiological studies (Ametrano et al., 2019). Deep-sea environments are characterized by the absence of sunlight irradiation and remain among the least explored regions of the earth. They are extreme environments because of large temperature fluctuations, with predominantly low temperatures (occasionally extremely high, >400°C near hydrothermal vents) and high hydrostatic pressure (up to 110 MPa). Deep-sea environmental gene libraries suggest that fungi are rare and non-diverse in high-pressure marine environments, in contrast to surface environments (Bass et al., 2007). Phylogenetic analyses have suggested the novel phylogenetic affiliation of a group of predominant deep-sea phylotypes within the phylum Ascomycota. Some of the amplified sequences were identified as common terrestrial fungal species, but the majority were novel sequences. Another novel phylotype is the phylum Chytridiomycota, with Rozella spp. as the closest related organism (Nagano et al., 2010). Yeast forms have also been reported to be the dominant fungi in some oceans, especially in depths from 1500 to 4000 m (Bass et al., 2007). In terms of evolution, the fungal diversity detected suggests that deep-sea environments are habitats hosting previously unexplored fungi and represent ancient ecosystems, thereby providing key insights into the early evolution of fungi and their ecological and physiological significance (Nagano et al., 2010). 18 Current Developments in Biotechnology and Bioengineering The discovered and isolation of anaerobic fungi (AF) in 1975 played an important role in general knowledge of fungi kingdom, being of the first isolation reports of fungi that not need oxygen to survive (Orpin, 1975). From then on, different AF has been isolated, identified mainly in gut ecosystems, and represent a key source of novel enzymes. These enzymes are related with degradation, fermentation, and bioaugmentation activity; became the most efficient source of plant fibber digestion enzymes (Morgavi et al., 2015). In general, some of the AF isolated belongs to the phylum Neocallimastigomycota. However, this phylum is understudied due to the limited information of full genomes, hard crop conditions, and the lack of genetics toolset to manipulate them. Nevertheless, loads of scientists have been doing the best effort to take advantage of AF potential as a enzymatic resource, especially those in which biomass degradation are related (Hooker et al., 2019). Recently, all of these efforts are reflected in the enhancement of biogas production by AF and algal biomass, producing 41% more methane (Sevcan et al., 2017). Furthermore, co-cultures of AF and methanogens improve biogas production and represent a decrease in the final cost of the process (Vinzelj et al., 2020). 7. Applications of fungi in biorefineries Lignocellulose represents the largest reservoir of carbon in nature, them, the bioconversion of renewable lignocellulosic biomass has become mandatory to face global warming and shortage of fossil fuels. The aforementioned metabolic and ecophysiological diversity of fungi makes them a valuable tool for their use in biorefineries. They or a cocktail of their enzymes as well as the use of genetically modified fungi can be used for biopre-treatment of lignocelluloses, to remove lignin and solubilize hemicellulose, making cellulose more accessible to hydrolytic enzymes (Camarero et al., 2014). In this context, filamentous fungi have the ability to extracellularly produce a large variety of nonspecific and specific enzymes capable of breaking down lignin, cellulose, and hemicellulose. In recent years, the development of techniques such as genome sequencing, transcriptomics, and proteomics have revealed the presence of enzymes of great interest for this industry, among which are xylanases, LPMOs, or endoglucanases. This capacity makes them microorganisms of special interest for their use in biotechnological processes such as the production of bioethanol from lignocellulosic materials. In addition, the new technologies of genome editing by CRISPR-Cas9 appears to be a novel tool in the design of fungal genomes to edit certain desired traits, such as tolerance to inhibitors, better tolerance to biofuels, thermotolerance, consumption of substrates, silenced, and targeted the competitive mechanisms and modification of strategic enzymes using in biofuel production (Javed et al., 2019). Genetic manipulation in the yeast genome through CRISPR-Cas9 (gene insertions and mutations) has demonstrated an important advance in the production of fatty acids as a precursor of biofuels (Ullah et al., 2021). Chapter 1 • World of fungi and fungal ecosystems 19 8. Conclusions and perspectives This chapter summarizes the main aspects of the physiological diversity of fungi related with applications in biotechnology. Understanding the basic aspects of fungal physiology and distribution is crucial when exploiting these microorganisms in biorefineries. The diversity of fungi allows specific uses for different purposes, since a combination of adaptation to different conditions and specific biotechnological treatments in some species make them a suitable option for biotechnological application. 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Zhang, N., Luo, J., Bhattacharya, D., 2017. Advances in fungal phylogenomics and their impact on fungal systematics. In: Townsend, J.P., Wang, Z. (Eds.), Advances in Genetics. vol. 100. Academic Press, pp. 309–328 (Chapter Eight). Zhou, D., Hyde, K.D., 2001. Host-specificity, host-exclusivity, and host-recurrence in saprobic fungi*. *Paper presented at the Asian mycological congress 2000 (AMC 2000) incorporating the 2nd AsiaPacific mycological congress on biodiversity and biotechnology, and held at the University of Hong Kong on 9-13 July 2000. Mycol. Res. 105 (12), 1449–1457. This page intentionally left blank 2 Fungal biotechnology Mohammadtaghi Asadollahzadeh, Marzieh Mohammadi, and Patrik Roland Lennartsson SWE DISH C ENTRE FOR RE SOUR CE R ECOVE RY, UNIVERS ITY O F BORÅ S, BOR ÅS , SW EDEN 1. Introduction As described in the first chapter of this book, filamentous fungi can be found almost everywhere on the planet where they fulfill a number of different ecological roles. Considering this vast diversity and that they are all heterotrophs, it should come as no surprise that filamentous fungi are able to both degrade and produce a plethora of different compounds. Fungal biotechnology is about exploiting these properties to produce whichever product is to be made, usually in a biorefinery, which is the topic for this book. Thus, this chapter will give an overview of the entire field with detailed descriptions in later chapters. In this chapter, we first provide an overview of the more technical aspects of fungal cultivations (Fungal cultivation and requirements followed by Fungal biorefineries) and then focus more on what can generally be produced by the different groups of fungi (Fungal metabolites and Fungal biomass). As the astute reader is probably already well aware of, biotechnological processes can both be considered as very simplistic, since ethanol fermentation has been carried out for thousands of years, and as highly complicated requiring very sophisticated control and state-of-the-art technology. This is also true for fungal biotechnology using filamentous fungi, which is what will be discussed here (by definition, yeast is also fungi but will not be included in this discussion). Fermentation with filamentous fungi ranges from small-scale fermented foods such as tempeh or beverages such as sake that have been produced using filamentous fungi for centuries, to fully industrial scale processes using the latest technology. The latter also includes the optimized traditional processes. A major part of the issue of complicated processes is the number of different factors (see Fig. 1 for a very brief overview) each with a very large number of different possibilities that interact with each other. Plenty of general trends can be found between different fungal species. For example, a metabolite that is produced under oxygen limited conditions for one species most likely requires oxygen limited conditions for another species, assuming the other species can produce the metabolite at all. However, general trends are not always true for all cases, and in some areas, identical conditions may lead to opposite reactions. For example, a few years ago Nyman et al. (2013) investigated pellet formation and found that addition Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00006-5 Copyright © 2023 Elsevier Inc. All rights reserved. 31 32 Current Developments in Biotechnology and Bioengineering FIG. 1 A brief overview of fungal biotechnology and the main process conditions to consider and which process outputs can generally be considered. of solids to the liquid medium decreased the likelihood of pellet formation for the investigated species, which was in direct contradiction to what was reported for most other species. Although fungal biotechnology brings a number of interesting possibilities, it also has special requirements or issues to deal with. Compared with yeast- or bacterial-based biotechnology, chief of these special requirements for fungal biotechnology is most likely caused by the filamentous growth of the fungi. These microscopic filaments can cause the fungi to attach to surfaces, entangle with each other, and influence the entire medium rheology. Thus, the filamentous growth always must be considered as it can influence the entirety of the process. This should be remembered as the rest of this chapter is read as well as the entire book. Chapter 2 • Fungal biotechnology 33 2. Fungal cultivation and requirements Fungi are known as decomposers but also as parasites of animals and plants, which can grow in almost all habitats and ecosystems on earth including soil, water, and vegetal environments. They thrive under very different environmental conditions and have variety of growth requirements like nutrients, pH, oxygen, osmotic conditions, and temperature (Chambergo and Valencia, 2016; Blackwell, 2011; Sugiharto, 2019). Under natural conditions of the environment, each microorganism usually adapt to the habitats most suitable for their needs. However, in the laboratory and industrial environments, a culture medium and reactor (or fermenter) must meet these requirements. The range of requirements depends on different factors and detailed investigations are needed to establish the most suitable chemical and physical requirements for fungal growth. 2.1 Fungal cultivation medium A substance on which a microorganism is grown on in the laboratory or industrial environment is called a medium and the microorganism growing on it, a culture. Culture medium contain all required components for growing and producing any primary or secondary products the cell has been designed to generate (Ikechi-Nwogu and Elenwo, 2012; Harvey and McNeil, 2008). A wide range of medium are used for growing fungi because of their diversity and numerous metabolic pathways. There are three general types of fungal culture medium based on chemical composition: natural, semisynthetic, or synthetic. Natural media are composed of various natural materials that are usually of plant or animal origin. The exact chemical composition is not known in natural medium but they are usually easy to prepare. Natural medium are used for many biotechnological processes because they are usually relatively cheap and available (Harvey and McNeil, 2008; Basu et al., 2015). Semi-synthetic medium are based on both natural ingredients and defined components. Potato dextrose agar (PDA), oatmeal agar (OMA), corn meal agar (CMA), yeast extract dextrose agar (YEDA), and peptone dextrose agar (PDA) are examples ( Jong and Birmingham, 2001). Synthetic media, on the other hand, contain ingredients of known composition and each component and its concentration are controlled. Synthetic medium are usually quite simple and also useful in research and laboratory works where experimental accuracy is paramount and data interpretation needs to be clear. Industrialists have not tended to use this kind of medium in fermentation processes because they are generally not cost effective at a large-scale (Harvey and McNeil, 2008; Jong and Birmingham, 2001). Medium used in bioprocessing can also be classified based on consistency (liquid or broth, semisolid and solid) and application or function (all-purpose medium, selective/differential medium, enrichment medium, and reducing medium) (Chauhan and Jindal, 2020). 2.1.1 Growth chemical requirements Despite the variety of medium in the type and combination of nutrients, the general composition of a medium remains the same with carbon source, nitrogen source, vitamins, 34 Current Developments in Biotechnology and Bioengineering and minerals (Chauhan and Jindal, 2020; Sood et al., 2011). Such essential nutrients can be divided into two categories: macronutrients, which are needed in large amounts, and micronutrients, which are needed in trace or small amounts. Macronutrients comprise sources of carbon, nitrogen, oxygen, hydrogen, sulfur, phosphorus, potassium, and magnesium; and micronutrients include trace elements like calcium, copper, iron, manganese, cobalt, molybdenum, and zinc (Chauhan and Jindal, 2020; Walker and White, 2017). The trace elements are generally part of enzymes and cofactors as well as help to maintain protein structure. These elements may not be necessary when natural or semi-synthetic medium are utilized. However, they must be added to pure synthetic medium or a medium in which a cell has an absolute requirement for a particular element or elements (Harvey and McNeil, 2008; Basu et al., 2015). Macronutrients usually help maintain the cell structure and metabolism. The physiological role of each macronutrients and their working concentration id presented in Table 1. A wide range of materials are used as carbon sources in culture media and the one chosen should both be appropriate to the microorganism and cost-effective (Harvey and McNeil, 2008). Sugars/carbohydrates are universally acceptable for fungal growth, and can range from simple monosaccharides like glucose to polysaccharides like starch and cellulose. In recent years, lignocellulosic materials have become more established as potential carbon sources due to their availability and low raw material cost. Following the carbon source, the nitrogen source is generally the next most plentiful substance in the cultivation medium (Kampen, 2014). Most industrially used microorganisms are able to use both inorganic and organic nitrogen sources. Inorganic nitrogen sources are generally NH3, NH4 + , NO3 , and NO2 . Components such as yeast extract, peptone, and whey are organic nitrogen sources and contain certain growth factors like amino acids or vitamins (Clarke, 2013; Stanbury et al., 2017a). Table 1 Physiological functions of macro-nutrients and their required concentration (Harvey and McNeil, 2008; Chauhan and Jindal, 2020; Kampen, 2014). Element Physiological function Carbon Nitrogen Hydrogen Oxygen Constituent of organic cell materials and energy source Constituent of proteins, nucleic acids, and coenzymes Constituent of cellular water and organic cell materials Constituent of cellular water and organic materials, as O2 electron acceptor in respiration of aerobes Constituent of amino acid (cysteine, cysteine, and methionine), some vitamins, e.g., biotin, as well as some coenzymes as CoA and cocarboxylase Constituent of phospholipids, coenzymes, and nucleic acids and the generation of energy (ATP, ADP) Principal intracellular cation and cofactor for the activity of certain enzymes This often acts as a cofactor for the activity of many enzymes, can play a significant role in membrane structure and function Sulfur Phosphorus Potassium Magnesium Required concentration (mol/L) > 10 10 3 – – 10 2 4 10 3–10 4 10 3–10 10 3–10 4 4 Chapter 2 • Fungal biotechnology 35 In addition to the carbon and nitrogen sources, carbon to nitrogen (C:N) ratio play an important role in fungal growth, sporulation, and production of mycelia. The C:N ratios required by a number of fungi have been reported in literature. The C:N ratios in semisynthetic media are closer to reality, assuming that “reality” is approximately equal to the C:N ratio of soil materials or about 9:1 or 12:1. In this range, the medium is appropriate for growth and for some synthesis of nitrogen containing by-products (Cooke, 1968). Sometimes, the culture medium should be supplemented with certain components such as growth factors, chelating agents, buffers, precursors, inhibitors, inducers, and antifoams to provide all requirements for optimum growth and to control some of the problems in the fermentation process. 2.1.2 Design and preparation of culture medium A successful fermentation process needs a proper medium design. Factors such as cost, availability of substrates, reliability of substrate supply, handling, storage, ease of preparation (and storage), and transportation of components make medium design complicated. Therefore, substantial research must be put into obtaining all the information needed to optimize the medium (Harvey and McNeil, 2008). In general, medium are designed based on the following simplified equation (oxygen is only present in aerobic bioprocesses): C + N + O2 + other nutrients ! biomass + productðsÞ + CO2 + H2 O + Heat In this equation, it is important to calculate the minimal quantities of nutrients which will be needed to produce a specific (and maximum) amount of biomass and required product yields. The medium should generally be designed to: • • • • • • • • Produce the maximum yield of product Produce the maximum concentration of biomass Obtain the maximum rate of product formation Minimize the yield of undesirable products Be of consistent quality and available throughout the year Cause minimal problems during medium sterilization Allow for easy aeration, agitation, downstream processing, waste treatment, etc. Be able to scale-up (Sood et al., 2011; Stanbury et al., 2017a) After the medium design and identification of its constituents, it is time to prepare it. The medium preparation is not just a scientific work but it requires detailed monitoring and planning. It is also important to find an appropriate preservation method and use the constituents optimally. When the culture medium has been made, it still (most often) has to be sterilized because of microbial contamination from air, receptacle or glassware, hands, etc. Although, all components present in the medium are usually sterilized together, some components like vitamins are sterilized separately in order to prevent losses (Harvey and McNeil, 2008). 36 Current Developments in Biotechnology and Bioengineering 2.2 Fungal fermentation process 2.2.1 Inoculum preparation The population of microorganisms or cells that are introduced in the fermentation medium or any other suitable medium is the inoculum. The inoculum preparation and optimization are carried out before the fermentation process starts. During the first stage of inoculum development, the inoculum as spores or hyphae (mycelia) is taken from the working stock culture and transferred to a suitable culture medium to initiate the fungal growth. Growth at this stage is influenced by the adaptation of inoculum to the new environment, inoculum size, inoculum morphology, and inoculum transfer strategy that further influences the final product (Sood et al., 2011; Harvey and McNeil, 2008). The inoculum performance can be improved by various modifications including DNA recombination, radiation, and chemical addition (Sood et al., 2011). The inoculum transfer to the next cultivation stage can be done as spore suspension, mycelia disc, mycelia suspension, and pre-inoculated substrates. Among these, spore inoculum is used in most fermentation processes due to far more “propagules” compared to vegetative inoculum. In addition, this approach is easier to operate aseptically and it may be applied on a large scale. Nevertheless, vegetative inoculum is well suited to solid substrate fermentation as their penetrating hyphal habit facilitates the colonization of solid substrates (Stanbury et al., 2017b). The inoculum can be polluted by the contamination of an undesirable organism or undesirable organisms, which can result in lower productivity or even complete fermentation failure. Therefore, various sterilization methods and offline and online monitoring methods are applied to detect and prevent contamination (Sood et al., 2011). 2.2.2 Types of fermentation processes Fermentation is controlled cultivation of microorganisms like fungi for conversion of renewable feedstock into useful products. There are two common fermentation techniques called submerged fermentation (SmF) and solid-state fermentation (SSF) systems that are applied to produce biomass and metabolites by amplifying their production from a laboratory to large scale (Moslamy, 2019). Both systems have their own benefits and drawbacks, and choosing the appropriate technique is crucial for achieving the best performance from the microorganisms during the fermentation process and recovery of the products. SSF is the cultivation of microorganisms on the surface and at the interior of a solid € lker et al., matrix, in the absence or near absence of free water (Soccol et al., 2017; Ho 2004). SSF is one of the oldest biotechnological processes known and traditional fermented foods like koji, tapai, tempe, soy sauce, annatto, miso, etc. have been produced by this € lker et al., 2004; Nigam et al., 2003). In process, especially in Asia, for many centuries (Ho recent years, SSF has gained attention from the academic and industrial sectors because of its potential for producing industrially important products such as animal feeds, bioethanol, and various enzymes (Soccol et al., 2017; Nigam et al., 2003). In addition, Chapter 2 • Fungal biotechnology 37 SSF is currently the best method of collecting fungal spores by aerial hyphae. The morphological, functional, and biochemical properties of spores produced in SSF differ distinctly from those obtained in SmF. It was found that more spores are obtained if a combination of SmF (for biomass production in a first step) and SSF (for subsequent spore production) € lker et al., 2004). is utilized (Ho The mycelial modes of growth and neutral physiological capabilities of filamentous fungi have made them a dominant microorganism in SSF processes. Considerable amounts of enzymes and other metabolites are synthesized by filamentous fungi in this condition (Soccol et al., 2017). The typical advantages of SSF processes include lower capital and operating costs, low energy requirements, simple and low-cost medium, lesser wastewater production, less effort in down-stream processing and stirring, less susceptible to bacterial contamination and substrate inhibition, better oxygen circulation, and higher productivity (Stanbury et al., 2017a; Mandal and Banerjee, 2019; Robinson et al., 2001). On the other hand, wild-type strains of fungi tend to perform better in SSF conditions than genetically modified fungi (Robinson et al., 2001). In spite of the advantages mentioned, there are also several disadvantages of SSF, which have discouraged the use of this technique for industrial production. The main drawbacks are difficulties of scale-up, low mixing efficiency, difficult control of process parameters (pH, heat, moisture, nutrient conditions, etc.), problems with heat build-up, higher impurity of the product, increased product recovery costs, relatively slow growth rate of microorganisms, and the process being limited to € lker et al., microorganisms that can survive and thrive in low moisture conditions (Ho 2004; Couto and Sanromán, 2006). The fungal growth and product formation take place in a vessel referred to as the bioreactor that is considered as the heart of all bioprocess operations. The bioreactor provides a well-monitored system to meet the needs of the biological reaction system so that a high yield of the bioproduct is achieved. The bioreactors in SSF have been classified into four categories based on the operation modes that are used: (1) reactors without forced aeration and mixing/agitation of the solid substrate (e.g., tray bioreactor), (2) reactors without mixing/agitation but with forced aeration (e.g., packed-bed bioreactor), (3) reactors with mixing/agitation but without forced aeration (e.g., rotating-drum and stirred-drum bioreactors), and (4) reactors with mixing/agitation and forced aeration (e.g., fluidized-bed, rocking-drum, and stirred-aerated bioreactors) (Ge et al., 2017; Zhong, 2011). Some versions are illustrated in Fig. 2. SmF involves inoculation of the microbial culture as a suspension in a liquid medium in which various nutrients are either dissolved or suspended as particulate solids in many commercial medium. These days, most of the commercial products including enzymes and secondary metabolites are produced through the SmF processes due to its advantages (Kapoor et al., 2016). The SmF possesses considerable advantages including ease of scale-up and automation, shorter required fermentation time, ease of product purification, and better control of the physical-chemical variables of the process. However, the major disadvantages 38 Current Developments in Biotechnology and Bioengineering FIG. 2 Schematic of bioreactors for SSF: tray bioreactor, packed-bed bioreactor, rotating-drum bioreactor, stirred packed bed bioreactor. associated with the SmF processes as compared to the SSF are lower productivity, high production cost, and complexity of medium, more effluent generation, complex fermentation equipment, large reactors needed, and risk of contamination (Kapoor et al., 2016; de Carvalho, 2016). There are three major fermentation modes in SmF processes, i.e., batch, fed-batch, and continuous cultivation (they exist for SSF processes as well), which can be operated either aerobic or anaerobic conditions. The development and optimization of the SmF processes involve understanding the three modes of fermenter operation so that the one best suited to the process can be determined (Kapoor et al., 2016; Macauley-Patrick and Finn, 2008). Batch fermentation is considered as the simplest mode of cultivation systems, as it is operated in a closed vessel where all of the nutrients required for the organism’s growth and product formation are added to the vessel, mostly under aseptic conditions, at the beginning of the fermentation process. Indeed, no extra feeding is used during the process; all is done as one batch. During batch fermentation, four typical phases can normally be observed: lag phase, exponential phase, stationary phase, and declination phase (Fig. 3). Fed-batch fermentation is a semi-open system in which one or more nutrients continuously or intermittently are introduced into the bioreactor after the start of cultivation, or from a certain point during the batch process and optimum concentrations of required nutrients are maintained by such intermittent feeding. Intracellular products of cell cultures that are stored within the cells are often produced by fed-batch cultures. In a continuous culture, the microorganisms are continuously fed with fresh medium and the nutrients consumed by the cells are removed from the system at the same rate. Therefore, factors such as culture volume, biomass or cell number, product and substrate concentrations, as well as the physical parameters of the system such as pH, temperature, and dissolved oxygen will be kept constant throughout the fermentation. This balanced growth is a very attractive tool in studies on growth and production kinetics or cell physiology (Zhong, 2011; Kapoor et al., 2016; Macauley-Patrick and Finn, 2008). Several types of bioreactors including stirred tank, airlift, bubble column, membrane, packed-bed, and fluidized-bed bioreactors, which are designed for the SmF processes. Chapter 2 • Fungal biotechnology 39 FIG. 3 The four phases seen in batch cultivation: lag phase during which the fungi get acclimatized, exponential phase during which exponential growth occurs, stationary phase during which no net change in biomass concentration happens, and declination phase during which the biomass concentration decreases. Novel bioreactors are constantly being developed for special applications and new forms of biocatalysts. In stirred tank reactors, mechanical devices like impellers and baffles provide efficient mixing and bubble dispersion, which allow for a good control of pH, temperature, airflow, and dissolved oxygen. This technique requires a relatively high input of energy per unit volume. Unlike stirred tank reactors, aeration, and mixing in bubble column and airlift reactors are achieved by air or gas sparging without mechanical devices. The patterns of liquid flow in airlift reactors differ from those in bubble column reactors. Packedbed reactors are operated with immobilized or particulate biocatalysts. The reactor consists of a tube, usually vertical, packed with catalyst particles. After medium feeding at either the top or bottom of the column, a continuous liquid phase between the particles is formed. This bioreactor considered as a promising tool for tissue engineering applications that support various cell lines for long incubation periods due to the immobilization of cells within matrices. In fluidized-bed bioreactors, biocatalysts such as enzymes or microbial cells are used to complete a variety of multiphase of chemical reactions. Despite the complexity of fluidized-bed bioreactors, it has been using in many industrial applications. When packed-beds are operated in upflow mode with catalyst beads of appropriate size and density, the bed expands at high liquid flow rates due to upward motion of the particles. This is the basis for operation of fluidized-bed reactors as illustrated in Fig. 4. Because particles in fluidized-beds are in constant motion, clogging of the bed and flow channeling are avoided so that air can be introduced directly into the column. This reactor can be used in waste treatment with sand or similar material supporting mixed microbial populations and vinegar production by flocculating organisms (Zhong, 2011; Doran, 2013; Moslamy, 2019). 40 Current Developments in Biotechnology and Bioengineering FIG. 4 Schematic of bioreactors for SmF: stirred tank bioreactor, bubble column bioreactor, airlift bioreactor, packedbed bioreactor, and fluidized-bed bioreactor. 2.3 Effects of process variables on growth and product formation Factors influencing the performance of a cultivation (or fermentation) process can be categorized into physical, chemical, or biological. The physical and chemical factors define the environment of microorganism, while the biological factors describe its behavior (Fazenda et al., 2008). The medium composition including type and concentration of nutrients affect fungal growth, metabolite production, and morphological differentiation. An evaluation was carried out on the cultivation of Aspergillus niger, Aspergillus flavus, Penicillium chrysogenum, Aspergillus terreus, Aspergillus glaucus, Fusarium oxysporium, and Rhizopus stolonifer on potato dextrose, soybean dextrose, sawdust sucrose, ofor (Detarium macrocarpum, a tree growing in Africa) sucrose, and groundnut dextrose broth. From the results Chapter 2 • Fungal biotechnology 41 obtained, soybean dextrose broth performed better than other broths for the growth of the fungi evaluated probably because it contains more vitamins and minerals vital to fungal growth. Groundnut dextrose broth was the second best medium, probably due to higher nutritional content, including the presence of cystine, thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid (vitamin B5), and folate (vitamin B9) (Ikechi-Nwogu and Elenwo, 2012). The comparison of nine agro-industrial and forestry by-products including (i) nut (almond and walnut 1:1 w:w) shells, (ii) beech sawdust, (iii) corn cobs, (iv) grape marc plus cotton gin trash (1:1, w:w), (v) olive mill by-products (leaves plus two-phase olive mill waste 1:1, w:w), (vi) extracted olive press-cake, (vii) pine needles, (viii) date palm leaves, and (ix) wheat straw fermented by Agrocybe cylindracea and Pleurotus ostreatus in SSF showed that grape marc waste plus cotton gin trash was the best performing medium for both fungi followed by olive mill by-products and pine needles for the former and latter species, respectively. Substrate composition had a marked effect on most cultivation parameters (Koutrotsios et al., 2014). Singh et al. (2020) found that glucose and sucrose when used as a carbon source resulted in higher biomass concentration of Pleurotus eryngii in comparison to other carbon sources. In addition, nitrogen sources favorable for the mycelial growth were observed to be yeast extract and peptone. The effect of physical factors including temperature, pH, oxygen concentration or oxygen transfer, aeration, and agitation rates on fungi performance and its productivity has been investigated by many researchers. This is discussed below. Although fungi can survive in a range of temperatures, but small variations in temperature can greatly reduce the productivity. The temperature range usually reported for fungal growth is 10–35 °C, with a few species capable of growth below or above this range (phsycrophillic to thermophilic). Increasing temperature generally results in higher metabolic rates, but decreases the solubility of dissolved oxygen in the medium (Fazenda et al., 2008). The optimal temperature for mycelium growth of oyster mushroom Pleurotus ostreatus and Pleurotus cystidiosus was 28 °C (Hoa and Wang, 2015). As Singh et al. (2020) reported, the initial pH of the medium did not influence the mycelial biomass concentration whereas the shaking frequency (i.e., mixing) and temperature variation had a positive influence on the mycelial biomass concentration. All microorganisms have an optimal pH at which they grow the best. Alteration of this pH value leads to undesirable growth. Most fungi are acidophilic and grow well between pH 4 and 6, but some species can grow in more acidic or alkaline conditions (around pH 3 or pH 8, respectively). Sar et al. (2020) found that changing the initial pH (6.1–6.5) to 5.0 had a negative influence on the amount of biomass produced from the edible filamentous fungus Rhizopus oryzae cultivated on fish industry side streams while medium supplementation had no influence. The maximum xylanase activity has been found to differ considerably dependant on the species producing it. In Aspergillus fischeri optimum pH was between pH 6 and 10, whereas the optimal pH for xylanase production by strains of Aspergillus fumigatus was pH 5 or less and maximal xylanase production by Aspergillus fumigatus occurred below pH 3.5. Moreover, a cultivation pH of up to 7.5 favored xylanase production by a Thermomyces lanuginosus and Aspergillus 42 Current Developments in Biotechnology and Bioengineering oryzae (Chipeta et al., 2008). A low initial pH of the medium (below 2.0) was necessary for increased yields in the citric acid fermentation by Aspergillus niger whereas the optimal pH for maximum gluconic acid production by Aspergillus niger was 6.5. Increasing the pH to 4.5 during the production phase reduced the final yield of citric acid by up to 80% (Papagianni, 2019). Oxygen transfer and maintaining a suitable oxygen concentration in the culture broth are always a concern in aerobic fermentation systems. When the supply of oxygen is limited, both cell growth and product formation can be severely affected (Zhong, 2011). Increased dissolved oxygen (DO) concentrations resulted in increased citric acid yield from Aspergillus niger fermentation, while even a short interruption in aeration had detrimental effects on the yield as it rerouted metabolism toward biomass formation (Papagianni, 2019). In SmF, aeration and agitation provide two related functions: (i) to supply dissolving oxygen needed by the growing hyphae, and (ii) to homogenize mycelial mass, nutrients and products within the culture broth. However, the shear forces created by agitation may damage cell structure, lead to morphological changes, and cause variations in growth rate and product formation. Therefore, the optimum agitation rate represents a balance between achieving adequate oxygen transfer into the medium and shear stress, both of which increase with increasing agitation rate (Zhong, 2011; Fazenda et al., 2008). It is widely accepted that inoculum concentration and age exert a major influence on the fungal fermentation profile. In fact, the amount, type (spore vs vegetative), age, and viability of the inoculum all may affect morphological state of the cells, especially in pellet production and the type of pellets produced (Fazenda et al., 2008). 3. Fungal biorefineries Fungal biorefineries are multi-output systems that convert biological feedstocks into a spectrum of high added value products, and are able to reduce the contamination of the environment. These systems have been the subject of a growing interest for the biorefinery concept because they represent a rich source of enzymes, organic acids, pigments, vitamins, volatile compounds, antibiotics, and other substances of relevance to the pharmaceutical, food, feed, agricultural, textile, pulp and paper, chemical, and biotechnological industries (Fig. 5). The most attractive group for fungal biorefineries are the so-called filamentous fungi, such as those belonging to the Ascomycetes, Basidiomycetes, and Zygomycetes groups. Filamentous fungi have some inherent important characteristics such as being able to produce a remarkable wealth of commercially interesting metabolites, a welldemonstrated growth ability on a broad substrate range, and well-developed methodology for genetic modification, which makes them great contributions within a wide range of industrial sectors (Chroumpi et al., 2020; Ferreira et al., 2013, 2016). Chapter 2 • Fungal biotechnology 43 FIG. 5 Industries profiting from the metabolic capacities of fungi. 3.1 Fungal biorefinery-based industries 3.1.1 Agriculture The importance of fungi in the agriculture industry can be considered from different aspects. One of the cost-effective and eco-friendly means for solubilizing insoluble phosphorous (P) in soils is to use phosphate-solubilizing fungi, especially plant growthpromoting fungal strains. Furthermore, fungi are among one of the novel and potential sources of biological control agents. Thus, these helps to increase the availability of phosphorous for plants and could be potent and promising alternatives to synthetic pesticides and chemical fertilizers, and such beneficial microorganisms are perfect candidates for sustainable agricultural production (Challa et al., 2019; Vyas and Bansal, 2018; Kaur and Sudhakara Reddy, 2017). Fungi belonging to the order Hypocreales are used to control 44 Current Developments in Biotechnology and Bioengineering pests (insects or phytopathogens). The biopesticides available in the market capable of controlling insects include Beauveria bassiana, Nomuraea rileyi, Metarhizium anisopliae, and Paecilomyces fumosoroseus (mitosporic or asexual or conidiogenous entomopathogenic fungi), whereas phytopathogens are inhibited using biocontrol agent Trichoderma viride (Challa et al., 2019). Fungi play a fascinating role in the composting processes, degrading recalcitrant compounds during composting, remediating soils contaminated by pollutants, stabilizing organic matter while releasing nutrients and essential elements that are beneficial for plant growth and fertility. Acremonium, Alternaria, Aspergillus, Chaetomium, Cladosporium, Emericella, Fusarium, Geotrichum, Mortierella, Mucor, Penicillium, Pseudallescheria, Scopulariopsis, and Trichoderma are the fungi most isolated from compost. Some fungi including Absidia, Aspergillus, Chaetomium, Coprinus, Mucor, Paecilomyces, Penicillium, Rhizomucor, Scytalidium, and Thermomyces can grow at elevated temperatures (>65 °C) and are isolated from thermophilic compost phases (Wright et al., 2016). 3.1.2 Food and feed Fungi have long been known to play a significant role in the food and feed industry. Fungi are used as a direct source of food (mushrooms and truffles as the edible fungi), processed food (bread, cheese and other bakery products), fermented foods (alcohols, beverages), fodder, etc. They can also be used to produce protein biomass such as single-cell protein € ttner et al., 2020; Challa et al., 2019; Ghorai et al., 2009). (SCP) (Hu The fungi and fungal biomass as food and feed are very nutritive as they are good source of protein, essential and nonessential amino acids, dietary fiber and vitamins, almost low in fat, free of cholesterol, and easy to digest. Thus, such beneficial properties, diet consciousness and increasing health awareness has increased the fungal food utilization on a global basis over the past few years. Particularly, the people who are vegetarian have resorted to eating mushrooms or processed foods, dietary supplements, and beverages of fungal origin (Devi et al., 2020; Kour et al., 2019; Ghorai et al., 2009). Quorn® mycoprotein is a commercial form of SCP created from filamentous fungus Fusarium venenatum. It is popular since it has many similar characteristics with meat, e.g., texture and appearance, and effectively competes with meat alternatives like soy and pea on € ttner et al., 2020; Challa et al., 2019; Kour et al., 2019). health grounds (Hu In addition, a number of ingredients used during food processing such as acidulants, enzymes, flavors, vitamins, colorants (pigments), and polyunsaturated fatty acids (PUFAs) are obtained from fungi through industrial fermentation (Copetti, 2019). Organic acids as acidulant and also as flavoring agents, buffers, preservatives, and technology adjuvants are widely used in modern food processing (Copetti, 2019). Filamentous fungi are considered as excellent sources of food-grade pigments. Some pigments commercially produced by filamentous fungi are Monascus pigments, natural red from Penicillium oxalicum, riboflavin from Ashbya gossypii, lycopene, and β-carotene from Blakeslea trispora (Challa et al., 2019; Copetti, 2019; Ghorai et al., 2009). Enzymes are used in the food industry for many different applications including production of dairy, meat, cereal, beverage, Chapter 2 • Fungal biotechnology 45 bakery and confectionery products, development and enhancement aroma, color, appearance, texture and flavor as well as improving quality of the final product while decreasing processing time and production costs (Copetti, 2019; Kour et al., 2019). Moreover, fungi, especially filamentous fungi, produce edible oils rich in PUFAs for dietary supplements and infant nutrition applications (Coradini et al., 2015). 3.1.3 Pharmaceutical Among microorganisms, fungi have significant ability to protect against a number of pathogenic bacteria and fungi due to the bioactive agents that they naturally produce as secondary metabolites. Penicillin was the first and the best-known discovery, which proved to inhibit the growth of Gram-positive bacteria (Devi et al., 2020; Gholami-Shabani et al., 2019). Several effective antibiotics and drugs with antifungal, nematocidal, antiprotozoal, antibacterial, antiplasmodial, and antiviral properties, as well as anti-inflammatory inhibitors, anti-tuberculosis and anticancer drugs, agonists or antagonists at adrenergic, dopaminergic, and serotonergic receptors, and hypercholesterolemia treatment agents are being produced using fungi (Devi et al., 2020; Chambergo and Valencia, 2016). For this, some human diseases are treated by fungi. For example, Agaricus campestris is used against sinusitis, inflammation, and tuberculosis. Laricifomes officinalis can be used for treating diarrhea and night sweating. Daedaleopsis flavida helps in the reduction of bilirubin and biliviridin and is very effective for the treatment of jaundice. The treatment of chronic gastritis, early tumors, and ulcers is done using Inonotus obliquus. Alkaloid secreted from Aspergillus, Penicillium, Pestalotiopsis, and Chromocleista is a β-carboline group containing secondary metabolites having antimicrobial, anti-HIV, and antiparasitic activities (Devi et al., 2020). Lovastatin as a cholesterol lowering drug is produced by a diverse range of filamentous fungi like Aspergillus sp., Aspergillus terreus, Monascus sp., Phoma sp., Gymnoascus umbrinus, Penicillium brevicompactum, Penicillium citrinum (Saberi et al., 2020; Chambergo and Valencia, 2016). The utilization of filamentous fungal enzymes is also growing in pharma industry, particularly ones that catalyze bio-oxidations. This includes oxidative functionalization of steroidal building blocks, and could be used to mimic reactions that take place in the human liver. Furthermore, transaminases, imine reductases, keto reductases, and lipases are the most relevant enabling enzyme classes for pharma research and development applications (Meyer et al., 2016). Some fungal metabolites such as xylitol and pigments are used as non-carcinogenic sweetener and coloring agents in pharmaceutical industry, respectively. Xylitol is also recommended in cases such as lipid metabolism disorder and respiratory infections, for the prevention of osteoporosis as well as for persons suffering with kidney and parental lesions. Pigments can be exploited for the treatment of cancer, diabetes, and other infectious diseases because of anticancer, antidiabetic, and antimicrobial properties. They are also proved to be efficient antioxidants and could be applied to bring down the level of free radicals. Fungal pigments also work as antibacterial, antifungal, and antiprotozoal (Dhiman and Mukherjee, 2018; Tirumale and Wani, 2018). 46 Current Developments in Biotechnology and Bioengineering 3.1.4 Pulp and paper Biotechnology has attracted growing attention in the pulp and paper industry in the last decade, since biotechnology provides tools to increase both the quality and the supply of feedstocks for pulp and paper, reduce manufacturing costs, and create novel high-value products (Bajpai, 2018). The various approaches to the usage of fungi to remove undesirable components of wood, pulp, and wastewater have been developed in the pulp and paper industry. White-rot fungi have a high selectivity for delignification, which can reduce cellulose loss to make fungal pre-treatment practical for biopulping (Chowdhary et al., 2018). Moreover, fungi have been used for removal and detoxification of extractives from wood to reduce pitch related problems to satisfactory level (Bajpai, 2018). Among the biological methods tried so far, fungal treatment technology using white-rot fungi appears to be the most promising for decolorization and detoxification of pulp and paper mill wastewaters especially bleach effluents (Bajpai, 2018). In addition, the number of applications of enzymes of fungal origin in pulp and paper manufacturing has steadily increased during recent years, and several have reached or are approaching commercial reality. These include enzyme-aided bleaching with xylanases, direct delignification with oxidative enzymes, deinking and fiber modification with cellulase-hemicellulase mixture, refining with cellulases, pitch reduction/removal with lipases, stickies control with esterases, freeness (or drainage) enhancement with cellulases rrez et al., 2011). and hemicellulases as well as enzymatic slime control (Bajpai, 2018; Gutie The use of fungi and their enzymes in different parts of the pulp and paper industry has demonstrated environmental, economic, and technical benefits due to reduction of energy and chemicals consumption as well as improvement of final product (or paper) quality. 3.1.5 Textile Textile industries use a large amount of synthetic dyes (pigments) during their manufacture process as these dyes are widely available at a reasonable price and produce a broad spectrum of colors, but they may cause skin allergy and other negative effects to human body as well as release toxic substances into the environment. The use of natural or microbial pigments with their better biodegradability and higher compatibility with the environment and human body coupled with fungal bioremediation of textile effluents have been proven to overcome these problems (Tirumale and Wani, 2018; Mukherjee et al., 2017). Filamentous fungi produce amazing pigments like carotenoids, melanins, flavins, phenazines, quinones, and monascins from different chemical classes (Venil et al., 2020). Bioremediation of synthetic dyes in textile waste effluents by fungi has been demonstrated to be more efficient, cost-effective, and environmentally friendly compared with other techniques. White-rot fungi such as Phanerochaete chrysosporium, Trametes versicolor, Pleurotus ostreatus, Pycnoporus sanguineus, Irpex flavus, Phellinus gilvus have shown a strong ability to degrade a wide range of synthetic dyes with different structures (Munck et al., 2018; Ma et al., 2014). Chapter 2 • Fungal biotechnology 47 Fabric from fungi is an attractive innovation in the textile industry as it can be antimicrobial, biodegradable, comfortable to wear, durable, eco-friendly, fire-resistant, flexible, non-toxic, skin-friendly, strong, suitable for sensitive skin, waterproof, and mended easily. Possibly, it could also be used to substitute animal leather and suede and be used for accessories, bags, and shoes (Challa et al., 2019). Bio-fabrication of renewable fibers from cell wall of zygomycetes fungus Rhizopus delemar cultivated on bread waste as a novel resource for production of sustainable textiles was investigated by Svensson et al. (2021). 3.1.6 Biotechnological industries for biofuels, biochemical, and biomaterial production The need to reduce fossil fuel dependence in the chemical and petrochemical industries due to their high prices and environmental concerns has led to the emergence of sustainable bio-based alternatives. In this regard, the proportion of bio-based products produced by biotechnology such as biofuels, biochemical, and biomaterials, particularly those obtained from fungi, has been increasing during recent years (Chambergo and Valencia, 2016). Many companies such as AB Enzymes, BASF, Bayer, Chr. Hansen, Dyadic International, DSM, DuPont, Kerry Group, Marlow Foods, Novozymes, Puratos, Syngenta, and Roal Oy are global leaders in using fungi for bulk manufacturing of organic acids, proteins, enzymes, and secondary metabolites, e.g., antibiotics (Meyer et al., 2020). The following are given an illustration of some bio-based products. Fungi can be a good source of biofuels and are explored globally for generation of various biofuels such as bioethanol, biodiesel, biogas, and so on. Filamentous fungi have attracted a lot of research interest due to their ability to produce a variety of lignocellulolytic enzymes and ferment both hexoses and pentoses to ethanol ( Joseph and Wang, 2018). Microbial lipids from oleaginous fungi, due to their plant-like oil composition, are now being studied to produce biodiesel in a sustainable and economical manner (Coradini et al., 2015). Anaerobic fungi have a great potential in the production of biogas from lignocellulosic waste due to the combination of their highly effective ability to hydrolyse lignocellulose (Saye et al., 2021; Chowdhary et al., 2018). Moreover, the fermentative production of novel biofuels such as butanol and isobutanol using fungi and yeast is being developed to overcome disadvantages of the common biofuels like ethanol and biodiesel (Coradini et al., 2015). Organic acids, which can be used as food additives, cosmetic ingredients, and chemical intermediates for the production of biodegradable polymers, are often produced by fila€ ttner et al., 2020). At present, citric acid and gluconic acid produced mentous fungi (Hu with the filamentous fungus Aspergillus niger are the two biggest commercial fungal products in terms of production volume (Troiano et al., 2020). Fungi are also capable of producing some innovative products. For example, they are excellent candidates to explore new eco-friendly methods for inorganic nanoparticles (NPs) production either intra- or extracellularly. Iron oxide NPs and gold NPs are two examples. The unique catalytic, optical, electronic, and photochemical properties of the NPs contribute toward a wide range of applications in the field of applied and basic research (Mahanty et al., 2019; Vágó et al., 2016). Mycelium-based composites, e.g., 48 Current Developments in Biotechnology and Bioengineering mycelium board biocomposites and mycelium/straw biocomposites, are a relatively new and environmentally sustainable class of materials that can replace petroleum-based products such as synthetic plastics (e.g., polystyrene) or other foams as well as natural materials such as cork and wood. They can be used in various applications such as automobile, aerospace, packaging and building industries, and sports instruments (Rafiee € sten, 2019). et al., 2021; Soh et al., 2020; Wo 3.2 Adding value to organic wastes through fungi Carbon-rich wastes are interesting raw materials for biorefineries, due to their high abundance and relatively low price. Indeed, there are vast amounts from different types of the wastes of industrial, agricultural and municipal origin that have little use but can be converted to higher value products. The production of valuable components from organic wastes, and the simultaneous elimination of the organic load are key steps toward mitigating economic and environmental hurdles (Chan et al., 2018). Among methods used for transformation of organic wastes into value-added components, the biochemical (or biotechnological) pathway has become an important research focus. There are many instances where fungi have been used for the conversion of various organic wastes into diverse value-added products. In fact, fungal bioconversion, through fermentative processes, has been revealed to be an eco-friendly and beneficial biotechnological approach for either the reutilization or the valorization of these wastes. Ethanol and fungal biomass production from waste or by-product streams generated in current industrial plants (e.g., the pulp and paper industry, the food industry, firstgeneration biofuel production plants) by using filamentous fungi have been investigated by several researchers (Asadollahzadeh et al., 2018; Mahboubi et al., 2017a,b; Bátori et al., 2015; Ferreira et al., 2012, 2014). Gmoser et al. (2020) produced new protein-enriched products using the edible filamentous fungi Neurospora intermedia and Rhizopus oryzae from stale bread and brewers spent grain. In the study of Souza Filho et al. (2018), a veganmycoprotein concentrate from pea-industry by-product using edible fungal strains of Ascomycota (Aspergillus oryzae, Fusarium venenatum, Monascus purpureus, Neurospora intermedia) and Zygomycota (Rhizopus oryzae) was obtained. Valorization of thin stillage, bakery waste, and food processing industry by-products (apple, pomegranate, black carrot, and red beet pulps) as nutrient sources for the fermentative production of biocolorant (pigment) by filamentous fungi has been developed (Gmoser et al., 2019; Bezirhan Arikan et al., 2020; Haque et al., 2016). Muniraj et al. (2015) showed the production of microbial lipids and γ-linolenic acid by Aspergillus flavus and Mucor rouxii (indicus), with potato processing wastewater as nutrient source. In addition, a variety of enzymes were produced from a number of organic wastes inoculated with filamentous fungi. 3.3 Strategies to improve investment and productivity in fungal biorefineries Fungal biorefinery operations may be made cost-competitive with petrochemical processes through a number of steps. Cheaper feedstocks, potentially lignocellulosic, and Chapter 2 • Fungal biotechnology 49 food processing residues can be used. Complete valorization of biomass can be carried out, including integration of multiple revenue streams and bioconversion steps. Furthermore, two or more distinct microorganisms can be used in the same culture to obtain a synergistic effect, and genetic engineering can be attempted (Troiano et al., 2020). The production of further value-added products, besides the main product, from by-products or waste-streams generated in already established industrial processes like dairy, paper and pulp, oil, fruit, sugar producing industries, and first-generation ethanol plants can lead to energy and cost savings as well as an increase in revenue (Ferreira et al., 2018). For example, in a starch-based ethanol facility, the thin stillage waste stream has been investigated for valorization into animal feed and additional ethanol using various filamentous fungi. Moreover, lignin extracted from the pre-treatment step when lignocellulosic feedstocks are used, can be used for purposes other than heat such as production of carbon fibers, resin, biocomposites, etc. Integration of first- and second-generation ethanol is an attractive way to reduce the investment costs and risks compared to a standalone second-generation process. This reduces a major part of the investment of necessary downstream infrastructure, as it is already available. Thus, in total the feasibility of commercial-scale second-generation bioethanol plants is potentially increased (Ferreira et al., 2018; Lennartsson et al., 2014). It is suggested that simplification of the biomass conversion process and consolidation of the three main steps of enzyme production, substrate hydrolysis, and fermentation into a single step, referred to as consolidated bioprocessing (CBP), can serve as a successful strategy to reduce capital and processing costs and enhance process efficiencies (Troiano et al., 2020; Salehi Jouzani and Taherzadeh, 2015). CBP has been discussed as one of the most promising fermentation approaches for bioethanol production from lignocellulosic biomass and has attracted international interest in recent years. In addition to ethanol, several additional compounds can potentially be produced through CBP by fungi and yeasts. The capability of filamentous fungi in deconstructing lignocellulosic biomass makes them potential components in CBP (Ali et al., 2016). Co-cultivation, which consists of two or more fungal partners or a fungus (fungi) with another microorganism such as a bacterium (bacteria) in the same culture, would be a potential strategy to decrease the number of steps involved in biorefining. The use of fungal co-cultures potentially also helps increase production yield, decrease operational costs, and avoid downstream processing costs (Troiano et al., 2020; Sperandio and Ferreira € sten, 2019). Pietrzak and Kawa-Rygielska (2019) found that a co-culture of Filho, 2019; Wo edible filamentous fungi (Aspergillus oryzae and Rhizopus oligosporus) with fodder yeast Candida utilis for backset water treatment was effective in improving the core production stage in an ethanol-production plant. The yield of biotechnological products can be much less when naturally available microorganisms are used. The modification of genetic structure of microorganisms is reported to increase their productivity. Genetic modifications in microorganisms can be prompted by various methods such as improvement of a classical strain by mutation and selection or by the use of recombinant DNA technology (Sood et al., 2011). Classical examples of successful strain improvement used in the industry can be found in the 50 Current Developments in Biotechnology and Bioengineering production of antibiotics and significantly increased titres, e.g., penicillin titres have been increased by more than a factor 1000 (Kavanagh, 2011). 4. Fungal metabolites Filamentous fungi are good producers of metabolites such as organic acid and ethanol, but also value-added products such as enzymes and pigments (Karimi et al., 2018). Traditionally, filamentous fungi have been classified to contain four groups (for a more recent classification, see Chapter 3 of this book): Ascomycota, Zygomycota, Basidiomycota, and Chytridiomycota (Ferreira et al., 2013). For biotechnological applications, the first three have been found to be of interest thus far. The following section aims to give an overview of the bulk chemicals that potentially could be produced by these fungi. 4.1 Ascomycetes Filamentous ascomycetes are, and have been, considered of interest to produce metabolites such as organic acids (citric, gluconic, and itaconic acid) and ethanol because of their enzymatic capabilities (Ferreira et al., 2016). Neurospora spp. and Fusarium spp. are mostly used in research for ethanol production and Aspergillus spp. are best known for the production of organic acids such as citric, gluconic, and itaconic acid (Karimi et al., 2018; Ferreira et al., 2016). Among the organic acids, especially production of citric acid is of interest due to a projected continuous growth in demand (Ferreira et al., 2016). 4.1.1 Ethanol A lot of the research interest for bioethanol production using filamentous fungi has been focused on converting lignocellulosic materials, which in general is limited by the high enzyme costs. In the 1980s, a few species such as Neurospora crassa and Fusarium oxysporum were reported as useful microbial catalysts for ethanol production directly from cellulose without the need of any external enzyme (Christakopoulos et al., 1989; Salehi Jouzani and Taherzadeh, 2015). Furthermore, the ability to produce ethanol from xylose is another advantage of utilizing filamentous fungi such as ascomycetes (Ferreira et al., 2015). In the case of Fusarium oxysporum it has been considered as a biocatalyst for conversion of brewer’s spent grain (BG), a by-product from breweries (Xiros and Christakopoulos, 2009). Fusarium oxysporum has also been investigated to produce ethanol directly from wheat straw (Panagiotou et al., 2005, 2011). Apparently, a glucose transporter belonging to Fusarium oxysporum was found to be interesting enough that it led to an investigation on its own (Ali et al., 2013). As mentioned earlier, Neurospora crassa is the most interesting microbial source for ethanol production among the Neurospora genus (Ferreira et al., 2016). For example, it has been considered for ethanol production using SSF of brewer’s spent grains (Xiros et al., 2008) and of sweet sorghum bagasse (Dogaris et al., 2012). A close relative, Neurospora intermedia, has also been considered for ethanol production from various by-products such as thin stillage (Ferreira et al., 2014, 2015) and wheat bran (Nair et al., 2015). Chapter 2 • Fungal biotechnology 51 4.1.2 Citric acid Citric acid (C6H8O7) is a weak organic acid and an intermediate of the tricarboxylic acid cycle. It is mainly used in the food (ca 70%) and pharmaceutical industry (ca 12%) (Singh Dhillon et al., 2011; Soccol et al., 2006; Sawant et al., 2018). Similar to bioethanol, the low price of citric acid makes low cost substrates such as orange processing waste and apple pomace very interesting (Dhillon et al., 2013; Singh Dhillon et al., 2011). Nowadays, Aspergillus niger is used for the production of citric acid and is being further developed for production from various novel substrates (Barrington et al., 2009; Angumeenal and Venkappayya, 2005; Wang et al., 2017). Both SSF and SmF seem to be of interest. 4.1.3 Gluconic acid Gluconic acid (C6H12O7) is an organic acid used a bulk chemical in many industries such as food, feed, pharmaceutical, paper, textile, and construction. It is derived from glucose through dehydrogenation catalyzed by glucose oxidase (Ferreira et al., 2016). Once again, Aspergillus niger is the most studied (Mukhopadhyay et al., 2005; Ikeda et al., 2006; Sharma et al., 2008). Up to now, various parameters such as type of carbon and nitrogen source, oxygen transport, and mode of cultivation (submerged or SSF) have been investigated to optimize the gluconic acid production from Aspergillus niger (Singh and Kumar, 2007; Ferreira et al., 2016). More recently, the thermostability of the glucose oxidase from Aspergillus niger has been investigated to produce gluconic acid more efficiently (Mu et al., 2019). 4.1.4 Itaconic acid Itaconic acid (C5H6O4) is an unsaturated dicarboxylic acid with considerable interest to use as a building block chemical, including as a co-monomer in the production of plastics and resins (Li et al., 2012). Itaconic acid is currently produced by fermentation of glucose or molasses by Aspergillus itaconicus and Aspergillus terreus (Blumhoff et al., 2013; Okabe et al., 2009). However, itaconic acid production by Aspergillus terreus is lower than its precursor citric acid: >80 g/L compared to >200 g/L (Ferreira et al., 2016; Zhao et al., 2018). Therefore, production of itaconic acid by Aspergillus niger using some genetic modification have been investigated (Li et al., 2012; Blumhoff et al., 2013; van der Straat et al., 2014). Various parameters for itaconic acid production such as type of substrate, oxygen source, € chs (2013) and and cultivation methodology have been reviewed by Klement and Bu Bafana and Pandey (2018). Furthermore, the medium components needed for production of itaconic acid has been investigated and the results showed similarity to that of citric acid production medium (Li et al., 2012). 4.2 Zygomycetes In zygomycetes, the final glycolysis product (pyruvate) can be directed to different pathways and can produce several value-added products based on the strain and cultivation medium type (Ferreira et al., 2013). Several species of zygomycetes are able to consume pentoses to produce ethanol (Omidvar et al., 2016). Some species can produce organic acids, such as Rhizopus sp. that have been used for production of fumaric and lactic acid (Zhang et al., 2007; Roa Engel et al., 2008). 52 Current Developments in Biotechnology and Bioengineering 4.2.1 Ethanol Ethanol is a well-studied metabolite produced during the fermentation process of zygomycetes (Mohammadi et al., 2012; Omidvar et al., 2016). Theoretically, 0.51 kg ethanol can be produced per kg of sugar monomer. However, several factors such as cultivation conditions such as nutrient concentration, oxygen supply, temperature and pH, and genetic stability influence the ethanol yield (Mohammadi et al., 2013; Aditiya et al., 2016). In a screening experiment, Millati et al. (2005) found ethanol yields in the range of 0.37–0.43 g/g glucose from different zygomycete species while Wikandari et al. (2012) reported yields in the range of 0.26–0.41 g/g. Anaerobic fermentation of Mucor indicus resulted in yields up to 0.46 g/g (Sues et al., 2005). These values represent the types of yields that can be expected, however, far from all zygomycetes are able to produce ethanol. 4.2.2 Lactic acid Lactic acid (C3H6O3) is the most abundant organic acid in nature and almost 2,000,000 metric tons were estimated from industrial production in 2020, of which ca 90% is derived from fermentation processes (Yuwa-amornpitak and Chookietwatana, 2018). L(+)-lactic acid and D( )-lactic acid are two isomers of lactic acid. L(+)-lactic acid is preferred in food, pharmaceutical, textile, cosmetic, and chemical industries (Ferreira et al., 2013). A significant amount of research has been performed about using Rhizopus strains to produce L-lactic acid instead of lactic acid bacteria (Zhang et al., 2007; Ahmad et al., 2020; Ferreira et al., 2013). Research on lactic acid production by zygomycetes is still an active research area, and some of the later findings are summarized in Table 2. Very briefly, the research seems to be most focused on using different species and strain to produce L-lactic acid from different sources, which most often are wastes or residues of some kind. Table 2 Recent investigations on the production of L-lactic acid by Rhizopus strains. Microorganism Medium/substrate Yield (g/g) or concentration (g/L) References Rhizopus microsporus (DMKU 33) Rhizopus oryzae (3.819) Rhizopus oryzae (NLX-M-1) Rhizopus oryzae (MTCC5384) Rhizopus microsporus (LTH23) Rhizopus oryzae (NRRL 395) Rhizopus oryzae (NRRL-395) Liquefied cassava starch 0.93 (g/g) Trakarnpaiboon et al. (2017) Sophora flavescens residues 47 (g/L) Ma et al. (2020) Xylo-oligosaccharides (XOS) manufacturing waste Paper sludge (PS) 0.60 (g/g) Zhang et al. (2015) 27 (g/L) Dhandapani et al. (2019) Cabbage glycerol media 4.0 (g/L) Solid pineapple waste (SPW) 0.10 (g/g) Yuwa-amornpitak and Chookietwatana (2018) Zain et al. (2021) Wheat wastewater 5.7 (g/L) € çeri et al. (2021) Go Chapter 2 • Fungal biotechnology 53 Specifically worth mentioning is that lactic acid fermentation by zygomycetes have carried out in 5 m3 bioreactor scale (Matsumoto and Furuta, 2018), and should thus be considered to be in the pilot scale. 4.2.3 Fumaric acid Fumaric acid (C4H4O4) is a non-toxic naturally occurring organic acid that can be produced chemically from maleic anhydride (produced from butane). However, the price of maleic anhydride as a petroleum derivative is increasing due to the rising petroleum prices (Sebastian et al., 2019a) and the environmental side effects of using fossil-based resources should be considered as well. However, fumaric acid is among the top 10 chemicals that could be produced by fermentation on industrial scale (Roa Engel et al., 2008). Fumaric acid is used in the food and pharmaceutical sectors, and has potential in the production of biodegradable polymers (Ferreira et al., 2013). Fungi in general could be considered for fumaric acid production (Sebastian et al., 2019a). In particular, the zygomycete Rhizopus oryzae has potential as a main producer as it has small nutritional demands for growth and fumaric acid production (Xin Li, 2020). With glucose as a carbon source, yields up to 0.70 g/g has been reported (Ferreira et al., 2013). Conversion of lignocellulosic biomasses has also been investigated for fumaric acid production, but the studies are infrequent and the yields are considerably lower with reported values of 0.35 and 0.43 g/g (Xu et al., 2010; Deng and Aita, 2018). A promising characteristic is that fumaric acid production has been reported to be successful without pH control, with pH values as low as 3.6 (Roa Engel et al., 2011). 4.3 Basidiomycetes Basidomycetes, or more specifically those classified as white-rot fungi, have been found to produce ethanol. However, the number of publications are rather limited, probably due to the general slow growth of basidiomycetes. 4.3.1 Ethanol One of the fungi worth mentioning is Trametes versicolor since it is able to directly convert hexose sugars, xylose, and untreated lignocellulosic biomass to ethanol. It can thus be suitable for fermentation of xylan-containing lignocellulosic biomass (Okamoto et al., 2014). Flammulina velutipes is a well-known basidiomycete mushroom in the food industry. It is also able to produce ethanol from glucose, mannose, sucrose, fructose, maltose, and cellobiose, but not galactose and pentose sugars. Furthermore, the fermentation time is long (6 days or even more) (Salehi Jouzani and Taherzadeh, 2015). Interestingly, ethanol yields of up to 0.36 g/g cellulose has been achieved from bagasse for this fungus (Maehara et al., 2013), which is quite high. Phlebia sp. MG 60, originally isolated from driftwood, has also been considered for ethanol production. It is interesting in the regard that it is able to utilize xylose, and convert lignocellulosic material with rather high yields, reaching 0.42 g/g from kraft pulp (Kamei et al., 2012, 2014; Khuong et al., 2014a,b). 54 Current Developments in Biotechnology and Bioengineering Other basidiomycetes that have been investigated include Peniophora cinerea, Trametes suaveolens, Lenzites betulinus (Okamoto et al., 2014; Im et al., 2016), but no noteworthy properties were discovered. Interestingly, Pleurotus ostreatus and Agaricus blazei, both normally used for mushroom production, have been found to be able to produce wines with 12.2% and 8% ethanol, respectively (Okamura et al., 2001). This is far higher than what is normally achieved by filamentous fungi that quite often seems to be limited by their ethanol tolerance. 4.4 Pigments and enzymes Filamentous fungi are known to produce various secondary metabolites that can be used for biotechnological purposes such as food, drug development, and cosmetics. Pigments including carotenoids, melanins, and flavins are among the important secondary metabolites from fungi due to their biological properties, e.g., antibacterial and antifungal (Nirlane da Costa Souza et al., 2016). Among the ascomycetes, Monascus spp. have been important sources for pigment production (Ferreira et al., 2016). The major pigments consist of yellow (monascin and ankaflavin), orange (monascorubrin and rubropunctatin), and red componds (monascorubramine and rubropunctamine). Other examples of pigments, e.g., yellow in the form of emodin and physcion, and red in the form of erythroglaucin, rubrocristin, and catenarin, are produced by some strains of Aspergillus spp. (Caro et al., 2015). As previously mentioned, filamentous fungi produce enzymes as well (Karimi et al., 2018). Zygomycetes fungi can produce a great diversity of hydrolytic enzymes including amylases, cellulases, xylanases, proteases, and lipases (Ferreira et al., 2013). Among the ascomycetes especially Aspergillus and Trichoderma spp. are very well known for industrial production of enzymes, including cellulases (Ferreira et al., 2016). Basidiomycetes are especially interesting for their ability to produce enzymes that degrade lignin and a potential source of cellulases (Kozhevnikova et al., 2017). 5. Fungal biomass Fungal biomass is rich in proteins and has a good composition of amino acids. The lipid content, with different fatty acids, and vitamin content is also worth mentioning. This makes fungal biomass a suitable source for animal feed (Karimi et al., 2018) and potentially human food. Furthermore, whenever the main product is ethanol, organic acid, or enzymes, filamentous biomass is also produced, which has a good potential to act as an addition valuable by-product. Compared with yeast, filamentous fungi also have the advantage that it can easily be separated from a liquid medium, only requiring a sieve like separator (Ferreira et al., 2016). 5.1 Ascomycetes 5.1.1 Human food Filamentous fungi, especially ascomycetes, are currently used in the production of human food. Especially Neurospora, Aspergillus, Monascus, and Fusarium are important in food Chapter 2 • Fungal biotechnology 55 applications containing species that are classified as Generally Recognized as Safe (GRAS) (Ferreira et al., 2016). Oncom, a traditional fermented food in Indonesia, is produced using Neurospora intermedia and is known to have a relatively high protein content (Shurtleff and Aoyagi, 1985). In Europe, Fusarium venenatum was investigated as a potential source to easily produce a palatable microbial source of protein from glucose or starch-based media for human consumption by the company Rank Hovis McDougall (O’Donnell et al., 1998; Wiebe, 2002). It resulted in Fusarium venenatum being commercially produced from a synthetic medium based on glucose, ammonium, and biotin, and is sold under the trade name “Quorn” (Wiebe, 2004). Another dish is red rice, which is produced using Monascus purpureus, and has known anti-hypertensive effects (Seraman et al., 2010). Aspergillus oryzae is well known in the production of koji among other things (Shurtleff and Aoyagi, 2012). Ascomycetes are also being investigated for more novel food products. For instance, Aspergillus oryzae, Fusarium venenatum, Monascus purpureus, and Neurospora intermedia have been investigated to convert by-products from the pea industry (Souza Filho et al., 2018). Neurospora intermedia has also been considered for conversion of leftover bread (Gmoser et al., 2020). These fungi grow fast and produce high amounts of protein-rich biomass. The total protein content increased from 16.5% to 21.1% in the final product. Minerals (Cu, Fe, Zn) vitamin E, and vitamin D2 all increased in concentration in comparison with untreated bread. The increase in mineral content can be attributed to a decrease in the total mass. One potential application of the fermented product is as a fungi burger patty, although the bitterness chewiness could still be improved. Many more examples can be found in the literature, and most seems to focus on using ascomycete strains that are already used for food production due to the safety of the final product and legal reasons. 5.1.2 Animal feed Protein rich fungal biomass seems like a good alternative to current soybean-based animal feed, especially if the fungal biomass is produced from by-product/waste stream. Other than protein content, amino acid composition, fatty acid content and composition, antioxidants, and pigments make fungal biomass a good choice for application as animal feed (Karimi et al., 2018). A major focus has been on ascomycetes, including Neurospora intermedia that has been extensively investigated to utilize by-products from bioethanol production (Ferreira et al., 2016) with the advantage that a co-product is ethanol. Aspergillus oryzae has also found research interest (Batori et al., 2015). 5.2 Zygomycetes Zygomycetes biomass contains considerable amounts of proteins, lipids, chitin, and chitosan (Ferreira et al., 2013). Zygomycetes isolated from different foods have been investigated to be considered for human consumption or animal feed (Lennartsson, 2012). Furthermore, the fungal biomass of zygomycetes can be used for heavy metal removal, 56 Current Developments in Biotechnology and Bioengineering as a source of chitosan, and superabsorbents production (Mohammadi et al., 2012; Ruholahi et al., 2016). Thus, the biomass of zygomycetes is investigated for human food, animal feed, and chitosan production. 5.2.1 Food and feed applications Commonly, zygomycetes contain about 40–50% protein and has thus become of interest for feed and food applications (Mohammadi et al., 2013; Karimi et al., 2018), since protein content is quite often crucial. The protein content of fungal biomass depends on the fungal species, harvesting, dewatering, drying methods, and cultivation medium and its composition such as nitrogen concentration (Karimi et al., 2018; Ferreira et al., 2013). Furthermore, many zygomycetes are classified as GRAS, which could reduce the amount of testing needed to be accepted for food production on large scale. One good example are the zygomycetes used in the production of tempeh, a traditional Indonesian food from fermented soybean by zygomycetes (Nout et al., 2007). The pleasant smell and taste, and easy separation of the zygomycetes biomass from the medium are other advantages of these fungi for feed and food purposes (Lennartsson, 2012). However, similar to other fungi, zygomycetes contain nucleic acids in levels that could limit their application in feed and food fields (Ferreira et al., 2013). This could be solved by treating the fungal biomass, or use the fungi for feed for animals that produce high levels of active uricase to metabolize nucleic acids without any health risk, such as salmonids (Karimi et al., 2018). Thus, it is not surprising that the research in the field has been focused on Rhizopus species related to tempeh fermentation. Examples include Rhizopus cultivation on paper pulp wastewater, e.g., spent sulfite liquor, to replace fishmeal in fish feed (Wikandari et al., 2012; Edebo, 2008), and Rhizopus cultivation on potato protein liquor (PPL), a side stream from the potato starch industry, for animal feed (Souza Filho et al., 2017). More examples can be found in the literature, but in general the research is not that well developed. 5.2.2 Chitosan The high amount of chitosan in the zygomycetes cell walls is one of the remarkable properties of these fungi with many potential applications (Ferreira et al., 2013; Abo Elsoud and El Kady, 2019). Chitosan is a linear polysaccharide containing randomly distributed glucosamine and N-acetyl glucosamine monomers. Chitosan is used in a variety of applications such as medicine, food, and chemical industry (Sathiyaseelan et al., 2017; Batista et al., 2018; Darwesh et al., 2018; Hamedi et al., 2018). Chitosan is often produced by chemical deacetylation of chitin from shellfish wastes. The zygomycetes cell wall is an alternative source of chitosan, which would require milder conditions to isolate (Mohammadi et al., 2012; Sebastian et al., 2019b). Mucor indicus is one of the zygomycetes species that have been investigated as a potential source of chitosan, since it can also produce ethanol (Mohammadi et al., 2013). The fungus is dimorphic (i.e., can grow as both yeast and filaments) and the amount of chitosan in the cell wall differ from one morphology to another. According to the research, 0.46 (g/g cell wall) of chitosan could be achieved when Mucor indicus grows Chapter 2 • Fungal biotechnology 57 as filaments, which is reduced to 0.23 (g/g cell wall) for yeast-like morphology (Mohammadi et al., 2012). Rhizopus oryzae has also been considered as a source of chitosan, as the fungus can be used for lactic and fumaric acid production (Liao et al., 2008; Liu et al., 2008). 5.3 Basidiomycetes In the case of basidiomycetes much of the focus has been on the mushrooms, which is slightly off topic and will thus only be discussed briefly. Generally, they are considered to have potential in various biotechnological applications such as production of food, dietary supplements and pharmaceutical substances (Asatiani et al., 2010). Antioxidant, immunostimulatory, antibacterial, and hypocholesterolemic properties are among the health-promoting properties of edible mushrooms (Bederska-Łojewska et al., 2017). Basidiomycetes also find potential applications due to their health benefits in other applications than direct human use. For example, many mushrooms such as Lentinula edodes, Agaricus bisporus, Agaricus blazei, Hericium caput-medusae, Pleurotus ostreatus, Pleurotus eryngii, Fomitella fraxinea, Flammulina velutipes, Ganoderma lucidum, Cordyceps inensis, and Cordyceps militaris could be used to improve poultry performance and health (Bederska-Łojewska et al., 2017). 6. Conclusions and perspectives Filamentous fungi have been used by humans for centuries and only recently have we started to fully unlock their potential. This ranges from materials, bulk chemicals, feed and food, enzymes, and pharmaceuticals (including antibiotics). Undoubtedly, there are many more potential applications that we have not even considered yet. Many fungi are also quite modest in their demands for nutrients, which makes industrial applications easier and potentially more profitable. 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Introduction Kingdom fungi constitute an exceedingly species-rich kingdom, covering almost 100,000 described and about 1.5–5.1 million undescribed species in diverse environments such as forest soil, phyllosphere, aquatic ecosystems, and soils of the pristine polar region (Jumpponen and Jones, 2009; Kagami et al., 2007; Durán et al., 2019). Fungi are important components of ecosystems. They play roles of decomposers, mutualists of plants and as parasites to various organisms (Talbot et al., 2008; Allen et al., 2003; Kohler et al., 2015). Humans utilized fungi in terms of applications in agriculture, pharmacology, the food industry, and environmental technologies (Rico-Munoz et al., 2019; Zjawiony, 2004; Cardoso and Kuyper, 2006; Wang and Chen, 2006). Thus, exploration of fungal diversity is worthy of effort and time not only for our ecosystem and ecological community but also to provide invaluable resources for various fields of applied microbiology (Table 1). Classical fungal taxonomy faced a chaotic and turbulent history. Whittaker (1969) proposed a fourth kingdom in the kingdom of life. Fungi that have been previously oscillating between kingdom Protista and Plantae were given a new place as a kingdom. The 2007 classification of kingdom Fungi was the result of collaborative research effort of dozens of mycologists, scientists, and individual projects working globally on fungal taxonomy. Formal recognition of fungal nomenclature including yeasts is governed by the International Code of Botanical Nomenclature (ICBN) as adopted by each International Botanical Congress. Assembling the Fungal Tree of Life (AFTOL) project was funded by the US National Science Foundation. The 2007 phylogenetic classification divided the fungi kingdom into 7 phyla, 10 subphyla, 35 classes, 12 subclasses, and 129 orders. Phylogenetic relationships (relation based on ancestry) are inferred from fossils, comparative morphology, and biochemistry. Constructing phylogenetic trees (evolutionary trees or cladograms) requires molecular data coupled to these traditional forms of data. To be formally recognized by taxonomists, an organism must be described in accordance with internationally accepted rules and given a Latin binomial. The presence of dual reproductive stages of fungal propagation, i.e., sexual (teleomorph stage) and asexual (anamorphs), has been used since century for nomenclature. Morphological details associated with sexual sporulation proved useful in higher fungal classification. The type of fruiting body (basidioma in basidiomycetes, ascoma in Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00017-X Copyright © 2023 Elsevier Inc. All rights reserved. 67 68 Current Developments in Biotechnology and Bioengineering Table 1 Some notable production strains. Fungi Division Products Rhizopus oligosporus Mucoromycota Aspergillus oryzae Ascomycota Penicillium camemberti Penicillium roqueforti Aspergillus oryzae, Saccharomyces cerevisiae Ascomycota Saccharomyces cerevisiae Ascomycota Ghorai et al. (2009) Ghorai et al. (2009) Ghorai et al. (2009) Soy sauce Ghorai et al. (2009) Rice wine (sake), Chinese liquor, Ghorai et al. animal feed (2009) Edible mushrooms, pharmaceutical, Ghorai et al. and nutraceutical by-products (2009) Vitamins (β-carotene) Cairns et al. (2019) Enzymes, organic acids Cairns et al. (2019) β-Lactam antibiotics Cairns et al. (2019) Enzymes Cairns et al. (2019) Enzymes Cairns et al. (2019) β-Lactam antibiotics, enzymes Cairns et al. (2019) Mycoprotein Cairns et al. (2019) Riboflavin Azizan et al. (2016) Riboflavin Azizan et al. (2016) Composite materials Cairns et al. (2019) Composite material, imitation Cairns et al. leather (2019) Textiles Cairns et al. (2019) Itaconic acid Cairns et al. (2019) Food, lovastatin Kala et al. (2020) Food, lovastatin Kala et al. (2020) Food, lovastatin Kala et al. (2020) Food, lovastatin Kala et al. (2020) Enzymes, food, and feed Ghorai et al. (2009) Ascomycota Agaricus bisporus, Volvariella volvacea, Pleurotus Basidiomycota ostreatus, Lentinula edodes and many more Blakeslea trispora Zygomycota Aspergillus niger Ascomycota Acremonium chrysogenum Ascomycota Thermotelomyces thermophila Ascomycota Tricoderma reesei Ascomycota Penicillium chrysogenum Ascomycota Fusarium venenatum Ascomycota Candida famata Ascomycota Ashbya gossyppi Ascomycota Pleurotus ostreatus Basidiomycota Ganoderma lucidum Basidiomycota Schizophyllum commune Basidiomycota Utilago maydis Basidiomycota Agaricus bisporus Basidiomycota Cantharellus cibarius Basidiomycota Imleria badia Basidiomycota Lentinula edodes Basidiomycota Termitomyces clypeatus Basidiomycota Tempeh (Indonesian fermented legume seeds) Miso, koji, sake, (fermented rice, beverages), enzymes Camembert and Roquefort cheese References Chapter 3 • Fungal biology 69 ascomycetes) or the type of ascus (a microscopic, unicellular, frequently globose, saccate, or cylindrical structure) is prominent sources for classification. Variations in ascus structure helps in the classification of these fungi, especially at the level of family and above. Earlier Fungi were usually classified in four divisions: the Chytridiomycota (chytrids), Zygomycota (bread molds), Ascomycota (yeasts and sac fungi), and the Basidiomycota (club fungi). 2. The fungal world “I thought a forest was made up entirely of trees, but now I know that the foundation lies below ground, in the fungi” … Derrick Jensen Fungi have shorter generation time, larger population size, more active haploid state, no embryonic state, higher chromosome plasticity, higher selective pressure for a reduced genome, and higher evolutionary rate compared to plants and animals (Naranjo-Ortiz and Gabaldon, 2020). 2.1 Growth of fungi Hyphal structures in fungi show polarized growth. Some unicellular fungi like Saccharomyces cerevisiae also demonstrate polarized growth but they do not produce hyphal structures. The components responsible for fungal polarized growth are well conserved. € rper which is composed of Hyphal structures contain one organelle called Spitzenko numerous vesicles originating from Golgi apparatus (Diepeveen et al., 2018). These vesicles are full of enzymes, lipids, and polysaccharides needed for the synthesis of membranes and cell wall. Those vesicles are coordinated via actin and microtubule cytoskeleton. Cell growth is associated with coordinated cycles or oscillations of exocytosis involving polarity markers, exocyst complex, and SNARE proteins (Takeshita, 2016). Dikarya groups are specialized to carry this machinery and studies reveal the conservative nature of this apparatus across all Dikarya. In Zygomycetes fungi, apical vesicle credent € rper (Fisher and Roberson, are less organized aggregates of vesicles compared to Spitzenko 2016). Hyphal structures are septate or nonseptate. Septa formation requires chitin rings and chitin synthase activity. True fungal network formation requires the ability to fuse the hyphal tips or anastomosis. This involves complex cell recognition mechanism while preventing fusion of genetically dissimilar hyphae during vegetative hyphal growth (NaranjoOrtiz and Gabaldon, 2020). Filamentous fungi generally form branches at nearly right angles to the parent hyphae (Carlile et al., 2001). Fungal growth is mostly determined by hyphal growth unit which is the total length of the mycelium divided by the total number of tips. Sometimes, fungal growth pattern is examined based on the mean hyphal segment length of the fungal network. Direction of fungal growth is indicated by the growth angle which can be defined as the difference between the angle of a segment and the angle of its preceding segment 70 Current Developments in Biotechnology and Bioengineering (Riquelme et al., 1998). Larger values for the mean growth angle are indicative of a complex network full of branches whereas a low value indicates that there is no or minimum change in the growth direction. Five distinguished phases of growth namely the lag, the first transition period, the log, the second transition, and the stationary phase were identified in Rhizopus microspores, Aspergillus fumigatus, and Scedosporium prolificans with varying growth rates (Meletiadis et al., 2001). Transition phases were long and log phases showed least variability with respect to growth rate among the strains. Although entry to the stationary phase or deceleration phase is critical considering its role in the production of industrially important secondary metabolites, this phase is least studied. In some cases, deviation from normal growth curve like hidden diauxic growth may happen due to reuse of released secondary metabolites like polyols, gluconate, or organic acids, etc., by the fungi (Schmitz et al., 2013). Currently, automated methods for fungal growth measurement are becoming popular. Typical sigmoidal growth curve was observed for economically important fungi Coniophora puteana and Rhizoctonia solani depending on mycelial area and number of tips under 16 different environmental conditions (De Ligne et al., 2019). Images of growing fungi at certain time interval were processed and analyzed via algorithms to study spatiotemporal growth dynamics. Environmental effects were measured depending on the Granger causality test, the Mann-Whitney test, and dynamic time warping. Fungal feature tracker, a modern tool, is developed to characterize morphology and growth of filamentous fungi quantitatively (Vidal-Diez de Ulzurrun et al., 2019). Modifications of polar growth and morphology are targeted for the industrial production of secondary metabolites from engineered filamentous fungi (Cairns et al., 2019). Different signaling pathways like the cAMP/PKA signaling pathways, the calcium/calcineurin signaling pathway and RAS signaling, etc., are pivotal in controlling the production rate. Several filamentous fungi are known to produce biofilms when grown on aqueous environment. Aspergillus sp., Alternaria sp., Botrytis sp., Cladosporium sp., and Penicillium sp. are capable of forming biofilms in environment as well as in a laboratory setup (Siqueira and Lima, 2013). Biomass or monolayer formation and exopolysaccharide production was directly co-related. Biofilm production was observed to follow five sequential stages: (i) propagule adsorption, (ii) active attachment to a surface, (iii) microcolony formation I, (iv) microcolony formation II, and (v) dispersal or planktonic phase. 2.2 Nutrition and transport in fungi Although today’s fungi originated from a flagellated unicellular organism with saprophytic or parasitic life style, they represent a wide variety of unicellular to complex multicellular organisms. They behave as osmotrophs with their surrounding environment with the help of numerous secreted proteins (enzymes) and secondary metabolites. Unicellular zoosporic fungi as saprophytic or having predatory-parasitic lifestyle. Those swimming cells normally attached themselves to an available substrate and phagocytic amoeboid extensions are produced which act as feeding structures (Oberwinkler, 2017). Many members of Chapter 3 • Fungal biology 71 Chytridiomycota having polycentric rhizoid structures belong to this category. Nutrient transport in filamentous fungi as mediated via cytoplasmic currents which pass cellular components including nuclei for growing hyphal tips (Naranjo-Ortiz and Gabaldon, 2020). Cytoplasmic waves are used to transport over long distances whereas cytoskeleton-based movement is essential for short-range movements or against the main cytoplasmic flow. They also narrated fungal sensory systems which are similar to that of plants. Key cellular events like reproduction, morphogenesis, virulence, and metabolisms are regulated via light-induced gene expressions. In those cases, fungi have well-regulated wavelength-ratio sensing mechanism and circadian clock in them. Zinc-finger transcription factors named white-collar complex proteins mediate changes in the transcriptome profile. Some yeast and parasitic fungi are devoid of those white-collar proteins (Adam et al., 2018). In some fungi, opsin-like proteins are also involved in regulation of sexual cycle and pathogenesis. Perception of gravity in fungi is controlled by units called statoliths which are composed of oxalate crystals and buoyant lipid structures (Kunzler, 2018; Medina-Castellanos et al., 2018). In major fungal groups like Ascomycota, Basidiomycota, Mucoromycotina, Mortierellomycotina, and Glomeromycota; buoyancy systems are present and they are conserved across long evolutionary distances. Filamentous and zoosporic fungi are found to detect electric fields and respond to it in Ca2+-dependent manner. Multicellular vegetative structures are called rhizomorphs in some fungi. These are vegetative hypha having thread-like aggregates which helps in nutrient distribution over large distances (Krizsan et al., 2019). 2.3 Fungal reproduction Reproduction is the fundamental characteristic of all life forms. It involves formation of daughter or new cells from parent. Fungi adopt different modes for reproduction like vegetative, asexual, parasexual, and sexual. Filamentous fungi can reproduce via sexual or asexual mode. Although sexual reproduction renders many advantages like increased genetic variation, more durable and resistant sexual fruiting bodies, etc., almost one-fifth of all fungi are known to reproduce asexually. Fungal sexual reproduction can be summarized in three steps (Lee et al., 2010). Compatible haploid cells fuse, i.e., plasmogamy, then nuclei of both the haploid cells fuse, i.e., karyogamy, and finally fused diploid cell produces four haploid spores via meiosis. Nuclear fusion initiates just after the cell fusion in the two basal fungal lineages, i.e., Chytridiomycetes and Zygomycetes as well as in some ascomycetes whereas the event of nuclear fusion is delayed after cell fusion in other ascomycetes and in basidiomycetes. The later groups have a stable dikaryotic hyphae stage (especially in basidiomyteces) throughout most of their hyphal growth stage and then nuclear fusion occurs leading to meiosis to finally produce haploid recombinant progeny. Size of the reproductive unit is defined as “subpopulations of nuclei that propagate together as spores, and function as reproductive individuals” and is dynamic and dependent on the environment (Ma et al., 2016). 72 Current Developments in Biotechnology and Bioengineering 2.3.1 Unicellular yeast form Vegetative mode of reproduction involves part of vegetative cells or somatic portion of the fungal hyphae/thallus. Higher fungi can accommodate two forms in their life—unicellular monokaryotic yeast form and filamentous dikaryotic form. Fungi adopting one of these forms are called monomorphic, while those taking up both forms are dimorphic. Yeast form is generated from the budding of a basidiospore and it reproduces asexually by budding, fission, or production of forcefully ejected ballistoconidia (Flegel, 1977). Various modes of vegetative reproduction in yeast are observed like multilateral budding, bipolar budding, unipolar budding, monopolar budding, binary fission, bud fission, budding from stalks, ballistoconidiogenesis, and pseudomycelia (Walker, 1998). Filamentous forms, as the terminology goes, consist of long branching tubular cells known as hyphae which may be septate via compartments or aseptate and grow at the apex (Morrow and Fraser, 2009). Parasexual cycle in wild mitosporic fungi occur through deoxyribonucleic acid (DNA) recombination. It may involve fusion of two haploid mycelia forming heterokaryotic mycelium with more than one genotype in uncontrolled proportions (Wallen and Perlin, 2018). In Candida albicans some DNA repair is performed through recombination in this life cycle. Instead of regular meiosis, tetraploid cells generated through mating of two (diploid) mating types go through concerted chromosome loss (CCL). This is believed to involve chromosome nondisjunction events during mitosis and the aneuploid cells show a wide variety of ploidies including haploid cells. One meiosis-specific recombinase Spo11 is found to be involved in the process. In C. albicans, homothallic mating is also observed (Bennett and Turgeon, 2016). A cell releases pheromone for both mating types which enable the cell to be self-fertile or inducing same sex mating. Unicellular ascomycetes such as yeast grow either as haploid or diploid cells but diploidy is predominant in wild (Lee et al., 2010). Haploid mating types can be either a or α. Mating type of the cell is decided by the genetic material at the mating-type locus. A single MAT locus is the deciding factor of mating-type identity. In case of unicellular ascomycetes like yeast Pichia pastoris; mating type switching occurs in the cell by flipflop inversion mechanism (Wallen and Perlin, 2018). Meiosis in diploid cells is triggered by DNA damage in nutrient limiting condition as through meiosis genetic materials got repaired. In pathogenic ascomycetes, no sexual reproduction is usually observed inside the host and in the wild as well, although they produce genetic components used for sexual reproduction, whereas pathogenic and saprophytic basidiomycetes reproduce sexually. Basidiomycetes yeast can produce conjugation tubes in presence of compatible mating type and eventually fuse to produce a dikaryotic hypha. They also undergo a complex form of mitosis via formation of clamp cells. Meiosis involves nuclear fusion with formation of tetrad of haploid basidiospores in basidium (Casselton and Olesnicky, 1998). Pathogenicity and parasitism are critically linked to the changeable forms of fungi and their modes of reproduction (Madhani and Fink, 1998; Nadal et al., 2008). Chapter 3 • Fungal biology 73 2.3.2 Chytridiomycota They reproduce via asexual mode through the flagellated zoospores from sporangia (James et al., 2006a). Those spores may be mono or polyflagellate and thallus developed from them may form either monocentric or one sporangium from single spore or polycentric or many sporangia from a single zoospore. Sporangia are thin walled but resting spores that germinate later on after a dormant period, are thick-walled structures. Zoosporangia are always produced asexually but resting spores may be formed by sexually or asexually. Meiosis may happen within the resting spores or during germination to form mature sporangium. Majority of the chytrids lack sexual cycle. Release of spores from sporangia is induced by the presence of haem or porphyrins release from ingested plant materials in the rumen. Those flagellated zoospores move toward soluble sugars and/or phenolic acids released from the stomata or lateral spikes of the ingested plant materials by beating of up to 8–17 flagella via chemotaxis. After attachment to the plant material, zoospores shed the flagella and form cyst. Germination from the cyst is mediated by formation of a germ tube from the opposite polar end of flagellar origin. In monocentric fungus, nucleus remains within the cyst which becomes larger to form zoosporangium, so the rhizoids are anucleate. In case of polycentric fungi, nuclei migrates into the rhizoidal portion and multiple sporangia are formed on each thallus. Prominent exceptions to this are several genera like Cyllamyces, etc. (Ozkose et al., 2001). The rhizoid structure penetrates the plant material and takes up nutrients from there for the development and maturation of multinucleate sporangia. These again produce a few (1–2) to many (50–80) zoospores. Induction of zoospore differentiation in the mature sporangium eventually leads to release of the spores after dissolution of the sporangial wall. These fungi may go through aerotolerant structure in their life cycle (Griffith et al., 2009). Sexual reproduction is unusual in case of chytrids. Sometimes rhizoids fuse to transfer nuclei between thalli. Alternately, some swimming mitospores behave as gametes and when two of them fuse, a single cell with two nuclei and two flagella is produced through plasmogamy. When the cell expands a thick-walled meiosporangium is formed which contains meiospores. Meiospores are produced through meiosis after fusion of nuclei. 2.3.3 Zygomycota One mode of asexual reproduction is through mitospores. The method is fast and economical although lack genetic variability in the offspring. Most common form of asexual reproduction involves formation of spores from the tip of the hyphae one by one. The spores are called conidia and corresponding hyphae is called as conidiophores. Aerial reproductive hypha is called sporangiophore. They swell at the tip to form a mitosporangium and the contents divide to form many mitospores inside the shelter. When the sporangium matures, it breaks open, as a result spores are freely released into the air and ready to get deposited in available surfaces. Sexual reproduction in zygomycetes involving formation of zygosporangium generated from the fusion of parental hyphae at the tip and subsequent hardening of the wall 74 Current Developments in Biotechnology and Bioengineering and production of a zygospore (Lee et al., 2010). Parental mycelia must be compatible in nature, if one is plus mating type, the other one must be minus mating type. Specific pheromone trisporic acid is required for sexual development which is produced from β-carotene in each mating type. Both parental hyphae meet at the tips. Behind the tips, cross walls are formed leading to the formation of structure gametangia which behaves as gametes. The walls dissolve and gametangia fuse via plasmogamy. The fusion cell is called zygosporangium where two nuclei form parental hyphae fuse and generate 2n zygote nuclei. The wall structure of the zygosporangium becomes hard and thick gradually to produce zygospore. 1n recombinant nuclei are generated from 2n nuclei through meiosis and germ sporangium is formed from swelling tip of hyphae grown from zygospore. From the sporangium, 1n meiospores are released into the air and they eventually germinate into hyphal structure when favorable environment is available. 2.3.4 Glomeromycota These are arbuscular mycorrhizae fungi. They produce asexual mitospores. Some species generate mitospores in the mycelial part that reaches to the soil while others produce mitospores inside the root structure between the cells or in the space enclosed by the walls of the host plant cells. Unlike other spores, mitospores are not made inside the sporangia (sac-like structure) but instead are actually the swollen tip of hyphae with cytoplasm and stored food. Their size may be as large as millimeter is diameter and they are distributed nearby by some sort of dispersal mechanism. 2.3.5 Ascomycota Asexual reproduction in the phylum Ascomycota begins with spores (n) (Bennett and Turgeon, 2016). When they get nutrient and favorable conditions, they germinate into mycelia. Hyphal tips form asexual conidia and conidia formation varies in different fungi. Some conidia are like blowing bubbles from a pipe while in other fungi conidiophores are split into short segments. Sexual reproduction cycle in Ascomycota requires two compatible mycelia, plus and minus mating types. Female structure, ascogonium, is larger compare to the male structure, antheridium. During the fusion process or plasmogamy, nuclei move from antheridium to the ascogonium. After fertilization, n + n hyphae generate from fertilized ascogonium and one fruiting structure called ascoma is formed via weaving of the 1n hyphae and n + n hyphae of the ascogonial parent. Mainly three different types of ascoma are found. Ascoma structure varies in different fungi. On one surface of ascoma many n + n hyphae make meosporangia or asci. Ascus generating hypha, ascogenous, bends its tip while growing and makes a hook like structure called crozier which contains nuclei (n + n). Mitotic division of the two nuclei and walls lead to the formation of three cells from the crozier. The penultimate cell containing two nuclei of opposite types is the young ascus. 2n zygote nucleus is formed within the ascus through karyogamy. The cell at the tip of the crozier while bent fuses with the stalk of the hyphae to generate n + n state. In this way, more asci are generated from the same Chapter 3 • Fungal biology 75 hyphae. Then, the zygote nuclei form four recombinant 1n nuclei via meiosis followed by mitotic division ultimately resulting eight meiospores in the cytoplasm. The spores are called ascospores and their release from ascus varies from fungus to fungus. They may be released passively or directly shot from the ascus as one spore at a time or all at once in a sticky mass. 2.3.6 Basidiomycota Basidiomycota fungi reproduce mainly via sexual mode. Asexual spores are also generated but lesser in extent. Culinary delicacy mushrooms are actually sexual fruiting bodies or basidiomata (basidioma in singular). Mating occurs when haploid meiospore in form of mycelium (1n) encounters one compatible 1n mycelium (like one plus and one minus or vice versa) (Coelho et al., 2017). Through plasmogamy, the tips of both the hyphae fuse and the fusion cell bears one nuclei from each parent. As the mycelium of basidiomycetes are septed in nature and in many cases, nuclei can pass through septa to other compartments and divide mitotically, so haploid mycelia are converted to single n + n mycelium. Fruiting bodies or basidiomata are formed in many numbers as fusion cell makes more branches of n + n hyphae. In mushrooms, dikaryotic hyphal development to form basiomata or fruiting bodies through signal exchange is dependent on the hyphal location in the basidiomata. Some dikaryotic hyphae make a stalk, some make protective cap, some produce gills, or spore producing part of the basidioma. Spores are generated from the tips of n + n hyphae present all over the surface of each gill. Swelling of basidium or the tipmost cell of all these hyphae results in 2n zygote nucleus by fusion of the paired nuclei through karyogamy. Four 1n nuclei are formed from the 2n zygote via meiosis. Spores are created through the pressure in the basidium as the wall of the basidium near each 1n nucleus generates a thin stalk or sterigma and the tip of the sterigma grows bigger into a spore. Through the pressure cytoplasm enters into sterigma along with a nucleus. Finally, four basidiospores with different genotypes are produced and spores are released after spore walls are hardened. Dikaryotic stage of life is much longer in basidiomycetes compare to ascomycetes. In mushrooms, dikaryons serve as both feeding mycelium as well as to produce fruiting body but in Ascomycetes dikaryons are short lived and produce fruiting bodies only. So, mushrooms can generate basidiomata many times in its life. Mushrooms maintain the dikaryotic stage by forming: (1) overlapping mitotic spindles, (2) clamp connection, and (3) perforated septum called dolipore septum. Spores are released from sterigma as ballistospores and drift downward to the open air. Two genetic MAT loci determine the fate of mating type in basidiomycota. One encodes for tightly linked pheromone and pheromone receptor P/R locus and the other encodes for HD locus or homeodomain-type transcription factors which decides viability after syngamy. Tetrapolar breeding system is generated when four mating types are produced through meiosis from two unlinked MAT loci. In some, a single MAT locus controls a bipolar system where both the P/R and HD loci are linked or one has lost its function in type determination. 76 Current Developments in Biotechnology and Bioengineering 2.4 Metabolic and genetic complexity in fungi Vast array of enzymes released from fungi enable them to take advantage of using various substrates. Gene duplication, gene exchange (horizontal transfer), and gene loss all contribute to metabolic diversity of fungi (Rokas et al., 2018). Numerous metabolites also help them while interacting with the other members of the ecosystem. They play active roles in cell signaling, pigmentation, osmotic protection, and preventing invaders as toxins, etc. Secondary metabolites are grouped mainly as small nuclear-encoded peptides, nonribosomal peptides, polyketides, terpenoids, and derivatives obtained from the shikimate pathway. Metabolically related as well as nonrelated clustered genes in fungi are diverse and abundant (Marcet-Houben and Gabaldon, 2019). Several regulatory molecules are involved in coordination of the expression of those clustered genes (Akhberdi et al., 2018). Most studied global regulator, the velvet complex proteins, regulate secondary metabolic pathways in Pezizomycotina although they are detected across all fungal groups. Those proteins have significant roles in sexual development of Ascomycota and Basidiomycota. However, S. cerevisiae, C. albicans, and other basidiomycetes leading biotrophic lifestyle are devoid of velvet complex whereas this is present in Yarrowia lipolytica. External factors like nutrient availability, light, pH, or injury, etc., and internal factors like cell type or sexual cycle, etc., regulate the production of metabolites (Keller, 2018). Anaerobic fungi from phylum Neocallimastigomycota living in the midgut or hindgut of gastrointestinal tract of mammalian herbivores, adapt to the anaerobic environment in various ways. They are devoid of mitochondria, cytochromes, and other biochemical features of the oxidative phosphorylation pathway (Youssef et al., 2013). They have special organelles named hydrogenosomes which help in generating energy via metabolism of glucose. These organelles are having similar features to that of mitochondria and believed to be coming from them as well (Muller et al., 2012). Hydrogenosomes contain hydrogenase which produces H2, CO2, formate, and acetate as waste product of metabolism. Those anaerobes produce lactate and ethanol along with the previous products from plant polysaccharides in the gastrointestinal tract of the herbivores through degradation and fermentation. In response to damage, fungi close their septa in order to reduce loss of cytoplasmic content. This also induces promotion of branching and sporulation along with other coordinated events like production of toxic metabolic compounds (Kunzler, 2018; MedinaCastellanos et al., 2018). Regular signaling pathways of eukaryotes like reactive oxygen species (ROS) play several significant roles in fungi. In addition to injury-signaling pathways, chemical mediators against biological intruders, ROS also signals in cell differentiation. ATP in extracellular space signals to leakage of cell. Oxypilin signaling and action potential-like electrical signals travelling across long distance are indicative of damage in fungi. Oxypilins coordinate a number of events in fungi like secondary metabolism, pathogenesis, the sex cycle, morphological switches, and defense against grazing. Complex multicellularity is observed exclusively in the fruiting body bearing fungi like Ascomycetes and Basidiomycetes. Fruiting body development involves overexpression of Chapter 3 • Fungal biology 77 many genes related to cell wall remodeling, DNA synthesis, ribosomes, and lipid metabolism directly or indirectly (Krizsan et al., 2019). Metabolic pathways are nowadays analyzed on the basis of reliable groups of orthologous proteins and mapping those groups on to the metabolic pathways described in KEGG and MetaCyc (Grossetete et al., 2010). Accurate functional annotation of industrially well-exploited fungal genomes like Saccharomyces cerevisiae, Candida albicans, and Yarrowinia lipolytica, etc., available in different databases is essential for selection of reliable set of orthologs. Inparanoid and OrthoMCL are generally used for identifying orthologs. Recently, one user friendly tool FUNGIpath, freely available in http://www.fungipath.u-psud.fr, is found to be more useful in searching orthologs, exploring metabolic pathways or a specific step in a pathway or a complete pathway compared to other databases like Swiss-Prot or KEGG. Fungi living with bacterial endosymbionts like Rhizopus microsporus (Mucoromycotina) and its endobacteria Burkholderia gave more insights to metabolic pathways during their mutually beneficial association (Lastovetsky et al., 2016). In presence of the symbiont, expression of lipid metabolic genes was changed with increase in the levels of triacylglycerol and phosphatidylethanolamine in the host. This upregulation was found to be associated with diacylglycerol kinase activity because its inhibition altered the fungal lipid profile resulting host-microbe interaction to antagonism instead of mutualism. So, the mutual interaction of the fungi with its endosymbionts had significant impact on the metabolic pathways. 2.5 Genome Zoosporic fungi have usually smaller genomes whereas anaerobic Neocallimastigomycotina and some Chytridiomycetes have genome size comparable to or larger than mushrooms (Oberwinkler, 2017). Some small genome chytrids are parasitic in nature whereas its saprophytic ancestor has larger genome which explains that reduction is somehow linked to its adaptation to the new lifestyle. Some Ascomycota and Basidiomycota members are also unicellular. In Basidiomycota, thallus reduction in primarily biotrophic parasites generated those unicellular forms which have high amount of compacted genome with reduced signaling pathways, secondary metabolic pathways and structural components. Polyketide synthetases and nonribosomal peptide synthetases play critical role in conidiation in Aspergillus and other filamentous Ascomycota but these clusters are absent or reduced in yeast and some biotrophic pathogens of plant (Riquelme et al., 2018). Various forms of genome may exist in fungal cell. Many yeasts, e.g., Saccharomyces cerevisiae exist independently as hybrids. Hybrids are defined as “fungal lineages that have emerged from mating between two lineages whose disparity exceeds that of those typically found across the most distance strains of well recognized species” (Peter et al., 2018). These hybrids are very common in industry setup (Mixao and Gabaldon, 2018). They may be generated due to the adaptation to a new environment or sometimes they are related to pathogenicity as evident from several plant pathogens from Ascomycetes 78 Current Developments in Biotechnology and Bioengineering and Basidiomycetes. Heterokaryosis occurs when two or more genetically distinct nuclear populations coexist within a syncytium. Keeping the nuclear population in control and passage the nuclei to newly formed hyphal branches in the fungal network need critical coordination. In Neurospora tetrasperma heterokaryon state is stably maintained for long period of time in spite of higher growth rate and asynchronous nuclear division. Aneuploidy refers to various ploidy levels within the same genome. This phase serves as transitory, intermediate state when the system adapts to certain conditions (Anderson et al., 2017). Polyploidy or genome duplication in fungi can be either autopolyploidy where all chromosomes have the same genotype during duplication or allopolyploidy where the chromosomes are genetically different (Gerstein et al., 2015). Autopolyploidy generates larger cell which is advantageous in certain situation like reducing the possibility of phagocytic predation. For instance, polyploid vegetative titan cells of Cryptococcus are resistant to vertebrate immune system. In halotolerant, black yeast Hortaea werneckii duplication of whole genome was responsible for the expansion of cationic transporters which were necessary for survival of the fungi in high salinity (Sinha et al., 2017). Closely 4940 core genes (present in all analyzed genomes) were estimated from in 7800 pangenome pool (complete gene pool of a single species) of S. cerevisiae strains from diverse ecosystems (Peter et al., 2018). 2860 flexible genes are located in subtelomeric regions and are associated to cell-cell interactions, secondary metabolism, and stress responses. This variability among strains is clearly indicative of horizontal gene transfer (HGT) between strains in the environment. Such evidence is available from other groups of fungi like Ascomycota. HGT mechanisms in fungi are not clearly understood. Simple transporters or enzymatic pathways and secondary metabolism-related genes are usually transferred through HGT compared to highly interconnected proteins. HGT may drastically change the lifestyle of the fungi as genus Metarrhizium was found to be entomopathogenic from a grass endophyte through HGT (Zhang et al., 2019). 3. Classification of fungi AFTOL project (http://www.aftol.org/) defines the exact phylogenetic relationships of the groups in the fungal kingdom. The modern-day taxonomy, divides true fungi it into nine major lineages: (1) Opisthosporidia, (2) Chytridiomycota, (3) Neocallimastigomycota, (4) Blastocladiomycota, (5) Zoopagomycota, (6) Glomeromycota, (7) Mucoromycota, (8) Ascomycota, and (9) Basidiomycota based on their sexual reproductive structures. Together, these lineages formed a monophyletic clade, the true fungi (Tedersoo and Smith, 2017). Opisthosporidia are called “fungi imperfecti”; are key saprotrophs and parasites of plants, animals and other fungi, playing important roles in ecosystems. 3.1 Opisthosporodia Opisthosporidia are not considered true fungi because of their phylogenetic position that place them as sister to true fungi, and some of their biological peculiarities contradict with Chapter 3 • Fungal biology 79 the classical definition of fungi. One such fact is that the trophonts of Aphelida and Cryptomycota (but not Microsporidia, which are extremely specialized parasites) show amoeba like phagocytosis by engulfing the whole host cytoplasm. Opisthoporadia roofs three main lineages: Aphelidea, Rozellidea, and Microsporidia (or the ARM clade). Species in this clade are mostly intracellular parasites or parasitoids of a wide range of eukaryotes. Karpov et al. (2013) included Aphelidea as the last major lineage to join the family and created the term Opisthosporidia by combining Opisthokont and sporae, in reference to the specialized penetration apparatus of the spore (in Microsporidia) and cyst (in the two other phyla) that characteristic for all three phyla Microsporidia, Cryptomycota, and Aphelida (Karpov et al., 2013). 3.2 Aphelidea Aphelids are a group of obligate endoparasitoids (the infected host cell is consumed and killed) of various algae and diatoms. To date four genera have been described in this group: Aphelidium, Amoebaphelidium, Paraphelidium (freshwater), and Pseudaphelidium (marine environments) (Karpov et al., 2017). Their life cycle consists of a mobile cell that is either flagellated (Aphelidium, Pseudaphelidium), amoeboid (Amoebaphelidium), or both (Paraphelidium). Presence of their posteriorly uniflagellate zoospores raises controversy over the classification of aphelids. The motile zoospore may be amoeboid. The zoospore may be round or oval, with or without pseudopodia (Table 2; Letcher and Powell, 2019). With only a few formally described species, environmental sampling suggests that Aphellidea is indeed a highly diverse and cosmopolitan clade (Karpov et al., 2014). 3.3 Rozellidea Rozella harbors a genus of flagellated parasitoids of zoosporic fungi (Chytridiomycota and Blastocladiomycota), Oomycetes, and some green algae (Gleason et al., 2012). Rozella species are zoosporic biotrophic parasites of oomycetes, chytrids, and Blastocladiomycota. Rozella indulges a zoosporic infectious stage that attaches to the host cell. A. Rozellidea also includes the recently described Paramicrosporidium and Nucleophaga, which are microsporidian-like parasites of amoebozoa (Corsaro et al., 2014). Sequence data obtained from environmental samples, mark their phylogenetical relation to Rozella and have been found present in virtually all aquatic environments, comprising a very high sequence divergence. Taxonomists interpreted such distribution and divergence as the existence of a highly species-rich and ecologically meaningful hidden clade, which could be comparable in diversity to the rest of true fungi. R. allomycis grows inside the host as naked protoplasm, and reproduces through the production of ephemeral zoosporangia or chitinous, thick-walled resting sporangia. Inside the host it produces the cell wall of the zoosporangium, which when matures bursts to form numerous zoospores with a single flagellum. When these zoospores, finds a suitable host, it retracts its flagellum, develops a cell wall, and injects its cytoplasm into the host (phagocytosing). The genome of Rozella allomycis, a parasitoid of the blastoclad Allomyces was published in 2013 (James et al., 2013). 80 Current Developments in Biotechnology and Bioengineering Table 2 Morphological characters describing spore (size and shape), spore type, flagellum, cyst, and host of Aphelidiaceae. Taxon Spore size (μm), shape Flagellum (μm) Cyst Resting spore size (μm) Host Not observed Chaetophora elegans 7–13  5–6.5, ellipsoidal Scenedesmus, chlorococcus algae (green algae) Kirchneriella obesa, Ankistrodesmus (green algae) Coleochaete soluta (green alga) Desmodesmus opoliensis (green alga) Morphological characters and hosts for aphelid taxa Aphelidium: Aph. chaetophorae Aph. chlorococcorum f. chlorococcorum Aph. chlorococcorum f. majus Aph. deformans Aph. desmodesmi Aph. melosirae 2.7–3, spherical 9 1.5–2, “stiletto” pseudopodia 8 2–3, spherical, conic “stiletto” anterior pseudopodium 2–3, spherical 14 Not known 2, spherical, subspherical,  6 angular numerous thin filopodia 4  6, pleomorphic  10 6–8 Aph. tribonematis 2–3, oval, numerous filopodia Paraphelidium: Pa. letcheri 8–10 2–2.5, spherical, with a lamellipodium and subfilopodia 2–2.5, oval, a broad anterior  7 lamellipodium; a few lateral and anterior subfilopodia Pa. tribonematis Amoeboaphelidium: 2 long Am. achnanthis Am. chlorellavorum 1–2, amoeboid Am. occidentale Not known Not known 7–10 1.3–2.7, spherical, subspherical, elongate Am. protococcorum 2–4, spherical to elongate,  7 numerous pseudopodia, thin trichipodia, thick lobopodia Am. radiatum 1–3, spherical, numerous Not filopodia known Pseudaphelidium: Ps. 3  5, elongate drebesii 15 Not observed Sessile or short stalk Stalked 5  8, ellipsoidal Not 12–30 diam, round observed to oval Stalked Not observed Sessile 12–14  10, oval Melosira varians (diatom) Tribonema gayanum, Sessile or 6–7, spherical, Botridiopsis residual body short intercedens (yellowoutside stalk green algae) Sessile or 6–8, spherical, single Tribonema gayanum wall, residual body short outside stalk Tribonema gayanum Stalked 8–10, ellipsoid, two-walled, residual bodies between the two walls Unknown Unknown Achnanthes lanceolata (diatom) Sessile 3–7, spherical Chlorella spp. (green alga) Stalked unknown Scenedesmus dimorphus Sessile 4–6  5–7, oval Scenedesmus, Protococcus, chlorococcus algae Stalked Unknown Sessile Unknown Adapted from Letcher, P.M., Powell, M.J., 2019. A taxonomic summary of Aphelidiaceae. IMA Fungus 10, 4. Kirchneriella, Ankistrodesmus, chlorococcus algae Thalassiosira punctigera (marine diatom) Chapter 3 • Fungal biology 81 Rozella and Microsporidia both have horizontally acquired Rickettsia-like NTT-ATP/ ADP transporters, but metchnikovellids, Mitosporidium, and Paramicrosporidium do not. The mitochondrial genomes of Mitosporidium and Rozella lack Complex I of the oxidative phosphorylation pathway and are AT-rich. On the contrary, Paramicrosporidium possess all genes of that pathway typically found in fungi. Another instance of potentially horizontally acquired genes is thymidine kinases found in Rozella and Microsporidia, but not in Paramicrosporidium. 3.4 Microsporidia No subdivision of this phylum group is proposed yet because of the lack of well-sampled multi-gene phylogenies within the group. Microsporidia may be a sister group of the rest of the Fungi, is uncertain due to incomplete sampling. Majority of the known genera of Microsporidia infect aquatic animals with host that varied from single-celled protists to vertebrates. Susceptible hosts of microsporidia span across wide taxonomic spectrum, from lower protists to higher mammals. Primarily, Microsporidia are parasites of invertebrates and vertebrates, endosymbionts of ciliates, hyperparasites in protists. Metchnikovellids are specialized parasites of gregarines (Apicomplexa), protistan gut symbionts of many invertebrates. Hyperspora aquatica is a hyperparasite of the paramyxid, Marteilia cochillia, a serious pathogen of European cockles. Microsporidia have very reduced genomes. Some of their characteristics are typical of prokaryotic genomes viz. presence of overlapping genes, mitochondria-derived organelles called mitosomes. Simple cellular morphology, absence of mitochondria and long-branch-attraction phylogenetic artifacts caused by their parasitic nature, led to the belief that Microsporidia was early-branching eukaryotes, whose divergence preceded the acquisition of mitochondria (Corradi and Keeling, 2009). They all form a specialized resistant spore containing a coiled polar filament surrounding the nucleus or diplokaryon and the sporoplasm (its associated cytoplasmic organelles) (Fig. 1). The spore is the only microsporidial form that is extracellular and is the infective stage. The microsporidia spores range from small, oval- or pyriform-shaped, highly resistant varies in length from approximately 1–12 μm. Microsporidia lack canonical Golgi apparatus and their mitochondria have been highly reduced to mitosomes. These mitosomes are unable to generate their own ATP through oxidative phosphorylation, requiring energy to be imported from the host via nucleotide transporters. Microsporidia also lack flagella and an apparent capacity for phagocytosis. Microsporidia undergoes two different phases, one known as proliferative phase and other the sporogonic phase. The only stage of microsporidia outside the host is the infective spores. Infection by microsporidia in economically important invertebrate hosts such as silkworm, honeybee, and shrimp as well as vertebrates such as fish can cause significant economic losses. 3.5 Chytridomycota (Chytrids) Members of the zoosporic true fungi are Blastocladiomycota, Chytridiomycota, and Neocallimastigomycota. Taxonomical identification of Chytrid considerably relies on a 82 Current Developments in Biotechnology and Bioengineering FIG. 1 Parts of microsporidian spore. (A) Anchoring disk; (B) lamellar-polaroplast; (C) outer exospore; (D) inner endospore; (E) vesicular polaroplast; (F) nucleus; (G) polar tube; and (H) vacuole (posterior). Parts of a specialized resistant spore containing a coiled polar filament surrounding the nucleus or diplokaryon and the sporoplasm (its associated cytoplasmic organelles). combination of ultrastructure and molecular data because majority of Chytridiomycota species are aquatic habitants and have rarely been cultured for studying and taxonomic purposes. Most were classified as “uncultured” and thus any speculations on a chytrid’s ecological role based on the literature is shifty due to the difficulty of assigning names to environmental sequences that match poorly to the databases. Chytrids are one of the early diverging fungal lineages and is considered to be one of the most ancestral groups of fungi. Their membership in the fungal kingdom is demonstrated by the presence of chitin cell walls, a posterior whiplash flagellum, absorptive nutrition, use of glycogen as an energy storage compound, synthesis of lysine by the α-amino adipic acid (AAA) pathway, Golgi apparatus with stacked cisternae, and nuclear envelope fenestrated at poles during mitosis. General morphological features of chytrids. 1. 2. 3. 4. Motile asexual zoospores (with a single posterior flagellum). Both a kinetosome and nonfunctional centriole. Nine flagellar props and a microbody-lipid globule complex in zoosporangia. Presence of a thallus that may be holocarpic (where thallus is involved in formation of the sporangium) or eucarpic (only part of the thallus is converted into the fruiting body), monocentric, polycentric, unicellular, or filamentous. 5. Sexual reproduction accompanies zygotic meiosis, that produces motile sexual zoogametes; sexual reproduction not oogamous. 6. Asexual reproduction by zoospores bearing a single posteriorly directed flagellum. 7. Zoospores containing a kinetosome and a nonflagellated centriole. Chapter 3 • Fungal biology 83 8. Thallus monocentric or rhizomycelial polycentric. Chytrids are important pathogens of plants (e.g., Synchitrium), animals (e.g., Batrachochytrium), parasites to several groups of algae (e.g., Chytridium, Dinomyces). Chytrids are efficient decomposers of highly recalcitrant organic matter, such as pollen (e.g., Spizellomyces, Rhyzophidium), cellulose (e.g., Rhizophlyctis), arthropod exoskeletons, and fungal spores. Due to its parasitoidism, existence chytrids are unculturable resulting in very limited sequence information on zoosporic fungi, which poses challenges in obtaining a robust chytrid tree of life (Grossart et al., 2016). A well-resolved phylogenetic backbone of the fungal tree of life is required to describe how the fungal nutritional toolkit has evolved over a billion years. However, with modern day tools and technology this situation is changing. With the use of single-cell-based techniques genomic environmental sampling is steadily increasing. Single-cell genomics method was used for the first time to uncultured mycoparasitic EDF from the Cryptomycota, Chytridiomycota, and Zoopagomycota (Ahrendt et al., 2018). The Chytridiomycota have attracted the attention of mycologists due to their potential for negatively affecting phytoplankton communities, and altering phytoplankton dynamics throughout the season. Kagami et al. showed that Chytridiomycota effectively channeled organic matter and energy to higher trophic levels, a mechanism which has been termed the “mycoloop” (Kagami et al., 2014). Chytridiomycota zoospores are consumed by zooplankton, especially by Daphnia as they serve as excellent food source in lakes, due to their nutritional quality (e.g., high contents of PUFAs and cholesterols) and their high abundance (ranging from 101 to 109 spores) (Kagami et al., 2004). In this way, zoosporic fungi play an important role in shaping aquatic ecosystems by altering food web dynamics, sinking fluxes, or system stability. The parasitic chytrids that infect cyanobacteria potentially improve the nutritional quality of cyanobacteria by adding sterol (Kagami et al., 2007) or by rendering them edible by fragmenting large filaments or colonies. Another example is the parasitic chytrid Dinomyces arenysensis, known to infect dinoflagellates (some of them toxic, such as Alexandrium spp.) in coastal areas and could serve as food for marine zooplankton or the saprotrophic Chytridiomycota that decompose pollen have the potential to facilitate zooplankton growth in lakes where resource subsidies (Masclaux et al., 2013). The roles of Chytridiomycota as parasites, such as parasitism of cyanobacteria and as causative agents of the global amphibian decline, are widely studied. Chytrids present a zoosporic dispersal stage usually growing nonflagellated stage. In spite of the presence of filopodia in several chytrid groups (e.g., Batrachochytrium), true phagocytosis has never been observed. True mycelial growth is restricted to certain genera within the Monoblepharidomycetes (Dee et al., 2015). Chytridiomycota are divided into two main classes: Chytridiomycetes and Monoblepharidomycetes. 3.5.1 Chytridiomycetes ORDER: CHYTRIDIALES; EXAMPLE GENERA: CHYTRIDIUM, CHYTRIOMYCES, NOWAKOWSKIELLA. 84 Current Developments in Biotechnology and Bioengineering FIG. 2 AGF protein domains. Functional annotation of HGT genes/pfams indicated that the majority (63.9%) of events encode metabolic functions, cellular processes, and signaling represent the second most represented HGT events (11.19%), while genes involved in information storage and processing only made up 4.69% of the HGT events identified. Chytridiomycetes is by far the largest class of zoosporic fungi with 1000 described species. Phylogenetic analyses and ultrastructure study of the zoosporic stage have raised several lineages to the level of orders. Characterized by asexually reproducing by zoospores bearing a single posteriorly directed flagellum; zoospores contain a kinetosome and a nonflagellated centriole; thallus may be monocentric or rhizomycelial polycentric (Fig. 2); sexual reproduction not oogamous. Chytridiomycetes have received considerable attention owing to the deadly parasite Batrachochytrium dendrobatidis that devastated considerable populations of amphibians worldwide (Berger et al., 2005). The genome of Batrachochytrium dendrobatidis, published in 2009, represented the first sequenced chytrid. The chytrid was placed in a new genus, Batrachochytrium under Phylum Chytridiomycota, Class Chytridiomycetes, and Order Chytridiales (Longcore et al., 1999). Researchers proposed two possible hypotheses to explain how a fungus that infects the superficial epidermis has the capacity to kill amphibians (frogs) (Berger et al., 1998). Initially, the chytrid might release proteolytic enzymes or other active compounds that are absorbed through the permeable skin of the frog and secondly, this damage to skin function results in disturbance of electrolyte flux and oxygen, water imbalance which results in death (Voyles et al., 2007). Batrachochytrium dendrobatidis Batrachochytrium dendrobatidis causes a lethal epidermal infection chytridiomycosis, a disease of amphibians, that leads to mass mortality, population declines and almost extinctions. The life cycle of Batrachochytrium dendrobatidis is a simple progression from zoospore to a thallus, which produces a single zoosporangium (a container for zoospores). The contents of the zoosporangium (also known as a sporangium) cleave into new Chapter 3 • Fungal biology 85 zoospores which exit the sporangium through one or more papillae. Sexual reproduction has not been observed. Batrachochytrium dendrobatidis discharges zoospores through an inoperculate opening and exhibits monocentric or colonial growth (Longcore et al., 1999). In amphibians, sporangia infect cells in the stratum granulosum and stratum corneum in the superficial epidermis layer. B. dendrobatidis has two primary life stages: a sessile, reproductive zoosporangium and a motile, uniflagellated zoospore released from the zoosporangium. The zoospores are known to be active only for a short period of time, and can travel short distances of one to two centimeters. The zoospores are capable of chemotaxis, and can move toward a variety of molecules that are present on the amphibian surface, such as sugars, proteins, and amino acids. 3.5.2 Monoblepharidomycetes Monoblepharidomycetes are a group of freshwaters, zoosporic fungi that can present either unicellular or mycelial growth. Monoblepharidomycetes chytrids form true hyphae, and present some typical cytological characteristics, such as the presence of centrioles but € rper, which points to an independent origin of these traits from the absence of Spitzenko other fungi (Dee et al., 2015). An oogonic sexual cycle (i.e., the presence of morphologically different gametes) is common in Monoblepharidomycetes, a unique feature among fungi. The Monoblepharidomycetes is the sister class to the Chytridiomycetes in the phylum Chytridiomycota. The six known genera have thalli that are either monocentric and without rhizoids or produce hyphae with an independent evolutionary origin from the hyphae of higher fungi. Monoblepharidomycetes (Chytridiomycota) displays an exceptional range of body types from crescent-shaped single cells to sprawling hyphae. Hyphae of Monoblepharidomycetes lack a complex aggregation of secretory vesicles at the hyphal apex (i.e., Spit€ rper), have centrosomes as primary microtubule organizing centers and have zenko stacked Golgi cisternae instead of tubular/fenestrated Golgi equivalents. The cytoplasmic distribution of actin in Monoblepharidomycetes is comparable to the arrangement observed previously in other filamentous fungi. Members of this class have a filamentous thallus that is either extensive or simple, unbranched. They often have a holdfast at the base. In contrast to other taxa in their phylum, they reproduce using autospores, although many reproduce through zoospores. Oogamous sexual reproduction may also occur. Asexually, they reproduce either by zoospores or autospores. Zoospores contain a kinetosome that is in parallel to a nonflagellated centriole, a striated disk partially extending around the kinetosome, microtubules radiate anteriorly from the striated disk, a ribosomal aggregation, and rumposome (fenestrated cisterna) adjacent to a microbody (Letcher et al., 2006). 3.6 Neocalimastigomycota CLASS: NEOCALLIMASTIGOMYCETES ORDER: NEOCALLIMASTIGALES; EXAMPLE NEOCALLIMASTIX, CYLLAMYCES SP. AND PECORAMYCES SP. 86 Current Developments in Biotechnology and Bioengineering Since their discovery by Orpin, 1974, members of the new phylum Neocallimastigomycota intrigued microbiologists as these organisms were reported to be found in digestive tracts of mammalian herbivores, with others potentially inhabiting other anaerobic niches. Taxonomic backbone contains eight genera of strictly anaerobic fungi. Members of this phylum are involved in the rumen function and animal digestion, and possess capacity to convert lignocellulose into bioenergy products (Solomon et al., 2016). Originally, these organisms were grouped under chytridiomycetous fungi but later assigned to higher phylum due to their life cycle (possess a vegetative structure from which zoospores are produced) and a chitin-containing cell walls. Later based on the type species Neocallimastix frontalis, Heath et al. (1983) formally classified them into a new family Neocallimastigaceae in the class Chytridiomycetous and order Spizellomycetales mainly due to the similarities of zoospore ultrastructure to some members of this order. Hibbett et al. (2007) later raised it to phylum containing anaerobic fungi, which are symbionts found in the digestive tracts of larger herbivores also known as the anaerobic gut fungi (AGF). Neocallimastigomycota dwell in the rumen and alimentary tract of herbivorous mammals, and reptiles (e.g., iguana) where they play important roles in the degradation of recalcitrated plant fiber. General characteristic features of Neocallimastogomycota. 1. Presence of monocentric or polycentric thallus. 2. Anaerobic and resides in the digestive system of herbivorous mammals and in other terrestrial and aquatic anaerobic environments. 3. Neocallimastigomycota lack mitochondria but instead contain hydrogenosomes in which the oxidation of NADH to NAD+, leads to formation of H2. 4. Horizontal gene transfer results in development of xylanase (from bacteria) and other glucanase activity. 5. Zoospores posteriorly unflagellate or polyflagellate zoospores. 6. Kinetosome-associated complex composed of a strut, skirt, spur flagellar ring, microtubules form a fan-like shape extending from spur and radiate around nucleus. 7. Flagellar props absent, nuclear envelope remains intact throughout mitosis. Neocallimastigomycota harbors a small group of flagellated, obligate anaerobic, nonparasitic fungi owing to a single family that comprises 18 recognized genera of which some may be paraphyletic (Hibbett et al., 2007). Neocallimastigomycota possess large genome sizes (101 Mb in Orpinomyces), very low GC content (as low as 17% in Orpinomyces), and high content of repetitive elements (Youssef et al., 2013). The low GC content is due to genetic drift triggered by the low effective population sizes, bottlenecks in vertical transmission, and the asexual life style of anaerobic fungi. AGF release asexual motile free zoospores into the herbivorous gut as part of their life cycle. Large AT-biased (78%–84%) genomes along with their fastidious growth condition impose challenges in the genomic and phylogenomic analyses of the AGF. These large genomes harbor-rich repertoire of carbohydrate-degrading enzymes shaped by gene expansions and horizontal gene et al., 2000). It is estimated the most recent common ancestransfer events (Garcia-Vallve tor of the AGF diverged millions years ago, a time frame that coincides with the evolution Chapter 3 • Fungal biology 87 of grasses (Poaceae), as well as the mammalian transition from insectivores to herbivores. This co-occurrence estimation suggests that AGF may have role in shaping the succession of mammalian herbivore transition by improving the efficiency of energy acquisition from recalcitrant plant materials. To endure in this anoxic and prokaryote-dominated environment, AGF members have undergone multiple structures and metabolic adaptations, such as the loss of the mitochondria replaced by a hydrogenosome, loss of respiratory capacities, and substitution of ergosterol with tetrahymanol in the cell membrane (van der Giezen, 2009). Interestingly, all known AGF taxa have a remarkable plant biomass degradation machinery, which aids in competing with other microbes for resources and establishing growth in the herbivorous anaerobic gut. Outside this environment, there is a single report of the presence of these fungi in the gut of a sea urchin based on the morphological identification (Thorsen, 1999). 3.6.1 AGF protein domains and homologous genes A comparative genomic analysis study between AGF and their nonrumen-associated chytrid relatives identified 40 Pfam domains that are unique to the AGF (accounting almost 0.67% of the total number of Pfams (5980) in the AGF pangenome-transcriptome). Functional annotation of HGT genes/pfams indicated that the majority (63.9%) of events encode metabolic functions such as extracellular polysaccharide degradation and central metabolic processes. Genes involved in cellular processes and signaling represent the second most represented HGT events (11.19%), while genes involved in information storage and processing only made up 4.69% of the HGT events identified (Fig. 2). Transcripts acquired by HGT represented >50% of transcripts in anywhere between 13 (Caecomyces) and 20 (Anaeromyces) GH families; 3 (Caecomyces) and 5 (Anaeromyces, Neocallimastix, Orpinomyces, and Feramyces) CE families; and 2 (Caecomyces and Feramyces) and 3 (Anaeromyces, Pecoramyces, Piromyces, Neocallimastix, and Orpinomyces) PL families. The AGF CAZyome encodes enzymes putatively mediating the degradation of 12 different polysaccharides. The predicted function of these domains included anaerobic ribonucleotide reductase (NRDD), metal transport and binding (FeoA and FeoB_C), carbohydrate binding (e.g., CBM_10, CBM-like, and Cthe_2159), glycoside hydrolase (e.g., Glyco_hydro_6 and Glyco_hydro_11), and atypical protein kinase (Cot H). In addition to these unique domains, many additional Pfams enriched the AGF genome. Polysaccharide degradation and monosaccharide fermentations domains like Chitin_binding_1, CBM_1, Cellulase, Glyco_hydro_10, Gly_radical, RicinB_lectin_2, Esterase, and Polysacc_deac_1 were found abundant. Phylogenetic analysis indicated that the AGF polysaccharide lyase domain is distinct and not orthologous to related enzymes in other fungi. In-depth analysis identified 106 Pfam domains that are not present in AGF genomes and transcriptomes but found in sister Chytridiomycota. Domains involved in the biosynthesis of nicotinic acid, uric acid, and photolyase, in purine catabolism, and in pathways of ureidoglycolate and kynurenine are absent in AGF species. Interestingly, most of these missing domains are related to oxidation reactions on cytochromes and mitochondria. Phylogenetic analyses support a horizontal transfer of certain Pfam domain such as Cthe_2159 domain (carbohydrate-binding domain-containing protein) transfer from rumen bacteria into 88 Current Developments in Biotechnology and Bioengineering AGF, followed by potential gene fusion to deliver eukaryotic specific functions. The majority of donors were anaerobic fermentative bacteria prevalent in the herbivorous gut with four bacterial phyla (Firmicutes, Proteobacteria, Bacteroidetes, and Spirochaetes) identified. 3.7 Blastocladiomycota Blastocladiomycota holds a very interesting position. Morphological and ecological similarities initially suggested a common ancestry with the core chytrids. Molecular analysis based on ribosomal DNA sequences and zoospore ultrastructural characters demonstrated it not monophyletic with Chytridiomycota but Blastocladiomycetes split more recently from the fungal backbone than the chytrids. The systematic classification of the Blastocladiomycetes was revised, and was given their own phylum Blastocladiomycota. Major evolutionary changes have accompanied the divergence of the Blastocladiales from the core chytrids. For example, the Blastocladiales have a life cycle with sporic meiosis whereas most core chytrids have zygotic meiosis (Letcher et al., 2006). However, the zoospore is functionally similar to those found among “core chytrids.” General characteristic features of blastoclaiomycota. 1. A single posteriorly directed flagellum, stored lipid, and glycogen reserves, branching thallus with pseudosepta. 2. A characteristic assemblage of lipids, microbodies, membrane cisterna called the sidebody complex. 3. A membrane-bounded ribosomal cap covering the anterior surface of a cone-shaped nucleus. 4. Blastocladiomycota present alternation of gametophytic and sporophytic generations. 5. Three types of life cycle (a) The Euallomyces life cycle; has alternating haploid gametophytic and diploid sporophytic generations. (b) The Cystogenes life cycle has a large, dominant, asexual sporophyte that produces thin-walled zoosporangia and resistant sporangia whereas the sexual gametophyte is a small, spherical, thin-walled cyst that produces variable numbers of isogametes that fuse in pairs forming biflagellate zoospores and the biflagellate zoospores develop into asexual thalli. (c) The Brachyallomyces life cycle, also called short-cycled, has no gametophytic or sexual thalli and reproduced only asexually. Two popular model organisms of Blastocladiomycota are Allomyces macrogynus and Blastocladiella emersonii, which are saprotophs with well-defined and well-studied alternation of generations. Other representatives of these genera are Physoderma (parasitic on higher plants), Blastocladiella, and Coelomomyces (obligate endoparasite of insects with alternating sporangia and gametangia stages in mosquito larvae and copepod hosts). CLASS: BLASTOCLADIOMYCETES of Blastocladiomycota contains a single-order, Blastocladiales, and four morphologically defined families bearing minor changes validated by molecular studies (Porter et al., 2011). Chapter 3 • Fungal biology Ø Ø Ø Ø 89 FAMILY: CATENARIACEAE contains both saprobes and pathogens (includes the genera Catenaria, Catenophlyctis, and Catenomyces). FAMILY: COELOMOMYCETACEAE contains pathogens of invertebrates. FAMILY: FAMILY BLASTOCLADIACEAE (genus Allomyces). FAMILY: SOROCHYTRIACEAE contains a pathogen of tardigrades. Later in Tedersoo et al. (2018), introduced another class; Class Physodermatomycetes, including Order Physodermatales. Family Physodermataceae contains obligate parasites of plants; (genera, Physoderma and Urophlyctis) an endobiotic polycentric thallus that produces thick-walled resting spores within the host plant. The thick-walled resting spore is meant for unfavorable conditions. Typically, blastocladian zoospores have a distinctive ribosomal nuclear cap, and in some species, a large side body containing lipid globules. Environmental sampling has detected ample Blastocladiomycota clades in aquatic environments. Its ability to grow in pure culture and its position as a relative of plant parasites make it a potentially interesting organism to study genes associated with parasitism. 3.8 Zygomycetous fungi Zygomycete fungi were classified under a single phylum, Zygomycota that reproduces sexually by zygospores and asexually by sporangia, except some show absence of multicellular sporocarps, and production of coenocytic hyphae. Phylogenetic classification includes 2 phyla, 6 subphyla, 4 classes, and 16 orders. Zygomycetous fungi formed two main lineages, one that is mostly parasites of opisthokonts (Zoopagomycota) and a second that is composed mostly of plant symbionts and saprotrophs (Glomeromycota + Mucoromycota; Table 3). Zoopagomycota comprises Entomophtoromycotina, Kickxellomycotina, and Zoopagomycotina. Phylum Mucoromycota comprises Glomeromycotina, Mortierellomycotina, and Mucoromycotina and is sister to phyla Dikarya. 3.9 Zoopagomycota This phylum is the earliest diverging group of nonflagellated fungi that includes three main lineages, i.e., ENTOMOPHTHOROMYCOTINA, ZOOPAGOMYCOTINA, and KICKXELLOMYCOTINA (Spatafora et al., 2016). Ø Ø Ø ENTOMOPHTHOROMYCOTINA: Monophylatic lineage representing insect pathogens mostly along with nematodes and mites. ZOOPAGOMYCOTINA: Obligate parasites of zygomycete fungi and microscopic soil animals like nematodes, rotifers, etc. KICKXELLOMYCOTINA: Includes four zygomycetes orders Asellariales, Dimargaritales, Harpeellales, and Kickxellales. Members of theses lineages have the ability to form true mycelia. Most members are either saprotrophs or parasites of metazoans, amoebae or other fungi, including highly specialized forms. Table 3 Phylogenic classification of zygomycete fungi. Zoopagomycota Mucoromycota Zygomycete fungi Subphyla Incertae sedis Life form/Host types Entomophthoro Mycotinaa Pathogens, saprobes, animal, arthropod Mucoro mycotina Saprotrophic mycorrhizalor parasitic plant Zygospore Zygospore Zygospore Trichospores, sporangia, merosporangia Bifurcate septa with lenticular plug Conidia Sporangia, sporangioles Sporangia Resembles chlamydo-spore Complete septa, bifurcate septa/ coenocytic; hyphal bodies €rper Spitzenko Coenocytic Coenocytic Coenocytic Apical vesicle crescent Spindle pole body Industrial enzyme (Tako et al., 2015) Apical vesicle crescent – Apical vesicle crescent – Polyunsaturated fatty acid (Wagner et al., 2013) Present (few) Mortierellales Biofertilizer (Aguilar-Paredes et al., 2020) Zoopago mycotina Kickxello mycotina Obligate parasites animal, fungi, nematode, rotifers, amoebae Animal, fungi Sexual reproduction Asexual reproduction Zygospore Sporangia conidia, arthrospores cllamydospores Hyphae type Coenocytic Hyphal tip morphology Mictotubules/ centrioles Biotechnology application Unsampled Fruiting body Order a Unsampled. Apical vesicle crescent Centriole-like Centriole-like Ecological study (amoebophagous fungi in permafrost soils) (NaranjoOrtiz and Gabaldon, 2019) Metal homeostasis (Shine et al., 2015) Biological control (Carla Baron et al., 2019) Absent Zoopagales Absent Asellariales, Kickxellales, Dimargatitales, Harpellales Absent b Basidiobolomycetes Entomophthoromycetes Neozygito-mycetes Present (few) Endognales, Mucorales, and Mortierellales Mortierello mycotina Saprotrophs, nonpathogenic except Mortierella wolfii, plant Zygospore Glomero mycotina Arbuscularmycorhizza, plant Unknown Present (few) Paraglomerales Diversisporales Glomerales Archaeosporales Raised to the rank of phylum as “Entomophthoromycota” in a scientific paper by Humber (2012). Raised to class Basidiobolomycetes. Adapted and modified from Spatafora, J., Chang, Y., Benny, G., Lazarus, K., Smith, M., Berbee, M., Bonito, G., Corradi, N., Grigoriev, I., Gryganskyi, A., James, T., O’Donnell, K., Roberson, R., Taylor, T., Uehling, J., Vilgalys, R., White, M., Stajich, J., 2016. A phylum-level phylogenetic classification of zygomycete fungi based on genome-scale data. Mycologia 108, 1028–1046. b Chapter 3 • Fungal biology 91 3.9.1 Entomophthoromycotina Entomophthoromycotina includes three classes (BASIDIOBOLOMYCETES, NEOZYGITOMYCETES ENTOMOPHTHOROMYCETES) (Humber, 2012). Ø Ø Ø BASIDIOBOLOMYCETES: representive genera Drechslerosporium, Schizangiella, Basidiobolu. NEOZYGITOMYCETES: representative genus Neozygites. ENTOMOPHTHOROMYCETES: representative genus Conidiobolus. All are saprobic and insect pathogenic fungi. The thallus may have either coenocytic or septate hyphae, which may fragment to form hyphal bodies or it may comprise only hyphal bodies. Asexual reproduction is through conidiogenesis from branched or unbranched conidiophores. Primary conidia are forcibly discharged and secondary conidia are either forcibly or passively released. Sexual reproduction is either by forming zygospores by gametangial copulation, involving hyphal compartments or hyphal bodies (Humber, 2012). Sterol data revealed sterols of different zoosporic and zygosporic forms exhibit structural diversity. Cholesterol and 24-ethyl-Δ5 sterols found in zoosporic taxa, and 24-methyl sterols in zygosporic fungi. Each of the three monophyletic lineages of zygosporic fungi has distinctive major sterols, ergosterol in Mucorales, 22-dihydroergosterol in Dimargaritales, Harpellales, and Kickxellales (DHK clade), and 24-methyl cholesterol in Entomophthorales. Entomophthoromycotina present 24-methyl cholesterol as their main membrane sterol (Weete et al., 2010). So far, Basidiobolus of class Basidiobolomycetes is the only genus in the Entomophthoromycotina to bear septate hyphae. Basidiobolus and Conidiobolus of class Entomophthoromycetes are unique among the zygomycetous fungi for possessing a true € rper. Ultrastructural image revealed a dense cluster of vesicles at the hyphal Spitzenko apex. The hyphal apex exhibited phase-dark inclusion exhibited independent motility within the hyphal apex and its presence and position were correlated to the rate and direction of hyphal growth. The hyphal apex of Basidiobolus sp. did not contain γ-tubulin (Manning et al., 2007). 3.9.2 Zoopagomycotina Zoopagomycotina include mycoparasites, predators, or parasites of small invertebrates and amoebae. Special haustoria structures are produced in association with hosts. The hyphal diameter is characteristically narrow in thalli that are branched or unbranched. Only limited species could be successfully maintained in axenic culture. Sexual reproduction, is by gametangial conjugation, forming globose zygospores on apposed differentiated or undifferentiated suspensor cells. Asexual reproduction is by arthrospores, chlamydospores, conidia, or multispored merosporangia that may be simple or branched (Walther et al., 2019). Zoopagomycotina comprises a single order, Zoopagales, that includes 5 families and around 20 genera. 92 Current Developments in Biotechnology and Bioengineering 3.9.3 Kickxellomycotina Subphylum Kickxellomycotina was created by combining poorly documented fungal groups united by the presence of septated mycelia that present unique septal pores with a lenticular plug. These pore plugs as well as the characteristics of the sporangia account for diagnostic traits for this group within the subphylum (Tretter et al., 2014). The group comprises four recognized orders: Ø Ø Ø Ø ORDER: KICKXELLALES; for example genera: Kickxella, Coemansia, Linderina, Spirodactylon. ORDER: DIMARGARITALES; for example genera: Dimargaris, Dispira, Tieghemiomyces. ORDER: HARPELLALES; for example genera: Harpella, Furculomyces, Legeriomyces, Smittium. ORDER: ASELLARIALES; for example genera: Asellaria, Orchesellaria. Mycelium is symmetrically divided into compartments by bifurcate septa that have lenticular occlusions. Sexual reproduction involves the formation of diversly shaped zygospores by gametangial conjugation of relatively undifferentiated sexual hyphal compartments. Sporophores may be produced from septate, simple, or branched somatic hyphae. Asexual reproduction involves the production of uni- or multispored merosporangia from sporocladium, sporiferous branchlets, or an undifferentiated sporophore apex. Species may be saprobes, mycoparasites, and symbionts of insects. The genus Coemansia of the Order Kickxellales (Kickxellaceae, Kickxellales); is a dung-associated saprotroph with intrincate asexual structures. It was the first sequenced Kickxellomycotina (Chuang et al., 2017). Septal ontogeny in Linderina pennispora is initiated by the ingrowth of material from the inner of the two layers of the cell wall. The growing point of the septum bifurcates and continues growth forming a lenticular cavity into which the septal plug is deposited. This type of septal development occurs in all septa except those produced between the pseudophialides and the merosporangia and spores. The latter septal type initially is formed as previously described, but then additional wall material is produced both above and below the septum. In the merosporangiospore, three layers of wall material are produced. The inner two layers give rise to the spore spines and at maturity all wall layers coalesce and form a single, thick spore wall. Extra wall material in the apex of the pseudophialide gives rise to a multilobed and mottled vacuole that shrivels just before spore liberation. Mature merosporangiospores are the only structures with spore spines. Aerial spines are produced on all structures except the merosporangiospores, although a relatively larger number of the aerial spines are produced on the aerial hyphae than on the pseudophialides and the sporocladia (Benny and Aldrich, 2011). 3.10 Glomeromycota Members of the Glomeromycota species are well known for forming arbuscular mycorrhizas (AMs) in the roots of vascular land plants. Morphological features are the soil-borne sporocarps (spore clusters) that are found in or near colonized plant roots. Earlier studies Chapter 3 • Fungal biology 93 placed Glomeromycota as the sister clade to Dikarya (Ascomycota + Basidiomycota + € βler et al., 2001). Later Glomeromycota was separated from Entorrhizomycota) (Schu the rest of Zygomycota based on ribosomal protein phylogenies. Species of Glomeromycotina produce coenocytic hyphae that harbor bacterial endosymbionts belonging to members of the family Gigasporaceae and Mollicutes (Mollicutesrelated endobacteria MRE) in their cytoplasm. During coevolution, MRE formed distinct, monophyletic evolutionary lineages within their fungal hosts with a 16S rRNA gene (16S) s et al., 2015). Asexually formed sequence divergence of up to 20% (Torres-Corte chlamydospore-like spores are borne on specialized hyphae. These spores are large, multinucleated, and filled with lipid and protein globules. Spore morphology defines the different groups. Most species produce spores directly in soil or in the roots, but several species in different lineages make macroscopic sporocarps. This phylum species live as obligate symbionts of land plants, forming a particular type of symbiosis termed arbuscular mycorrhizae. Arbuscules are distinguishing structures of AMF and serve as the site of nutrient exchange and transfer in arbuscular mycorrhizae. They are modified, highly branched haustorium-like cells that are produced in cortical plant root cells. The fungal mycelia grow inside the root of the plant penetrating the host cells. The host plant exerts influence over the proliferation of intercellular hyphal and arbuscule formation. The fungus helps the plant in acquiring of phosphorus, nitrogen, and water in exchange for photosynthesis-derived metabolites. The mycelium is always nonseptate and presents anastomoses (Redecker et al., 2013). Members of this group have 24-ethyl-cholesterol as the main membrane sterol, apparently lacking ergosterol. Ø Ø Ø Ø ORDER: ORDER: ORDER: ORDER: ARCHAEOSPORALES; for example genera: Archaeospora, Geosiphon. DIVERSISPORALES; for example genera: Acaulosporaceae, Diversispora, Pacispora. GLOMERALES; for example genus: Glomus. PARAGLOMERALES; for example genus: Paraglomus. Recent molecular studies have suggested a separate phylum is appropriate for the AM fungi, the Glomeromycota, and this is the position taken by the AFTOL study. The ICBN requires the name of a family or order to be formed from the genitive singular of a legitimate name of an included genus. The genitive of the type genus Glomus is Glomeris, and so the name of the family should be Glomeraceae and order Glomerales (rather than “Glomales”). 3.11 Mucoromycota Phylum Mucoromycota, currently listed along with clade zygomycetes under incertae sedis comprises three orders under subdivision Mucoromycotina. Sexual reproduction is through zygospore production by gametangial conjugation. Zygospores tend to be globose, smooth or ornamented, and produced on opposed or apposed suspensor cells with or without appendages. Asexual reproduction involves chlamydospores and spores produced in sporangia and sporangioles. Hyphae has large diameter and coenocytic. 94 Current Developments in Biotechnology and Bioengineering Ø ORDER: MUCORALES; for example genera: Mucor, Parasitella, Phycomyces, Pilobolus, Rhizopus. ORDER: ENDOGONALES; for example genera: Endogone, Peridiospora, Sclerogone, Youngiomyces. ORDER: MORTIERELLALES; for example genera: Mortierella, Dissophora, Modicella. Ø Ø Mucoromycota is characterized by plant-associated nutritional modes (plant symbionts, decomposers of plant debris, plant pathogens, etc.) but have rare ecological interactions with animals (as opportunistic infections). 3.11.1 Mortierellomycotina Mortierellomycotina are diferentiated from Mucoromycotina by the morphology of the zygospore and the absence of a columella which is centrally vacuolated part of hyphae bearing spores, typically a basally inflated sporangiophore (Smith et al., 2013). Species of Mortierella live as saprotrophs in soil, on decaying leaves and other organic materials. Most species of Mortierellomycotina form microscopic colonies but few in the genus Modicella make multicellular sporocarps. Compared with Mucor-like fungi, the mitosporangia are typically smaller and contain fewer spores and lack a columella. Mortierellomycotina reproduce asexually by sporangia and form zygospores (naked or surrounded by nest like hyphae) that are the developed through plasmogamy between gametangia belonging to complementary mating types. Mortierella species are producers of fatty acids especially polyunsaturated fatty-acid like, arachidonic acid, and they frequently harbor bacterial endosymbionts (Higashiyama et al., 2002; Sato et al., 2010). 3.11.2 Mucoromycotina Mucoromycotina subphyla taxonomic placement is still considered as incertae sedis by some mycologists and these groups were originally clustered as Zygomycota. Mucoromycotina fungi represent the majority of zygomycetous fungi in pure culture. Species can be isolated from soil, dung, plant debris, and sugar-rich plant parts (e.g., fruits). Sexual reproduction within Mucoromycotina is by prototypical zygospore formation. It involves the production of zygospores by apposed gametangia within a simple sequestrate or enclosed sporocarp. Asexual reproduction involves the copious production of sporangia and/or sporangioles. Primary cell walls component of Mucoromycotina is chitosan, a deacetylated form of chitin lida et al., 2015). Presence of an inflated swelling of a sporangiophore termed a columella (Me is synapomorphic for the subphylum. Porous, plasmodesmata-containing septa sometimes appear in reproductive structures and senescent hyphae. Mucorales members often referred to as pin molds, produce sporangia held up on hyphae, called sporangiophores. Sporangiophores are upright (simple or ramified) hyphae that support sac-like sporangia filled with asexual sporangiospores. The sporangiospores germinate to form the haploid hyphae of a new mycelium. Chapter 3 • Fungal biology 95 Endogonales covers both ectomycorrhizal and saprobic species. Current findings suggest that ectomycorrhizae have probably evolved twice within Endogonales (Tedersoo and Smith, 2013), once as an independent origin of mycorrhizae relative to the arbuscular mycorrhizae of Glomeromycotina and second as ectomycorrhizae of Dikarya. Like many of Mucoromycota, they harbor endohyphal bacteria. 3.12 Dikarya The name was derived from the Greek word di- (two) and karyon (nut or kernel, interpreted by biologists to refer to nuclei). Instead of usual pattern of phylum name ending in mycota such as Dikaryomycota or Neomycota, the clade was named Dikaryon following the phylogeny-based classification of Hibbett et al. (2007) which has been adopted both in Ainsworth & Bisby’s Dictionary of the Fungi and the GenBank taxonomy (http://www.ncbi.nlm.nih.gov/guide/taxonomy). Monophyly of Dikarya is strongly documented by independent and combined analyses of nuclear ribosomal RNA genes, RNA polymerase II subunits, and whole genomes sequencing (James et al., 2006b). Dikarya are characterized to have a sexual cycle that includes hyphal fusion uncoupled with meiosis, which produces hyphae that contain two independent nuclear populations (dikaryotic hyphae). Beside these features like the presence of septate hyphae, ergosterol as the building block of the membrane sterol, and several lineages that are able to form multicellular reproductive or vegetative structures such as cytoplasmic fusion of two haploid monokaryotic hyphae giving rise to dikaryotic condition defines the putative synapomorphy of this group nomenclature. Clamp connections of Basidiomycota and croziers of Ascomycota, are structures that function in the distribution and allocation of nuclei to daughter cells following mitosis in dikaryotic hyphae, are homologous and represents an additional synapomorphy. Evenly septate hyphae are typical synapomorph to this group, because Mucoromycota, its sister taxon, have predominantly coenocytic hyphae. From the above description, Nagy et al. (2014) proposed that if clamps/croziers and septate hyphae of Basidiomycota and Ascomycota are homologous, then the ancestor of Dikarya must have been filamentous (Nagy et al., 2014). 3.12.1 Ascomycota Derived from the Greek askos (sac) + mykes (fungus) the name Ascomycota rather than the synonyms Ascomycetes (class) and Ascomycotina (subphylum), was termed following the phylogeny-based classification of Hibbett et al. (2007), similar to Basidiomycota. Ascomycota comprises three mutually exclusive subclades (Schoch et al., 2009). Taphrinomycotina, Saccharomycotina, and Pezizomycotina pezizomycotina include all ascomaproducing taxa. The fossil record of Ascomycota dates back to Devonian period, with Paleopyrenomycites and Prototaxites taitii dating from the Middle Ordovician until the Late Devonian molecular phylogenies of the fossil record have estimated the origin of Ascomycota around 0.40–1.3 billion years before present (Taylor and Berbee, 2006). 96 Current Developments in Biotechnology and Bioengineering General characteristic features of Ascomycota. Ascomycota includes diverse organisms from unicellular yeasts to complex cup fungi. Half of all members of the phylum Ascomycota form symbiotic associations with algae to form lichens (Kumar et al., 2011). 1. Sexual reproduction leads to formation of meiospores (ascospores) within sac-shaped meiosporangia (asci) by the process of free cell formation. 2. Free cell formation involves the production of an enveloping membrane system, which is derived from either the ascus plasmalemma or the nuclear envelope and delimits ascospore initials. 3. Ascomycota are devoid of flagella and exhibit intranuclear mitosis with spindle pole bodies instead of centrioles. 4. Most Ascomycota have filamentous septate hyphae fenced by septal walls that have septal pores. 5. The cell walls of the ascomycetes contain chitin and β-glucans. A unique character of the Ascomycota (but not present in all ascomycetes) is the presence of membrane bound structure with a crystaline protein matrix called Woronin bodies on each side of the septa. Asexual reproduction produces mitospores or vegetative reproductive spores called the conidiospores. 3.12.2 Basidiomycota Why did the mushroom come to the party? because he’s a fungi! Derived from the Latin word -basis (means base, support) plus -idium, refers to the basidium, a “little pedestal,” on which the basidiospores develop. The most pronounced difference between the ascomycota and basidiomycota is the extended dikaryotic phase of some of the basiomycota. Similar to ascomycota the name basidiomycota was adapted by Hibbet et al., as well as in Ainsworth & Bisby’s Dictionary of the Fungi, and the GenBank taxonomy (http://www. ncbi.nlm.nih.gov/guide/taxonomy) (Hibbett et al., 2007; Kirk et al., 2008). Three major subdivision, Pucciniomycotina (rusts—Pucciniales and relatives), Ustilaginomycotina (smuts—Ustilaginales and relatives), and Agaricomycotina which includes mushrooms Agaricomycetes, jelly fungi (Auriculariales, Dacrymycetales, Tremellales) and others have been allotted under this and is strongly supported by phylogenetic analyses of multilocus molecular data (James et al., 2006b). Genome-based datasets strongly support the monophyletic descend of Basidomycota which was also corroborated in an analysis of nonmolecular characters by Zhao et al. (2017) and Nagy et al. (2016). In gist Basidiomycota is form of extended, free-living dikaryotic mycelium where the production of meiospores on basidia is putative synapomorphies. General characteristic features of Basidomycota. 1. Basidiomycota reproduces sexually via the formation of specialized club-shaped end cells called basidia that normally bear external meiospores (usually four). These Chapter 3 • Fungal biology 2. 3. 4. 5. 6. 97 specialized spores are called basidiospores borne on distinctive basidiocarps or basidioma. The basidiospores are the dispersive spores and they are forced out by hydrostatic pressure between the sterigma and the basidiospore on the basidium. In these cases, they are called ballistospores. The hymenium is the tissue layer of the fungi fruiting body from which the basidia arise. Mycelium that grows from a basidiospore is haploid. Haploid mycelia fuse via plasmogamy until the hyphae contains a pair of compatible nuclei (dikaryon), hence called dikaryotic. This dikaryotic stage can last for years and maintenance of the dikaryotic status in dikaryons in Basidiomycota is facilitated by the formation of clamp connections. Dikaryotic mycelium grows as an expanding circle and feeds on its substrate for organic matter and nutrients during its expansion. With time, the older dikaryotic mycelia expand in circles as a ring (called fairy ring), which also becomes the source of the basidiocarps. The Basidiomycotina is the most diverse subphylum in the Basidiomycota. Tree ear is an example of this taxa. Basidiomycotina can be differentiated on the basis of their basidiocarps, which show great disparity in form. The Basidiomycota fungi range from common edible mushroom forms to some of deadly plant pathogens. The symbiotic basidiomycota such as rusts (Subphylum Urediniomycotina) and smuts (Subphylum Ustilagomycotina), attack wheat and other crops and are main groups of plant pathogens. The rusts (e.g., wheat rust and white pine blister rust) alternate between two hosts (heteroecious) and have five different kinds of spores (macrocyclic) in their life cycles. In Puccinia graminis, karyogamy occurs in the teliospore (fourth spore) after which it undergoes meiosis with each four cells bearing one basidiospore each. The basidiospores then after disperse and restart the infection process (Begerow et al., 2006). Not all symbiotic Basidiomycota are harmful to their partners. For instance, some Basidiomycota form ectomycorrhizae, which are symbiotic associations with the roots of vascular plants. Ectomycorrhizal Basidiomycota help to transfer mineral nutrients from the soil to the plant, and in exchange they receive carbon source like sugars produced in photosynthesis. The basidiomycete toxin phalloidin (from the mushroom Amanita phalloides) binds actin, which is a component of microfilaments. This prevents depolymerization of actin fibers. Gradually phalloidin destroy the liver cells and causes nephrosis. 4. Conclusions and perspectives Ever since the discovery of George Beadle and Edward Tatum “one gene, one enzyme” hypothesis based on experiments on bread mold Neurospora crassa, fungi continued to be the preferred model organism for genetic experiments largely because they are less expensive than any other eukaryotic organism and also because of ease of culture, definite 98 Current Developments in Biotechnology and Bioengineering life cycles, short generation time gap, etc. Fungi comprise a highly heterogenous community from unicellular microscopic entity yeast to complex multicellular macroscopic filamentous structures. They represent parasitic anaerobes in the animal gut as well as saprophytic aerobes in wild. Their taxonomic origin has been revised numerous times in recent past. Some chytrid members are given new phylum status whereas old phylums are dissolved. Phylum AGF/Neocallimastigomycota, distinct from the chytrid fungi, possess several unique traits that make their study fascinating yet challenging to mycologists. Much of the interest in these fungi relates to the genes/enzymes important for biorefining and biofuel production, notably xylose isomerases and glycosyl hydrolases (xylanases, cellulases). In Prague, Kate and her colleagues have explored the use of anaerobic fungi to improve the hydrolytic phase of biogas production. They have also investigated which fungi are present in the cow manure used to prime the biogas fermentations. Most exploited industrial strains are mainly from Ascomycota and Basidiomycota. Moreover, some members of Ascomycota like unicellular S. cerevisiae and heterothallic filamentous Neurospora crassa are overanalyzed as model organisms. 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Prod. 67 (2), 300–310. 4 Mycotoxins Manikhardaa, Hanifah Nuryani Lioeb, Rachma Wikandaria, and Endang Sutriswati Rahayua a DEPARTMENT OF FOOD AND AGRICULTURAL PRODUCT T ECHNOLOGY, UNI VERSITAS GADJ AH M A D A , Y OG Y A K A R T A, I NDO N E S I A b DEPARTME NT OF FOOD SCIENCE AND TECHNOLOGY, IPB UNIVERSITY, BOGOR, INDONESIA 1. Introduction Mycotoxins, first coined in 1962 after an incident involving aflatoxin (AF) and costing the death of approximately 100,000 turkey poults in London, are defined as toxin synthesized by filamentous fungi species as secondary metabolites that might pose a health hazard to human or vertebrates of other animal groups in low concentration and usually consists of low molecular compounds (Ashiq, 2015; Bennett et al., 2003). However, it is important to note that not all fungal toxic secondary metabolite is defined as mycotoxin. For example, an antibiotic is a fungal product that is mainly poisonous to bacteria. The same goes for other fungal toxins harmful to plants, but they are not classified as mycotoxins. The presence of filamentous fungi also does not always imply mycotoxin occurrence because the fungi might also grow without secreting toxins. Compared to other fungal by-products, mycotoxins have a wide selection of hosts and targets that cross-plant species. Currently, about 500 mycotoxins are recognized. Mycotoxins classification is usually based on the fungal producers, chemical configurations, and (or) action mechanism. However, one type of mycotoxins can be produced by several different species. Similarly, a single species of fungi might generate one or several types of mycotoxins. Furthermore, a systematic definition of modified mycotoxins and “masked mycotoxins” had been proposed into four hierarchical levels to distinguish the unbound and unmodified mycotoxins from biologically or chemically modified mycotoxins and matrix-bound mycotoxins (Rychlik et al., 2014). Molds are a major spoilage agent of foods and feedstuffs. They cause the reduction of crop yield and quality with significant economic losses and contamination of grains with mycotoxins that are harmful to people and livestock. Mycotoxins contamination in food and feed still becomes a headline due to the negative impact on the health and economy of the country. Moldy food is easily recognized by the naked eye from the color of conidia, spore, mycelia of the fungi. However, some could not be easily detected and need further analysis in the laboratory, such as direct or dilution plating methods. Among the different molds or fungi, there are toxigenic and nontoxigenic molds/fungi. Toxigenic ones produce mycotoxins. Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00008-9 Copyright © 2023 Elsevier Inc. All rights reserved. 105 106 Current Developments in Biotechnology and Bioengineering The presence of mycotoxin produced by filamentous fungi in food or feed could exert adverse health effects. Though the appearance of moldy food could be observed visually, mycotoxin contamination is barely detected by the naked eyes. The ingestion of mycotoxins can be direct via consuming contaminated products or indirect via consuming the mycotoxin residue in animal products. The lethal effect of mycotoxins can occur shortly after exposure at certain levels. While at lower levels, several disorders or immunity impairment can occur depending on the type and concentration of the mycotoxin exposure, the health status of the individual ingesting the mycotoxin, alcohol consumption, duration of exposure, and coexposure to other toxins (Ashiq, 2015; Bennett et al., 2003). Thus, the severity of mycotoxin poisoning can be compounded by vitamin deficiency, caloric deprivation, alcohol abuse, and infectious disease status. This phenomenon denotes that mycotoxins affect people’s health (Weidenborner, 2008). The list of some mycotoxins and their health effects are summarized in Table 1. Some studies also had reviewed the mycotoxin effect on reproductive health in Africa (Eze et al., 2018), and epidemiological studies of mycotoxin exposure and cancer risk (Claeys et al., 2020). Table 1 Health risk/clinical manifestation of mycotoxins. Mycotoxins Health effect Concentration Incidence References Aflatoxins (B1, Liver lesions, cirrhosis, primary B2, B3, G1, hepatocellular carcinoma, G2, M1, P1) Kwashiorkor, Reye’s syndrome, immunosuppression The adverse effect of AF is usually due to chronic exposure to one’s diet Fumonisins Esophageal carcinoma, neural tube defect 1–5 μg/kg (9 samples), 5–20 μg/ kg (3 samples), 31 μg/ kg (1 sample) Fukal et al. (1990) and Reddy et al. (2010) Ochratoxin A 25–27 μg/kg Incidence: 13/37 Country: Czechoslovakia 1/10–6/10 death rates of hepatitis in China and Africa Incidence: 14/32 country: Spain, USA, South Africa, China Incidence: 4/24 Country: Bulgaria Endemic nephropathy, urothelial tumors 4.8–85.5 μg/L Deoxynivalenol Nausea, vomiting, abdominal pain, 5–100 μg/kg (5 samples) diarrhea, dizziness, headache, immunosuppression Zearalenone Premature puberty in girls, cervical 2–8 μg/kg cancer, breast cancer Patulin Damage of gastrointestinal, respiratory systems, DNA, many enzymes 2.6 μg/kg Reddy et al. (2010), Shepard (2006), and Torres et al. (1998) Petkova-Bocharova and Castegnaro (1985) and Reddy et al. (2010) Eskola et al. (2000), Incidence: 5/7 country: Finland, Reddy et al. (2010), and Shepard (2006) India Claeys et al. (2020), Incidence: 1/7 Country: Finland, Eskola et al. (2000), and Reddy et al. US (2010) Bergner-Lang et al. Incidence: 1/16 (1983), Piva et al. Country: (2006), and Reddy Germany, Italy et al. (2010) Chapter 4 • Mycotoxins 107 In the case of AF exposure in Indonesia, a preliminary study of AF risk assessment had been conducted in the Temanggung region, a corn-based food consumption. Almost every day, people in this area consume corn in the form of sekelan (corn-based food) as carbohydrates sources. AFB1 in ready-to-eat sekelan was about 0.96–0.98 ppb (lower than the LOD 1.75 ppb, using enzyme-linked immunosorbent assay (ELISA) methods). With the daily consumption rate of sekelan about 117.1–358.7 g/person per day, it was estimated that the AFB1 exposure value was 79 ng/kg bodyweight per day. According to JECFA (1998), estimation for cancer liver was 0.01–0.30 cases/year per 100.000 people. Therefore estimation for liver cancer, with a population of 8.000 people in the region, is about 0.06–1.80 cases/year per population (Mahdy et al., 2013). The exposure of mycotoxins in developing countries is possibly higher than that of developed countries since developed countries implement strict regulations for mycotoxins. Therefore, this situation forces developing countries to keep their most contaminated food domestically for their consumption while exporting their best crops (Wu, 2006). For instance, AF exposure for Indonesian people is about 9–122 ng/kg body weight per day , and for South Asian people, in general, is about 30–100 ng/kg body weight per day . For comparison, AF exposure for European (in general) is 0–4 ng/kg body weight per day . Liver cancer, is the third leading cause of cancer death worldwide (WHO, 2004). Mycotoxin contamination implies considerable economic loss due to losses in human health, animals and livestock well-being, as well as agricultural commodities (Hussein and Brasel, 2001). Because the lesser stock of commodities complies with the tolerable levels of mycotoxins, it leads to accumulation of rejected products, while conceivably it would necessitate higher cost to provide mycotoxin-safe foods (Ashiq, 2015). Therefore, mycotoxins are responsible for millions of dollars in annual economic losses (Table 2). The impacts of fungi and mycotoxins, apparently AFs in maize and peanut that is still relevant to other fungal and mycotoxins contaminated food had been specified in a report (Lubulwa and Davis, 1994), i.e., (a) the decline in agricultural products quality; (b) decomposition of the agricultural products; (c) negative health impacts on humans who consume mycotoxin-contaminated food; (d) livestock well-being and its impact on the productivity; and (e) loss of trade markets due to regulations restricting the trade of mycotoxin-contaminated products. Feed contaminated by mycotoxins is worsening the health of the animal or even lethal, thus causing significant losses in animal production and animal welfare and might result in the toxins carrying over to the animal-derived products such as meat, milk, or eggs. However, the level might be lower than that found in plant-derived products (Fink-Gremmels, 2006). Strict regulation is needed to maintain human health. On the other hand, too rigid regulation might be resulted in higher economic losses due to rejection. Implementation of more strict regulations from food importers does not have a significant effect on their health. For instance, reducing the limit of AF from 20 to 10 μg/kg only decreases the risk of mortality due to hepatitis B and C by two in a billion annually (Henry et al., 1999). Nevertheless, it significantly impacts the exporting countries as more products are rejected, and they need to keep their contaminated product for themselves. Hence, it is important 108 Current Developments in Biotechnology and Bioengineering Table 2 Mycotoxins Aflatoxins Economic impact related to mycotoxin incidence. Cases/ commodities Maize, wheat, rice, sorghum, groundnuts, tree nuts, figs Economic impact ($) References In 2009, maize prices in Kenya plummeted by half. EU mycotoxins regulation was predicted to reduce 64% of African export for agricultural products, approximately 670 million US$ in value yearly. Gambia’s cumulative financial loss on domestic and international trade from 2000 to 2014 was about 23 million US$, on average, about 1.52 million US$ yearly. Presumably, 1.2 billion US$ loss is caused by aflatoxin contamination globally per year, with 38% of this loss (450 million US$) came from African nations N.A. Bankole and Adebanjo (2003), ECOACAP (2014), IITA (2012), Marechera and Ndwiga (2015), and Reddy et al. (2010) Deoxynivalenol Maize, wheat, cereals, cereal products Fumonisins Maize, maize The international implementation of products, sorghum 2 mg/kg fumonisin regulation in food would cause export losses to corn exporting nations (USA, China, Argentine) around $20 million to $40 million annually. If the standard of 0.5 mg/kg fumonisin in corn is applied, the global annual market loss will increase to $200 million through product rejection Ochratoxin A Cereals, dried vine N.A. fruit, wine, coffee Patulin Apples, apple juice N.A. N.A. Zearalenone Maize, wheat cereals, cereal products Reddy et al. (2010) Reddy et al. (2010) and Wu (2006) Reddy et al. (2010) Reddy et al. (2010) Reddy et al. (2010) and Shepard (2006) to do a risk assessment in order to determine the regulation limit of mycotoxin, so it will be beneficial for both food importers and exporters. 2. Major groups of mycotoxins: chemistry and processing stability Among 500 mycotoxins are recognized to date, the common mycotoxins in foods are described in Table 3. The major mycotoxins producing fungal genera were Aspergillus, Table 3 Type, producing fungi, and stability of different mycotoxins. Mycotoxin Chemical structure Producing fungi Aspergillus flavus, A. parasiticus, A. bombycis, A. ochraceus, A. pseudotamari Aflatoxin B1, aflatoxin B2, aflatoxin B3 (parasiticol), aflatoxin D1, aflatoxin G1, and their derivates (aflatoxin M1, aflatoxin P1, aflatoxin Q1, and aflatoxicol) Aflatoxin B1 (AFB1) Aflatoxin M1 (AFM1) Aflatoxin G1 (AFG1) Aflatoxin P1 (AFP1) Aflatoxicol Chemistry and processing stability References The AFs are heterocyclic compounds with dihydrodifurano or tetrahydrodifurano moieties attached to a substituted coumarin moiety. These toxins are highly oxygenated. They are also highly fluorescent under UV light and could be recognized by their fluorescing characteristics. Both AFB1 and AFB2 showed blue fluorescence, and AFG1 and AFG2 showed yellowgreen fluorescence More than 20 kinds of AFs and their derivatives had been recognized. AFB1 has the highest prevalence among the AFs group. AFB2a is the metabolites resulted from AFB1 detoxification with the least acute toxicity among the major aflatoxins. AFG2a is derived as the metabolite product of AFG1 The metabolite derivatives of AFB1 are aflatoxicol A, AFM1, AFP1, and AFQ1, among others. Aflatoxicol A might also be synthesized biologically in Tetrahymena pyriformis, Dactylium dendroides, and Rhizopus spp. AFM1 is secreted in urine and milk. AFM1 has Bennett et al. (2003), Castells et al. (2005), Cole and Cox (1981), Park et al. (2005), Raters and Matissek (2008), and Reddy et al. (2010) Continued Table 3 Type, producing fungi, and stability of different mycotoxins—cont’d Mycotoxin Chemical structure Alternaria toxins (alternariol, altenuisol, alternariol methyl ether, altenuene, altenusin, dehydroaltenusin, altertoxin I, altertoxin II) Producing fungi Alternaria spp., A. tenuis, A. dauco, A. cucumerina Alternariol (AOH) Altenuene (ALT) Chemistry and processing stability References less effect than AFB1. AFP1 is the main urinary metabolite of AFB1 with less toxicity. AFQ1 is the major metabolites from AFB1 and less toxic AFs are almost completely degraded at heating temperatures of 160°C and above. An average AFB1 reduction of 34% was exhibited in normal cooking of contaminated rice. The extrusion process reported reducing total AFs around 50%–95% or more Dry baking reduces Alternaria toxins better than wet baking. Alternariol monomethyl ether (AME) is the most stable compared to alternariol (AOH) and altenuene (ALT), with ALT being the least stable. In sunflower flour, rising temperature to 100°C barely degraded AOH and AME; keeping the temperature at 121°C for 60 min could decrease the toxins. However, the heat-treated material exhibited adverse effects in rats. Alternaria toxins are very stable and hard to break down in fruits and their processed products Cole and Cox (1981), Fernández-Cruz et al. (2010), Lee et al. (2015), Logrieco et al. (2009), Ostry (2008), and Siegel et al. (2010) Cytochalasins (cytochalasins group consists of cytochalasins E, cytochalasins G, cytochalasins A, cytochalasins B, cytochalasins F, cytochalasins H, cytochalasins C, and cytochalasins D); Cytochalasin A chaetoglobosin group consists of chaetoglobosin A, chaetoglobosin B, chaetoglobosin C, chaetoglobosin D, chaetoglobosin E, chaetoglobosin F, chaetoglobosin G, chaetoglobosin J, and chaetoglobosin K; deoxaphomin, proxiphomin, Cytochalasin C protophomin, zygosporin D, zygosporin E, zygosporin F, zygosporin G Rosellinia necutrix, Aspergillus clavatus, Nigrosabulum spp., Helminthosporium dermatiodeum, Phoma spp., Hormiscium spp., Phomopsis spp., Metarrhizium anisopliae, Zygosporium masonii, Chaetomium globosum, Diplodia macrospora Citrinin Penicillium citrinum Thorn, P. implicatum Biourge, P. lividum Westling, P. fellutanum Biourge, P. jenseni Zaleski, P. citrioviride Biourge, P. expansum, P. notatum, P. viridicatum Westling, P. steckii, Aspergillus terreus Thorn, A. niveus Blockwitz, A. candidus Citrinin Cytochalasins are characterized by the phenylalanine or tryptophan moiety attached to a perhydrosoindole moiety that is connected to a polyketide ring system comprising a lactone (cytochalasin A), a carbocyclic (cytochalasin C), and a cyclic carbonate (cytochalasin E) moiety. This group can be divided into several subgroups based on their chemical structures. Cytochalasins inhibit cytoplasmic cleavage that leads to massive polynucleate cells Citrinin is a quinone methide with two intramolecular hydrogen bonds. Citrinin can be broken down in acidic or alkaline solution and by heating. The degradations of citrinin in wet and dry conditions occur at >100 and >175°C, respectively. The concentration of citrinin in Monascus is reduced by half after 20 min of boiling, which indicates that citrinin is heat sensitive and unstable in an aqueous solution. This might be correlated with the low level of citrinin in processed foods Cole and Cox (1981), Fernández-Cruz et al. (2010), Lee et al. (2015), Logrieco et al. (2009), Ostry (2008), and Siegel et al. (2010) Ali et al. (2015b), Berde and €rmer (1978), Cole and Stu Cox (1981), Fajardo et al. (1995), Hamuel (2015), Krska et al. (2008), Merkel et al. (2012), Pierri et al. (1982), Silva et al. (2021), Wang et al. (2017), and Xu et al. (2006) Continued Table 3 Type, producing fungi, and stability of different mycotoxins—cont’d Mycotoxin Chemical structure Ergot alkaloids (Ergocornine, Ergocristine, α-Ergokryptine, Ergometrine, Ergosine, Ergotamine) Ergotamine Producing fungi Chemistry and processing stability References Hussein and Brasel (2001) Acremonium sp., Claviceps This group of mycotoxins purpurea has a comparable structure to lysergic acid diethylamide, a hallucinogenic medicine. There are three types of ergot alkaloids (EA), including clavine, watersoluble, and water-insoluble lysergic acid. EAs contain a C9]C10 double bond readily exhibits epimerization. The left-hand rotation is called ergopeptines (e.g., ergotamine), whereas the right-hand rotation is called ergopeptinine (e.g., ergotaminine). Epimerization of EA could occur at cold storage for 14 days in barley and rye extracts. EA could be decomposed by 2%–30%, and the epimeric ratio change during the baking of cookies. In general, EAs are quite stable as it still presents after cooking. For instance, processing flour into pan bread only gives a slight effect on EA. However, cooking in an alkali solution could enhance the removal of EA Fumonisins (fumonisin B1 (FB1), hydrolyzed (HBF1), fumonisin B2, fumonisin B3, 3-epi-fumonisin B3) F. verticillioides, F. proliferatum Fumonisin B1 Fumonisin B2 Fumonisin B3 Fumonisin possesses a longchain hydrocarbon comparable to that of sphingosine and sphinganine, thus determines their toxicity mode of action. The most toxic of the group is fumonisin B1 (FB1). Fumonisin removal depends on the time and temperature of processing. Though appearing to have heatstable properties, and even its bioavailability might increase during heat treatment, the fumonisin level seems to decline as the processing temperatures raised. In the cooking processes at 125°C or lower (i.e., baking and canning), the decrease of fumonisin is low (25%–30%), while the processes at 175°C and higher (i.e., frying and hot extrusion), losses are greater (90% or more). Besides heating, fumonisin levels could be reduced by baking, frying, roasting, nixtamalization, and extrusion cooking of foods Castells et al. (2005), Cole and Cox (1981), De Girolamo et al. (2016), Hussein and Brasel (2001), and Reddy et al. (2010) Continued Table 3 Type, producing fungi, and stability of different mycotoxins—cont’d Mycotoxin Chemical structure Fusarium cyclodepsipeptide beauvericins (BEAs), enniatins (ENNs) consist of enniatin A1 and enniatin B1, beauvenniatins (BEAs), and allobeauvericins (ALLOBEAs) Producing fungi BEAs, ENNs, BEAEs, and allobeauvericins (ALLOBEAs) consist of three N-methyl amino acids and three hydroxy acid groups. The biosynthesis of cyclodepsipeptides involves a multidomain nonribosomal peptide synthase (NRPS) which is consisted of enzymatic modules used to prolong the proteinogenic and nonproteinogenic amino acids, as well as carboxyl and hydroxy acids. ENNs could be reduced by thermal treatment and common industrial processes, including bread-making, beer-making, brewing or malting processes, and cooking. However, this mycotoxin cannot be completely removed during processing FUSA is a bicyclic Fusarium proliferatum, sesterterpene. It is F. subglutinans, F. antophilum, F. begoniae, synthesized from acetyl-CoA F. bulbicola, F. circinatum, subunits through the isoprenoid pathway via F. concentricum, common terpene F. succisae, F. udum intermediates. It can be degraded at 80, 120, and 180°C under wet conditions. However, the complete degradation occurred at 240°C only under dry conditions. Besides heating, FUSA can F. acuminatum, F. concentricum, F. proliferatum, F. verticillioides, F. oxysporum, F. tricinctum Beauvericins (BEA) Enniatin A1 (ENN A1) Fusaproliferin (FUSA) Fusaproliferin (FUSA) Chemistry and processing stability References Bushley and Turgeon (2010), Gallo et al. (2013), Herrmann et al. (1996), Huang et al. (2020), Meca et al. (2012), Schoevers et al. (2016), Tolosa et al. (2017), Urbaniak et al. (2020), and Žužek et al. (2016) Logrieco et al. (1996), Manetti et al. (1995), Moretti et al. (2007), Ritieni et al. (1997, 1999), and Santini et al. (1996) Fusarin C Fusarin C Moniliformin (MON) Moniliformin Ochratoxins (ochratoxin A, ochratoxin B, mellein, 4-hydroxymellein, 4-hydroxyochratoxin A, ochratoxin C) Ochratoxin A (OTA) Mellein be reduced by treating the samples with a saturated solution of dichloroisocyanuric acid F. verticillioides, Fusarins possess a polyketide F. oxysporum backbone and are distinguished by their different substitution at the 2-pyrrolidone part. Fusarin C was observed to be acutely toxic. The degradation of Fusarin C occurs rapidly as the pH increased and losses stability on light exposure Moniliformin consists of a Fusarium moniliforme sodium or potassium salt of Sheldon, F. moniliforme 1-hydroxycyclobut-1-ene-3, var. subglutimans, 4-dione. This toxin can F. graminearum, contaminate the next F. fusarioides, F. proliferatum, Gibberella batches of the plants and could remain in the soil for fujikuroi years A 27% reduction of MON in the extrusion process of corn grits was observed. Heat treatment for 60 min at 175° C combined with pH 10 also showed to reduce MON Aspergillus ochraceus Wilh. This group is characterized by the presence of 3,4(NRRL 3174) dihydro-3-methyl A. carbonarius, A. sulphureus (NRRL 4077), isocoumarin moiety attached to L-βA. melleus (NRRL 3519; 3520), A. oniki, Penicillium phenylalanine by an amide verrucosum, P. viridicatum bond. Ochratoxin A (OTA) and its methyl and ethyl (ATCC 18411) esters are the major toxic compounds from this group, while the rest of the members showed little or no toxicity Bryden et al. (2001), Desjardins (2006), Han et al. (2014), Leslie and Summerell (2006), and Zhu and Jeffrey (1992) Castells et al. (2005), Cole and Cox (1981), and Pineda-Valdes and Bullerman (2000) Castells et al. (2005), Cole and Cox (1981), and Skudamore and Banks (2004) Continued Table 3 Mycotoxin Type, producing fungi, and stability of different mycotoxins—cont’d Chemical structure Producing fungi Ochratoxin C Patulin Patulin Penicillium expansum (P. leucopus), P. patulum Bainier (P. urticae; P. griseofulvum), P. claviforme P. equinum (P. terrestre) P. novae-zeelandiae, P. lapidosum, P. granulatum, (P. divergens), P. lanosum, P. melinii, P. cyclopium, P. cyaneo-fulvum, P. roqueforti, Aspergillus clavatus, A. giganteus, A. terreus, Byssochlamys nivea (Gymnoascus spp.) Chemistry and processing stability References OTA seems to be heat stable up to 180°C, thus reduction of OTA in baked wheat products found to be 66%. While soaking and boiling beans reduced the amount of OTA up to 64% and 45%, respectively. The extrusion process on the wheat whole meal decreased 8%–39% of OTA. The removal of bran and offal part of the contaminated wheat could also reduce OTA in bread making, while the heat treatment during baking showed less effect on the toxin reduction Patulin is dissolved in water, alcohols, acetone, ethyl acetate, chloroform; while showing little solubility in ethyl ether, benzene; and hardly dissolved in petroleum ether. Though it is less stable in polar solvents, its biological potency is lost at alkaline pH. Yeast fermentation showed successful degradation of patulin. Physical selection by excluding rotten and lower quality apples can lessen the concentration of patulin. The presence of vitamin C can also gradually reduce patulin in apple juice Cole and Cox (1981), Peraica et al. (2002), Reddy et al. (2010), Scott (1998), and Skudamore and Banks (2004) Penicillic acid (PA) Penicillic acid PR-Imine PR-Imine Penicillium lividum, P. puberulum, P. griseum, P. simplicissimum, P. cyclopium, P. thomii P. roqueforti (P. suavolens), P. martensii, P. fenelliae, P. aurantio-virens, P. janthinellum, P. viridicatum, P. palitans, P. baarnense, P. madriti, P. lilacinum, P. canescens, P. chrysogenum, P. olivinoviride, Aspergillus ochraceus, A. sulphureus, A. melleus, A. scelrotiorum, A. alliaceus, A. ostianus, Paecilomyces ehrlichii Penicillium roqueforti Penicillic acid (PA) present in corns with high moisture preserved at cold temperatures. PA showed low oral toxicity and easily decomposed at 100°C Bianchini and Bullerman (2014), Cole and Cox (1981), Ismaiel and Papenbrock (2015), and Li et al. (2015) PR toxin consists of a bicyclic sesquiterpene with functional groups attached, namely, (CH3COO ), aldehyde (–CHO), and ketone complemented with two epoxide rings. Though having a toxic effect, PR toxin is unstable and could be transformed into less toxic derivatives like PR imine, PR amide, and/or PR acid, depending on the circumstances in the blue cheese making at the low O2 level Chang et al. (1993), Hymery et al. (2014), Siemens and Zawistowski (1993), and Wei et al. (1975) Continued Table 3 Mycotoxin Type, producing fungi, and stability of different mycotoxins—cont’d Chemical structure Roquefortines (chlororugulovasine A, chlororugulovasine Β, rugulovasine A, rugulovasine Β, fumigaclavine A (SM-2), roquefortine A (isofumigaclavine A), fumigaclavine Β, roquefortine Β (isofumigaclavine B), fumigaclavine C (SM-1), Roquefortine A roquefortine C) Rubratoxin (rubratoxin A, rubratoxin B) Rubratoxin A Sterigmatocystins (Sterigmatocystin, dihydrosterigmatocystin, O-methylsterigmatocystin, dihydro-Omethylsterigmatocystin, aspertoxin, 5-methoxysterigmatocystin, Sterimagtocystin dihydrodemethylsterigmatocystin) Producing fungi Chemistry and processing stability References Aspergillus fumigatus, Penicillium islandicum, P. concavorugulosum, P. roquefortii, Rhizopus arrhizus Roquefortines are alkaloids Cole and Cox (1981) usually found in the production of Roquefort cheese (or another variant of blue cheese) Penicillium rubrum Rubratoxins consist of complex nonadrides with lactone rings and anhydrides. Rubratoxin A is relatively stable for 2 years. Treatment with low pH at 60°C for 30 min showed no substantial degradation. However, degradation of rubratoxin B through yeast fermentation has been found to be effective Aspergillus versicolor (Vuill.) Sterigmatocystins (STs) are distinguished by a xanthone Tiraboschi, A. nidulans moiety attached to a (Eidam) Wint., Bipolaris dihydrofurano or sorokiniana, A. aurantiotetrahydrodurano moiety. hrunneus, A. quadrilineatus, A. ustus The variations in Bainier, A. variecolor, Also unsaturation of positions 2 and 3 of the difurano ring an intermediate in the biosynthesis of aflatoxins by system and substitution A. parasiticus and A. flavus group on positions 6, 7, and Bokhari and Aly (2009), Cole and Cox (1981), Septien et al. (1993), Takahashi et al. (1984), and Veršilovskis and Bartkevics (2012) Bokhari and Aly (2009), Cole and Cox (1981), Septien et al. (1993), Takahashi et al. (1984), and Veršilovskis and Bartkevics (2012) Dihydrosterimagtocystin Tremorgen (fumitremorgins consist of fumitremorgin A, fumitremorgin Β, fumitremorgin C (SM-Q), verruculogen, 15-acetoxy verruculogen, TR-2; Penitrems consist of penitrem A, and penitrem Β, lolitrem B, paspalitrems consist of paxilline, paspaline, paspalicine, paspalinine, paspalitrem A, paspalitrem Β and aflatrem; tryptoquivalines (tryptoquivaline, nortryptoquivalone (tryptoquivalone), nortryptoquivaline deoxytryptoquivaline deoxynortryptoquivalone and deoxynortryptoquivaline; tryptoquivaline E, tryptoquivaline F, tryptoquivaline G, tryptoquivaline H, tryptoquivaline I, tryptoquivaline J, tryptoquivaline Fumitremorgins A Penitrem A 10 of the xanthone system and/or a substitution on position 3 of the difurano system define the differences among STs. ST is the most acutely toxic and carcinogenic among the member. This toxin dissolves in chloroform and pyridine. Some processing steps like milling, baking, and roasting might lower the toxin concentration. However, in bread manufacturing ST was found to be stable Tremorgens with indole Penicillium spp., moiety from tryptophan can P. verruculosum, be divided into four P. crustosum, P. palitans, subgroups according to their P. puberulum, chemical structures, i.e., P. spinolosum, Claviceps spp., C. paspali, Aspergillus penitrems, fumitremorgins, paspalitrems, and spp., A. fumigatus, tryptoquivalines group. A. caespitosus, A. tenuis, Some metabolites that are A. lolii chemically correlated but do not exhibit tremorgenicity also comprised tremorgens, namely tetramic acid groups and other related nontremorgenic metabolites Cole and Cox (1981), ElBanna et al. (1983), Hussein and Brasel (2001), Reddy et al. (2010), Scott et al. (1984), and Visconti et al. (2004) Paspalitrem A Continued Table 3 Mycotoxin Type, producing fungi, and stability of different mycotoxins—cont’d Chemical structure L, tryptoquivaline M, and tryptoquivaline N; related nontremorgenic metabolites (deoxybrevianamide Ε, preechinulin, neoechinulin, neoechinulin Ε, neoechinulin D, cryptoechinulin G, neoechinulin Tryptoquivaline A, neoechinulin Β, neoechinulin C, isoechinulin A, isoechinulin Β, isoechinulin C, cryptoechinulin A, echinulin, and austamide; Tetramic acid group Tenuazonic acid Cyclopiazonic acid Cyclopiazonic acid Cyclopiazonic acid imine Bissecodehydrocyclopiazonic acid Trichothecenes (12,13epoxytrichothec-9-enes (trichodermol or roridin C, verrucarol, scirpentriol, T-2 tetraol, trichodermin, T2-toxin monoacetoxyscirpenol, diacetoxyscirpenol (DAS), neosolaniol, neosolaniol monoacetate, HT-2 toxin, diacetyl HT-2 toxin or T-2 triol, T-2 toxin, 4,15-diacetylverrucarol, 7αT-2 triol hydroxydiacetoxyscirpenol, 7α,8αdihydroxydiacetoxyscirpenol, Producing fungi Chemistry and processing stability References Myrothecium roridum, M. verrucaria, Fusarium roseum, F. equiseti, F. poae, F. sporotrichioides, F. sulphureum, F. tricinctum, F. sambucinum, F. lateritium, F. graminearum, F. semitectum, F. diversisporum, F. scirpi, F. solani, F. avenaceum, F. culmorum, F. langsethiae, Giberella This group is distinguished by sesquiterpenes with the presence of a 12,13epoxytrichothec-9-ene ring system with distinctive constituents on positions 3, 4, 7, 8, and 15. The members of this group are divided into four subgroups based on their chemical structure depend on the presence of carbonyl function at C-8 and macrocyclic ester bridge Bretz et al. (2005), Castells et al. (2005), Cole and Cox (1981), Krska et al. (2014), Kuchenbuch et al. (2018), and Ueno et al. (1983) calonectrin, 15-deacetylcalonectrin, acetyl T-2 toxin, crotocin, crotocol, trichothecene, 4β,8α-15triacetoxy-12,13epoxytrichothec-9-ene-3α-72diol, and triacetoxyscirpenol), T-2 tetraol 8-ketotrichothecenes (deoxynivalenol (DON), nivalenol (NIV), deoxynivalenol monoacetate, fusarenon-X, trichotecin, nivalenol diacetate, trichothecolone, and trichodermone), macrocyclic diester of verrucarol (satratoxin G, Deoxynivalenol (DON) satratoxin H, roridin A, roridin D, roridin E, roridin H, vertisporin, isororidin, 7β,8β-epoxyisororidin E, 7β,8β-epoxyroridin H, 7β,8β,20 ,30 -diepoxyroridin H, and baccharin) and macrocyclic triesters of verrucarol (verrucarin Nivalenol (NIV) A, verrucarin B, 20 dehydroverrucarin A, verrucarin J, and verrucarin K) Satratoxin H intricans, Trichoderma lignorum, T. viride, Calonectria nivalis, Trichothecium roseum, Cephalosporium crotocinigenum, Verticimonosporium diffractum, Cylindrocarpon spp., Phomopsis spp. T-2 toxin and DAS were considered the most toxic of the group. In contrast to DON and nivalenol that soluble in polar solvents, T-2 and DAS are soluble in nonpolar ones Trichothecene group also exhibits biological activities, namely antibacterial, antiviral, antifungal, cytostatic, insecticidal, phytotoxic, and animal and human toxicity to some extent. This group also shows cytotoxicity to mammalian cell culture Trichothecenes display inhibitory activity to protein and DNA synthesis. DON is observed to be highly stable during baking. DON degradation in pasta increases with an increasing percentage of water used. The extrusion process reported reducing 42%– 99.5% DON. Nivalenol (NIV) could be degraded with long time heating at high temperatures Curtobacterium sp. strain 114-2 could transform T-2 toxin and HT-2 toxin into their less toxic derivative, T-2 triol. T-2 Degradation up to 45% in T-2 toxin and 20% in HT-2 toxin were observed in the thermal process of biscuit manufacturing Continued Table 3 Type, producing fungi, and stability of different mycotoxins—cont’d Mycotoxin Versicolorins (versicolorin A, versicolorin B, versicolorin C, averufin, norsolorinic acid, versiconal hemiacetal acetate, versiconol acetate, versiconol, nidurufin, dimethylnidurufin, aversin, O-methylaversin) Chemical structure Producing fungi Aspergillus versicolor, A. parasiticus, A. ustus, A. nidulans Versicolorin A Versicolorin C Versiconol acetate Viomellein Aspergillus sulphureus, A. melleus, A. ochraceus, Penicillium viridicatum, P. cyclopium Chemistry and processing stability References Versicolorin exhibits little or Cole and Cox (1981) no acute toxicity to vertebrae. The presence of dhydrofurano and tetrahydrofuran moieties is similar to that of aflatoxins and sterigmatocystins. The number and position of substituents on the anthraquinone ring distinguished the members in this group. Some members of versicolorins are also mentioned as precursors of aflatoxins (e.g., versicolorin C, norsolorinic acid, averufin, versiconal hemiacetal acetate, versiconol acetate, versiconol, nidurufin, dimethylnidurufin, aversin, O-methylaversin) Cole and Cox (1981) Viomellein Xanthomegnin Xanthomegnin Aspergillus ochraceus, A. melleus, A. sulphureus, Penicillium viridicatum, P. cyclopium, Trichophyton rubrum, T. megnini, T. violaceum, Microsporum cookei Xanthomegnin could cause Cole and Cox (1981) and nephropathy and mortality Gupta et al. (2000) in animals. This toxin gives of red color underneath T. rubrum culture. The presence of this toxin can be found in infected skin and nails Zearalenone (ZEA) Zearalenone (ZEA) Fusarium roseum (F. graminearum, Gibberella zeae) F. tricinctum F. lateritium F. oxysporum F. culmorum F. moniliforme F. equiseti F. gibbosum F. avenaceum F. nivale F. sambucinum var. Coeruleum Zearalenone (ZEA) could affect the female reproduction system, namely hyperestrogenism, as well as males. ZEA is thermally stable, but extrusion cooking of cereals found to partially degrade ZEA. Moreover, the extrusion process of corn grits could decrease 66%– 83% of ZEA Castells et al. (2005), Cole and Cox (1981), Gupta et al. (2018), and Numanoglu et al. (2012) 124 Current Developments in Biotechnology and Bioengineering Penicillium, Fusarium, and some Alternaria (Reddy et al., 2010). The major mycotoxins in foods were AFs, fumonisins (FMNs), zearalenone (ZEA), ochratoxin A (OTA), citrinin (CIT), and trichothecenes (TCT). Some members of TCTs are deoxynivalenol (DON) and nivalenol (NIV) (Shepard, 2006; Pickova et al., 2020). Mycotoxins can also become airborne spread, namely AFs (A. flavus, A. parasiticus), DON, and other TCTs (Fusarium spp.), ochratoxins (A. ochraceus), sterigmatocystin (A. versicolor), and satratoxins (S. chartarum) (Hintikka et al., 2006). Many mycotoxins tend to accumulate in the lipid fraction of animals or plants due to their lipophilic nature (Hussein and Brasel, 2001). AF is the most extensively studied mycotoxins, which could be found in many commodities. This toxin poses a threat as carcinogenic, teratogenic, genotoxic, immunosuppressive, acute diseases, and even mortality at certain doses and conditions; therefore, a strict rule was applied regarding AFs in international trade. The four major occurring AFs are AF B1, B2, G1, and G2. AFB1, known as the major AF, synthesized by the toxigenic strains and also the most potent carcinogenic. Some other AFs like AFB2a, AFG2a, AFQ1, and AFP1 are biotransformation derivatives from the major AFs (Table 3). A. flavus group, which potentials in producing AF, carries nor-1, aflR, and omtB genes, is responsible for AF metabolism (Rahayu et al., 2016). 3. Occurrence of mycotoxins in food Since decades ago, surveys related to mycotoxins contamination in some commodities have been conducted. Table 4 presents the occurrence of mycotoxin in various commodities, including cereals, legumes, fruits, spices, etc. As presented in Table 4, various commodities are infected by various toxins such as AF, ochratoxin, FMN, ZEA, DON, patulin, TCTs, and sterigmatocystin. AF, ochratoxin, and FMN are the most commonly found mycotoxins in those commodities. AF contaminations are found in the range of 0.26–1632 μg/kg. Meanwhile, the OTA and FMN are in the range of 0.03–907.5 μg/kg and 0.374–4537 μg/kg, respectively. According to RASSF, mycotoxin became the most reported hazard, with 534 notifications in 2019. Cereals are important carbohydrate and protein sources. Because they are highly nutritious, they are susceptible to mold growth and mycotoxin contamination. The mycotoxins in the cereals are dominated by DON, ZEA, and fumonisin, AF, and ochratoxin. These toxins are produced by Fusarium spp, Aspergillus spp, and Penicillium spp, which are commonly found in a warm-climate region. The mycotoxin could be produced at different stages, including vegetation of the plant by field fungi, harvesting, and storage of the plant by storage fungi. The mycotoxin contamination in the raw material can be transferred to the final product. The incidences global occurrence of DON, ZEA, and FMN in cereals ranging from 50% to 76%, 15% to 50%, 39% to 95%, respectively, were reported (Lee and Ryu, 2017). In addition, van der Fels-Klerx reported that the occurrence of DON in North West European cereal grains commodities in 11% samples were over the legal limit set by European Commission (van der Fels-Klerx et al., 2012). The co-occurrence of mycotoxin, in which one sample contains more than one type of toxin, is found in several Chapter 4 • Mycotoxins 125 Table 4 Occurrence of mycotoxin in various commodities. Commodities Type of mycotoxin Level (μg/kg) References Cereals Maize Rice grain Corn Cereals (raw materials and derived products) Wheat-based products Half maize, half rye bread Maize bread Wheat bread Maize flour Rye flour Wheat flour Corn Corn Maize grain Maize grits Maize flour Cornflakes Sweet maize Wheat grain Wheat flour Wheat Maize milling products Grains for human consumption Wheat milling products Bread and rolls Pasta Breakfast cereals Fine bakery wares Sweet corn Brewing barley Wheat Wheat and wheat flour Barley Oat Rye and rye flour Maize Biscuits Bread Pasta Breakfast cereals Brewing barley Aflatoxin B1 Aflatoxin B1 Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Fumonisins Fumonisin B1 Fumonisin B1 Fumonisin B1 Fumonisin B1 Fumonisin B1 Fumonisin B1 Fumonisin B1 Fumonisin B1 Zearalenone Zearalenone Zearalenone Zearalenone Zearalenone Zearalenone Zearalenone Zearalenone Zearalenone Zearalenone Deoxynivalenol Deoxynivalenol Deoxynivalenol Deoxynivalenol Deoxynivalenol Deoxynivalenol Deoxynivalenol Deoxynivalenol Deoxynivalenol Deoxynivalenol Deoxynivalenol 0.26 26–33 0.093 0.29 1.36–21.17 0.89 0.42 0.12 0.67–2.75 0.77 0.26–0.85 71,121 0.374 346.4 347.6 408.5 31.5 12.4 71.2 64.4 3049 14 5.7 13 5.2 5.8 5.7 7.7 4.8 100–2300 41,157 205 37 95 42 594 50.6 88.9 141.2 198.8 310–15,500 EFSA (2007) Serrano et al. (2012) Simatupang et al. (2014) SCOOP (2002a) Kumar et al. (2012) Paı́ga et al. (2012) Paı́ga et al. (2012) Paı́ga et al. (2012) Spahiu et al. (2018) Spahiu et al. (2018) Spahiu et al. (2018) Lee and Ryu (2017) Rahayu et al. (2015) SCOOP (2003) SCOOP (2003) SCOOP (2003) SCOOP (2003) SCOOP (2003) SCOOP (2003) SCOOP (2003) Lee and Ryu (2017) EFSA (2011) EFSA (2011) EFSA (2011) EFSA (2011) EFSA (2011) EFSA (2011) EFSA (2011) EFSA (2011) Piacentini et al. (2018) Ji et al. (2014) SCOOP (2003) SCOOP (2003) SCOOP (2003) SCOOP (2003) SCOOP (2003) SCOOP (2003) SCOOP (2003) SCOOP (2003) SCOOP (2003) Piacentini et al. (2018) Aflatoxin B1 Aflatoxin B1 Aflatoxin B1 1.46 22.2 0.95 EFSA (2007) EFSA (2007) EFSA (2007) Legumes Almonds Brazil nuts Hazelnuts Continued 126 Current Developments in Biotechnology and Bioengineering Table 4 Occurrence of mycotoxin in various commodities—cont’d Commodities Type of mycotoxin Level (μg/kg) References Cashews Peanuts Pistachios Soybean Soybean Aflatoxin B1 Aflatoxin B1 Aflatoxin B1 Ochratoxin A Fumonisin B1 0.42 1.93 16.8 0.045 0.067 EFSA (2007) EFSA (2007) EFSA (2007) Simatupang et al. (2014) Rahayu et al. (2015) Ochratoxin A Patulin Patulin Zearalenone 2.30 15.6 4.9 72 SCOOP (2002a) SCOOP (2002b) SCOOP (2002b) EFSA (2011) Aflatoxin B1 Aflatoxin B1 Aflatoxin B1 Aflatoxin B1 Aflatoxin B1 Aflatoxin B1 Aflatoxin B1 Aflatoxin B1 Aflatoxin B1 Aflatoxin B1 Aflatoxin B1 Aflatoxin B1 Aflatoxin B1 Aflatoxin B1 Aflatoxin B2 Aflatoxin B2 Aflatoxin B2 Aflatoxin B2 Aflatoxin B2 Aflatoxin B2 Aflatoxin G1 Aflatoxin G1 Aflatoxin G1 Aflatoxin G1 Aflatoxin G1 Aflatoxin G1 Aflatoxin G1 Aflatoxin G1 Aflatoxin G2 Aflatoxin G2 Aflatoxin G2 Aflatoxin G2 Aflatoxin G2 Ochratoxin A Ochratoxin A Ochratoxin A 1.46 1632.2 39.3–139.5 155.7 75.8 57.0 56.8 39.8 27.4 26.5 21.7 18.2 16.8 11.0 2.6–33.3 μ 9.9 2.5 2.3 1.7 1.6 318.1 157.5 41.2 31.5 12.9 10.5 8.1 7.0 45.4 16.0 7.6 1.5 0.4 1.15 907.5 177.4 EFSA (2007) Dharmaputra et al. (2015) Wikandari et al. (2020) Gambacorta et al. (2018) Zahra et al. (2018) Migahed et al. (2017) Migahed et al. (2017) Khazaeli et al. (2017) Migahed et al. (2017) Azzoune et al. (2015) Ali et al. (2015a) Migahed et al. (2017) Migahed et al. (2017) Ali et al. (2015a) Wikandari et al. (2020) Gambacorta et al. (2018) Migahed et al. (2017) Ali et al. (2015a) Ali et al. (2015a) Ali et al. (2015a) Gambacorta et al. (2018) Migahed et al. (2017) Migahed et al. (2017) Migahed et al. (2017) Migahed et al. (2017) Migahed et al. (2017) Migahed et al. (2017) Alsharif et al. (2019) Gambacorta et al. (2018) Migahed et al. (2017) Migahed et al. (2017) ray (2015) Karaaslan and Arslang ray (2015) Karaaslan and Arslang SCOOP (2002a) Manda et al. (2016) Gambacorta et al. (2018) Fruits Dried fruits Apple juice Apple puree Vegetable oils Spices Spices Nutmeg Chili Paprika Black pepper Licorice Black cumin Ginger Parsley Saffron Fennel Mustard Thyme Coriander Chili Paprika Parsley Fennel Turmeric Coriander Paprika Anise Thyme Black pepper Rosemary Mustard Parsley Chili Paprika Black pepper Mustard Chili Cinnamon Spices Chili Paprika Chapter 4 • Mycotoxins 127 Table 4 Occurrence of mycotoxin in various commodities—cont’d Commodities Type of mycotoxin Level (μg/kg) References Black pepper Cardamom Nutmeg Licorice Cumin Cinnamon Ginger Curry Turmeric Garlic white pepper Onion Garlic Mint Paprika Dawadawa Black pepper Thyme Licorice Nutmeg Onion Chili Paprika Dawadawa Paprika Licorice Paprika Dawadawa Paprika Paprika Dawadawa Thyme Licorice Oregano Paprika Thyme Black pepper Chili Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Fumonisin B1 Fumonisin B1 Fumonisin B1 Fumonisin B1 Fumonisin B1 Fumonisin B1 Fumonisin B1 Fumonisin B1 Fumonisin B1 Fumonisin B2 Fumonisin B2 Fumonisin B2 Fumonisin B2 Trichothecenes Trichothecenes Trichothecenes Trichothecenes Trichothecenes Zearalenone Zearalenone Zearalenone Zearalenone Sterigmatocystin Sterigmatocystin Sterigmatocystin Sterigmatocystin Sterigmatocystin 79.0 78.0 60.7 36.7 20.4 16.1 12.7 9.6 8.5 5.1 4.9 591.0 540.0 256.0 243.9 165.0 135.0 125.0 39.3 25.0 4537.0 425.0 176.9 170.0 59.8 11.0 243.9 32.0 27.1 53.6 86.0 209.0 8.8 28.0 18.0 14 49.0 32 Jacxsens and De Meulenaer (2016) Gherbawy and Shebany (2018) Ostry et al. (2015) Ostry et al. (2015) Ali et al. (2015a) Jalili (2016) Ostry et al. (2015) Ali et al. (2015a) Jalili (2016) El Darra et al. (2019) Nguegwouo et al. (2018) Motloung et al. (2018) Tonti et al. (2017) €rer Soyogul et al. (2016) Gu Gambacorta et al. (2018) Chilaka et al. (2018) Jacxsens and De Meulenaer (2016) €rer Soyogul et al. (2016) Gu Huang et al. (2018) Reinholds et al. (2017) Motloung et al. (2018) Motloung et al. (2018) Gambacorta et al. (2018) Chilaka et al. (2018) Gambacorta et al. (2018) Huang et al. (2018) Gambacorta et al. (2018) Chilaka et al. (2018) Gambacorta et al. (2018) Gambacorta et al. (2018) Chilaka et al. (2018) Reinholds et al. (2017) Huang et al. (2018) Reinholds et al. (2017) Motloung et al. (2018) Reinholds et al. (2017) Jacxsens and De Meulenaer (2016) Jacxsens and De Meulenaer (2016) Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Zearalenone Zearalenone 0.20 1.62 0.72 0.03 0.36 0.24 9.0 1.0 SCOOP (2002a) SCOOP (2002a) SCOOP (2002a) SCOOP (2002a) SCOOP (2002a) SCOOP (2002a) EFSA (2011) EFSA (2011) Others Meat products Green coffee Roasted coffee Beer Wine Cocoa and derived products Biscuits Beer 128 Current Developments in Biotechnology and Bioengineering FIG. 1 Mycotoxigenic fungi and their mycotoxin in different samples. samples. The mixture of toxins is reported to have additive or synergetic effects. Thus, they have a more adverse effect on human health (Alborch et al., 2012). Spices are important agricultural commodities, particularly in developing countries. Asia is the top spices producer, which contributes up to 95.1% of global spices (Thanushree et al., 2019). As presented in Table 4, spices contain a diverse type of mycotoxin at contamination levels ranging from 1.46 to 4537 μg/kg. The dominant mycotoxins in spices are AFs and ochratoxins. The incidence of mycotoxins in spices ranging from 47% to 100% (Zinedine et al., 2006). According to RASFF, from 2015 to 2019, mycotoxin notification of spices was in the third rank with 219 notifications. Among the notification, approximately 80.2% and 19.8% belong to AF and ochratoxin, respectively. The mycotoxin-contaminated spices were dominated by chili (51.3%) and nutmeg (20.5%). Table 4 indicates that the occurrence of mycotoxin contamination is relatively high in many types of spices as the maximum limits of total AF and ochratoxin A in spices are 10 and 15–80 μg/kg, respectively, according to EU legislation. The occurrence of mycotoxin is related to the presence of mycotoxigenic mold. Several fungi have been isolated and identified based on morphological and molecular characteristics. Key genes responsible for mycotoxin metabolisms are usually used in this study to confirm the potency of strains in mycotoxin metabolisms. Fig. 1 shows a picture of mold growth in media using the direct plating method, identified mold species, and mycotoxins. 3.1 International regulation Due to the harmful effect of mycotoxin both on human and animal health, many countries have established regulations related to mycotoxin in food and feed. In 1995, CODEX set a Chapter 4 • Mycotoxins 129 standard related to mycotoxin, including regulation of AFs, DON, ochratoxins, FMN, and patulin. The number of countries with known specific regulations on mycotoxin has significantly increased from 33 countries in 1981 to 100 countries (corresponding to 85% of world inhabitants) in 2003 (Van Egmond et al., 2007). In addition, the percentage of the inhabitants of each region with known regulation varies from 59% in Africa (15 countries), 88% in Asia (26 countries), 99% in Europe (39 countries), 91% in Latin America (19 countries) to 100% in North America (2 countries) (Food and Agriculture Organization, 2004). The regulation of mycotoxin varies between countries, commodities, and types of mycotoxin. The type of mycotoxins which are regulated includes AFs (B1, B2, G1, and G2), AF M1, TCTs (DON, diacetoxyscirpenol, T-2 toxin, and HT-2 toxin), FMNs (B1, B2, and B3), agaric acid, ergot alkaloids, OTA, patulin, phomopsins, sterigmatocystin, and ZEA (Van Egmond et al., 2007). The maximum limit of each type of mycotoxin is summarized in Table 5. Several factors considered in setting limits for mycotoxins include scientific factors and socioeconomic issues. The scientific factors are the availability of toxicological data and occurrence data and detailed knowledge about possibilities for sampling and analysis. Comparing the regulation in 1995, the regulations in 2003 cover more types of mycotoxins, commodities, and products, whereas the tolerance limits generally tend to decrease. Not only the maximum limit, but the regulation also covers more detailed information regarding official procedures for sampling and analytical methodology. The mycotoxin test procedures consist of several steps, including taking a given size of the sample from the lot, comminution, removal of subsample, mycotoxin extraction, and quantification. The size of the incremental sample depending on the weight of the lot. For instance, for a lot of cereals and cereals products less than 50 kg, the number of incremental samples is 3–100 with the weight of the aggregate sample of 1–10 kg. Meanwhile, for lot weight of 50 tons and 300 tons, the number of incremental samples is 100 with the weight of the aggregate sample of 10 kg (European Comission, 2014). There are several analytical techniques to determine the mycotoxin include fluorometry, ELISAspectrophotometry, GC-MS, GC, liquid chromatography (LC), thin-layer chromatography Table 5 Worldwide regulation limit of different types of mycotoxin (Food and Agriculture Organization, 2004). Type of mycotoxins Worldwide regulation limit (μg/kg) Limit by most countries (μg/kg) Aflatoxin B1 Aflatoxin total Aflatoxin M1 in milk Patulin in fruits Ochratoxin A in cereals Deoxynivalenol in wheat and cereals Zearalenone Fumonisin 1–20 0–35 0.05–15 5–100 3–50 300–2000 50–1000 1000–3000 2 (applied by 29 out of 61 countries) 4 (applied by 29 out of 76 countries) 0.05 (applied by 34 out of 60 countries) 50 (applied by 44 out of 48 countries) 5 (applied by 29 out of 37 countries) 750 (applied by 19 out of 37 countries) 1000 (applied by 8 out of 17 countries) 1000 (applied by 4 out of 6 countries) 130 Current Developments in Biotechnology and Bioengineering (TLC), and minicolumn chromatography. Among them, LC and TLC are the most frequently used for mycotoxin regulatory analysis (Food and Agriculture Organization, 2004). 4. Toxigenic fungi and factors affecting mycotoxins production Mold contamination and toxins production in agricultural commodities occur during preharvest, postharvest, storage, and supply chain. Fungal species that produces toxins are called toxigenic fungi. The phenotypic and metabolic plasticity of toxigenic fungi enables them to grow a various important crops and to thrive on different environmental conditions (Moretti et al., 2017). Most mold contaminants are present in agricultural products, i.e., in cereals (e.g., maize and rice), legumes (e.g., peanut and soybean), coffee, cocoa, and spices (e.g., nutmeg and chili). Moreover, molds can also be found to contaminate marine products such as dried salted fish. Some molds contaminated certain agricultural commodities over others due to the differences in the substrate types. For example, peanuts were highly contaminated by AF producer Aspergillus flavus, while maize beside A. flavus was also contaminated by Fusarium. Ochratoxin producing A. niger was dominant in cocoa and coffee beans. Various classifications are used in categorizing factors that affect the incidence of mycotoxigenic mold and mycotoxins production in the food chain. These factors are divided into three main groups, i.e., intrinsic factor and extrinsic factor. However, the human factor is also very important, particularly the awareness related to mycotoxins and their hazards. 4.1 Intrinsic factors Intrinsic factors are those inherent to the type and properties of foods or the substrate for fungal growth that can be divided into three categories: chemical, biological, and physical factors. Chemical factors involve moisture content, substrate type, plant type, and nutrient composition. Biological factor comprises the structure and defense mechanism of the plant. Meanwhile, physical factors include water activity, pH, and activity of electrons (Eh). Biosynthesis of mycotoxins can be derived through polyketide route (e.g., AFs and patulin), terpene route (e.g., TCTs) or amino acids (e.g., ergot toxins and gliotoxins) among others. Mycotoxins are secondary metabolites that are by-products of secondary metabolism or biosynthesis. Generally, fungi are likely to perform secondary metabolism when they are in suboptimal conditions, because in optimal condition the fungi would perform their primary metabolism (Steyn, 1980). Casquete et al. (2017) observed that the AFs production started around 2 days after the end of lag phase. In our study, after 10 days of inoculation on soybean, A. flavus could produce AFs B1 between 12 and 381 ng/g at 20°C, 1 to 935 ng/g at 30°C, and from not detected to 60 ng/g at 40°C with humidity range of 70%–90%. At 10 day of 20, 30, and 40°C of 80%– 90%, sporulation has been occurred (observed in our research, data not reported in the journal). The 10 days growth was at the maximum growth stage before entering the lag Chapter 4 • Mycotoxins 131 phase (Pratiwi et al., 2015). Other study also showed that the 10-day growth was at the maximum stage of the fungal growth (Ahmed et al., 2016), this condition give a fact that AF B1 production can be easily observed at the fungal maximum growth. It is important to note that the mycotoxins production depends on several factors, such as pH, temperature and water activity. For instance, at room temperature (25°C) with water activity of 0.95 and pH 5, the AFs started to be produced around 2 days after lag phase. However, under similar condition, no mycotoxins are detected at temperature of 15°C (Casquete et al., 2017). Other factors should be considered in mycotoxin production, namely microbial interaction, growth factors, growth kinetics, and toxins concentrations. 4.2 Extrinsic factors The extrinsic factors affecting mycotoxin production include duration of fungal growth, aeration, humidity, temperature, storage conditions (Ashiq, 2015) soil condition (type of soil and fungal diversity), climates (dry or rainy season), and agricultural treatment (fertilization, irrigation, plant density, and time of harvest). In addition, stress factors such as lack of water, insect infestation, and other pests’ attack could trigger the formation of toxins (Sanchis and Magan, 2004). Each fungal species requires different optimum conditions for growth and mycotoxin production (Council for Agricultural Sciences and Technology (CAST), 2003). However, in general, hot and humid are the significant factors contributing to mycotoxin production (Ashiq, 2015). 5. Prevention and reduction of mycotoxins Codex Alimentarius prepares general guidelines or standard operating procedures for good farming and livestock practices, good harvest and post-harvest practices, good storage practices, as well as good manufacturing practices (GMPs). These good practices were socialized to farmers, retailers, and food processors through training on management control of AF. This management control was based on real problems identified starting from the farm, preharvest, harvest, postharvest, storage, processing up to market or consumers (food safety from farm to table). The most effective effort to control mycotoxins contamination in the food process is the use of good raw material, as possible, free mycotoxins, which can be obtained with appropriate pre- and postharvest handling. Implementation of good practices should be done at every stage of the supply chain. Haque et al. also had proposed a control strategy for mycotoxin contamination (Haque et al., 2020). Each mycotoxin type might require different risk management strategies, particularly on preharvest and dietary levels. Hazard analysis and critical control point (HACCP) requirements must be complied with to ensure the safety of the final product. The implementation of good practices should be accompanied by monitoring and evaluation. Several performance indicators are targeted, such as increasing the productivity, quality, and safety (low AF level) of the commodities, increasing market share, increasing awareness of the AF problem, and income of farmers. 132 Current Developments in Biotechnology and Bioengineering 5.1 Prevention of mycotoxins 5.1.1 Good agricultural practices Good agricultural practice (GAP) is a primary line against mycotoxin contamination in the field, followed by GMPs for handling and storage after harvesting. Codex Alimentarius has released a code of practice for the prevention and reduction of mycotoxin contamination in cereals in 2014 (Of et al., 2014). The guidance includes planting, preharvest, postharvest, storage, and transport from storage. For planting, it is recommended to rotate the crops, using fungal-resistant seeds or resistant varieties, analyzing the need to use fertilizer, removing old seeds, determining a suitable time for planting, preventing water stress in plants, and avoiding overcrowding of the plants. In addition, it is also suggested to begin with good soil preparation, application of competitive nontoxigenic fungal strains to the soil, adequate fertilizer and irrigation, and good pest and disease controls are required for healthy plants (Adeyeye, 2016; Dorner and Cole, 2002; Sarrocco et al., 2019). Insect infestation causes damage to the grain, thus accelerating mold infection and mycotoxin production. The application of insecticides and fungicides can prevent mold growth and mycotoxin production. In addition, weeds can also be vectors for mold, especially soil-borne pathogens such as F. graminearum and F. moniliforme. Weeds can be mechanically removed using herbicides or other safer ways. During preharvest, it is recommended to apply GAP to ensure that cultivars are adapted to local environments, to breed for insect resistance, to forecast mycotoxin formation, timely harvesting, to control weeds using mechanical, registered herbicides, or other safe methods, to minimize mechanical damage, to ensure the adequate supply of water, to ensure the equipment used for harvesting are functional, and to harvest at low moisture and full maturity. At this stage, irrigation and soil conditions should be controlled since drought stress and soil fertility greatly affect the intensity of mold attack and the production of mycotoxins. Managing the temperature and soil moisture plays an important role in the control of mycotoxin contamination. High water content and soil moisture are excellent for spore germination and mold proliferation. In peanut plants, drought stress in the reproductive stage is very sensitive to A. flavus and AF contamination. While in corn plants, drought stress and high humidity level are ideal for the occurrence of proliferation F. moniliforme and FMN production. Harvest is preferably conducted in the dry season and after the seed is fully matured. Young seeds or grains contain lots of water support for mold growth. Water content at harvest time should be adjusted to a certain range, for corn 23%–25%, sorghum 12%–17%, soybean 11%–15%, and groundnut 35%–50% (Maryam, 2006). Too early or too late harvest increased contamination of mold in agricultural produce. Equipment used during harvest or for transportation to the drying and storage area must be cleaned to minimize the population of insects and molds. In addition, it is suggested to implement GMP and HACCP, avoid mechanical damage, and cleaning the seed. Chapter 4 • Mycotoxins 133 Agricultural produce should be dried as soon as possible within a period of not more than 24–28 h after harvesting until it reaches adequate moisture content for storage to prevent the growth of mold and the production of mycotoxins. Drying can be done traditionally by employing sunlight, hanging the produce in the open air or indoors with little heating/curing, especially for products that are easily infected with molds, and by using a drying machine. Sortation is done through visual observation, by separating good produce from damaged ones. Produce could be mechanically damaged or deteriorated by insects, mold infections, or rotten. Agricultural produce should be dried to certain moisture content for storage. In temperate countries, the ideal moisture content is <13% for storage over 9 months, whereas for short storage, the moisture content can be up to 14%. However, for tropical countries with high temperatures and humidity, ideal moisture content ranges is from 7% to 9%, especially for commodities stored for more than 3 months (Maryam, 2006). The agricultural product is stored in a storage warehouse with good air circulation, where possible, temperature and humidity are measured regularly during the storage period. An increase in temperature of 2–3°C may indicate the presence of mold or insect infestation. For packaged products, it is preferable to use packs that have pores for air circulation and are placed on a board. For transportation, transport containers should be dry and sterile from fungi, insects, or other contaminants, use airtight containers to avoid additional moisture, and use insect and rodent-proof containers. 5.1.2 Controlling the fungi growth and the storage condition Controlling the toxigenic fungi growth can be conducted by controlling the temperature and humidity of storage conditions. A combination of storage temperature and humidity could exhibit different effects on fungal growth and mycotoxins production. However, the soybeans storage at a temperature of 30°C and 70% humidity is more effective in inhibiting the toxigenic Aspergillus flavus growth and its AF B1 production (the most toxic AF) than storage at 20°C and 70% humidity. Storage at 30°C cannot be combined with humidity above 70% (80% or 90%) due to the rapid fungi growth and increasing mycotoxin production. For the same reason, 90% of humidity cannot be applied at any storage temperatures (20–40°C) (Pratiwi et al., 2015). However, storage at 40°C at any humidity (70%–90%) could totally inhibit the growth of Fusarium verticillioides on maize and soybeans, and only storage at 20–30°C temperatures and 90% humidity allowed FMN B1 production from Fusarium verticillioides growth in maize and soybeans (Rahayu et al., 2015). To avoid OTA, production in maize and soybeans from the growth of toxigenic Aspergillus ochraceus, storage at 70%–80% and a temperature of 30°C should be applied (Simatupang et al., 2014). This is because storage at 20°C at any humidity (70%–90%) could allow the OTA production. 5.1.3 Networking Networking between academia (researchers), government, businesses (factories, traders, retailers, collectors), as well as communities, i.e., farmers, should be established to run the 134 Current Developments in Biotechnology and Bioengineering program on mycotoxin prevention and control. Through an integrated program, every strategy is discussed and set up based on the occurrence of mold, mycotoxin contaminations, and their impact on health and the economy. The researchers could develop a low-cost and portable device for rapid detection of mycotoxin and reduction method of mycotoxin level. The researchers or academia could conduct training and supervising to educate and monitor good practices and raise awareness for local farmers and small traders. The government, through relevant agencies, monitors the quality of every food product in the market. In addition, the government could also provide incentives for farmers to apply GAP and tax reduction for food business who applies GMP. The active role of the food industry is needed to provide appropriate incentives so that farmers are motivated to supply high-quality raw materials. 5.2 Reduction of mycotoxins Reduction of mycotoxins can be applied by the reduction of fungal infection or by the reduction of mycotoxins which are already present in foods. Physical, biological, and chemical methods for toxigenic fungi growth inhibition or mycotoxin reduction are described below. Each method has advantages and disadvantages. The advantage is mainly to obtain foods with mycotoxin levels below regulation limits. Meanwhile, the disadvantages are regarding the safety of applied methods, loss of food nutritional value, and reduction in food palatability. 5.2.1 Reduction by physical methods The common physical method to reduce mycotoxins in seeds or kernels is by sorting the damaged seeds and kernels (Stasiewicz et al., 2017). Seeds with mold, broken parts, discoloration, and fluorescence appeared under UV 365 nm are discarded. Physical methods by the use of adsorbents that could be applied for a higher reduction of mycotoxins have been applied for animal feed to bind mycotoxins in the gastrointestinal tract of animals in order to decrease the bioavailability of mycotoxins in feed. However, these physical methods could reduce mycotoxins in liquid foods. Single adsorbent that is a nanocomposite of magnetic graphene oxide and chitosan has been proven to effectively adsorb more than 60% of AF B1, ZEA, and OTA at pH 5 with an initial concentration of 50 ng/L mycotoxins (adsorbed by 0.03 g of adsorbent) (Pirouz et al., 2018). Another adsorbent, such as clay named sepiolite, could adsorb up to 60 g/kg of AF B1 in aqueous cornmeal, which was more effective than other clay, named montmorillonites. Activated charcoal could also reduce more than 70% of mycotoxins from Fusarium sp., such as ZEA and DON (Sabater-Vilar et al., 2007). Another physical method to reduce mycotoxins in food is by irradiation using a UV light for several hours. UV-B light is more effective than UV-A light to reduce AF B1 production by Aspergillus parasiticus. However, UV-A light is more effective than UV-B light to reduce OTA production by Aspergillus carbonarius in fruit (grape) and nut (pistachio) with applying 16 h/day of irradiation for 2–3 weeks (Garcı́a-Cela et al., 2015). In addition, the use of UV-C light for 3 h could reduce AFs, OTA, DON, and ZEA in rice grain varieties (Ferreira et al., 2021). Chapter 4 • Mycotoxins 135 Gamma-ray irradiation applied at a dose of 30 kGy to a dry food could reduce OTA. Nevertheless, the reduction was relatively low (around 20% of OTA). The irradiation to wet food was also not effectively applied (Calado et al., 2018). However, the dose of gamma-ray irradiation could reduce about 50% of OTA and AF B1 in peppers ( Jalili et al., 2012). Another physical method by hydrothermal has been tried to reduce almost 50% of AFs and FMN B1 in cornmeal (Massarolo et al., 2020). The hydrothermal applied was at 120°C for 40 min. However, the temperatures of the food ranged from 70 to 85°C. Thermal processing, like roasting, also reduces some mycotoxins to a certain extent, though most mycotoxins are stable under heating under 80–121°C (Ashiq, 2015). Mycotoxins are generally heat-stable; therefore, heating treatments, such as boiling, roasting, and frying, could not destroy all the mycotoxins which already exist in raw material. 5.2.2 Reduction by biological methods The reduction of mycotoxins in food by microorganisms has been shown by the actions of fungi, yeasts, and bacteria. Through the microbial growths on food, the microbes could degrade the different mycotoxins in food. Fermentation on peanut products contaminated by AFs had been attempted to reduce the toxins without significant change in nutritive and physical properties. However, this method can alter the food characteristics, thus affecting consumer preference. AFs could be reduced by the growth of Aspergillus sp., Rhizopus sp., Mucor sp., and Penicillium sp. as reviewed by Ji et al. (2016). Rhizopus sp. also could reduce ZEA ( Ji et al., 2016). A yeast (Saccharomyces cerevisiae) could inhibit in general more than 40% of the growth of toxigenic Aspergillus flavus, Aspergillus ochraceus, Fusarium verticillioides in maize and soybeans (Rahayu et al., 2015). Bacteria, through lactic acid fermentation, could effectively reduce AFs and FMNs in maize grain and its porridge product (Ademola et al., 2021); also, one paper reviewed that bacteria from Bacillus sp. and Actinomycetales could reduce AFs and DON ( Ji et al., 2016). Another biological method applied is by using enzymes that can change the chemical structure of mycotoxins into non- or less-toxic ones. This method could be a choice for the reduction of mycotoxins in grains. Acetylation or glycosylation by enzymes could alter DON into a non- or less toxic compound, as reviewed by Ji et al. (2016). Application of laccase enzyme produced by several microorganisms, applied for several hours, could degrade more than 60% of AFs ( Ji et al., 2016). 5.2.3 Reduction by chemical methods Chemical methods used for mycotoxins reduction are by using certain acids, alkalis, oxidizing agents, and ozonation, as reviewed by Marshall et al. (2020). In the case of maize, nixtamalization during processing could significantly reduce AF in the raw material. Ozonation at concentrations at around 20–50 mg/L for about 1 h could reduce more than 50% of AFs in cereals. The antifungal application can inhibit the growth of toxigenic fungi as well as their mycotoxin production. An example of the application of bacillomycin D at 75 μg/g wheat to F. graminearum can effectively inhibit more than 90% of the mold growth and reduce more than 70% of DON production (Sun et al., 2018). 136 Current Developments in Biotechnology and Bioengineering The use of a spice essential oil in the active packaging of food to inhibit the growth of mycotoxin-producing fungi or to inhibit mycotoxin production during mold growth has been proven by a laboratory experiment. The minimum inhibitory concentration of 2% of cinnamon essential oil was proven for the reduction, and at 4% of the oil in the packaging film could act as fungicidal (Manso et al., 2014). 6. Detection and determination of mycotoxins Detection and determination of mycotoxins are reported using spectroscopic methods and chromatographic methods. Among spectroscopic methods, the use of UV-vis spectrophotometer and fluorometer is commonly linked to ELISA; meanwhile, for chromatographic methods, the use of LC method is more common than gas chromatography method and thin-layer chromatography method (Shi et al., 2018). The LC method with fluorescence and with mass spectrometry detection could reach the sub ppb level of mycotoxin detection. One paper had reviewed some techniques in mycotoxins analysis (Alshannaq and Yu, 2017). 6.1 Detection and determination by the spectrometric method The spectroscopic method is coupled with the ELISA method, allowing analysis done in less than 30 min since a sample weighed. ELISA reader used at the end of the analysis to measure the absorbance is actually a spectrophotometer, either a UV-vis spectrophotometer or a fluorometer (Zheng et al., 2006). ELISA-spectrometric method for analysis of AFs total in corn has a detection limit at 2.5 ppb, with an accuracy of 94%–123% and precision of 5%–16%, using visible wavelength detection at 450 nm in a UV-vis spectrophotometer or UV-vis-spectrometer. Immunoaffinity column (IAC) cleans-up spectrometric method for AF analysis in corn with detection by using a fluorometer can detect at a lower concentration than that using UV-vis spectrophotometer. The method could possibly detect the aflatoxin as low as 1 ppb, with an accuracy range of 105%–123% and a precision range of 12%–17%. Meanwhile, the solid-phase extraction (SPE) clean-up-fluorometric method could detect AFs as low as 5 ppb, with an accuracy of 92%–102% and precision of 9%–20%, as reviewed by Zheng et al. (2006). The analysis time needed with the two fluorometric methods is less than 15 min. The disadvantage of all spectrometric methods described is that individual AFs (AFs B1, B2, G1, and G2) cannot be determined. The analysis results are expressed as total AFs. The spectrometric method could also be applied to different mycotoxins, with different ELISA-spectrometric methods (Zhan et al., 2016). 6.2 Detection and determination by chromatographic method Determination of individual AFs (AFs B1, B2, G1, and G2), OTA and FMN B1 in cereal and legume using HPLC-fluorescence, with detection limits at subppb levels, has been reported by Arzandeh et al. (2010), Pratiwi et al. (2015), Rahayu et al. (2015), and Chapter 4 • Mycotoxins 137 Simatupang et al. (2014). The determination involved the use of different IAC columns for sample clean-up in the sample preparation step prior to HPLC analysis. Fluorescence detection was set at excitation 365 nm and emission 435 nm for AFs analysis, excitation 333 nm and emission 477 nm for OTA analysis, and at excitation 335 and emission 440 nm for FMN B1 analysis. For simultaneous different mycotoxins analysis, HPLC or UHPLC with mass spectrometric (MS) detection (using double MS or tandem MS), called as LC-MS-MS method, can be conducted (Shi et al., 2018), without any derivatization of analytes. The simultaneous analysis of mycotoxins in food by LC-MS-MS has been applied for multiple mycotoxins determination in cereals, fruits, and legumes. To operate the sophisticated instrument and to process the different signals (signals of chromatogram and signals of mass spectra), it needs a qualified operator. The instrument also has subppb levels of the detection limit for the multiple mycotoxins. Thus, this analytical technique is a cutting-edge method recently. 6.3 Advance technology There are several advanced technologies that could be developed to improve the detection of mycotoxin, thus increase the effectiveness of mycotoxin control in the future. A low-cost portable device for rapid detection, which is user-friendly and reliable, would help farmers and traders to identify the contaminated crops to prevent further spread of contamination both on-farm and off-farm. Another device that can be developed is GPS-based tools which could remotely map the infected crops, thus prevent further infection in the field. Predictive modeling of mycotoxins was also under development. Other innovative technologies, for example, the use of competitive filamentous fungi or other biological control, nanobiotechnology, antibodymediated technology, and genetic modification, can further be explored (Haque et al., 2020; Sarrocco et al., 2019). 7. Conclusions and perspectives Despite the wide application of filamentous fungi in the food industry, these fungi are able to produce mycotoxin in certain conditions, in which mycotoxins are detrimental to human health. However, more countries aware of these threats; therefore, the regulation regarding mycotoxin-contaminated food becomes more comprehensive (comprising type of mycotoxins, commodities, and sampling procedures), and the range of tolerable mycotoxin limit has been narrowed. To manage the mycotoxin threat, an integrated database to create harmonization between countries, development of rapid analysis to facilitate the detection of mycotoxin contamination in the food supply chain, implementation of GAP and GMP to have better control of mycotoxin in the future, and the networking and cooperation between stakeholders are needed. 138 Current Developments in Biotechnology and Bioengineering For sustained management control of mycotoxins along the food chain, there are several recommendations including integrated control system based on HACCP approach, implementation of GAP and GMP; implementation of good practices along the supply chain of agricultural products (on-farm, harvesting, drying, storage); incentive program to motivate farmers in increasing good practices to meet the quality and safety standards; improvement of awareness related to mycotoxin contaminants and health effects to the societies (farmers, producers, consumers), consistently and continuously; improvement the detection of mycotoxins, utilization of sensitive and reliable methods; risk assessment and enforcement of the existing legislation on food safety; research and development related to mycotoxins control in collaboration between university, government, and international parties; international conferences and workshops to increase knowledge and technical ability for the related societies; improvement of health and economic status could contribute could reduce the risk of mycotoxins contamination since the health status of an individual would influence the detoxification mechanism. 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Taherzadeha a SWE DISH C ENTRE FOR RE SOUR CE R ECOVE RY, UNIVERS ITY O F BORÅ S, BOR ÅS , SW EDEN b DE PARTME NT OF CE LL AND MOL ECULAR B IOLOGY & MICR OBI OL OGY, FACULT Y O F BIOLOGICAL SCIENCE AND TECHNOLOGY, UNI VERSITY O F ISFAHAN, ISFAHAN, IRAN c GUJARAT P OL L UT I ON CONTROL BOARD, GANDHINAGAR, GUJARAT, INDIA 1. Introduction The kingdom Fungi includes ubiquitous and adaptable microorganisms playing a predominant role in our life and has benefits in the fields of biotechnology, genetics, biochemistry, food industry, bioremediation, medical and plant mycology, environmental ecology, soil biology, and taxonomical studies. Fundamental information is required on how a fungus can be isolated, purified, classified, and employed for well-defined goals to achieve countless fungal applications. In this regard, knowledge of fungal classification, growth physiology, preservation, and growth monitoring in the lab will be supportive. In general, the classification of fungi is a very controversial task, and there are important arguments among taxonomists determining a phylogenetic definition based on the 18S rRNA gene and other scientists who rely on significant ecological, physiological, or morphological features. Latterly, closely related species may not discriminate easily, and the employed techniques may take more time to identify a new fungal taxon (Santos et al., 2010; Dube, 2013; Yakop et al., 2019). Moreover, phenotype-based identification may not ensure enough sensitivity and specificity for classifying a fungal taxon (Gherbawy and Voigt, 2010). Based on the latest molecular classification, eight phyla belong to the kingdom Fungi, including Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Glomeromycota, Microsporidia, Neocallimastigomycota, and Zygomycota (Dube, 2013; Yakop et al., 2019). Though, Hibbett et al. (2007) described the subkingdom Dikarya consisting of phyla Ascomycota and Basidiomycota. Regarding the phylum Zygomycota, several ambiguous points about groups positioned in this phylum caused some fungal classification systems not to accept it as a phylum (Hibbett et al., 2007; Moore et al., 2020). However, recent classification systems recognized it as a novel phylum. Among fungal phyla, the phylum Glomeromycota contains genera with symbiotic relationships with plants, while Neocallimastigomycota and Chytridiomycota include animal pathogens (Gherbawy and Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00014-4 Copyright © 2023 Elsevier Inc. All rights reserved. 149 150 Current Developments in Biotechnology and Bioengineering Voigt, 2010). Remarkably, most industrial filamentous fungal genera and species belong to the phyla Ascomycota, Basidiomycota, or Zygomycota (Moore et al., 2020). In the kingdom Fungi, the term “filamentous fungi” is used for those fungal species that produce filament-like hyphae. Nearly, for most the kingdom Fungi members, filamentous fungi can be referred to. This term has been applied in contradistinction to “yeasts” that are unicellular fungi. However, hyphal growth is the main characteristic of fungal cells that leads to mycelia formation in which water and nutrient transport, intercellular relation€ es and Fischer, 2006). ships, and aerial structures occur (Hawksworth et al., 1988; Ku The first step for using filamentous fungi in the various biotechnological processes is to isolate them from their natural habitats. After identification, it is necessary to maintain fungal isolates under appropriate conditions in protected places such as biological resource centers or culture collections (Dube, 2013; Benz and Schipper, 2020). Nowadays, the significance of filamentous fungi in biological processes is quite clear, and many approaches developed to monitor their growth under experimental or scale-up conditions. These procedures have significant effects on the yield and bio-economy of the processes using filamentous fungal isolates (Cairns et al., 2019). Comprehensively, filamentous fungi taxonomy, morphology, and physiology have allowed us to prepare optimal conditions in the bench and large scales to achieve high productivity and yield with the lowest costs. 2. Applications of filamentous fungi Filamentous fungi, the main functionalists of natural ecosystems, have attracted the attention of scientists to use them in bioprocessing and biotechnological applications. The ancient usage of filamentous fungi is concerned with man’s food. In Asia, several fermented foods and drinks are based on filamentous fungi such as Aspergillus, Monascus, Mucor, Penicillium, and Rhizopus (Hawksworth et al., 1988; Hyde et al., 2019). Due to the remarkable metabolic and secretory capacity, robustness, and excessive diversity of filamentous fungi in the utilization of metabolites, they are noteworthy in the production of extraand intracellular enzymes, organic acids, polysaccharides, mycoproteins, vitamins, significant antibiotics, and bioactive compounds such as polyketids, isoprenoids, statins, € es and antitumors, plant growth hormones, steroids, alkaloids, etc. (Punt et al., 2002; Ku Fischer, 2006; Svahn et al., 2012; Hyde et al., 2019). For instance, organic acids produced by filamentous fungi such as Aspergillus (malic, citric, and itaconic acids) and Rhizopus (lactic acid) can be applied as food conservatives, pharmaceutical, and cosmetics solvents, chemical intermediates, or primary blocks of biodegradable polymers (Liaud et al., 2014). Genetic engineering, database mining, synthetic biology, whole-genome sequencing, “-omics” tools, and molecular approaches such as CRISPR/Cas9 have enabled us to discover and modify the genome of filamentous fungi to access the potential abilities of these microorganisms with viable and industrial relevance. Moreover, various molecular techniques make it possible to express extra copies of genes involved in the production of enzymes, metabolites, or other compounds (Punt et al., 2002; Richter et al., 2014; Chapter 5 • Sampling, preservation, and growth monitoring 151 Scazzocchio, 2014; Meyer et al., 2016). For example, Aspergillus nidulans was engineered to carry a gene cluster of Aspergillus terreus that encoded geodin, an antifungal compound (Richter et al., 2014). These developments and progress result in fungal strains with extra production capacity; however, genetically modified organisms (GMOs) have some disadvantages that may limit their usage (Punt et al., 2002; Karalis et al., 2020). In addition, filamentous fungi have been frequently used in waste treatment, biodegradation of environmental pollutants, detoxification, biofuels production, and transformation of organic materials (Hawksworth et al., 1988; Richter et al., 2014; Meyer et al., 2016). Recently, filamentous fungal biomass is considered a potential substance in the textile industry and the construction of buildings. Additionally, it is proven that fungal biomass can be a novel substitute for petroleum-based plastics (Cairns et al., 2019). Nevertheless, some species may be pathogenic for humans, plants, and animals (Santos et al., 2010; Hyde et al., 2019). 3. Isolation, identification, and sampling of filamentous fungi Filamentous fungi can be found everywhere, even in unexpected places. Humidity and darkness are favorable conditions for fungal growth as they can grow over or through any things, including man-made products, and cause damages to the equipment. Due to high metabolic capability, these microorganisms can grow on a wide variety of carbon and energy sources. In addition, filamentous fungi are resistant to dryness and high osmotic conditions (da Silva et al., 2003; Dube, 2013). Before doing any research on the fungi, it is typically required to isolate them from their habitats. Isolation, sampling, and cultivation methods mostly depend on the environments that filamentous fungi are living. Moreover, the fungal type is another significant factor for choosing isolation methods by mycologists. For example, isolation of parasitic strains needs some precautions and protective procedures to decrease the injury to the hosts. However, the isolation of saprophytes is easier than pathogenic strains. Furthermore, if filamentous fungi can produce spores, it would make them simple to be isolated, purified, and identified (Booth, 1971; Dube, 2013). 3.1 Isolation and purification of filamentous fungi It is not too difficult to culture filamentous fungi in the laboratory; however, it may sometimes not be so easy to grow special fungi. The simplest method used by researchers is slide culture which is very useful for isolation, purification, and monitoring of fungal culture. This method can be used to isolate and purify those fungi that do not form spores under laboratory conditions. To do slide culture, a suitable solid plate with 2 mm agar thickness can be applied. By means of a sterile scalpel, 6 mm squares should be cut and placed on a sterile glass slide. Every slide of squares can be inoculated, and agar should be covered with a sterile coverslip. Afterward, slide culture is ready to be set in an empty sterile petri dish containing a wet filter paper (soaked in water or 20% glycerol) 152 Current Developments in Biotechnology and Bioengineering in the bottom. These conditions provide a mounted chamber to support fungal growth under optimal temperature until mycelium formation. In addition to simplicity, one of the advantages of this technique is its adjustability based on laboratory conditions and type of fungal strain. After fungal growth and microscopic observation, the grown mycelia should be transferred to a fresh and adequate medium to prevent dryness, lack of nutrients, and contamination, because that agar square cannot support an optimal fungal growth (Booth, 1971; Su et al., 2012; Bhat, 2017). It should not be neglected that many factors in the laboratory may affect fungal growth, spore germination, or dormant state duration. Also, sometimes, spores may lack their germination ability, and under such situations, it is better to provide conditions as close as their natural habitats. Stimulators, such as heat, cold, light, acids, enzymes, etc., may aid in reducing spore dormancy and trigger its germination. For example, heat treatment could trigger spore germination in the genera Anthracobia, Aspergillus, Neurospora, € es and Fischer, 2006; Su et al., 2012). and Penicillium (Booth, 1971; Ku During the isolation and purification procedures, culture media compositions play a critical role in obtaining a pure culture of fungal strains. Cornmeal agar is one of the routine media that supports fungal growth well, and its preparation is easy and affordable. As well, sabouraud dextrose medium, oatmeal medium, malt extract medium, potato carrot medium, neopeptone-glucose-rose Bengal medium, and oomycete selective medium are generally used in the laboratory working with fungi (Kinsey et al., 1998). In an enrichment medium, mycelium production may exceed and suppress spore formation. Therefore, mycologists favor using oligotrophic media to stimulate sporulation. Some materials may be helpful to support fungal growth, their fructification, or spore formation in the agar plates, including wheat straw, herbaceous stems, filter paper, sawdust, etc. For instance, wheat straw is an appropriate composition for conidiophore formation in the genera Sporodesmium and Curvularia, while filter paper strips and sawdust are suitable €es and Fischer, 2006). for Chaetomium and Basidiomycetes, respectively (Booth, 1971; Ku Nevertheless, using a single medium cannot be aided in the isolation of all types of fungi, and the selection of media depends on various parameters such as type of fungus, taken samples, or media availability. Despite the easy and fast growth of fungi, some substances like heavy metals may be toxic to fungi. So, it is very important to prepare media carefully and avoid various contaminations. Moreover, considering origin sources and environmental conditions of isolates is required in most studies on fungi. Another factor that affects fungal growth is their requirement for high concentrations of carbohydrates. Xeromyces bisporus, one of the most xerophilic microorganisms, grows only on 60% sucrose. In addition to media compositions, the pH of the media directly affects fungal pigmentation under laboratory conditions (Booth, 1971; Leong et al., 2015). Ordinarily, isolation and identification of fungi are performed on solid media; however, liquid media can be a preferable choice when fungal mycelia are needed for physiological study, enzymes, antibiotics or pigments extraction, and bio-assay assessments. In most liquid media, the compositions are the same as solid media without agar. For enhanced Chapter 5 • Sampling, preservation, and growth monitoring 153 mycelial growth in the liquid media, flasks should be agitated continuously to distribute nutrients and oxygen throughout the medium. Moreover, using rotary shakers is preferred to magnetic stirrers and reciprocal shakers for this purpose. Although agitation is a significant factor in filamentous fungal growth, agitation speed and duration should be checked for every strain to obtain optimal conditions for higher productivity and growth based on € es and Fischer, the research goals (Hawksworth et al., 1988; El-Enshasy et al., 2006; Ku 2006). Aspergillus niger, one of the most well-known filamentous fungi, shows high morphological variations from pellet to filamentous forms in liquid media. The pelleted mass of A. niger can reduce fluid viscosity that consequences in better mass transfer in the liquid media and simple downstream processing; although this form of growth may limit penetration of oxygen and nutrients into the inner parts of the pellet. Particularly, agitation speed influences A. niger morphology and protein secretion. For instance, in batch culture of A. niger, intermediary and high agitation rates result in the highest recombinant protein productivity and improved extracellular enzyme production, respectively (Booth, 1971; El-Enshasy et al., 2006). Additionally, the agitation rate plays an essential role in the growth and productivity of Trichoderma reesei, which is a significant cell factory for cellulase production. This factor mainly impacts the scale-up of cellulase production in this filamentous fungus due to some struggles such as complex rheology of the liquid medium, increased viscosity, shear-thinning (non-Newtonian behavior of liquid medium), spatial heterogeneities, and low rate of oxygen mass transfer (Gabelle et al., 2012; Hardy et al., 2017). Above and beyond, liquid media can be employed to enrich samples with few filamentous fungi. For this purpose, an appropriate amount of a selected sample is added to an enriched medium such as sabouraud dextrose broth or potato carrot broth. After incubation, a subculture is plated onto the solid petri dish with the same compositions as the enriched medium until fungal colonies appear (Kinsey et al., 1998). 3.1.1 Spore isolation Asexual spore formation is a usual phenomenon among filamentous fungi. This reproductive state of the fungal cell is known as an anamorph. They can be formed in conidiophores, sporangia, acervuli, or pycnidia (Dube, 2013). Isolation of these spores is a proper way to grow them under laboratory conditions. Based on sporulation stages, fungal spores can be isolated by various methods. There are two stages in which spore isolation can be performed: (1) before spore germination and (2) during spore germination. The easiest technique is to use a moisturized chamber or petri dish to create an optimal environment for sporulation of the fungal samples. In this method, it is important to keep the chamber moist, so it is suggested to set some filter paper disks at the bottom of the chamber and moisturize it with sterile distilled water. Regularly water spraying prevents dryness €es and Fischer, 2006; Dube, 2013). After sporulaof the chamber and fungal samples (Ku tion, conidia, sporangia, and other forms can be simply picked up by a sterile needle and transferred to the sterile water droplets for further isolation and purification procedures (Booth, 1971; Dube, 2013). Recently, Libor et al. (2019) designed a new chamber for the 154 Current Developments in Biotechnology and Bioengineering isolation of rare fungi. The fungal one-step IsolatioN Device (FIND) is a suitable device for the isolation of marine and land species. This device is a multi-chambered microplate containing agar, and only a mycelium or spore can grow in this device under approving growth conditions (Libor et al., 2019). Mostly, microscopy is routinely used to observe and check filamentous fungal spores, mycelia, hyphae, and their growth stage. Direct observation can be performed by various techniques in the laboratory (Kinsey et al., 1998; Dube, 2013). In one of these techniques, a coverslip is placed on the surface of a solid petri dish containing a young culture of a filamentous fungal strain. By growth development, hyphae and mycelia can grow superficially on the coverslip, and conidia, sporangia, or pycnidia can be observed without difficulty under a light microscope. In addition, hanging drop culture and slide culture are particularly valuable to inspect spore formation in various filamentous fungal species such as the genus Glomerella (Booth, 1971; Dube, 2013). 3.1.2 Isolation of single spore Once the fungal strains are isolated, it is significant to ensure that the fungal cultures are pure and only one strain has been isolated. So, it is recommended to re-isolate the strain by the single spore technique. The dilution series is prepared from main spore suspension and 1 mL from appropriately diluted suspension spread in solid or semi-solid growth media to purify a single strain grows up from one single spore. Mostly, a final dilution of 1/10,000 is suggested in this procedure. It is preferred to check the number of spores in the suspension by a light microscope and counting slide. This technique is very suitable for spore-forming species except for the genera Trichoderma and Penicillium that freely sporulate and need more diluted suspensions. The capillary tube method, dry needle method, and mechanical method using the cutter, micro-manipulator, or blade are other techniques used by mycologists to isolate a single spore of various fungal species (Booth, € es and Fischer, 2006). 1971; Ku 3.1.3 Sampling and isolation of filamentous fungi from environments As it is known, fungi are present everywhere, even in Antarctica (Kostadinova et al., 2009), and by putting an open petri dish filled with nutrient agar, we can isolate air fungal spores. In this method, fast-growing filamentous fungi can prohibit other fungal species which grow slowly. Hence, it is significant to choose selective media or antifungal antibiotics to limit the fast-growing species like soil saprophytes in order to isolate the desired species € es and Fischer, 2006; Dube, 2013). (Ku One of the best environments for the isolation of filamentous fungi is soil. The most frequent filamentous fungal genera found in the different soils (forest soil, red soil, calcareous soil, etc.) belong to Aspergillus, Penicillium, Fusarium, and Trichoderma. Many mycologists developed effective methods to isolate fungal species, especially rare filamentous fungi, from various soils. As soils are very favorable environments for fungal growth, proper treatments should be performed to reduce the number of strains per gram of samples. For instance, by using 60% ethanol, most fungi of the soil samples can be limited, and alcohol-resistant Ascomycetes will be isolated (Booth, 1971; Dube, 2013; Yakop et al., 2019). Chapter 5 • Sampling, preservation, and growth monitoring 155 Furthermore, marine environments such as mangrove forests, estuarine, coastal areas, gulfs, or deep-sea vents are considered other diverse natural resources for isolation of marine filamentous fungi from various places, including sediments, water, grass, and marine animals such as cnidarian, sponges, corals, fish, or mollusk. It is well-defined that marine filamentous fungi possess high potentials such as diverse metabolic capability or resistance to high pressures and salt concentrations to be used for bioactive compounds production and degradation of the marine pollutants. Sampling from a specific environment can be done at different times (e.g., summer and winter). This mode helps the researchers know about ecological changes in the mycobiota of that location (Booth, 1971; Da Silva et al., 2008; Dube, 2013). Water distribution systems are other environments in which filamentous fungi can be isolated. The genera such as Trichoderma, Penicillium, Aspergillus, and Cladosporium are very common but other genera and species are found. The presence of filamentous fungi in the water may cause health concerns due to their ability for toxin production. Therefore, studying fungal communities, their occurrence, and frequency in the water distribution systems is critical. However, sampling, isolation, and enumeration of filamentous fungi from water are problematic. Water sampling should be done with sterile bottles containing sodium thiosulfate to neutralize probable chlorine. Samples can be kept at 4 °C or immediately be examined in the laboratory. Moreover, swab sampling can be performed whenever the interior part of a pipeline is subjected to study. In this method, more care should be taken to prevent cross-contamination. As well, inoculation of growth media with the swab can be done as quickly as possible. However, the examined locations in the swab sampling techniques are relatively small and may not represent the fungal communities of those areas (Kinsey et al., 1998). One of the widely used methods for the isolation of filamentous fungi from water is membrane filtration. Membrane filters can be transferred to the czapek medium or sabouraud dextrose medium. The first one contains sucrose and inorganic nitrogen, which is suitable for the species of Aspergillus and Penicillium. The second medium has a high concentration of glucose and organic nitrogen used for the samples with low nutrient contents. In this method, growth may occur rapidly, and solid media cover with fast-growing species. So, it is important to monitor the media in short intervals, e.g., every day till 1 week, and as the new colonies appear, they should be transferred to new media. Moreover, hydrophobic spores of Aspergillus and Penicillium may interact with the filter materials. Consequently, it is recommended to apply more than one method for sampling and isolation to obtain precise and reproducible results. In addition to membrane filtration, pour plate and spread plate methods are recommended to isolate and enumerate water fungal species. In the pour plate method, the sample is less likely to be lost on the spreader; however, the heat shock of the medium in this method may influence the results (Booth, 1971; Kinsey et al., 1998). In addition to isolation sites and their characteristics, several factors affect fungal growth. One of these factors that can participate in the isolation of fungal species is growth temperature; however, most fungi are mesophiles. Moreover, the psychrophilic fungi have an optimal growth temperature near the mesophilic range (Booth, 1971). 156 Current Developments in Biotechnology and Bioengineering Based on the research purposes and isolation places, isolation protocols can be different. For example, Da silva et al. (2008) isolated marine filamentous fungi from cnidarian samples. Taken samples were washed with sterile seawater to avoid external contamination and homogenized properly. Prepared samples were cultured onto the marine agar and malt extract agar supplemented with 3% NaCl to resemble seawater salt concentration. Streptomycin sulfate was added to both media to prevent bacterial growth. Sometimes, it is required to prepare decimal serial dilutions from the taken samples to prevent overlapping growth in the solid media (da Silva et al., 2003, 2008). Using chemicals, specific substrates, or agents is a practical way for the isolation of fungal strains with specific features such as filamentous fungal strains degrading lignocellulosic materials or oleaginous filamentous fungi. This old but effective method which is known as the baiting technique is preferred by most researchers working with fungi (Rynearson and Peterson, 1965; Muhsin and Hadi, 2002; Hamm et al., 2020). In this method, particular substrates are placed in the desired environment to enrich the fungal population. Over time, this bait can act as a trap to collect fungal strains, especially zoosporic filamentous fungi. Afterward, the bait samples will be ready for further isolation and purification of desired filamentous fungi (Booth, 1971; Simpanya and Baxter, 1996). Sometimes, in an environmental sample or a laboratory one, the number of fungal strains is low. Hence, it is necessary to increase fungal concentration by centrifugation. However, it should be taken care not to increase centrifugation speed as it may affect cell viability (Kinsey et al., 1998). 3.1.4 Sampling and isolation of filamentous fungi from fermented products Many foods, particularly oriental and Asian foods and beverages such as tempeh, sufu,  ketan, Oncom, Katsuobushi, Huan-jiu, Yakju and takju, hamanatto, Red kojic rice, Tape Sake, Chiang, etc., are produced due to fungal fermentation. These products can be subjected to the isolation of various filamentous fungal strains (Table 1). Some fungi of Mucorales, such as Rhizopus spp., Mucor indicus, and Mucor circinelloides, are common filamentous fungi used for this purpose. The order Eurotiales, including Penicillium and Aspergillus, is another common fungal order found in fermented foods. Furthermore, the most well-known genus of the order Sordariales, Neurospora, is broadly used in fermented food biotechnology (Nout and Aidoo, 2002; Dube, 2013; Chen et al., 2014). Isolation and identification of fungal communities from fermented foods and beverages can reveal notable information about the fermentation process, intermediate and final produced compounds, fungal relationships, and physiological aspects of fungi under specific conditions. However, culture-independent methods such as denaturing gradient gel electrophoresis (DGGE) can provide worthy information and complement other results obtained in the culture-dependent methods. It should be considered some fermented products have more than one fermentation stage, and the fungal communities and numbers may change depending on which stage has been considered (Chen et al., 2014). The first step in the isolation and enumeration of filamentous fungal strains from fermented products is to homogenize samples. Normal saline and peptone water can be used Chapter 5 • Sampling, preservation, and growth monitoring 157 Table 1 Fermented products that are resources for the isolation of particular filamentous fungi. Raw substances Soybeans or peanut solids Milk Fermented products Soybeans and wheat Omchom Blue-veined cheeses Camembert cheeses Soy sauce Rice Sake Soy beans Hamanatto Cassava fibers Coconut Soy beans Oncom Tempe bongkrek Tempe kedele Bonito fish Katsuobushi Rice Yakju Takju Huan-jiu Schochu Bai-jiu Sweet potato, barley, or rice Wheat, rice, or kaoliang (sorghum) Barley and millet Rice, wheat flour, and soy beans Soy beans, wheat Barley, wheat, rice, and soy beans Soy bean curd (tofu) Sorghum, wheat, barley, or peas Chiang Jnard Kochujang Chiang-yu Shi-tche Shoyu Chiang Taoco Miso Tou-fu-ru Jiu Isolated fungal species References Neurospora intermedia Penicillium roqueforti Dube (2013) and Chen et al. (2014) Penicillium camemberti Aspergillus flavus var. oryzae Aspergillus sojae A. flavus var. oryzae A. flavus var. oryzae Mucor spp. Neurospora sitophila Rhizopus spp. Rhizopus oligosporus Rhizopus oryzae Mucor indicus Aspergillus glaucus Aspergillus melleus Aspergillus reptans Aspergillus candidus A. flavus var. oryzae A. sojae Rhizopus sp. Aspergillus spp. Rhizopus spp. Mucor circinelloides R. oryzae A. flavus var. oryzae A. sojae A. flavus var. oryzae A. sojae R. oryzae A. flavus var. oryzae A. sojae R. oryzae Actinomucor sp. Mucor sp. Aspergillus sp. Mucor sp. Rhizopus sp. Trichoderma sp. Absidia sp. Monascus sp. Nout and Aidoo (2002) and Dube (2013) Nout and Aidoo (2002) Nout and Aidoo (2002) Continued 158 Current Developments in Biotechnology and Bioengineering Table 1 Fermented products that are resources for the isolation of particular filamentous fungi—cont’d Raw substances Rice Meat Fermented products Rice Red kojic rice Dry-fermented sausages Cheongju Cassava Gathot Isolated fungal species Monascus purpureus Penicillium nalgiovense biotype 6 Aspergillus sp. Lichtheimia sp. Rhizopus sp. Mucor sp. Acremonium charticola Rhizopus oryzae References Canel et al. (2013) Yang et al. (2011) Sugiharto et al. (2015) for sample homogenization. After the preparation of appropriate serial number dilution, solid plates contained suitable growth media are inoculated and incubated at the desired temperature until fungal growth appearing (Moreira et al., 2001; Nout and Aidoo, 2002; Lv et al., 2012). A list of common culture media used for isolation and enumeration of mycobiota from fermented products is shown in Table 2. 3.2 Sampling procedures for filamentous fungi From a microbiological point of view, fungal growth in the laboratory is not complicated as it can grow well in the simplest culture media. However, some fastidious filamentous fungi may not grow successfully under routine laboratory conditions. Sometimes, adopted fungi in the laboratory lose their sexual fructification ability (teleomorph state of a fungal cell) and may grow asexually. Moreover, some fungi do not show their natural morphology under laboratory conditions; therefore, growth monitoring and checking main morphological characteristics are substantial to ensure the strains’ viability, efficacy, and survival. In both bench scale and large scale experiments using fungi, sampling techniques are of special importance and reveals ongoing experiment conditions and fungal strain situations. Hence, it is a considerable concern to use different methods and techniques to check the fungal growth and prepare optimal conditions for each fungal isolate in the laboratory to observe every stage of their growth (Malik et al., 2013; Abdul Manan and Webb, 2018). Obviously, filamentous fungi show very different morphological features based on the growth conditions, and therefore, sampling techniques may vary regarding this issue. Moreover, based on the experiment types and final goal, sampling of a fungal culture can be altered from another culture. For instance, Abdul Manan and Webb (2018) took the sample from spore and grown mycelium on the solid medium to check the color of Aspergillus awamori and Aspergillus oryzae (Abdul Manan and Webb, 2018). Chapter 5 • Sampling, preservation, and growth monitoring 159 Table 2 The common culture media used for isolation and enumeration of mycobiota from fermented products. Culture media Rose Bengal agar Dichloran 18% glycerol agar Malt extract agar Czapek yeast extract agar Czapek-Dox Agar Potato dextrose agar Isolated filamentous fungal genera Fermented products Aspergillus Microascus Monascus Paecilomyces Penicillium Rhizopus Lichtheimia Rhizopus Mucor Syncephalastrum Aspergillus Penicillium Aspergillus Mucor Cladosporium Scopulariopsis Geotrichum Eurotium Mucor Rhizopus Paecilomyces Penicillium Aspergillus Monascus References Chinese Maotai-flavor liquor Chen et al. (2014) Nuruk Yang et al. (2011) and Carroll et al. (2017) Dried sausages made in Argentina Canel et al. (2013) Chinese yellow rice wine starter Lv et al. (2012) 3.3 Identification of filamentous fungi Identifying a fungal taxon is valuable for taxonomists and is required when a fungal strain has significant applications in the medicine, pharmaceutical, or food industries. Currently, polyphasic taxonomy can be a comprehensive attitude for providing high-quality and accurate data to aid taxonomists in the identification of a fungal taxon. In point of fact, the polyphasic approach combines old-fashioned and novel methods, and the results obtained from these methods give rise to almost complete identification of a fungal taxon (Malik et al., 2013). For the classification of a filamentous fungal taxon, molecular identification can be performed simultaneously with morphological identification. The selection of a molecular approach for initial identification depends on the working samples. For instance, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is used to identify natural morphotypes of ectomycorrhizal fungi. The conserved regions of the genome used in the molecular identification of filamentous fungi are internal transcribed 160 Current Developments in Biotechnology and Bioengineering spacers (ITS1 and ITS2) located in the ribosomal operon. ITS amplification and sequencing can be benefited in the taxonomic levels from family to species and preferred for lower rank levels in the fungal taxonomy (Martin and Rygiewicz, 2005; Gherbawy and Voigt, 2010). Furthermore, the small subunit of the 18S rDNA (SSU) and large subunit of the 28S rDNA (LSU) are commonly used in phylogenetic studies to classify taxa into the class, genus, and species levels. A full-length of SSU or a length of ca. 600 base pairs (bp) can be enough for taxonomic studies. But, it should be pointed out that in the SSU, variable domains do not provide sufficient information and, larger parts of the gene must be sequenced to gain more resolution for species identification. Besides, the SSU and LSU do not provide high resolution for lower rank levels of the taxa. In addition to ITS1, ITS2, SSU, and LSU, rpb1, rb2, tef, beta-tubulin, 5.8S rRNA genes, and many other protein-encoding genes can be used in filamentous fungal phylogeny. The 5.8S rDNA has a significant role in determining the relationship in the order level as it is small and does not have much variability (Gherbawy and Voigt, 2010). Moreover, repetitive genome sequences (rep) have been employed for the identification of Aspergillus species (Hansen et al., 2008). Various molecular approaches such as RFLP, random amplification of polymorphic DNA (RAPD), DNA fingerprinting, amplified fragment length polymorphism (AFLP), proteomics, whole-genome sequencing, and ribotyping are currently used in the polyphasic taxonomy, especially for the strains that cannot be easily described by traditional methods. In addition to molecular techniques, biochemical characteristics, and morphological features, spectral analysis such as matrix assisted laser desorption/ionization timeof-flight mass spectrometry (MALDI-TOF MS), flow cytometry, and Fourier-transform infrared (FTIR) are required for the rapid identification of filamentous fungi. However, identification based on morphological characteristics, particularly spore-containing structures, has remained valid for these microorganisms due to more distinguishing morphology than bacteria. Throughout, it is still arduous to identify filamentous fungi by only one approach (Santos et al., 2010; Malik et al., 2013; Bleichrodt and Read, 2019; Leite et al., 2020). 4. Preservation of filamentous fungi It is a fact that the nature of all organisms will change and their features and functions maybe gone astray with time and filamentous fungi are no exception. In general, the simplest solution for slowing down or stopping the biological clock is to decrease the temperature of biological materials and their surrounded environment. The too-low temperatures have been proved to provide a state that extends the viability and functionality of biological materials and microorganisms. Moreover, sublimation of water under vacuum conditions from viable microorganisms or biological materials in a frozen state is another technique that helps to maintain them for a long time without any damages. The first technique is known as cryopreservation which is widely used to store various types of microorganisms, and the latter is known as freeze-drying or lyophilization Chapter 5 • Sampling, preservation, and growth monitoring 161 method (Espinel-Ingroff et al., 2004; Ayala-Zermeño et al., 2017; Wolkers and Oldenhof, 2021). Based on the living cells and many other factors, preservation protocols should be adjusted. It is a common mistake that generalizes one preservation protocol to all cells or microorganisms. With increasing progress in biotechnology, genetic engineering, metabolic engineering, industrial microbiology, mycology, and teaching purposes, the need to preserve applicable microbial strains, especially filamentous fungi and yeasts, has been greater than before. The response to this need led to the establishment of microbial culture collections that are secure places for the long-term storage of all types of microorganisms, including filamentous fungal strains. These centers maintain microorganisms and provide microbial strains for researchers and validate authenticity, purity, and stability of microorganisms (Ayala-Zermeño et al., 2017; Castro-Rios and Bermeo-Escobar, 2021). In this regard, some filamentous fungi are too infrequent, and the re-isolation of them from the environment may not be affordable. Therefore, deposition of all isolates especially, rare filamentous fungi in the culture collections, is a necessity. Furthermore, it is an urge that associated researchers get sure about the safety and conservation of their fungal strains for further studies, as references strains or confirmation of the obtained results. Interestingly, most biochemical efforts had been made based on the strains, which are not accessible anymore (Hawksworth et al., 1988; Benz and Schipper, 2020; Wolkers and Oldenhof, 2021). In general, there are two usual types of conservation; short-term and long-term preservation. In the short-term preservation, fungal strains are maintained as actively growing form at room temperature or in the refrigerators. In the second type, fungal isolates can be preserved in very low temperatures in ultra-freezers or in liquid nitrogen tanks. Freezedrying can be applied for some filamentous fungi as a long-term method. Short-term preservation is considered a facile method that is applicable for ongoing research and investigations in the industries or at the universities. Overall, using an appropriate short-term or long-term method can diminish the risk of contamination, accidental loss, passage numbers, physiological changes, morphological alteration, and genetic variations. However, other factors such as growth temperature, oxygen requirement, humidity, or culture media compositions can affect the viability and survival of the fungal strains after preservation. These factors may vary from one strain to another one. The main requirement that should be considered carefully is a suitable and fresh culture of any strains unless mentioned. Because sometimes, old culture is needed to harvest resistant spores. Comprehensively, there is not only one successful protocol for the maintenance of all fungal strains, and it is preferred to use several methods based on the facilities, cell type, and laboratory infrastructures (Lakshman et al., 2018; Wolkers and Oldenhof, 2021). 4.1 Short-term preservation In short-term preservation techniques, cells are metabolically active, but the rate of metabolism has reduced, and the cells can be conserved for up to 1 year (Romano 162 Current Developments in Biotechnology and Bioengineering et al., 2005a,b; Al-Bedak et al., 2019; Ilyas and Soeka, 2019). Subculturing (serial transfer) of fungal strains is considered a short-term method. However, it is not a practical solution when there are many fungal strains in the laboratory or a culture collection. Also, this method is time-consuming and prone to trouble mistakes that may result in culture loss, pleomorphic growth, genetic instability and mutation, or productivity reduction (Homolka et al., 2001; Kitamoto et al., 2002; Ayala-Zermeño et al., 2017). Preservation of fungi in sterile distilled water at ambient temperature was introduced by Castellani in 1939 (Castellani, 1963) and confirmed by Borman et al. (2006) for longterm preservation (Borman et al., 2006). But it seemed this easy method could not ensure the viability and characteristics stability of all fungal strains. Therefore, other short-term preservation protocols such as preservation of fungal slants covered with mineral oil or paraffin (slackened technique) are suggested. These covered slants can be kept at the refrigerator (4 °C) (Kitamoto et al., 2002; Espinel-Ingroff et al., 2004; Voyron et al., 2009). Moreover, sterile saline, soil, silica gel, filter paper discs, cotton balls, woods, agar strips, plant tissue, sand, or grains have been used for the short-term preservation (less than 2 years) of filamentous fungi at room temperature or in the refrigerator. Silica gel is a suitable carrier and minimizing the metabolic activity rate of fungal cells, which allows them to be preserved (Ayala-Zermeño et al., 2017; Al-Bedak et al., 2019). This method can be employed for conidial filamentous fungi and some entomopathogenic strains (Ayala-Zermeño et al., 2017; Lakshman et al., 2018). Advisably, it is recommended not to use short-term preservation protocols for pathogenic filamentous fungi, particularly pathogens with biosafety level 3 (BSL-3) (Borman et al., 2006). The BSL-3 agents may cause serious diseases or be potentially lethal. They can transfer by air and infect humans via the inhalation route. Consequently, working with these agents necessitates specific procedures, practices, and different laboratories with particular equipment. The most important filamentous fungi with BSL-3 include Blastomyces dermatitidis, Coccidioides immitis, and Sporothrix schenckii species complex, which belong to the phylum Ascomycota (Chosewood and Wilson, 2020). 4.2 Long-term preservation In contrast to the short-term preservation methods, in long-term protocols, microbial cells are metabolically inactive. In this kind of preservation, cells can stay safe without any change for a long time, sometimes more than decades. Several protocols are classified as long-term preservation techniques. The most widely used ones include cryopreservation and freeze-drying, which are applicable for filamentous fungi. Cryopreservation is employed for most filamentous fungal strains, while few genera can be lyophilized (Voyron et al., 2009; Ilyas and Soeka, 2019; Wolkers and Oldenhof, 2021). It is expected, in long-term preservation, fungal cells will stay in a viable form for up to years if influencing parameters are kept constant during the preservation period. It means, if the samples are maintained in the liquid nitrogen ( 196 °C), no fluctuation should occur in the temperature. Therefore, it is critical to ensure fungal cells are preserved under controlled Chapter 5 • Sampling, preservation, and growth monitoring 163 conditions and checked regularly to assess viability and survival of preserved fungal strains (Lakshman et al., 2018; Wolkers and Oldenhof, 2021). 4.2.1 Cryopreservation of filamentous fungi Reducing the temperature of filamentous fungi decreases the metabolism rate of cells and may be lethal for them due to internal ice formation. For this reason, cryopreservation protocols should be customized for each filamentous fungal species to decrease damage possibility. In general, cryopreservation means the use of ultra-low temperatures for the maintenance of microbial cells. Ultra-low temperatures can be employed from 70 °C to 196 °C for spore-forming and non-sporulating filamentous fungi (Homolka et al., 2001; Castro-Rios and Bermeo-Escobar, 2021; Wolkers and Oldenhof, 2021). At first glance, it seems that cryopreservation is a simplistic method; however, many factors such as cell type, cell size, culture conditions, growth phase, equilibration time with cryoprotectant, cooling and warming rates, freezing temperature, storage duration, recovery conditions, etc., affect this technique and successful preservation (Ayala-Zermeño et al., 2017; Wolkers and Oldenhof, 2021). One of these factors is related to the choosing of chemicals and compounds that protect cells from freezing. These materials are known as cryoprotectants or cryoprotective agents (CPAs). The role of CPAs in the successful maintenance of filamentous fungi is undeniable. Any changes in the environmental factors of a living cell may result in a stress response. One of the main stresses that filamentous fungi may encounter is low temperature or freezing (Missous et al., 2007). A wide variety of cryoprotectants vary from carbohydrates to alcohol and other substances such as 1,2-ethanediol, dimethyl sulfoxide (DMSO), or polyvinyl pyrrolidine. Based on the properties of cryoprotectants, they can be classified into various groups; low-molecular-weight or high-molecular-weight, penetrating or non-penetrating (Hubálek, 2003). However, research findings and our personal experience have shown glycerol was a suitable slowly penetrating cryoprotectant for most filamentous fungi. It is shown, by using 10% glycerol, a high viability rate and low rate of phenotypic and genotypic changes were observed considerably (Kitamoto et al., 2002; Paul et al., 2015; Rohadi et al., 2020). In a protocol used by Paul et al. (2015), glycerol was added in a ratio of 1:1 to the cell suspension of filamentous fungi to preserve them at 4 °C (Paul et al., 2015). In addition to glycerol, DMSO is used as a rapidly penetrating cryoprotectant for filamentous fungi such as Neurospora crassa and some Basidiomycetes. But, many filamentous fungi are usually sensitive to high concentrations of DMSO. Other cryoprotectants such as serum, skim milk, polyvinylpyrrolidone, or a mixture of them with defined concentrations have been used for the preservation of filamentous fungi (Hubálek, 2003; Lakshman et al., 2018; Castro-Rios and Bermeo-Escobar, 2021). Table 3 shows a list of all cryoprotectants used for the cryopreservation of fungi (both yeasts and filamentous fungi). Other factors that may influence cryopreservation included cooling and warming rates. The optimal cooling rate occurs when the temperature of the sample decreases by 1 °C in a minute. In contrast, slow warming rates during thawing may cause the recrystallization of 164 Current Developments in Biotechnology and Bioengineering Table 3 Most and lowest frequent cryoprotectants and additives used for fungi cryopreservation (both yeasts and filamentous fungi) (Hubálek, 2003; Wolkers and Oldenhof, 2021). Compound Chemical group Frequency Glycerol DMSO Malt extract Blood serum Sucrose Terehalose Glucose Skim milk Ethylene glycol Methanol Gelatine Propylene glycol Ethanol Polyethylene glycol Polyvinyl alcohol Polyvinylpyrrolidone Peptone Yeast extract Trypticase soy Sorbitol Mannitol Dulcitol Inositol Dextran Hydroxyethyl starch Ficoll Lactose Polyol Organosulfur Reducing sugars Protein Disaccharide Most frequent Monosaccharide Lactose and protein Alcohol Protein Alcohol Lowest frequent Polyether Polymer Polymer Protein hydrolysate Yeast hydrolysate Enzymatic digest of soybean meal Sugar alcohol Carbocyclic sugar Polysaccharide Disaccharide ice. Therefore, for the high viability of fungal cells, it is recommended to cool samples gradually and thaw them rapidly (Rohadi et al., 2020; Wolkers and Oldenhof, 2021). Freezing temperatures that employed in the cryopreservation protocols may vary from 20 °C (common freezer), 70 °C (ultra-freezer), and 130 °C (vaporous nitrogen) to 196 °C (liquid nitrogen). The choice of these temperatures is related to fungal cell type, laboratory equipment, cryoprotectant type, etc. The 20 °C is usually used for short-term cryopreservation, and other temperatures are applied for long-term cryopreservation. The storage at 130 °C is preferable to 196 °C because in the vapor phase, the penetration of liquid nitrogen into the cryotubes and cryovials cannot occur, and the possibility of vial explosion after removing the vials from the storage tank decrease (Espinel-Ingroff et al., 2004; Wolkers and Oldenhof, 2021). However, regularly supplying liquid nitrogen and its high price are the disadvantages of this method. Also, working with liquid nitrogen Chapter 5 • Sampling, preservation, and growth monitoring 165 needs more care as it evaporates and replaces the oxygen content of the air as this phenomenon may cause anesthesia in lab experts. Hence, some laboratories and culture collections prefer to use ultra-freezers ( 70 °C) for work lot vials, while seed and master lot vials are kept in the liquid nitrogen tanks (Kitamoto et al., 2002; Lakshman et al., 2018). Cryopreservation of fungi started in American Type Culture Collection (ATCC) in the 1960s and the successful protocols widespread among other culture collections throughout the world. In general, a suspension of filamentous fungal mycelia or their spores with suitable cryoprotectant is aliquoted in the glass, cryovials, or cryotubes (Homolka et al., 2001; Espinel-Ingroff et al., 2004; Lakshman et al., 2018). In addition to mycelia or spores suspension, agar pieces cut from actively growing mycelia culture can be immersed in the appropriate cryoprotectant and preserved in ultra-low temperature (Homolka et al., 2001). In Fig. 1, an overview of filamentous fungi cryopreservation in the laboratory has been presented. Moreover, carrier beads made from polystyrene or porous ceramic have been used for some spore-forming filamentous fungi such as Aspergillus fumigatus. The porous shape of the beads facilitates filamentous fungi adhesion to the surface. Cellular adhesion may prevent the cells from freezing injury. Homolka et al. (2001) employed perlite carrier, a volcanic mineral, for cryopreservation of non-asexual spore-forming Basidiomycetes in liquid nitrogen. They tested this carrier on 60 filamentous fungal strains from 36 species, and no changes were observed after cryopreservation. However, cryopreservation of Basidiomycetes is not as easy as other fungal groups (Homolka et al., 2001, 2006; Voyron et al., 2009; Lakshman et al., 2018). In Table 4, a brief of some employed protocols for cryopreservation of filamentous fungi has been shown. In bead protocol, fungal cultures are mixed well with cryoprotectant suspension included sterile beads. Paying attention to the amount of cryoprotectant suspension and FIG. 1 A general overview of filamentous fungi cryopreservation steps. 166 Current Developments in Biotechnology and Bioengineering Table 4 Cryopreservation protocols and associated steps for conservation of filamentous fungi. Cooling program Sample form Cryoprotectant type and concentration (%) Agar plugs cut from active culture Cell suspension 5%, 10%, 15%, and 20% glycerol with basal sawdust medium 15% glycerol Agar plugs cut from active culture Grown fungal cells in cryotubes with perlite Cell suspension 10% glycerol Storage in References No cooling program applied Deep freezer Kitamoto et al. (2002) Cooling from 20 to 10 °C with a Peltier cooler-heater during 20–40 min 7/NDa 35/45–60 Deep freezer Liquid nitrogen tank Missous et al. (2007) Homolka et al. (2006) Deep freezer Voyron et al. (2009) Liquid nitrogen tank Deep freezer Ayala-Zermeño et al. (2017) Cooling with a rate of 1 °C/min to 70 °C by a rate-freezer 10% glycerol Room temperature/60 4/240 10% glycerol or 10% trehalose Agar plugs cut from active culture b Initial cooling temperature (° C)/duration (min) 5% glycerol Agar plugs cut from active culture Agar plugs cut from active culture Agar plugs cut from active culture a Pre-cooling temperature (° C)/duration (min) 10% glycerol 4/720 10% glycerol with 5% trehalose 4/144 20% glycerol with 20% skim milk 4/30 Cooling in Mr. Frostyb at 80/120 70 4/overnight 70/overnight Liquid nitrogen tank Ilyas and Soeka (2019) and Rohadi et al. (2020) Personal data ND, not determined. A freezing container contained within isopropyl alcohol to cool the samples near to theoretical cooling rate of 1 °C/min. bead numbers is very critical. If a proportional amount of cryoprotectant suspension and numbers of beads present, inoculated beads won’t stick together during freezing. The inoculated beads should be kept in the ultra-freezer ( 70 °C) overnight, and after the freezing step, they are ready to be preserved at much lower temperatures in liquid nitrogen tanks (Espinel-Ingroff et al., 2004; Lakshman et al., 2018). 4.2.2 Freeze drying of filamentous fungi Freeze-drying or lyophilization is a long-term preservation method in which frozen samples are dried under deep vacuum conditions (dehydration of frozen samples). In this Chapter 5 • Sampling, preservation, and growth monitoring 167 process, cell or spore suspension is frozen at very low temperatures. Then, the frozen sample is subjected to sublimation and drying under vacuum until more than 97% of water content evaporated. Several factors can affect the successful lyophilization of filamentous fungi regarding stability and viability; (1) cell type, (2) applied lyoprotectant, (3) cooling rate during freezing, (4) primary and secondary drying temperatures, (5) warming rate, (6) storage conditions after lyophilization, (7) residual humidity in the dried sample, and (8) storage duration. Additionally, growth conditions of the strains before freezing, media or solution used for rehydration, and growth conditions after rehydrating lyophilized strains are important factors to be considered (Voyron et al., 2009; Wolkers and Oldenhof, 2021). Freeze-drying has some outstanding advantages in comparison to the other long-term preservation methods that include long shelf-life, simple storage, comfortable transportation, and stability. Despite these advantages, it is a time-consuming method, and cumbersome employed protocols need exclusive equipment and infrastructures such as a vacuum pump, ultra-freezer, constrictor, oxygen torch with a hot flame, temperature-controlled shelves, and freeze-drier (Espinel-Ingroff et al., 2004; Castro-Rios and Bermeo-Escobar, 2021). In brief, a freeze-drying cycle includes three steps: (1) Freezing of the shelves and samples, (2) primary drying (reducing pressure in the chamber and increasing temperature in the shelves and samples), and (3) secondary or final drying (high vacuum and temperature) (De Meyer et al., 2015). In 1945, this technique was introduced by Raper and Alexander for the preservation of fungi (Raper and Alexander, 1945). Among filamentous fungi, spore-forming fungi can tolerate lyophilization but, Basidiomycetes and non-sporulating strains are more sensitive to freeze-drying conditions (Homolka et al., 2001; Toegel et al., 2010; Ayala-Zermeño et al., 2017). Based on our personal experience in the laboratory and literature, many species of this group do not produce asexual resilient spores and mostly grow as mycelium. The mycelial structure is not resistant to harsh conditions; hence, freeze-drying of these filamentous fungi is rather complicated and limited to some species. The culture media that stimulate sporulation and minimal mycelia formation are suitable for the growth of fungal strains which are to be lyophilized (Voyron et al., 2009; Wolkers and Oldenhof, 2021). Lyoprotectants like cryoprotectants are protective substances that prevent cells from damages during the lyophilization process. The main lyoprotectants used for the freeze-drying of filamentous fungi include 10% skim milk suspension, 1% sodium glutamate solution, peptone, serum, and various carbohydrates (sucrose, trehalose, or myoinositol) or a mixture of them (Ayala-Zermeño et al., 2017; Wolkers and Oldenhof, 2021). But the latter is not suitable enough due to its foamability, sensitivity to contamination, and high price (Wolkers and Oldenhof, 2021). 4.3 Quality control of preserved filamentous fungi Prosperous conservation of filamentous fungi depends on cellular viability and survival. As well, fewer or no changes should occur in the cell properties such as metabolite or enzyme production. Therefore, it is significant to check and control cellular viability, purity, genotype stability, and main properties with routine and molecular assays after 168 Current Developments in Biotechnology and Bioengineering preservation in reasonable intervals. For instance, through AFLP, genetic variations in the preserved samples are determined (Voyron et al., 2009; Wolkers and Oldenhof, 2021). The intervals can be set immediately after preservation, 1, 3, 6, or 12 months after storage. Based on the obtained results from the initial assessments, the subsequent intervals for quality control of the preserved samples can be determined. Another important point regarding quality control of the preserved samples referred to increased incubation time after reactivation and recovery. It seems microbial cells, including filamentous fungal strains; require a long lag phase (sometimes more than 1 week) for their cellular retrieval (Espinel-Ingroff et al., 2004; Ilyas and Soeka, 2019). Assessment of cell survival, growth, sporulation, and morphology can be performed by various tests in the laboratory. Besides, metabolite and enzyme production of preserved fungal strains are compared with unpreserved samples to estimate the effect of preservation protocols on the preserved strains (Homolka et al., 2001; Wolkers and Oldenhof, 2021). To control the quality of cryopreserved samples, they should be reactivated. For this purpose, preserved cryovial(s) is rapidly transferred from ultra-low temperatures to a warm water bath (37 °C or 60 °C) to be thawed (Ilyas and Soeka, 2019; Wolkers and Oldenhof, 2021). It is recommended to clean the cryovial(s) surface with a routine disinfectant (70% ethanol). After thawing, the cryovial(s) content is cultured on the appropriate media (both solid and liquid media) and incubated under optimal growth conditions until measurable fungal colony appears. Remarkably, the viability of fungal cells is the first issue that can be checked (Homolka et al., 2006; Voyron et al., 2009; Wolkers and Oldenhof, 2021). The recovery test of lyophilized filamentous fungi can be done immediately after sealing the ampoules under vacuum conditions or at appropriate intervals. Lyophilized ampoules containing a dried suspension of the fungal cells are broken under aseptic conditions. Then, a suitable liquid medium or ringer solution is added to the dries cake to resuspend it. Afterward, an appropriate amount of this suspension is added to growth media, and inoculated media are incubated under optimal growth conditions until fungus appears (Wolkers and Oldenhof, 2021). 5. Growth monitoring of filamentous fungi It is indubitable that filamentous fungi are responsible for many fermentation processes in various industries. The fermentation process aimed to produce desired bio-products, either microbial cells or metabolites, at a high possible yield rate. This purpose cannot be achieved except with a good understanding of filamentous fungi physiology and providing optimum environmental conditions. The optimal growth of fungi is an indicator of c et al., 2000). Moreover, product formation during fungal fermenthe culture state (Golobi tation is associated with the pellet morphology of the filamentous fungal cells. During the fermentation process, filamentous fungal mycelia show morphological polymorphisms from dispersed hyphae to compact pellets and loose clumps (Cairns et al., 2019). Hence, Chapter 5 • Sampling, preservation, and growth monitoring 169 continuous, high-quality monitoring of fungal growth can show if all physical, chemical, and biological parameters such as oxygen concentration, temperature, pH, and other factors, including nutrient/substrate concentrations, have been provided for fungi in the fermentation process (Chisti, 2014). In general, growth monitoring makes the opportunity to realize and correct any deviation from specified optimum conditions. Defining biomass concentration is the most basic and often essential for growth monitoring in biotechnological processes (Banerjee et al., 1993). As a whole, methods available for biomass measurement are categorized into direct and indirect ways. The biomass can be monitored by its concentration or metabolic activity (Biechele et al., 2015). The difference between the direct and indirect methods measurement principally is in the technique and speed of the assessment. Regarding the fermentation scale, laboratory, or industrial scale, monitoring might be done offline by taking the sample from the culture or online using the sensors and control unit. 5.1 Direct methods for biomass evaluation 5.1.1 Gravimetric method The most prolonged established method for biomass measurement is the gravimetric technique, which determines mycelial dry weight (Sonnleitner et al., 1992). In this procedure, the sample is taken from the culture and separated through the filter or centrifugation. Then, biomass is weighed while the cells get entirely dried. This method is accurate and usually consider as a reference method for comparing all other procedures. However, this method has some drawbacks. For example, mycelial dry-weight measurement, although accurate, is tedious and time-consuming and does not apply to large-scale fermentation. In addition, if the medium contains suspended solids which usually exist in a practical fermentor, the dry weight does not represent the biomass concentration (Madrid and Felice, 2005). Preferentially the suspended solids would have to be removed before any of the direct methods can be employed. Finally, this method cannot figure out dead cells from active ones, while active biomass is essential for defining growth rate and productivity. 5.1.2 Cell count Besides the gravimetric method, cell or nuclei count is the other offline cell measurement. In this method, the total number of cells is measured by diluting the original sample and observation the counting chamber under a light microscope. It also can be measured automatically with a cell counter such as a flow cytometer or coulter counter. In one study, Steudler et al. (2015) have described the flow cytometry method based on counting fungal nuclei as a method for fungal biomass determination during solid-state fermentation (SSF) (Steudler et al., 2015). The obvious disadvantage of this method is the delay between sampling and analysis that may increase the risk of contamination. Real-time monitoring of important variables such as biomass concentration is one of the principal aims in the biotechnological process. This type of control and monitoring is essential for high efficiency, reproducibility, and productivity (Landgrebe et al., 2010). 170 Current Developments in Biotechnology and Bioengineering The usage of the sensors in online monitoring offers a substantial advantage in bioprocess. They are non-invasive, no sampling from the process is required, and several compounds can be measured together. As well, there is no time delay for delivering the information, so they are good candidates for continuous monitoring of the progress of culture. However, probes have to fulfil certain requirements such as calibration, being precise at low and high-density cultures, and linear dependency. In addition, they should be autoclavable, able to endure high pressure and temperature, stable in corrosion, and biologically inactive (Kiviharju et al., 2008). In recent years, several methods for online cell concentration monitoring using probes have been developed. Generally, these methods are based on optical, capacitance, fluorescence, and spectroscopy technique (Robinson, 2014). 5.1.3 Optical density Optical density (OD) is the easiest and most frequent method for monitoring biomass concentration. The linear correlation between OD and cell concentration caused to development of the online method for measuring OD (Zhang et al., 2019). Different types of optical measurements are based on scattering, reflection, transmission, and absorption of light. The scattering and transmission measurements are used for medium with low to moderate turbidity, while for high turbidity, the reflection measurement is applicable. The size of the measured organism defines the wavelength area. An object smaller than 3 mm is measured in visible wavelength; however, detecting a larger object in the nearinfrared region is preferred. OD application depends on measurement specifications and culture conditions (Myers et al., 2013). The accuracy and sensitivity of this method increased with the extent of fragmentation of fungal hyphae. Banerjee et al. (1993) have reported OD as a method for measuring the biomass concentration of Neurospora sitophila during all growth phases after homogenization (Banerjee et al., 1993). OD instruments (optical probes) are sensitive to fouling, air bubbles, and suspended solids. In addition, they cannot differentiate between dead, viable, or inactive cells c et al., 2006). These are the major concerns regarding using this method for mea(Vojinovi suring biomass concentration. Over time, new solutions have been developed to solve these problems. For instance, the negative effect of air bubbles can minimize the insertion of degassing chamber or fine mesh on the sensor tip, which is permeable to cells but not to bubbles (Biechele et al., 2015). Nevertheless, optical techniques still are the most applicable method for measuring cell concentration. It has been confirmed by Fan et al. (2016) which used an optical sensor for the online monitoring of biomass concentration in a membrane bioreactor system for lactic acid production (Fan et al., 2016). Moreover, Junker et al. (1994) investigated the optical probe for cell density measurement in microbial and animal cell cultures (Junker et al., 1994). 5.1.4 Imaging and microscopy An interesting microscopy technique to process monitoring directly inside the reactor is in-situ microscopy (ISM). This microscope can take images from suspended organisms in Chapter 5 • Sampling, preservation, and growth monitoring 171 the reactor to analyze cell concentration, cell size, and cell morphology. Typical ISM involves a direct light source, 25 mm stainless steel tube, two charged-coupled device (CCD) cameras, and a measuring chamber. Generally, the measuring chamber is opened and closed regularly, and a defined volume of culture comes through the chamber and is trapped (Horta et al., 2015). After a few seconds, when the movement of entered culture is stopped, they are investigated with the microscope. The images that the microscope has taken can be visualized through a CCD camera to acquire high quality. The pictures are evaluated by digital image processing software to cell concentration, cell size, cell volume, and morphology (Bittner et al., 1998). The main advantage of ISM is that changes in cell morphology do not interfere as in OD measurements. Expensive instrumentation and complicated functioning are disadvantageous of this technique (Vojinovic et al., 2006). Several studies have been investigated using ISM in bioprocesses. In one study, the accuracy of ISM has been approved by comparison with the other benchmark methods such as hemocytometer (Vecht-Lifshitz and Ison, 1992). Besides, Zhang et al. (2019) have monitored fungal biomass by commercial imaging systems, i.e., oCelloScope (Zhang et al., 2019). The oCelloScope is an automated live-cell imaging technique and quantifies fungal growth in the image from spores to hyphae. The oCelloScope is composed of a digital camera, an illumination unit, and a lens. This microscope supports different types of sample containers such as microscope slides and 96 microtiter well plates. The optical axis of the oscilloscope is leaned 6.25 degree relative to the horizontal plane, and this leaning provides more ease of operation at high and low concentrations of the analyte. Sequences of images are taken along the horizontal plane to make a Z-stack image containing the focus and the out-of-focus images. The time-lapse videos of the acquired images, the growth curve, and the quantitative analysis of morphological structure are generated using UniExplorer software (Solutions, 2021). 5.1.5 Impedance and capacitance techniques One of the promising techniques for online cell concentration measuring in the broth culture is capacitance measurement. There is a linear correlation between viable cell concentration and capacitance (Horta et al., 2015). This method works by providing an alternating electrical field. By applying the electrical field to the culture, the ions are forced to move. The positive ions will flow toward the field, while the negative ions will push in the opposite direction. Both inside and outside the cell, the ions can move until they encountered the non-conductive plasma membrane. This membrane behaves like a physical barrier and prevents further movement. The separation of negative and positive charges led to polarization at the poles of the cells. This polarization alters the permittivity of the broth and, following changing capacitance or impedance of the solution. Since the dead cells are not polarizable because of the disrupted membrane, they are invisible to this method, so the capacitance directly shows the viable cell concentration in the broth (Carvell and Dowd, 2006). With the ability to realize viable cells, this method can be used for other purposes, such as defining the specific growth rate and controlling the feeding 172 Current Developments in Biotechnology and Bioengineering flow rate in the fed-batch fermentation (Horta et al., 2015). The great advantage of capacitance measurement rather than the other methods like OD is that air bubbles and the other non-biological particles cannot interfere with the result as they do not have a plasma membrane (Biechele et al., 2015). However, the capacitance changes are not directly correlated with cell concentration when there is an ionizable compound such as salts in the culture. Hence, this method has reliable results in the fermentation broth with low conductivity (Gencer and Mutharasan, 1979). 5.1.6 Fluorescence techniques Fluorescence techniques are widely used in research and biotechnological processes to measure substrate, biomass concentration, and media characterization (Faassen and Hitzmann, 2015) Several molecules can be classified as fluorophores that are employed to monitor bioprocessing. The most common fluorophores include nicotinamide adenine dinucleotide (phosphate) [NAD(P)H], aromatic amino acids (tryptophan), adenosine triphosphate (ATP), vitamins, coenzymes, and pyruvate (Biechele et al., 2015). After absorption of light with a specific frequency by these molecules, the electron elevated from the vibrational level in the ground state to the vibrational level of the excited state. As these molecules return to the basic energy level, the photon is released at the other frequency. So, each fluorophore has a specific pair of wavelengths for excitation and emission. Traditionally, the most common fluorescence-active compound for biomass estimation is NAD(P)H. This fluorophore is measured at wavelength 450 nm after exciting at 360 nm. Using one wavelength for excitation and another for emission is called 1D fluorescence (Ulber et al., 2003). In many studies on different microorganisms, the linear correlation between NADPH €rk et al., 2002). Zabfluorescence signal and biomass concentration has been approved (Sta riskie and Humphrey (1978) have used culture fluorescence for online estimation of viable biomass during the cultivation of Saccharomyces cerevisiae. The accurate biomass estimation based on this method occurs when there is a constant concentration of NADPH in the cells and when there is no compound or metabolite which absorbs light at excitation or emission wavelength (Zabriskie and Humphrey, 1978; Kiviharju et al., 2008). During cultivation time, the signals from the other fluorescence components, air bubbles, and scattering particles cause interference with NADPH fluorescence measurement. By using two-dimensional (2D-) fluorescence, some interferes problem has been solved. Since This technique has multiple excitation-emission wavelengths, it enables to detection of different types of fluorophores, which can be related to biomass, such as NAD(P)H, tryptophan, riboflavin, flavin dinucleotide (FAD), flavin mononucleotide (FMN), and pyridoxine (Olsson and Nielsen, 1997). Chemometric tools such as partial least squares (PLS) or principal component analysis (PCA) are needed to accurately process data in this method (Beutel and Henkel, 2011). 5.1.7 Near-infrared spectroscopy technique Near-infrared spectroscopy (NIR) provides outstanding information in bioprocess monitoring. It is widely used for detailed monitoring and estimation of the chemical Chapter 5 • Sampling, preservation, and growth monitoring 173 composition of substrate, biomass, and metabolite concentrations in biotechnology processes (Beutel and Henkel, 2011). This technique is based on vibrating XdH bonding the high dipole moment in the molecule and measuring the absorbance in the NIR range, at 700 to 2300 nm. Since biologically important bonds such as CdH, NdH, OdH, and SdH are found in most compounds in bioprocess, NIR can measure the concentration of different types of organic components in complex media. In comparison to the OD technique, NIR enables gathering more process component data and biomass concentration. The NIR wavelength area, correlated with biomass concentration, is 2300 nm in at-line systems (Kiviharju et al., 2008). Abundant articles are found regarding near-infrared spectroscopy for bioprocess monitoring. Navrátil et al. (2005) provided information about NIR spectroscopy for online monitoring of biomass, glucose, and acetate in fed-batch cultivation (Navrátil et al., 2005). Liu et al. (2010) investigated the variability of biomass chemical composition by the NIR technique. Interpretation of spectra is the main challenge in NIR spectroscopy. This issue happens because of the large number of recorded wavelengths and overlapping absorbance. Hence, an advanced analytical system such as Fourier transformations and PLS are necessary for obtaining accurate data from the spectrum (Rinnan et al., 2009; Liu et al., 2010). 5.2 Indirect methods for biomass evaluation 5.2.1 Adenosine triphosphate measurement Measurement of ATP has been used as an indirect index of biomass concentration. As there is a rapid consumption of ATP in dead cells after cell injury or nutrient depletion, ATP quantification considers as a method for defining active cells. In this method, the constant amount of ATP in the active biomass cell is measured with bioluminescence. The principle of ATP measurement is based on its reaction with luciferin, catalyzed by luciferase in the presence of oxygen. With hydrolyzation of each molecule ATP, a photon of light is emitted and detected through high-performance liquid chromatography (HPLC) and ion-exchange chromatography, with ultraviolet (UV) detection. This method has been successfully employed for measuring active biomass cells in a variety of systems (Gaunt et al., 1985; Suberkropp et al., 1993; Abelho, 2001). Although ATP determination is a reproducible and reliable technique, it is too sensitive. This sensitivity is related to the sampling and ATP extraction and caused this method to be time-dependent. Hence, there is a necessity to process the sample instantly after collection to prevent physiochemical changes in the cell. 5.2.2 Chitin measurement Another method for fungal biomass estimation is chitin assay. Chitins, a polymer of N-acetyl-D-glucosamine, is a main component of the fungal cell wall and may hence be used as an indicator for measuring fungal biomass, both viable and dead cells. Wallander et al. (2013) have used chitin as a biomarker to quantify fungal biomass. The chitin assay is easily performed by acid hydrolysis of the polymer and followed determination 174 Current Developments in Biotechnology and Bioengineering of glucosamine. Glucosamine can be identified by colorimetric methods such as assaying amino sugars or chromatographic techniques (Singh et al., 1994; Wallander et al., 2013). The chitin content of fungal biomass differs according to mycelia’s age and physiological state, and growth conditions. So, converting chitin to biomass cannot be maintained constantly. In addition, the existence of the other hexosamines causes interferes with the chitin measurement (Singh et al., 1994). 5.2.3 Calorimetric measurement Heat flow is an indicator for vital organisms. It represents microbial activity for the quantity of substrate consumption and metabolite production. Monitoring microbial heat evolution can be used indirectly to estimate biomass concentration and oxygen uptake rate (OUR). In addition, it can be used as an indicative measurement to realize the proper time for feeding the nutrient in the fed-batch process (Sivaprakasam et al., 2011). The heat yield relates to the rate of catabolism and differs during the growth process. After inoculation, when the fungi are in the lag phase, there is no catabolic reaction. Therefore, the heat evaluation remains inappreciable. However, the heat production alters in the exponential phase with limiting substrate concentration. Calorimetry refers to the measurement of heat released by the biological activity of the organisms. There have been many calorimetric studies on yeast where the heat released follows the biomass production curve (Brettel et al., 1980; Singh et al., 1994). In the other study, the heat yield was estimated from the calorimetric technique due to the biomass growth of Aspergillus tamarii (Dhandapani et al., 2012). The challenge of this method is that how the overall heat production of the process is measured. Therefore, heat originated from cellular metabolism and growth must be calculated separately. 5.2.4 CO2 production and oxygen uptake rate Monitoring fungal growth based on either construction material of the cell wall such as chitin, ergosterol, or suspended constitute in the cytoplasm such as RNA and DNA are not always applicable. For example, using heterogenous or complex substrate like wheat flour for fungi causes interference in detection of selective substance since they are present in both wheat and fungal cell (Koutinas et al., 2003). Among the indirect method, measuring OUR and carbon dioxide (CO2) production is a definite solution for the mentioned issue since they directly reveal the metabolic activity of the cells. The ratio between CO2 evolution rate (CER) and OUR by the cell is called the respiratory quotient. It is nearly equal to one when a desired aerobic state with considerable cell biomass exists. The CER and OUR can be proportional to the cell mass and are measured from the exhaust gas. Using mass spectrophotometry for analyzing exhaust gas has been described in S. cerevisiae (Buckland et al., 1985). The other method for measuring exhaust gas for monitoring fungal growth is integrated magneto-acoustic and photoacoustic spectroscopy (MA/PAS). Oxygen is measured through MA, and PAS is used for measuring CO2 (Koutinas et al., 2003). This method is accurate and is very stable over a long period. Chapter 5 • Sampling, preservation, and growth monitoring 175 To date, different methods have been developed to estimate biomass concentration in the fermentation process. Various techniques give prominence to differentiate biomass properties, such as metabolic activity or total concentration, cell number, and cell viability. An unavoidable outcome is that all the different methods cannot be appropriate to all microorganisms and processes. Thus, it is necessary to expand our knowledge about the measurement limitations, methods principles, and the correlation of the technique used with the variable that needs to be specified. 6. Conclusions and perspectives The kingdom Fungi includes a wide variety of different groups as well as filamentous fungi, but it does not mean that it is impossible to study them. Despite the difficulties associated with the investigation of filamentous fungi, many benefits arise from these microorganisms that encourage researchers and scientists to explore more details about them. For a better understanding, our limited knowledge about filamentous fungal biology should be overcome by associating various fields from basic mycology to nextgeneration sequencing (NGS). Moreover, interdisciplinary training of researchers and technicians could be supportive of exploiting the filamentous fungal world. Isolation and sampling protocols can be optimized based on filamentous fungi habitats, type of fungal strains, and research target. For a long time, filamentous fungi have been used in non-industrial and industrial processing. Hence, it is critical to check fungal strain authenticity, growth conditions, final products quality, biosafety of fungal-based foods, and every detail that may help us to increase filamentous fungi applicability forsustainable biotechnology, foods and feeds suppliers, agriculture, and pharmaceutical industry. Nowadays, many novel analytical and molecular methods have been developed to validate researches related to filamentous fungal growth. 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Zabriskie, D., Humphrey, A., 1978. Estimation of fermentation biomass concentration by measuring culture fluorescence. Appl. Environ. Microbiol. 35 (2), 337–343. Zhang, X., et al., 2019. Advances in online methods for monitoring microbial growth. Biosens. Bioelectron. 126, 433–447. 6 Industrial wastes as feedstock for filamentous fungi growth Pooja Sharmaa,b a ENVIRONMENTAL R ESEARCH INSTITUTE, NATIONAL UNIVERSITY OF SINGAPORE, SINGAPORE, SINGAPORE b ENERGY AND ENVIRONMENTAL SUSTAINABILITY FOR MEGACITIES (E2S2) PHASE II, CAMPUS FOR RESEARCH EXCELLENCE AND TECHNOLOGICAL ENTERPRISE (CREATE), SINGAPORE, SINGAPORE 1. Introduction There are many types of hydrolytic enzymes secreted by filamentous fungi, which is why they are the primary source of enzymes used in lignocellulose hydrolysis. However, several filamentous fungi can ferment a wide range of substrates and produce several metabolic products, including ethanol. Human activities that are more rapid use more energy and produce more waste. As a result, these fungi can be studied to see whether they can be used to turn waste into energy (Ferreira et al., 2016; Sharma et al., 2021a). Owing to widespread human exploration of natural resources and waste, similar facts apply to additional elements like phosphorus and sulfur nitrogen. Filamentous fungi are a varied collection of eukaryotic organisms that may flourish in a range of environments, including biological waste, plant, soil, and algal biomass. This can also grow on a range of small substrates and produce a variety of interesting metabolites and enzymes, scientists’ interest in employing it as a biotechnology innovation species (Meyer, 2008; Sharma et al., 2020). Several important industrial substances are currently produced employing filamentous fungi as cell factories, including proteins, exopolysaccharides, organic acids, secondary metabolites, and enzymes (Nielsen and Nielsen, 2017). Fungi such as Trichoderma reesei, Aspergillus oryzae, and Aspergillus niger, are becoming essential for industrial enzyme production (Frisvad et al., 2018). More than half of the enzymes used in industrial applications are fungal enzymes remarking filamentous fungi’s significance as cell factories. The prospects of fungal biotechnology are important to use molecular-level study or gene editing in fungas €kela €, 2020). Filamentous fungi have attracted stains (Garrigues et al., 2020; de Vries and Ma a lot of attention in the industrial-scale production of numerous value-added products such as enzymes, pigments, organic acids, and other metabolites due to their capacity to flourish on a variety of organic-rich substrates (Sudhakaran et al., 2013; Teodosiu et al., 2018). The use of industrial wastes as fungal growing media has recently attracted a lot of research attention to lower the costs of large-scale fungal product processing while also reducing the environmental impact of industrial wastes. Filamentous fungi synthesize and secrete many enzymes, including proteases and lipases, that allow them to break Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00013-2 Copyright © 2023 Elsevier Inc. All rights reserved. 181 182 Current Developments in Biotechnology and Bioengineering down, consume, and thrive on a range of surfaces. Filamentous fungi can produce metabolites including carboxylic acids and ethanol, as well as antibiotics and pigments (Gmoser € ffler, 2018). Furthermore, since fungal biomass is high in proet al., 2017; Anke and Schu teins, fatty acids, and vitamins, it may be used as a human food or animal feed source. In Eastern and Southeastern Asian countries like China, Japan, Indonesia, and fungal fermented human foods like tofu, miso, and tempeh are traditionally used (Lennartsson, 2012a,b). As a result, the nutrient composition of fungal biomass makes it a promising substitute or substitution for traditional animal feed, like fish feed. Cultivated edible filamentous fungi Neurospora intermedia and Aspergillus oryzae on a variety of dairy wastes and by-products (Mahboubi et al., 2016). Some filamentous fungi that secrete amylases, such as those from the genera Neurospora, Rhizopus, and Aspergillus, may perform simultaneous saccharification and fermentation (SSF) on starchy polymers and different crops (Zhang et al., 2008; Liang et al., 2012). Soybean, rice, grains, legumes, potato, maize stover, wheat bran, and other agricultural and forestry (lignocellulosic material) residues have all been used in the production of various fungal metabolites and enzymes. Lignocellulosic residues such as wasted sulfite liquor from the pulp and paper industry, stillage from the bioethanol industry, molasses (from sugar beet and sugar cane), sucrose, and starchbased effluents have all been explored as possible fungal culture mediums (Asadollahzadeh et al., 2018). Several of these substances, like cyclosporine, lovastatin, and penicillin, are vital to human health (Garrigues et al., 2020). Fungi range in size from single cells to long chains of cells stretching for miles. Fungal cells are oval (3–8 m, 5–15 m) and have large trichomes that contain organelles and intracellular granules and structures. Filamentous fungi-like Ascomycetes and Zygomycetes families have played important in industries. A previously published reported that the value-added items made by zygomycetes on a variety of substances (Ferreira et al., 2016). Ascomycetes are bigger microbes that have received a lot of press importance recently (Ferreira, 2015). Penicillium chrysogenum and Aspergillus oryzae, are the most industrially investigated filamentous fungi and their contribution to the synthesis of the antibiotic (Gibbs et al., 2000). Many value-added products are made by Aspergillus spp., including enzymes including protease, catalase, amylase, lipase, lactase, and phytase. Trichoderma sp. produced both cellulase and xylanase (Pandey et al., 2015). Furthermore, Aspergillus sp. produced a significant portion of commercial organic acid production like gluconic acid, citric acid, and itaconic acid, as well as the production malic and oxalic acid (Pandey et al., 2015). Moreover, chitosan can be made by hydrolyzing chitin from Aspergillus sp. cell walls, and these ascomycetes can also produce keratinase hydrolysates, which can be used to manufacture superabsorbents (Zamani, 2010). Monascus sp., Aspergillus sp., Fusarium sp., and Neurospora sp., have been sources of various human food products and pigments for the food industry. Unicellular ascomycetes like Pichia spp., Yarrowia spp., and Saccharomyces spp., have been confirmed to be potential sources of organic acids like—lactic acid, ketoglutaric acid, pyruvic acid, and malic acid, and polysaccharides such as glucan, proteins such as polyunsaturated fatty acids, collagen, and sterols. A large number of Chapter 6 • Industrial wastes as feedstock 183 Filamentous fungi Use waste and residue for growth 1. 2. 3. 4. 5. 6. 7. 8. Potato peel Wheat straw Rice hulls Waste office paper Tea waste Kitchen waste Sugarcane bagasse Banana peel Biomass HYDROLYSIS 1. Pentoses 2. Hexoses Metabolite Enzymes FIG. 1 Use of waste and residue for the growth of filamentous fungi and their value-added products. carbohydrates, proteins, and micronutrients can be identified in starch wastewater. Such nutrients can be recovered either through purification methods or by the cultivation of microbes for the generation of useful biomaterials instead of being transported to a wastewater treatment facility for microbial removal (Nasr et al., 2015). Filamentous fungi are recognized for their ability to secrete enormous numbers of various hydrolytic enzymes, making them the primary source of enzymes for the hydrolysis of lignocellulose (Fig. 1). 2. Microbiology of filamentous fungi Filamentous fungi are saprophytic microorganisms that secrete a variety of enzymes that help decompose and recycle complex biopolymers from living cells. Fungi are eukaryotes that come in a variety of shapes and sizes (Gravesen et al., 1994). Filamentous fungi are very well sources of a diverse range of secondary metabolites with diverse biological functions. Many fungi are saprophytes (those that thrive on decomposing organic matter), while some are parasites. Fungi are absorptive heterotrophs, meaning they digest organic matter by cells secrete enzymes onto the substances and then consuming easily accessible organic compounds. Fungi were found to grow at temperatures between 30°C and 35°C (Maheswari and Chandra, 2000). Fungal hyphae have a small volume but a wide surface area, which helps fungi absorb the additional nutrient. Filamentous fungi are noted for their ability to secrete vast quantities of various hydrolytic enzymes, making them the primary source of enzymes for the hydrolysis of lignocellulose. Some filamentous fungi can ferment a wide variety of substrates and produce a wide range of metabolic products, like ethanol (Vallero, 2015). Moreover, gradients in substrate concentration, enzymes, water 184 Current Developments in Biotechnology and Bioengineering content, and temperature, and also the presence of a substrate-air interface, may result from fungal hyphal development (Biesebeke et al., 2002). Some filamentous fungi are obligatory aerobes, which mean they need oxygen to function. Aeration and agitation are being used to replenish oxygen in the culture medium. Filamentous fungi developed in waste materials, for example, offer an intriguing option to processed sugar. Antibiotics, enzymes, organic acids, and ethanol are just a few of the substances that filamentous fungi can produce (Ferreira et al., 2016; Nair and Taherzadeh, 2016). Filamentous fungi are a diverse collection of eukaryotic organisms that may thrive in a wide range of conditions, including biological waste, algal biomass, soil, and plant. It also can grow on several low-cost substrates and produce a diverse range of fascinating metabolites and enzymes, which has piqued the scientific community at large interest in using them as biotechnology production organisms (Meyer, 2008). Filamentous fungi are found all over the world, and they play an important role in natural ecosystems €es, 2015). and environmental stability (Ku Fungi are significant main decomposers of organic matter as well as outstanding chemical engineers, generating a vast range of natural compounds, including antibiotics and mycotoxins, some of which have potent toxic properties. The genes involved in these metabolic processes can be physically connected on chromosomes to create gene clusters in fungal genomes. This extraordinary metabolic diversity is essential to the wide range of ecological tactics used by mushrooms, but little is known about the evolutionary mechanisms that lead to it (Wisecaver et al., 2014). 3. Diversity of filamentous fungi Fungi are a distinct kingdom from plants, protists, bacteria, mammals, and one of the main classes of eukaryotes. Fungi are estimated to number between 2.2 and 3.8 million € cking, 2017). Zygospecies, with only 120,000 having been confirmed (Hawksworth and Lu mycota, Chytridiomycota, Ascomycota, and Basidiomycota are the four classes of fungi that have been classified. Filamentous fungi are multicellular branched fungi with hyphae (thread-like elongated structures) (Karimi et al., 2018). Filamentous fungi are abundant in nature and play a crucial role in ecosystem balance through biodegradation, nutrient cycling, and symbiosis partnerships. Filamentous fungi are taxonomically diversified, but representatives of three classes, basidiomycetes, ascomycetes, and zygomycetes, are most typically found in pollution mitigation research studies or industrial exploitation, using high-quality medium formulations (Troiano et al., 2020). Their dominant ecological position, broad usage in research, and commercial exploitation are all linked to their macroscopic filamentous development, evolutionary variety, the array of extracellular and intracellular enzymes, range of potential value-added products, creation of surfactants, cell wall sorption, and synergistic possibilities in co-culture strategies. Filamentous ascomycetes can also play a minor part in industrial processes that are already in existence. For example, filamentous ascomycetes can transform side-streams from the ethanol production process, such as thin stillage and full stillage, from sugar-rich or starch-rich crops into Chapter 6 • Industrial wastes as feedstock 185 value-added products (Ferreira, 2015). Filamentous fungi are endophytic microbes that secrete a variety of enzymes that assist breakdown and reuse of complicated biopolymers from both mammalian tissues. The majority of these enzymes are hydrolytic, and these play an important role in fungal feeding by releasing carbon and nitrogen from insoluble polymers derived from other species’ metabolism. 4. Wastes, residuals, and wastewater as nutrients Filamentous ascomycetes such as Fusarium sp., Aspergillus sp., Neurospora sp., and Monascus sp. are adaptable fungi that can grow on a wide range of substances. Carbohydrates include monomers like hexose and pentose as well as disaccharides like cellobiose, lactose, and sucrose (Angumeenal and Venkappayya, 2005; Bari et al., 2009; Bansal et al., 2014). In addition to the monomers generated during the hydrolysis of lignocellulosic materials, ascomycetes can grow on carbohydrate polysaccharides like arabinan, glucan, and xylan. It is using solid-state fermentation to grow on non-pretreated substrates like wheat bran or wheat germ (Panagiotou et al., 2011). Industrial wastes are produced in large quantities of minerals by industrial processes and can be used in a variety of appli€ m, cations if they are technically and environmentally friendly (Sorvari and Wahlstro 2014). Some bio-based materials differ from synthetic polymers in that they can be degraded into natural metabolic products by fungi (Davis and Song, 2006). They are similar to biodegradable polysaccharides such as starch, cellulose, lignin, and chitin, proteins such as gelatine, casein, wheat gluten, silk, and wool, and lipids such as plant oils and animal fats. Natural rubber and some polyesters produced by microorganisms, polyhydroxyalkanoates (PHAs), and poly-3-hydroxybutyrate (PHB) or synthesized from bio-derived monomers, polylactic acid is further examples of economically viable, biodegradable natural products (PLA). The growth flexibility of these filamentous ascomycetes is assumed to be due to the varied enzymes they can create depending on the substrates they develop on. Its ability suggests that these fungi have a high potential to play key roles in “waste biorefineries” by valorizing waste materials from various industries. Filamentous ascomycetes can grow on agricultural waste like wheat bran, rice hulls, corn straw, corn cobs, and sugarcane bagasse (Jabasingh and Nachiyar, 2011; de Almeida et al., 2014). These materials can allow the entire crop to be used for biotechnological reasons while minimizing the demand for sugar-rich crops or starch-containing cereals and will be used for human food substitutes. Filamentous ascomycetes can be used to recover food waste such as banana, lemon, pineapple, beet, bulb, and potato peels, as well as empty palm oil fruit bunches and apple pomace (Dhillon et al., 2011). Filamentous fungi can grow on waste office paper, tea waste, lactose-rich wastes from the dairy industry like cheese whey, cream, and even spent grains from the brewing industry (Ikeda et al., 2006; Xiros and Christakopoulos, 2009). Nonetheless, the fee of employing ascomycetes to manufacture value-added products from lignocellulosic materials will undoubtedly be restricted by the degree of expressed enzymes, which will dictate the time required for degradation. 186 Current Developments in Biotechnology and Bioengineering Identifying microbes with naturally robust enzymatic machinery capable of rapidly breaking and fermentation, particularly refractory materials has become a major scientific problem. The filamentous fungi may also have a role in currently running manufacturing processes. Using minimal standard regulation and the entire investigations of the ethanol production line, the filamentous fungi can change sugar- or sugar field land into valueadded products (Lennartsson et al., 2014). If ethanol side-streams are converted to ethanol and biomass for feed by filamentous ascomycetes, the distillation column and dryer, are already usable. Filamentous fungi can use waste materials to produce enzymes, biomass, organic acids, ethanol, and metabolic flexibility for feed applications. The use of wastewater is especially significant for lowering the cost of existing industrial items or developing new processes that value-added products. Acremonium, Rhodotorula, Candida, Geotrichum, Cladosporium, Sporothrix, Geotrichum candidum, Penicillium, Trichophyton, and Scopulariopsis have all been identified as common fungal taxa found in activated sludge, aerobic granulated sludge (AGS), reduced the pollution load in wastewater (Table 1). The zygomycetes are a tiny, ecologically diverse, paraphyletic, or polyphyletic group of primarily terrestrial fungi that are found near the bottom of the fungal food web (Taylor et al., 2014). Moreover, the composition of the Zygomycete cell wall differs from that of the major Eumycota since chitosan performs the function of glucans as essential structural components. Chitosan, together with chitin, forms the backbone of the cell wall, which is encased in polyglucuronic acid and mannoproteins. Chytridiomycota, Zygomycota, Ascomycota, and Basidiomycota are the four phyla that make up the kingdom Fungi. Zygomycota is divided into two classes: Trichomycetes and Zygomycetes, which are saprophytes Table 1 Role of filamentous fungi compounds in the industrial application (Meyer, 2008; Linder et al., 2005; Olempska-Beer et al., 2006). Filamentous fungi Name of compounds Industrial application Aspergillus niger Aspergillus niger Aspergillus oryzae Taxomyces andrenae Penicillium chrysogenum Acremonium chrysogenum Claviceps purpurea Fusarium graminearum Aureobasidium pullulans Aspergillus oryzae, Aspergillus niger, Rhizopus delemar Mucor miehei Trichoderma konignii, Aspergillus terreus Trichoderma viride Aspergillus niger α-amylase Citric acid Kojic acid Taxol Penicillin Cephalosporin Ergot alkaloids Zeranol Pullulan Proteases Rennin Xylanases Itaconic acid Cellobiohydrolase Glucose oxidase Food industry Beverage and food industry Food industry Pharmaceutical industry Pharmaceutical industry Pharmaceutical industry Pharmaceutical industry Livestock farming Food industry Detergent industry Food industry Pulp, paper, textile, and bakery industry Polymer industry Textile industry Textile industry Chapter 6 • Industrial wastes as feedstock 187 that grow on dead organic debris and are found all over the world. Zygomycetes have been employed in food production for a long time. They are pathogens of plants, animals, and other fungi, requiring the selection of suitable strains (Lennartsson, 2012a,b). 5. Role of filamentous fungi in pollution reduction Anthropogenic sources are filling wastewater treatment plants and the environment with a growing number of organic and inorganic substances, causing health and environmental problems (Fig. 2). Filamentous fungi, their filamentous growth, an array of extracellular and intracellular enzymes, surfactant activity, cell wall biosorption capabilities, and mutualistic momentum, may aid in changing public views of contaminants. Mycoremediation is a kind of bioremediation that uses fungi to remove, degrade, or reduce the toxicity of a variety of pollutants from a range of surfaces. An overwhelming amount of research supports the use of filamentous fungi for pollution mitigation; however, no full-scale applications have been developed. Pollution of the environment is a reality and the processing of ordinary side-streams and micropollutant-rich wastewaters. To address knowledge gaps in the field and suggest a comprehensive approach, governmental authorities, industries, and academics must collaborate to develop Filamentous Fungi Industrial waste 1. 2. 3. 4. 5. 6. Wastewater containing micropollutants 1. Alipha c hydrocarbons 2. Polycyclic aroma c hydrocarbons 3. Hydrocarbons 4. Herbicides Sugar Lignocellulose Minerals Fat Proteins Inhibitors Value-added products FIG. 2 Filamentous fungi use nutrients from different sources of wastewater. 188 Current Developments in Biotechnology and Bioengineering filamentous fungal processes. The biochemical variety has been incorporated into manufacturing processes that supply a variety of goods value of industrial waste streams and micropollutants removal (Ferreira et al., 2013, 2016; Naghdi et al., 2018; Daccò et al., 2020). The biochemical variety has been incorporated into manufacturing processes that supply a variety of goods, as well as research projects aimed at maximizing the value of industrial waste streams. Its features include filamentous and macroscopic growth, a wide range of enzymes, surfactant synthesis, cell wall sorption ability, a wide range of valueadded products that can be created, and synergistic output when co-cultured with other microbes. These properties have an overall advantage over unicellular microbes due to better biomass recovery from the medium, reduced substrate specificity, increased substrate bioavailability, and the potential for a greater diversity of activities. They also have a benefit over physicochemical methods for wastewater treatment in that they can manufacture value-added goods while using fewer resources and chemicals. Simulating filamentous fungi’s strong position in environmental nutrient cycles, which is inorganic and organic matter, which can have harmful environmental consequences, are seen as prospects to achieve a resource-efficient. Investigators are now giving a summary of the challenges faced with filamentous fungi when grown in ordinary side streams, as well as the ability of filamentous fungi to remove micropollutants from wastewaters. Given the multifaceted role filamentous fungi can play in pollution mitigation, information integration across application matrices is critical for developing new, cost-effective technologies. Filamentous fungi found in heavy metal-contaminated environments offer a lot of potential for bioremediation, but they are often underutilized. Microbes consume toxins and, due to their innate adhesive properties, can survive under metal stress. Mycoremediation relates to the use of high or low fungi for heavy metal removal (Vimala and Das, 2009; Ayangbenro and Babalola, 2017). Fungal colonization is critical in the industrial world because fungi have thermotolerant and pH-tolerant extremophilic enzymes (Neifar et al., 2015). Heavy metals can be successfully absorbed from the soil by microbes (Sharma et al., 2021c,d,f). The isolation of Penicillium, Fusarium, and Aspergillus from metal stress conditions has been documented in numerous investigations. Heavy metals can be effectively removed by these microbes (Iram et al., 2012). Due to the negative charges of functional groups found in cell wall components, fungal cells have a large surface area and outstanding metal-binding abilities (Khan et al., 2019). Fungi also have numerous antioxidant systems, metal transporters, metal buffering molecules, metal transformation enzymes, vacuolar sequestration capacities, and produce metal precipitating chemicals; nevertheless, filamentous fungi have few mechanisms characterized (Danesh et al., 2013; Robinson et al., 2021; Kumar and Dwivedi, 2021). Metal resistance in filamentous fungi was already related to their isolation sites, the toxicity of the metal tested, the amount of the metal in the medium, and the competence of the isolated. Many metal-tolerant filamentous fungi have been isolated from heavy metal contaminated soils, including Trichoderma, Rhizopus, Penicillium, Fusarium, and Aspergillus (Zafar et al., 2007) as described in Table 2. To protect the new possibilities affected, it is becoming increasingly vital to develop diverse approaches for removing microplastics from wastewater, which Chapter 6 • Industrial wastes as feedstock Table 2 189 Different nutrient source for fungus growth from waste. Nutrient media Strains Reference Rice hull hydrolyzate Pre-treated molasses wastewater Bunches of palm empty fruit Cheese whey Sugarcane bagasse residue Wheat bran, corn straw Sugarcane molasses Rice straw Mortierella isabellina ATHUM 2935 Aspergillus sp. Aspergillus tubingensis Mucor circinelloides URM 4182 Aspergillus terreus Phanerochaete chrysosporium ATCC 24725 Mucor circinelloides URM 4182 Colletotrichum sp. DM06 Economou et al. (2011) Yang et al., (2019) Cheirsilp and Kitcha (2015) Braz et al. (2020) Kamat et al. (2013) Liu and Qu (2019) Bento et al. (2020) Dey et al. (2011) would otherwise end up in the oceans through pipelines, ships, and other environmental problems. Bioaccumulation capacities of Pb, Cr, and Cd in Trichoderma harzianum and Komagataella phaffi have also been reported by Liaquat et al. (2020). Heavy metals can be removed from the soil effectively by fungi. Researchers are trying to figure out how these microbes can help with remediation. Several findings demonstrate that microorganisms can bind metals on their surfaces and have biosorption or bioaccumulation abilities. Several studies have also looked at the significance of genes like hydrophobin in giving metal resistance to different fungi. Rhizopus spp., Trichoderma spp., Penicillium spp., and Aspergillus spp. have all been shown to have similar bioremediation pathways (Sim et al., 2016; Puglisi et al., 2012; Iskandar et al., 2011). Penicillium simplicissimum can remove Copper (Cu) and Lead (Pb) from the liquid medium through a biosorption mechanism. Through a biosorption process, Trichoderma asperellum may withstand Cadmium (Cd), Pb, Zinc (Zn), Copper (Cu), and Chromium (Cr). According to some studies, exposing microorganisms to high metal concentrations gradually improves their metal tolerance and boosts their ability to remove heavy metals (Kumar et al., 2011). 6. Removal of microplastic using filamentous fungi Microplastics are emerging pollutants that have gained considerable attention in recent decades because of their negative effects on human organisms and the ecosystem (Mammo et al., 2020). Alternative processing methods for eliminating heavy metal ions from polluted areas are considered bioremediation. Bioremediation is the use of naturally occurring living organisms to convert hazardous contaminants into less hazardous forms (Sharma et al., 2022a, 2022b, 2022c, 2022d). Filamentous fungi then degrade or detoxify the harmful substances to human health and the environment (Siddiquee et al., 2015). Because of the increasing plastic trash in the environment, plastic, one of the greatest innovations of the last century, has become a curse to the existence of both aquatic and terrestrial living forms. Primary microplastics and secondary microplastics are the two major families of microplastics (Morsi et al., 2020; Ossai et al., 2020). Micropollutants 190 Current Developments in Biotechnology and Bioengineering are produced by inadequately treated wastewaters from industries, hospitals, and homes, as well as informal dumping, landfill and agricultural runoffs, spills, and combustion processes (Daccò et al., 2020; Kanaujiya et al., 2019; Collado et al., 2019; Li et al., 2015). Micropollutants include heavy metals, pharmaceutically active compounds, aliphatic and polycyclic aromatic hydrocarbons, surfactants in clothes and personal care items, herbicides, hormones, and dyes. Advanced physicochemical techniques have been offered as a cost-effective and environmentally safe alternative to filamentous fungal biodegradation. The discovery of lignocellulose enzymes from white-rot basidiomycetes fungi with limited substrate specificity triggered a rush of research into employing these filamentous fungi or their enzymes to eliminate a broad spectrum of microplastics. Ascomycetes and Zygomycetes, as well as intracellular enzymes in fungal bioremediation € es, 2015; Mir-Tutusaus et al., 2018; Ijoma and Tekere, 2017; Naghdi et al., 2018; Huang (Ku et al., 2018). The usage of filamentous fungi as a primary catalyst in lignocellulose-based biorefineries is a critical step toward commercial viability. Microplastic additions to soil have variable effects on the overall microbial community composition and activity, which is most likely a function of microplastic content and chemical composition (Yu et al., 2020). The exact mechanism is thought to be changed in soil qualities, particularly bulk density, which increases aeration and hence stimulates aerobic bacteria; or, more broadly, microplastics cause a shift in the microbiomes (Liu et al., 2017). Ren et al. (2020) investigated the impacts of two different microplastic particle sizes (150 and 13 m) on microbial communities and discovered that MP particle size affects richness and diversity, with smaller particles tending to increase these characteristics. The microbial community structure changed when microplastics were added to the soil; for example, Actinobacteria increased in soils with microplastics, whereas other groups such as Proteobacteria or Acidobacteria, and for smaller particles, some fungal groups such as Basidiomycota and Chytridiomycota decreased (Ren et al., 2020). Other investigators have reported similar observations, such as Actinobacteria dominating and Proteobacteria being reduced (Huang et al., 2019; Zhang et al., 2019). The composition of the soil microbial population can have a significant impact, for example, there are “mycorrhiza helper bacteria” (Pseudomonas sp., Burkholderia sp.) that facilitate root colonization or hyphal growth from spores (Viollet et al., 2017) and nitrogen-fixing bacteria can help to maximize nutrient acquisition in the host. Proteobacteria are a type of bacteria that can be affected by microplastic contamination. Their presence can modify the structural assemblages and boost root colonization in contaminated soils (Dagher et al., 2020). 7. Conclusions and perspectives Filamentous fungi are well-known producers of a diverse range of secondary metabolites with diverse biological processes. Several of these compounds, like cyclosporine and penicillin, is important for human health. 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Viollet, A., Pivato, B., Mougel, C., Cleyet-Marel, J.C., Gubry-Rangin, C., Lemanceau, P., Mazurier, S., 2017. Pseudomonas fluorescens C7R12 type III secretion system impacts mycorrhization of Medicago truncatula and associated microbial communities. Mycorrhiza 27 (1), 23–33. Wisecaver, J.H., Slot, J.C., Rokas, A., 2014. The evolution of fungal metabolic pathways. PLoS Genet. 10 (12), e1004816. Xiros, C., Christakopoulos, P., 2009. Enhanced ethanol production from brewer’s spent grain by a Fusarium oxysporum consolidated system. Biotechnol. Biofuels 2 (1), 1–12. Yang, S., Gao, B., Jang, A., Shon, H.K., Yue, Q., 2019. Municipal wastewater treatment by forward osmosis using seawater concentrate as draw solution. Chemosphere 237, 124485. Yu, H., Fan, P., Hou, J., Dang, Q., Cui, D., Xi, B., Tan, W., 2020. Inhibitory effect of microplastics on soil extracellular enzymatic activities by changing soil properties and direct adsorption: an investigation at the aggregate-fraction level. Environ. Pollut. 267, 115544. Zafar, S., Aqil, F., Ahmad, I., 2007. Metal tolerance and biosorption potential of filamentous fungi isolated from metal contaminated agricultural soil. Bioresour. Technol. 98 (13), 2557–2561. Zamani, A., 2010. Superabsorbent Polymers From the Cell Wall of Zygomycetes Fungi (Doctoral dissertation). Chalmers University of Technology. 196 Current Developments in Biotechnology and Bioengineering Zhang, Z.Y., Jin, B., Kelly, J.M., 2008. Production of L (+)-lactic acid using acid-adapted precultures of Rhizopus arrhizus in a stirred tank reactor. Appl. Biochem. Biotechnol. 149 (3), 265–276. Zhang, M., Zhao, Y., Qin, X., Jia, W., Chai, L., Huang, M., Huang, Y., 2019. Microplastics from mulching film is a distinct habitat for bacteria in farmland soil. Sci. Total Environ. 688, 470–478. Further reading Awasthi, M.K., Sarsaiya, S., Patel, A., Juneja, A., Singh, R.P., Yan, B., Awasthi, S.K., Jain, A., Liu, T., Duan, Y., Pandey, A., 2020. Refining biomass residues for sustainable energy and bio-products: an assessment of technology, its importance, and strategic applications in circular bio-economy. Renew. Sustain. Energy Rev. 127, 109876. € rlich, de Souza Machado, A.A., Lau, C.W., Kloas, W., Bergmann, J., Bachelier, J.B., Faltin, E., Becker, R., Go A.S., Rillig, M.C., 2019. Microplastics can change soil properties and affect plant performance. Environ. Sci. Technol. 53 (10), 6044–6052. El-Metwally, M.M., El-Sharkawy, A.M., El-Morsy, A.A., El-Dohlob, S.M., 2014. Statistical optimization of rapid production of cellulases from aspergillus niger MA1 and its application in bioethanol production from rice hulls. Afr. J. Biotechnol. 13 (36). Li, W., Zheng, P., Guo, J., Ji, J., Zhang, M., Zhang, Z., Zhan, E., Abbas, G., 2014. Characteristics of selfalkalization in high-rate denitrifying automatic circulation (DAC) reactor fed with methanol and sodium acetate. Bioresour. Technol. 154, 44–50. Sharma, P., 2021. Efficiency of bacteria and bacterial assisted phytoremediation of heavy metals: an update. Bioresour. Technol., 124835. Sharma, P., Rath, S.K., 2021. Potential applications of fungi in the remediation of toxic effluents from pulp and paper industries. In: Fungi Bio-Prospects in Sustainable Agriculture, Environment and NanoTechnology. Academic Press, pp. 193–211. Sharma, P., Singh, S.P., 2021. Role of the endogenous fungal metabolites in the plant growth improvement and stress tolerance. In: Fungi Bio-Prospects in Sustainable Agriculture, Environment and Nanotechnology. Academic Press, pp. 381–401. Sharma, P., Purchase, D., Chandra, R., 2021b. Residual pollutants in treated pulp paper mill wastewater and their phytotoxicity and cytotoxicity in Allium cepa. Environ. Geochem. Health, 1–22. Sharma, P., Tripathi, S., Vadakedath, N., Chandra, R., 2021e. In-situ toxicity assessment of pulp and paper industry wastewater on Trigonella foenum-graecum L: potential source of cytotoxicity and chromosomal damage. Environ. Technol. Innov. 21, 101251. 7 Filamentous fungal morphology in industrial aspects Anil Kumar Patela,b, Ruchi Agrawalc, Cheng-Di Donga, Chiu-Wen Chena, Reeta Rani Singhaniaa, and Ashok Pandeyd,e a DEPARTMENT OF MARINE ENVIRONMENTA L E NGI NE E R I N G , N A T I ON A L K A OHS I UNG UNIVERSITY OF SCIENCE AND TECHNOLOGY, KAOHSIUNG CITY, T AI WAN b INST IT UT E O F AQUATIC SCIENC E AND TECHNOLOGY, NAT IO NAL KAOHSIUNG UNIVERSITY OF SCIENCE AND TECHNOLOGY, KAOHSIUNG CITY, T AI WAN c THE ENERGY AND RESOURCES I NSTITUTE, TERI GRAM, GWAL PAHARI, HAR YANA, INDI A d CENTRE FOR I NNOVAT ION AND TRANSLATIONAL RESEARCH, CSIR-INDIAN INSTI TU TE O F TO XI CO LOGY RESEARCH, LUCKNOW, INDIA e SUSTAINABILITY C LUSTER, S CHOO L O F ENGINEERING, UNIVERSITY O F PETROLEUM AND ENERGY STUDIES, DEHRADUN, INDIA 1. Introduction Filamentous fungi are ruling over the commercial production of biological compounds due to their capacity of higher protein and other metabolites secretion in the culture medium. Controllable, reliable, and economically feasible bioprocess for filamentous fungi is of utmost importance for the large-scale production of the wide range of value-added products. With the advent of new omics technologies, such as genomics, transcriptomics, proteomics, metabolomics, metagenomics, next-generation sequencing technologies, genetic engineering, etc., more new biological compounds have been detected and purified from filamentous fungi. Most of the commercial products of filamentous fungi are industrial biotechnological bulk products unlike biopharmaceutical products and are thus not subject to tight regulatory rights (Posch et al., 2012). Filamentous fungi are involved in producing diverse range of enzymes, organic acids, sweeteners, pigments, bio-herbicides, biopesticides, biofertilizers, antibiotics, vitamins, and even pharmaceutical products. More than 9000 different bioactive compounds have been reported to be produced by fungi during last decade (Brakhage and Schroeckh, 2011). Aspergillus is among the most exploited filamentous fungi for industrial production of number of various enzymes such as cellulase, amylase, lipase, laccase, protease, etc., hence, it finds places in food industries, detergent industries, pharmaceutical industries, biofuel industries, etc. Aspergillus oryzae is the most employed microorganisms for enzyme production by enzyme giant “Novozymes.” It has immense capacity for producing enzymes and is considered completely harmless for humans (Novozymes, 2022). Over several decades the filamentous fungi as well as industrial bioprocess have gone through rigorous improvement and modification that presently the industrial production Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00015-6 Copyright © 2023 Elsevier Inc. All rights reserved. 197 198 Current Developments in Biotechnology and Bioengineering of homologous proteins is reported to be 100 g/L in the cultivation medium (Cherry and Fidantsef, 2003). With the developments in the field of genetic engineering and molecular biology, these filamentous fungi are explored more for the production of heterologous protein (Singhania et al., 2017a,b,c; Singh et al., 2017). Aspergillus oryzae and Aspergillus niger has a long history for being used in fermentation and are hence “Generally Regarded as Safe” (GRAS) strain which have been exploited largely as host for producing food grade enzymes/proteins. Multiple copy of gene for a protein is introduced to get higher protein level. Though, heterologous expression has been widely accepted for higher level of secretion or secretion with improved properties; there are a number of limitations such as low level of transcription, instability of mRNA, inefficient translation, and posttranscriptional bottlenecks, and sometimes degradation of the protein of interest (Gouka et al., 1997). “Heterologous expression” refers to the expression of a gene or a part of the gene from one organism into another organism which is referred as a host and the host does not originally harbors that gene of interest. The gene of interest is inserted into the host by recombinant deoxyribonucleic acid (rDNA) technology. There are possibilities that after insertion the foreign gene get integrated to the host DNA and get expressed or may not get integrated also. It may also be possible that either the integration is permanent or temporary. The host organism can be any cell such as bacterium, yeast, fungi, plant or mammalian cell. This host is called the expression system. Thus, heterologous expression differs from homologous expression in a sense that here the host is different from the original organism that harbors the gene. Thus, especially with reference to fungi, it is an important technique which allows expression of proteins in other organisms or in other fast-growing fungi itself so that there can be hyper secretion. Most of the industrial enzymes are produced by heterologous expression. Details of the technology can be found elsewhere. The accumulation or presence of the aberrant proteins in the endoplasmic reticulum (ER) becomes fatal to the cell, and the eukaryotic cell to react with these misfolded proteins expresses several genes which are related to protein folding and its degradation, which is called the unfolded protein response (UPR) (Carvalho et al., 2010). UPR is caused due to protein folding stress in eukaryotes and is another limitation of heterologous expression. High level of proteases is again a constrain. Using a protein deficient strain for host could be useful. The improvement of production of heterologous protein has gained significance in the field of fungal molecular biology and fungal fermentation. Typically, fungal enzyme genes are present in clusters which code multi-domain and multi-modular enzymes, such as polyketide synthases, prenyltransferases, non-ribosomal peptide synthases, and terpene cyclases (www.scribd.com). 2. Industrial bioprocess with filamentous fungi Filamentous fungi have been explored for both kinds of bioprocesses as solid-state fermentation (SSF) as well as submerged fermentation (SmF). SSF closely imitates the natural environment of filamentous fungi having low water activity which is the reason for getting higher productivities. SSF also offers several benefits like cheaper infrastructure such as tray reactors is needed instead of sophisticated bioreactors required in case of Chapter 7 • Filamentous fungal morphology in industrial aspects 199 SmF and also the cost of labor is comparatively less. Contamination issues by bacteria is also minimum due to less water activity unsuitable for bacteria to grow. Pandey et al. (2006, 2015, 2017) have done extensive work on fungal biotechnology and fungal biorefinery, with special focus on fungal enzymes. Recently, Singh et al. (2019) presented state-ofart developments and technological perspectives for various industrial enzymes, including those derived from fungi. One of the most important groups of industrial enzymes obtained from fungi is cellulases required for biomass hydrolysis (Sukumaran et al., 2021). Although cellulases research has focused on production titers, cost reduction and enzymes characteristics since more than four decades, desirable enzymes are yet to be developed with techno-economic feasibility and desired properties (Binod et al., 2019; Patel et al., 2019; Singhania et al., 2017a,b,c). Chitinases are another group of fungal enzymes having potential industrial applications, including crop protections (Mathew et al., 2021). Other fungal enzymes of industrial relevance include phytase (Ushasree et al., 2017, 2019), xylanase (Chadha et al., 2019), amylases (Sindhu et al., 2018; Patel et al., 2017), pectinases (Rebello et al., 2017), lipases (Stergiou et al., 2013), etc. In SSF, the filamentous fungi form tubular germinative hyphae after germinating from spores that elongate at the tips. The fungal mycelium is compared to cytoplasmic mass that moves within a tube in a directed flow to the tip of the hyphae. A hydrostatic pressure (turgor) is generated by the osmotic uptake of water which maintains the tubular form of the hyphae and provides the mechanical force for penetration of the hyphae into solid substrate (Trinci et al., 1994). In the biotechnological processes, the SSF has been not been well explored due to technical issues of scale-up which is mainly characterized with the issue of mass transfer, inability to control pH and temperature, challenges in downstream processing due to impurities in the product that can be overcome with the data of the physiology of fungi growing on solid substrates and application of this knowledge in the development of bioreactors adapted to exploit this potential of the filamentous fungi. Also, the ease of automation and operation at large scale is always associated with SmF which makes SmF suitable for commercial production processes (Singhania et al., 2019). It cannot be denied that even SSF which was earlier called as a bioprocess for low-value high-volume products have made its place even in pharmaceutical industries producing high-value products utilized in life saving drugs as well (Kumar et al., 2021). In this chapter, morphology of filamentous fungi in SmF has been emphasized as majority of the industrial process developed are in SmF. Filamentous fungi may grow in three forms in liquid culture, that is as suspension, clumps, or pellets. Suspension usually is referred as homogenous growth as usually it is suspended uniformly in the medium whereas clumps signifies heterogenous loosely packed mycelia which differs in shape and size. Pellets are closely packed mycelia and the size of the pellet differs which decides its productivity. Designing a bioprocess with fungi needs a vast knowledge of morphologies of fungi. Time consuming traditional bioprocess design and empirical strategies is adapted usually for filamentous fungi bioprocess due to complex interactions between process technology, fungi morphology and overall performance. Common empirical methods lack 200 Current Developments in Biotechnology and Bioengineering scientific insight into process technology and key process parameters leading eventually into a suboptimal bioprocess development which may have high chances of failure. It is therefore necessary that bioprocess engineers pursue two overall goals which could be summarized doubling the productivity as well as reducing the bioprocess development time by half (50%) which will lead to development of efficient economic feasible bioprocess. Advancements in bioprocesses as well as genetic manipulation have resulted in significant improvement in production of heterologous protein from filamentous fungi. The manipulations at genetic level opens a new arena for the improvement of strains by introducing desired characteristics in natural strains. For commercialization of any bioprocess, it is inevitable to understand and control the biological system which may not be very easy. 3. Important factors for industrialization of bioprocess with filamentous fungi Three important factors that affect industrial bioprocess development for fungi: (1) strain and inoculum, (2) morphology, and (3) rheology. 3.1 Strain screening and inoculum Identification of the suitable strain is the first step to develop any bioprocess. During screening of potential fungal strain for particular metabolite; only higher productivities if targeted may lead to failure during scale-up process. Hence, for fungi bioprocess from the very beginning, importance must be given to morphological characters of the fungi, its growth pattern, etc. Complex growth pattern such as the tendency to grow adhering to the wall may result in no reproducibility due to heterogeneity (localized growth such as clumps) in distribution and difficulties for scale-up process. There are few studies which shows that reducing the headspace in the bioreactor may reduce heterogeneity in growth of fungi (Siebenberg et al., 2010; Meyer et al., 2010; Posch et al., 2012). In this chapter, we will majorly refer suspended or uniformly dispersed form of mycelia as well as pellets. Homogenous (equally distributed like a suspension) growth of fungi is usually preferred for industrial bioprocess development as heterogenous growth may not deliver the desired productivity when scaled-up in the way as in the small scale. Micro titer plates as well as miniature bioreactors are used for high throughput screening and morphological characteristics of fungi are closely observed. Growth medium also plays an important role for the morphological characteristics of the fungi. For inoculum preparation, in case of fungi, spores are used as inoculum as we can count the spores and is comparatively more homogenous than mycelia. Spores concentration, viability as well as its efficiency for fast germination are few characteristics which are required to be monitored for careful selection of inoculum. Fresh batch of inoculum is needed to be prepared with optimization each time to have high viability. It is necessary to know the germination time of spores to know the exact time point to transfer the seed into production fermenter. Chapter 7 • Filamentous fungal morphology in industrial aspects 201 3.2 Morphology It is an important characteristic to be studied during development of fungal bioprocess. Filamentous fungi pass through many morphological changes during growth as shown in Fig. 1. A variety of factors will influence fungal morphology that is normally characterized by mycelial suspensions, clumps, and pellets, and a good control of these parameters is crucial for successful bioprocesses. There is a complex relationship between the morphology of these fungi with the viscosity of the cultivation broth, transport phenomena, and related productivity. The morphological characteristics vary greatly between freely suspended mycelia and distinct pellets of aggregated biomass. Both of these morphologies have a distinct influence on medium/broth rheology. Hence, the advantages and disadvantages for mycelial or pellet cultivation have to be carefully balanced out. Fungal morphology is often a bottleneck of productivity in industrial production due to inadequate understanding of the morphogenesis of filamentous fungi. To obtain an optimized production process, it is of great importance to gain a better understanding of the molecular and cell biology of these fungi as well as the relevant approaches in biochemical engineering. Specific problems often arise from fungal growth forms such as mass transfer in fungal pellet forms. Fungi may belong to coagulating type strains or noncoagulating type which is a phenomenon used to describe conidia aggregation. Aggregation may be affected by osmolality and pH of the medium immediately after inoculation or after germination of spores it may also get affected by agitation and aeration in addition to pH of the medium. Pellets are formed either by one spore that germinates to form hyphae get branched and entangled, also the dispersed mycelia clump together into packed round structure, which increases in size with time to some extent. Reduced inoculum size is also reported to cause pellet formation (Sohoni et al., 2012). This type of morphology has some benefits as well as limitations. At times, a pellet type of morphology is preferred in industrial cultivations because of the non-viscous rheology of the broth and ease of downstream processing. A list of products have been given with the type of morphologies exhibited by fungi in Table 1. The subsequent separation of the pellets from the cultivation broth is very simpler than in mycelial cultivations but the mass transfer is difficult and the mycelia which are in outer a.Spores b.Hyphae c.Mycelia FIG. 1 Morphological development in filamentous fungal culture. d.Pellets 202 Current Developments in Biotechnology and Bioengineering Table 1 Different morphology of fungi exhibited during bioprocess for production of metabolites. Fungi Morphology/factors affecting Product Bioreactor References Neurospora intermedia Mycelial pellet form (2.38  0.12 to 2.86  0.38 mm) Ethanol and glycerol Nair et al. (2016) Monascus purpureus Trichoderma reesei Mycelium hyphal growth Red pigment Shake flask submerged culture SSF Bulbous cells, hyphal growth Lignocellulosic biomass degrading enzymes such as hemicellulose and cellulase A. terreus Lovastatin and geodin production Cellulase Shake flasks A. oryzae Inoculum concentration determines the pellet size and growth rate Large pellets at low inoculum levels, small flocs at higher concentration, decrease of pellet size with increase of concentration, increased synthesis with small flocs due to high inoculum concentration Mycelial growth SSF using pretreated softwood kraft pulp and lodgepole pine SmF in shake flasks α-Amylase A. niger Pellets Glucoamylase A. niger Mycelial and pellet growth Phytase A. niger Pellets Glucoamylase Carlsen et al. (1996) Grimm et al. (2005) Vats et al. (2004) Lin et al. (2008) A. niger Mycelial growth A. niger Decrease in pellet size with increasing conidia concentration Rising the spore inoculum level from 104 to 109 spores per ml, a clear transition from pelleted to dispersed forms occurs, it demonstrates that adjusting the spore inoculum level controls effectively mycelial morphology Pellets, a correlation between osmolarity, pellet morphology and fermentation yields was reported Fructooligosaccharides (FOS) and β-fructofuranosidase (FFase) Glucoamylase-GFP fusion protein Citric acid Stirred tank bioreactor Stirred tank bioreactor Stirred tank bioreactor Stirred tank bioreactor Shake flasks Shake flasks Xu et al. (2000) Stirred tank bioreactor Papagianni and Mattey (2006) Stirred tank bioreactor Wucherpfennig et al. (2011) T. reesei A. niger A. niger Fructofuranosidase, glucoamylase Manan et al. (2017) Novy et al. (2021) Bizukojc and Ledakowicz (2009) Domingues et al. (2000) Driouch et al. (2010) Chapter 7 • Filamentous fungal morphology in industrial aspects 203 cover are only able to get oxygen and nutrient whereas the cells in the core region starve due to lack of mass transfer leading to the death of inner mycelia/cells. Also, as the mycelial tips are known to be involved in secretion of protein, pellet form is not desired for such bioprocess. Even mycelial growth of filamentous fungi allows homogenous growth like bacteria; allowing comparative easier mass transfer and is preferred for protein production in industries. Thus, the knowledge of coagulation characteristics of fungal inoculum is necessary for bioprocess development for industries. The use of freely suspended or dispersed morphology in SmF has gained popularity because this morphology enhanced growth and production. Table 1 gives dispersed/suspended or pellet growth for production of various metabolites such as enzymes. Filamentous growth increases viscosity in the broth increasing the temperature causes concentration gradient due to transfer limitations within the bioreactor. To ensure high protein secretion and at the same time a low viscosity of the cultivation broth, it is desired by the industry to tailor-make the morphology of filamentous fungi (McIntyre et al., 2001). The morphological type and the related physiology strongly depend on environmental conditions in the bioreactor which can be controlled by regulation of the process parameters such as inoculum concentration and spore viability, pH value, temperature, and dissolved oxygen concentration, as well as aeration- and stirringinduced mechanical stress (Deckwer et al., 2006). Fungal morphology does not only influence the productivity of the process, but due to its impact on rheology it exerts influence on mixing and mass transfer within the bioreactor. Both of these morphologies have a distinct influence on broth rheology. 3.3 Rheology Rheology has a strong complex interconnection with morphology and biomass concentration of fungi (Riley et al., 2000). Rheology is an extremely important attribute for industrial bioprocess development with filamentous fungi. It is highly dependent on fungal morphology as freely dispersed growth of mycelia causes high rheology and with increase in biomass concentration, rheology increases causing high viscosity thereby causing hinderance to mass transfer. Thus, pellet morphology is preferred in production due to less viscosity in the medium; however, at critical diameter pellet core cells lyse due to unavailability of oxygen and nutrient caused by limitation of mass transfer. Pellets with diameter smaller than 400 μm consists of metabolically active layer only where all the cells are supplied with sufficient oxygen; however, in large pellets inner cells autolyse due to insufficient supply of oxygen inside. Hydrodynamics inside the reactor influences rheology to a great extent. Fluid dynamics plays an important role to prevent rupture of pellets due to collision, etc. so as to control rheology. The complication of the bioprocess lies in interdependence of one parameter on another. To facilitate the mass transfer agitation is required but high agitation may also cause shear stress which due to which the fungal filaments in pellet form may break and release in the broth leading to increased viscosity. However, it is also true 204 Current Developments in Biotechnology and Bioengineering that viscosity is less in regions with higher shear rates, near the impellers. Again, increased viscosity leads to insufficient mass transfer and oxygen limitation (Kelly et al., 2006). To overcome fungal mycelial breakage knowledge of hyphal strength as well as mechanical force exerted on them during turbulent flow is must. Energy dissipation by impellers and aeration is also crucial as higher energy dissipation causes strong erosion which may lead to change the fungal culture morphology as well (Kelly et al., 2006). Rheology analysis is important, however in fungal bioprocess traditional methods for rheology measurement may not be used as the broth follow non-Newtonian motion during advanced stage of bioprocess as is more heterogenous. Inline viscometers are available for measuring rheology of heterogenous broth, but rheology is not measured commonly in real-time for filamentous bioprocess. For rheology measurement novel miniaturized devices including vibrating devices, electromagnetic acoustic resonators and ultrasound spectroscopic tools are still in their infancy; however, it describes a potent online rheology measuring tool for filamentous broth viscosity (Posch et al., 2012). Once these factors are under control an industrial bioprocess for fungi can be setup. Airlift fermenters are designed to overcome issues of shear stress for filamentous fungi which is common with stirred tank bioreactor. Airlift bioreactors are more suitable for filamentous fungi as it does not have impeller for mixing which can overcome the issue of shear stress. Airlift fermenters are gas liquid bioreactor which is based on the draught tube principle where aeration and agitation is done via compressed air. Usually, the filamentous fungi require 0.05–0.4 vol of air/vol of liquid/min oxygen supply for optimum growth. In this reactor, the aerator is a glass grid which helps to pass the humidified air, which is useful for mixing and oxygenation. Airlift reactors are in use for culturing filamentous microorganisms especially fungi but they have their own drawbacks as well. The formation of a dead zone due to insufficient mixing and non-uniform nutrient supply caused by high biomass density is a major drawback. These limitations badly affect the growth of filamentous fungi and secondary metabolite production (Rawat et al., 2019). Filamentous fungi are notorious which are ruling over the industries for many bioproducts and the major ones are enzymes. Applications of fungal enzymes in various industries are presented in Table 2 in brief; however, we have focused in this chapter on pulp industry as well as biofuel industry. 4. Applications of filamentous fungi in pulp and biofuel industries 4.1 Pulp industry 4.1.1 Bio-debarking High energy is consumed during debarking process of the wood chips prior to the production of pulp and paper. The presence of bark residues in the raw material might reduce the quality of the paper significantly due to the darkening of the pulp and paper product due to the high content of pectins, lignin, and hemicelluloses in the bark structure. Chapter 7 • Filamentous fungal morphology in industrial aspects Table 2 205 Applications of filamentous fungi in various industrial sectors. Industrial products Filamentous fungi Applications References Antibiotics Penicillium chrysogenum Cephalosporium acremonium Penicillium patulum Pharma/drug industries Demain and Martens (2017), €sten (2019) H€ader (2021), and Wo Aspergillus niger Food and feed industries Aspergillus awamori Food industries Champreda et al. (2019), H€ader (2021), Ouedraogo and Tsang (2021), Rodrigues and Odaneth (2021), Srivastava et al. (2020), Tandon et al. (2021), Trono (2019), €sten (2019) and Wo Penicillins G and V Cephalosporin C Griseofulvin Penicillin N Pleuromutilin Cyclosporin A Enzymes Cyclosporin A and B Glucose oxidase, pectinase, and phytase Xylanase and invertase α-Amylase and glucoamylase Cellulase and hemicellulase Chitinase Mycotoxins Other native fungal products Emericellopsis sp. Tolepocladium inflatum Cylinrocarpum lucidum Aspergillus oryzae Trichoderma reesei, Penicillium sp., Aspergillus sp., Myceliopthora thermophila Trichoderma harzianum Lipase Aspergillus sp. Aflatoxins, citrinin, and ochratoxin Trichothecenes and zearalanone Citrinin, ochratoxin Riboflavin Aspergillus sp. Citric and gluconic acid Kojic acid and biotin Aspergillus niger Food and biofuel industries Agriculture industries as biocontrol agent and cosmetics Food and detergent industries Pharma/drug industries Devi et al. (2020), Lange (2010), Navale et al. (2021), Ranjan et al. €sten (2019) (2021), and Wo Fusarium sp. Penicillium sp. Ashbya gossypii Aspergillus oryzae Nutraceutical industries Food industries Karaffa and Kubicek (2020), €sten Wierckx et al. (2020), and Wo (2019) Food industries, Pharma/drug industries Continued 206 Current Developments in Biotechnology and Bioengineering Table 2 Applications of filamentous fungi in various industrial sectors—cont’d Industrial products Filamentous fungi Applications Itaconic acid Aspergillus terreus Pullulan Aureobasidium pullulans Fusarium culmorum Claviceps purpurea Giberella fujikuroi Building blocks of plastics, resins Food industries Biotin Ergot alkaloid Gibberellic acid Linoleic acid β-Carotene Recombinant heterologous proteins Human interleukin-6 Tissue plasminogen activator (tPA) Human lactoferrin Bovine prochymosin Martierella isabellina Phycomyces blakesleanus Aspergillus niger and A. nidulans Aspergillus niger References Pharma/drug industries Plant growth hormone, agriculture industries Nutraceutical industries Health and medicine H€ader (2021), Khare et al. (2020), Ouedraogo and Tsang (2021), € ck (1997), Ward Radzio and Ku (2012), Wei et al. (2021), and € sten (2019) Wo Aspergillus oryzae, A. awamori, and A. nidulans Aspergillus awamori Lignocellulolytic enzymes such as pectinases, xylanases, and laccases have been found to partially reduce the energy requirements of the debarking process. It has been estimated that the treatment with pectinases might reduce the energy consumption by 80% (Chahal et al., 2020). It also improved the yields by reducing the loss of the raw material during the conventional debarking procedure. Thus, fungal enzyme pretreatment has potential to not only to increase the efficiency of the debarking and save the capital investment but it may also be utilized for the debarking of the most challenging substrates (Bajpai, 2018). 4.1.2 Bio-pitching The pitch includes the lipophilic extractives present in the biomass physio-chemical structure. It includes the fatty acids, fatty alcohols, resins acids, hydrocarbons, steroids, terpenoids, and triglycerides types of chemical moieties. It could be present in about 2–8% in the chemical composition of the biomass depending upon the plant species and the time of the year. It has been reported that presence of pitch in the paper causes the reduction in the paper quality and escalates the equipment maintenance and operating costs during paper manufacturing. The natural process of seasoning of the wood is used to reduce the pitch content in wood, but it causes the reduction in the process yields, low brightness and high processing times. Certain chemicals such as alum, talc, ionic and Chapter 7 • Filamentous fungal morphology in industrial aspects 207 non-ionic dispersants, cationic polymers, and other chemical additives have also been used for the dispersion and adsorption of pitch deposits during pulping and papermaking processes. Controlled seasoning with short duration of time and selected fungi has been proved an effective approach for removal of extractives. Several sapstain and white-rot fungi (WRF) have been tested for removal of wood extractives from different softwoods and hardwoods. Therefore, treatment with the fungal derived enzyme cocktail such as lipase, laccase, sterol esterase, and lipooxygenases is now being proposed to reduce the pitch-related issues in paper manufacturing process (Bajpai, 2018). 4.1.3 Bio-retting Process to produce fibers from the flax stems is known as retting. It is also known as the procedure for the loosening or the separation of the fiber bundles from the cuticularized epidermis and the woody core cells and subdivision to smaller bundles and ultimate fibers. There are both chemical and biological methods for carrying out the retting. The chemical methods depend upon the ethylenediaminetetraacetic acid (EDTA), alkali and steam explosion treatment approaches. The two biological methods are known as water retting and dew retting. The water retting method is based upon mostly anaerobic bacteria treatment such as Clostridium sp. to produce pectinases and ret. flax dew retting is dependent upon treatment with aerobic fungi. However, both of these methods suffer from inconsistent quality concerns and high costs. Thus, now a days pectinase or enzyme treatment is more popular to carry out the bioretting and produce high quality fibers. The Penicillium capsulatum, is the preferred fungal strain for the pectinase production and bioretting treatment. In addition to it several commercial enzyme manufacturers like Novozyme, have developed enzyme-based solutions for the bioretting such as Viscozyme, Multifect, Texazyme for the industrial applications (Bajpai, 2018). 4.1.4 Biopulping During conventional pulping process, debarking of the wood bark is carried out followed by delignification by chemical cooking process by utilizing the caustic soda and sulfur at high temperature and pressure. Instead of the chemical pulping process, the bio-based pulping is dependent upon filamentous fungi to produce lignin and xylan degrading enzymes to produce pulp fibers for paper manufacturing. During biopulping, the debarked wood chips or lignocellulosic biomass is inoculated with selected fungal species under warm and humid conditions at 18–25°C for about 15 days. The fungal hyphae penetrate and colonize the chip surfaces with the help of secreted lignocellulolytic enzymes mainly laccases, ligninases, peroxidases, and xylanases, etc. The fungal treatment leads to the formation of loosening of the fibrous structure of biomass to produce an easy to process pulp for paper production. The fungal strains selected for the biopulping process should possess following physiochemical characteristics such as fast growth rate, ability to colonize on soft and hard wood along with agricultural residues such as rice straw, high lignolytic, and xylanolytic enzyme production with little or no cellulase production and non-pathogenic in nature. The WRF 208 Current Developments in Biotechnology and Bioengineering have been proven beneficial for biopulping process are considered as Phanerochaete chrysosporium, Ceriporiopsis subvermispora, Coriolopsis rigida, Coriolus versicolor var. antarcticus, Peniophora sp., Phanerochaete sordida, Pycnoporus sanguineus, Steccherinum sp., Trametes elegans, and Trametes villosa. There are several benefits of biopulping such as reduced chemicals and energy demand for pulp and paper manufacturing process; it is sustainable and environment friendly in nature, improved paper quality and strength. However, it also suffers with certain disadvantages like it is difficult to scale-up at industry levels, long incubation period for delignification, sluggish rate of reaction, difficult to maintain process conditions for fungal inoculation, aeration and heat dissipation, pigmentation during pulping process and screening and selection of single microbe for biopulping is a challenge. Orozco Colonia et al. (2019) evaluated the effects of biopulping and biobleaching process on oil palm empty fruit bunches using the xylanase and lignin peroxidase enzyme cocktail produced by the filamentous fungal strain, i.e., Aspergillus sp. LPB-5 by solid state fermentation approach. The strain produced about 54.32 U/g of xylanase, 13.41 U/g lignin peroxidase with minimal cellulase activity and 88% pulp yields were obtained. Singhal et al. (2015) have studied the biopulping of the sugarcane bagasse with the enzyme secreted by the filamentous fungal strain Cryptococcus albidus under partially sterilized conditions to access its potential industrial application in the pulp and paper industry. This strain depicted several advantages over other strains with negligible amount of cellulase production, surface colonization, pit formation over bagasse as a substrate and nil cellulose degradation as per the scanning electron microscopy (SEM) and Fouriertransformation infrared spectroscopy (FT-IR) analysis. The fungal treatment of bagasse also reduced the Kappa number by 42% which depicted a substantial reduction in lignin content. Here, the Kappa number is a measurement of standard potassium permanganate solution that the pulp will consume. The measurement is inflated by the presence of hexenuronic acids in the pulp which is formed during chemical pulping of hemicellulose. Biopulping was demonstrated at pilot-scale in a 50 tons plant using Eucalyptus grandis wooden chips using a fungal strain Ceriporiopsis subvermispora (Ferraz et al., 2008). It showed that biopulping process is initiated by the biodegradation of the lignin; however, the no increase in aromatic hydroxyls was observed with the breaking of aryl-ether linkages, showing that the ether-cleavage-products remain as quinone-type structures. Further, it was postulated that the MnP-initiated lipid peroxidation reactions caused the depolymerization of the non-phenolic lignin intermediates. However, cellulose degradation was minimal due to the non-occurrence of the Fenton’s reaction during bio-based pulping process. 4.1.5 Biobleaching Bleaching is a process of removing the residual lignin present in the pulp after pulping process. It is carried out to improve the brightness of the paper and getting rid of the undesired brown pigmentation due to the presence of residual lignin after the pulping process. The chemical based bleaching process is carried out with the chlorine based chemicals Chapter 7 • Filamentous fungal morphology in industrial aspects 209 (chlorine, chlorine dioxide, hydrogen peroxide, oxygen, and ozone). These chemicals are hazardous in nature and generate chlorinated organics as waste which is highly toxic to flora and fauna. Thus, now biobleaching is being considered as an environment friendly alternative to chemical-based bleaching process (Biko et al., 2020). Orozco Colonia et al. (2019) did compare the biological, thermal and chemical pretreatment of oil palm empty fruit bunches followed by the enzymatic biobleaching. It showed that, a permutation of biopulping and biobleaching resulted in 37% lignin removal, 26% hemicellulose extraction, with 74% pulp yields and 37% digestibility. In comparison to it, the biobleaching of alkali treated biomass (oil palm empty fruit bunches) resulted in 82% hemicelluloses, 94% lignin removal and 74% digestibility. The genetically engineered Pichia pastoris strain comprising the XynA gene from fungal strain Thermomyces lanuginosus under the control of GAP promoter was used to produce high quantities of xylanases (139–177 IUmL 1). The xylanase production was scaled up in a 5 L fermenter in 72 h. The xylanase enzyme was used to enhance the brightness of bagasse pulp by biobleaching approach with 50 IU of crude xylanase used per gram of pulp (Birijlall et al., 2011). The biobleaching and the xylanase potential of 98 fungal species isolated from the Eastern Ghats of India was evaluated by the Sridevi et al. (2017). The selected fungi (Trichoderma asperellum) secreted 981.1 UmL 1 of xylanase via SmF. The secreted xylanase decreased the Kappa number by 4.2 points and increased brightness by 4.0 points. FTIR and SEM studies revealed loosening of pulp fibers after enzyme treatment. In another study by Hamedi et al. (2020) have shown that the biobleaching by the Streptomyces rutgersensis UTMC 2445 strain improved the brightness by 7%, at 30°C for 6 h, however, the 60 min treatment reduced the consumption of bleaching chemicals by 12.5% and improved the brightness by 55%. 4.2 Biofuels industry Second generation bioethanol or 2G bioethanol produced from non-food and non-feed lignocellulosic biomass has attracted wide attention globally as an alternative liquid fuel having the potential to replace the fossil derived gasoline. It offers several advantages such as low carbon and greenhouse gas emissions, low pollution, upliftment of rural economy and societal development and improved sustainability. Production of 2G bioethanol involve four major fundamental steps such biomass pretreatment, enzyme saccharification, fermentation, and distillation. Pretreatment is the primary step to disintegrate the orderly and highly complex structure of biomass to make it amenable to get hydrolyzed by the selected biocatalysts or enzymes in the second step to produce fermentable sugars by the hydrolysis of the polysaccharides (cellulose and hemicellulose) (Agrawal et al., 2021; Patel et al., 2020, 2019; Singhania et al., 2017a,b,c, 2015). In the third and fourth steps fermentation by yeast and ethanol distillation is conducted to obtain high purity ethanol. Out of these four steps, pretreatment and enzymatic saccharification or hydrolysis are the most capital and operational cost intensive processes, respectively. 210 Current Developments in Biotechnology and Bioengineering The enzymatic hydrolysis of pretreated biomass is carried out by the enzymes mainly secreted by the filamentous fungi. These enzymes mainly include cellulases (exoglucanase, endoglucanase, β-glucosidase, lyticpolysaccharide monooxygenases or LPMOs), hemicellulases (xylanase, pectinases, arabinofuranosidase, esterases) (Agrawal et al., 2013, 2017; Singhania et al., 2021). Nowadays fungal derived, lignin degrading enzymes like laccases, ligninases, peroxidases and oxido-reductases are also being evaluated to carry out the biological pretreatment of the lignocellulosic biomass (Agrawal et al., 2017). A table depicting the fungal enzyme, their mechanism of action, fungal source and the commercial product available in the market from fungi are shown in Table 3. It showed that, although big commercial enzyme manufacturers such as Novozymes, Denmark offer enzyme cocktails-based solutions for the pulp-paper and biofuels industry, but only a few selected enzymes are available commercially in high purity. Mostly, cellulases and its component enzymes are available in the market with only a few xylanases and hemicellulases. This created a large void area and offers novel opportunities to develop, produce, and market new enzyme products for future research and innovation. Table 3 Application of fungal derived enzymes in biofuel industry. Enzyme (with CAZy & EC) Cellobiohydrolase: GH7 (3.2.1.176) GH6 (3.2.1.91) Endo β 1,4 glucanase: GH5, GH7, GH12 GH45 (3.2.1.4) Endo β 1,4 xylanase: GH10, GH11, GH 7 (3.2.1.8) Endo β 1,4 mannanase: GH5, GH26, GH134 GH5, GH7, GH45 (3.2.1.78) β-Glucosidase: GH3 (3.2.1.21) Xyloglucanase: GH12, GH74 (3.2.1.151) β-Xylosidase: GH3 (3.2.1.37) β-Mannosidase: GH2 (3.2.1.25) Mechanism of action Fungal strain Liberating cellobiose from reducing ends Liberating cellobiose from non-reducing ends Cleaving β 1-4 linkage Cellobiohydrolase I (Megazyme, Trichoderma reesei, Ireland) Trichoderma longibrachiatum, Penicillium sp., Aspergillus sp. Trichoderma β-Glucanase (Sigma, Merck, Germany) longibrachiatum Cleaving β 1-4 linkage Cleaving β 1-4 linkage Trichoderma viride, (xylan) Trichoderma longibrachiatum, Thermomyces lanuginosus Cleaving β 1-4 linkage(glucomannan) Aspergillus niger Cleaving the D-glucose Aspergillus niger from non-reducing ends Cleaving β 1-4 linkage Fusarium equiseti, (xyloglucan) Aspergillus terreus, Cephalosporium sp. Liberating xylose Penicillium janthinellum Liberating mannose Commercially available enzyme from fungi Aspergillus awamori Endo-1,4-β-Xylanase M1 (Megazyme, Ireland), endo-1,4-β-Xylanase from Trichoderma longibrachiatum (Sigma, Merck, Germany), Xylanase (Sigma, Merck, Germany) Endo-1,4 β-mannanase (Megazyme, Ireland) β-Glucosidase (Megazyme, Ireland) Chapter 7 • Filamentous fungal morphology in industrial aspects Table 3 211 Application of fungal derived enzymes in biofuel industry—cont’d Enzyme (with CAZy & EC) Mechanism of action Fungal strain Commercially available enzyme from fungi Hemicellulose debranching enzymes α-Arabino furanosidase: GH43, GH51 GH54, GH 62, (3.2.1.55) α-Galactosidase GH27, GH 36 (3.2.1.22) Cleaving arabinosyl linkages Trichoderma reesei, Aspergillus sp. Cleaving D-galactosyl linkages α-Glucuronidase GH67 (3.2.1.139), GH115 (3.2.1.131) Cleaving D-glucuronic acid linkages Aspergillus oryzae, Trichoderma, Penicillium sp. Aspergillus fumigates Deacetylases Acetyl xylan & acetyl mannan esterases: CE1-6 (3.1.1.6) &CE16 (3.1.1.72) Feruloylesterase: CE1(3.1.1.73) Glucuronoyl esterase: (GE) CE15 (3.1.1) Hydrolyze acetyl groups Trichoderma, Penicillium, Aspergillus Aspergillus niger, Cleaving Aspergillus awamori hydroxycinnamoyl linkages Cleaving ester linkages Schizophyllum commune Lytic polysaccharide monooxygenase (LPMO) AA9 (1.14.99.54) AA9 (1.14.99.56) AA9 (1.14.99.6) AA10(1.14.99.54) AA10(1.14.99.53) AA10(1.14.99.54), (1.14.99.56) AA11(1.14.99.53) AA13 (1.14.99.55) AA14 (1.14.99.6) AA15 (1.14.99.54) AA16 (1.14.99.54) Aspergillus niger Cleaving cellulose at C1/C4 sites Cleaving cellulose at the C4 sites Cleaving β1-4 linkages Removing xylan Cleaving cellulose at C1 site Cleaving chitin at C1 site and cellulose at C1/ C4 and chitin at C1 site Cleaving chitin at C1 site Cleaving chitin at C1 site Hydrolyzing starch Hydrolyzing xylan Cleaving cellulose at C1 site Cleaving cellulose at C1 site Modified from Agrawal, R., Verma, A., Singhania, R.R., Varjani, S., Di Dong, C., Kumar Patel, A., 2021. Current understanding of the inhibition factors and their mechanism of action for the lignocellulosic biomass hydrolysis. Bioresour. Technol. 332, 125042. 212 Current Developments in Biotechnology and Bioengineering 5. Conclusions and perspectives Filamentous fungi have proven their credibility in industrial applications due to its immense potential to produce higher titres of extracellular enzymes. In lab scale studies morphological features does not look to be so important; however, when the studies are moved to pilot or industrial scale than the morphological aspect of fungi becomes the utmost important. Homogenous growth of filamentous fungi increases the viscosity of the medium which poses challenges for mass transfer, though the secretion of protein may be higher. In pellets form, the viscosity of medium remains controlled however the size of the pellet matters hugely for the mass transfer to the mycelium present in the extreme core of the pellet. For higher pellet size, the innermost hyphae die due to lack of mass transfer or oxygen supply. It necessitates to research on how to control the size of the pellets so as to have least viscosity and maximum mass transfer so as to obtain maximum product. Several factors like inoculum concentration, carbon source and its concentration, agitation and aeration affects morphology of filamentous fungi which can be very well exploited for the benefit of the bioprocess by carefully monitoring and designing the bioprocess. Hence it is necessary while developing a bioprocess for filamentous fungi, morphology must be kept as an important criterion during the screening itself to avoid failure of the bioprocess at advanced stage. Rheology must be studied carefully which is highly dependent on morphology of filamentous fungi and the bioprocess. Carbon source and its concentration in the bioprocess as well as agitation, aeration affect rheology inside the reactor. Impellers and its speed need to be specific so as not to cause shear stress and at the same time must ensure proper mass transfer/mixing. Airlift fermenters could be more suitable for filamentous fungi as it does not have impellers for mixing which can overcome the issues of shear stress, however mixing is an issue in this bioreactor. With the advent of genetic engineering tools; genetic modification in fungi became easier and lead do the development of robust strain for hyper production of desired metabolites. Hence, it can be concluded that morphological aspects must be included in earlier phase studies for developing a bioprocess for industrial products by filamentous fungi to ensure success. Pellets as well as dispersed morphology of fungi have their own advantages and it needs careful monitoring to tailor the morphology of filamentous fungi desired according to the bioprocess for metabolites production. References Agrawal, R., Satlewal, A., Verma, A., 2013. 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Elsevier, Amsterdam, pp. 473–497 (Chapter 13). Singhania, R.R., Adsul, M., Pandey, A., Patel, A.K., 2017a. Cellulases. In: Current Developments in Biotechnology and Bioengineering. Elsevier, pp. 73–101 (Chapter 4). Singhania, R.R., Patel, A.K., Pandey, A., Ganansounou, E., 2017b. Genetic modification: a tool for enhancing beta-glucosidase production for biofuel application. Bioresour. Technol. 245, 1352–1361. Singhania, R.R., Patel, A.K., Saini, R., Pandey, A., 2017c. Industrial enzymes: β-glucosidases. In: Current Developments in Biotechnology and Bioengineering. Elsevier, pp. 103–125 (Chapter 5). Singhania, R.R., Patel, A.K., Gottumukkala, L.D., Rajasree, K., Soccol, C.R., Pandey, A., 2019. Solid-state fermentation: current trends and future prospects. In: El-Mansi, E.M.T., Nielsen, J., Mousdale, D., Allman, T., Carlson, R. (Eds.), Fermentation Microbiology and Biotechnology, fourth ed. CRC Press, Taylor and Francis, New York, pp. 243–254. Singhania, R.R., Dixit, P., Patel, A.K., Kuo, C.H., Chen, C.W., Dong, C.D., 2021. LPMOs: a boost to catalyse lignocellulose deconstruction. Bioresour. Technol. 335, 125261. Sohoni, S.V., Bapat, P.M., Lantz, A.E., 2012. Robust, small-scale cultivation platform for Streptomyces coelicolor. Microb. Cell Fact. 11, 9. Sridevi, A., Ramanjaneyulu, G., Suvarnalatha Devi, P., 2017. Biobleaching of paper pulp with xylanase produced by Trichoderma asperellum. 3 Biotech 7 (4), 266. Srivastava, N., Mishra, P.K., Upadhyay, S.N., 2020. Laccase: use in removal of lignin in cellulosic biomass. In: Srivastava, N., Mishra, P.K., Upadhyay, S.N. (Eds.), Industrial Enzymes for Biofuels Production. Elsevier, pp. 133–157 (Chapter 7). Stergiou, P.Y., Foukis, A., Filippou, M., Koukouritaki, M., Parapouli, M., Theodorou, L.G., Hatziloukas, E., Afendra, A., Pandey, A., Papamichael, E.M., 2013. Advances in lipase-catalyzed esterification reactions. Biotechnol. Adv. 31, 1846–1859. Sukumaran, R.K., Christopher, M., Valappil, P.K., Athira Raj, S.R., Mathew, R.M., Sankar, M., Puthiyamadam, A., Prasannakumari, V.A., Aswathi, A., Rebinro, V., Abraham, A., Pandey, A., 2021. Addressing challenges in production of cellulases for biomass hydrolysis: targeted interventions into the genetics of cellulase producing fungi. Bioresour. Technol. 329, 124746. Tandon, S., Sharma, A., Singh, S., Sharma, S., Sarma, S.J., 2021. Therapeutic enzymes: discoveries, production and applications. J. Drug Delivery Sci. Technol. 63, 102455. Trinci, A.P.J., Wiebe, M.G., Robson, G.D., 1994. The mycelium as an integrated entity. In: Wessels, J.G.H., Meinhardt, F. (Eds.), Growth, Differentiation and Sexuality. Springer Berlin Heidelberg, Berlin, Heidelberg, pp. 175–193. Trono, D., 2019. Recombinant enzymes in the food and pharmaceutical industries. In: Singh, R.S., Singhania, R.R., Pandey, A., Larroche, C. (Eds.), Advances in Enzyme Technology. Elsevier, pp. 349–387 (Chapter 13). Ushasree, M.V., Shyam, K., Vidya, J., Pandey, A., 2017. Microbial phytase: impact of advances in genetic engineering in revolutionizing its properties and applications. Bioresour. Technol. 245, 1790–1799. Ushasree, M.V., Jiaswal, A.K., Shyam, K., Pandey, A., 2019. Thermostable phytase in feed and fuel industries. Bioresour. Technol. 278, 400–407. Vats, P., Sahoo, D.K., Banerjee, U.C., 2004. Production of phytase (myo-inositolhexakisphosphate phosphohydrolase) by Aspergillus niger van Teighem in laboratory-scale fermenter. Biotechnol. Prog. 20 (3), 737–743. Ward, O.P., 2012. Production of recombinant proteins by filamentous fungi. Biotechnol. Adv. 30 (5), 1119–1139. Chapter 7 • Filamentous fungal morphology in industrial aspects 217 Wei, H., Wu, M., Fan, A., Su, H., 2021. Recombinant protein production in the filamentous fungus Trichoderma. Chin. J. Chem. Eng. 30, 74–81. € beck, P.S., Steiger, M.G., Mira, N.P., Punt, P.J., 2020. Metabolic specialization in Wierckx, N., Agrimi, G., Lu itaconic acid production: a tale of two fungi. Curr. Opin. Biotechnol. 62, 153–159. € sten, H.A.B., 2019. Filamentous fungi for the production of enzymes, chemicals and materials. Curr. Wo Opin. Biotechnol. 59, 65–70. Wucherpfennig, T., Hestler, T., Krull, R., 2011. Morphology engineering—osmolality and its effect on Aspergillus niger morphology and productivity. Microb. Cell Fact. 10 (1), 58. Xu, J., Wang, L., Ridgway, D., Gu, T., Moo-Young, M., 2000. Increased heterologous protein production in Aspergillus niger fermentation through extracellular proteases inhibition by pelleted growth. Biotechnol. Prog. 16 (2), 222–227. Further reading Jouzani, G.S., Aghbashlo, M., Tabatabaei, M., 2020. Biofuels: types, promises, challenges, and role of fungi. In: Jouzani, G.S., Tabatabaei, M., Aghbashlo, M. (Eds.), Fungi in Fuel Biotechnology. Springer Nature, Switzerland, ISBN: 978-3-030-44487-7, pp. 1–14, https://doi.org/10.1007/978-3-030-44488-4. Patel, A.K., Singhania, R.R., Pandey, A., 2016. Novel enzymatic processes applied to the food industry. Curr. Opin. Food Sci. 7 (1), 64–72. This page intentionally left blank 8 Bioreactors and engineering of filamentous fungi cultivation Daniel G. Gomes, Eduardo Coelho, Rui Silva, Lucı́lia Domingues,
A. Teixeira and Jose CEB —CENT RE OF B IOLOGI CAL ENGINE ERING, UNIVERSITY O F MINHO, B RAGA, PORTUGAL 1. Introduction Filamentous fungi represent a wide group of organisms that on an industrial context can play very distinct biotechnological functions, ranging from the production of a specific antibiotic to the degradation of pollutants in waste waters. Hence, depending on the organism that is being used, as well as the metabolic state required for the process, operation conditions should be adjusted to achieve an optimized process. The cultivation apparatus, commonly known as bioreactor, represents one of the most critical elements for the efficiency of the biological process since different configurations can provide very distinct profiles of aeration, mixing, oxygen and substrate/products diffusion, shear stress, among others, all important determinants in a biological process. This is even more relevant given the growth phenotype adopted by these fungi. Differently from other common organisms used by the industry, such as the budding yeast Saccharomyces cerevisiae or the bacteria Escherichia coli, filamentous fungi develop hyphal structures that will translate into new challenges when compared with “traditional” unicellular organisms. While extended hyphae would favor oxygen/nutrients transfer in comparison with aggregated pellets, the former are also usually associated to superior viscosities (Cai et al., 2014; Zhao et al., 2021). In this sense, given the high sensibility of hyphal structures, particular aspects of bioreactor design such as the shear forces should be carefully considered. For the particular case of cultivating filamentous fungi, bioreactors can be framed under two main categories: submerged fermentation (SmF) or solid-state fermentation (SSF). While SSF has clear benefits for some kind of processes, this still represents a relatively recent technology with many technological limitations when considering an industrial implementation. On the other hand, SmF has already been largely studied and it is now widely implemented on an industry level, corresponding in many cases to already well established and optimized processes. Understanding the main specifications and limitations of each reactor design can be critical when designing a new process employing a filamentous fungus as it will facilitate to achieve an optimized operation from an efficiency and economic standpoint. For this analysis, cells morphology may also play a Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00018-1 Copyright © 2023 Elsevier Inc. All rights reserved. 219 220 Current Developments in Biotechnology and Bioengineering critical role since the reactor design can significantly dictate the morphology of cells under specific conditions, which in turn could have a major impact on their efficiency. The next sections will address the most critical aspects on the design of bioreactors for the cultivation of filamentous fungi, providing a brief description of the main designs currently employed. Because submerged fermentations clearly represent the technology more often used by the current industry, this discussion will be focused on this particular configuration. This will be further complemented with an extensive discussion on the relation between different bioreactor configurations and the morphologies that can be adopted by fungi. 2. Engineering fundamentals in bioreactor design Reactor design on an industrial scale is a complex exercise that should consider multiple technical-operational and economic issues. From a technical standpoint, the reaction apparatus should comply with medium characteristics (e.g., viscosity, concentration of insoluble solids) but also with critical specifications of the microorganism (e.g., maximum operating shear stress, temperature) and of the biological process being conducted (e.g., oxygen demand, mass transfer rates, etc.). On the other hand, key operational parameters, such as agitation, gains an increased importance due to their considerable cost contribution when considering an industrial scale. In the particular case of filamentous fungi-based processes, some of these aspects become even more relevant as the morphologies typically adopted by these organisms (i.e., pellets, hyphal growth) can originate undesired phenomena of inefficient mixing and biofilm formation on the agitation apparatus, or on the other hand, inappropriate mixing can also potentiate damaging/disruption of hyphal structures (Quintanilla et al., 2015). Although multiple variables should be considered, there are a few that stand out and will be here briefly addressed. Because SmF still is the predominant type of process for industrial filamentous fungi cultivation, this discussion will be mainly based on its principles. However, some of these aspects are discussed under a context of SSF on the next chapter. 2.1 Medium viscosity and rheology One of the most important parameters on the cultivation of filamentous fungi is the viscosity of the medium, and ultimately its rheological properties. Viscosity can be defined as the resistance of a given fluid to flow, which in the context of an agitated system refers to its resistance to shear and tensile stress. According to the forces being transmitted by the agitation system to the liquid (cultivation medium) and specific physical properties such as temperature, a given medium viscosity will be obtained, which will dictate the rheological profile. When growing filamentous fungi, one of the first elements to consider in terms of viscosity changes during the process is the fungal biomass being produced (Abdella et al., 2016). Regardless, the extent of biomass formation, it will always result in an increase of medium viscosity since it refers to a solid element being formed. Hence, the Chapter 8 • Bioreactors for filamentous fungi 221 fermentation medium here characteristically follows a non-Newtonian behavior (Bliatsiou et al., 2020), which means that viscosity changes according to the shear stress applied (Goudar et al., 1999), as previously depicted by the Ostwald-de Waele relationship (Eq. 1). μapp ¼ τ K γn ¼ γ γ (1) where μapp is the apparent viscosity, τ is the shear stress, γ is the shear rate, K is the flow consistency index and n is the flow behavior index. The complexity of viscosity variation further increases when one considers that the ever varying physiological response of the fungal species during the entire duration of the process may also affect medium viscosity (Veiter et al., 2018). One final factor to consider is the additional challenge imposed by the utilization of lignocellulosic materials (LCMs) as substrate (Haddadin et al., 2009; Lan et al., 2013; Panagiotou et al., 2003; Xiros et al., 2008), an attractive procedure on industrial operations since it usually refers to lowcost materials (Gomes et al., 2016). LCMs typically present low-densities and high waterretention capacities (Gomes et al., 2018), resulting on high viscosity suspensions and the operational challenges associated to that (Koppram et al., 2014). As for the fungal biomass, the contribution from the LCMS on medium viscosity will vary along the process as the material is being hydrolyzed; this conversion may either result on the production of small soluble molecules, with minimal impact on medium viscosity, to the production of fungal biomass, which itself results on a viscosity increase. As we can see, this intricate complex process on which viscous elements are being synthesized as others are being hydrolyzed leads to a more difficult control of the overall process since numerous critical variables affected by viscosity are difficult to predict, and consequently control. 2.2 Power consumed for agitation On an industrial scale, the energy consumed for agitation represents one of the most important contributors in operational costs. This gets particular relevance in operations with filamentous fungi, where an efficient agitation would be critical not only for a proper aeration but also to counteract highly viscous regimes commonly found on these processes (Nørregaard et al., 2014). For systems with mechanical agitation, such as the traditional stirred tank reactor, this could represent a considerable consumption of energy not only involved on the agitation process, but also on temperature control from counteracting the heat generated by the mixing impellers ( Jaszczur et al., 2020). For this particular context, the power consumed for agitation (P), also known as power draw, can be generally estimated by Eq. (2): P ¼ N P ρN 3 D5+ (2) where NP is the power number, ρ is the broth viscosity, N is the velocity of impellers and D is the impeller diameter. 222 Current Developments in Biotechnology and Bioengineering The dimensionless power number is case specific, depending on the selected impeller but also the turbulence regime, which itself is dictated by the Reynolds number (Eq. 3). Re ¼ ρND2 μ (3) A most relevant aspect to consider is that for aerated processes, which is the most common configuration adopted by filamentous fungi, the presence of air usually causes a reduction of the power number (Albaek et al., 2008), here referred as degassed power number (PG), which ultimately results on an inferior power consumption. The extent to which this reduction occurs will however depend on the specification of the process as not only the type of impeller but also the flow of air can affect this reduction (Stocks, 2005). 2.3 Medium aeration and kLa One of the most critical aspects in aerobic submerged filamentous fungi fermentations (the most common process configuration adopted by industry) is the supply of oxygen, being inclusively intimately related with the selection of the reactor configuration. Since filamentous fungi have a high demand of oxygen, their productivity (g product per medium volume per hour) is closely dependent of oxygen availability for cells (Petersen et al., 2008). As opposing to a soluble component on the liquid medium (e.g., glucose), oxygen represents a greater challenge since it has a low solubility in water and its transport to cells requires a more complex mechanism involving its transfer across the interface between the air bubbles and the medium, which itself could be affected by numerous factors (e.g., viscosity; surface area, turbulence, etc.). Because the considerable oxygen demand and the challenging transfer process, oxygen frequently becomes a limiting element in aerobic submerged fungal-fermentations (Klein et al., 2002). The concentration of dissolved oxygen inside a bioreactor (CL,O2) can be described by Eq. (4):  dC L,O2 ¼ kL a C 0G,O2 dt C L,O2  dC L,O2 ¼ kL a C 0G,O2 dt  qO2 X C L,O2  DC L,O2 qO2 X (4) (5) 0 is the concentration of oxygen for saturation conditions, qO2 is the specific rate where CG,O 2 of oxygen consumption by cells, X is the concentration of biomass, and D is the dilution rate. On a batch culture, which refers to a typical configuration in fungi-based processes, Eq. (4) can be simplified to Eq. (5), which states that the dynamics of oxygen availability will be determined by the oxygen consumed by cells from the liquid media, but most importantly, the rate of oxygen transfer from gas-to-liquid phase. This important parameter is critically determined by the volumetric coefficient of oxygen transfer between phases—kLa—which itself could be affected by a very complex set of variables. Although there is not a particular expression to describe this parameter, Eq. (6) has been widely proposed for that (Brown Chapter 8 • Bioreactors for filamentous fungi 223 et al., 2004) with Nørregaard et al. (2014) recently proposing a modified version for filamentous fungi, which takes into account medium viscosity (Eq. 7).   P a U gas b V (6)   P a U gas b μapp c V (7) kL a ¼ C kL a ¼ C where P is the power consumed for agitation, V is the volume of medium, Ugas is the superficial velocity of the gas, with a, b, c, and C being empirical constants to be determined. From a practical standpoint, kLa is intrinsically connected to two main operation aspects: agitation and medium properties. In what refers to the role of agitation, adding to its function on a homogeneous distribution of air bubbles on the reactor, one may also consider the contribution that agitation-based shear forces have on the generation of smaller air bubbles, hence resulting on a superior specific superficial area for oxygen transfer (Laakkonen et al., 2007). On the other hand, medium properties such as viscosity can significantly affect the equilibrium of dispersion-coalescence phenomena, which directly affects the specific superficial area, but can also affect the mass transfer coefficient (kL). As mentioned above, viscosity represents an especial challenge given its dynamic behavior over the entire course of a filamentous fungal fermentation, therefore, an efficient process control would be required to control this variable ensure a specific process performance. 2.4 Shear forces and shear stress In the operation of biological reactors, a specific aspect that should be carefully controlled is the shear stress undertaken by the biological elements in place, either enzymes or cells. Shear stress translates the sum of shear forces exerted on a specific region/element, and for the case of microorganisms, it is a particularly sensitive aspect since some types of cells are highly susceptible to shear forces (e.g., animal cells) (Gooch and Frangos, 1993). Shear forces are also relevant for some aspects of industrial processes namely cells immobilization, being usually an undesirable trait. For the particular case of filamentous fungi, shear forces have been one of the most critical elements determining cells morphology, contributing to more or less compacted hyphal aggregates (Mitard and Riba, 1988). On the other hand, distinct compactions of hyphal agglomerates have been associated with different mass transfer limitations and oxygen availabilities (Mantzouridou et al., 2002). Although the potentially damaging effect of shear forces is an aspect to consider, shear forces should also be generated in suitable levels to enable an adequate rheological profile for fungi cultivation. Shear forces applied to the liquid medium will importantly dictate its viscosity, which in turn can affect gas-liquid and liquid-liquid mass transfer, among other mechanisms. Shear forces may also dictate the formation of stagnant/dead zones since particular parts of the reactor may be subjected to an insufficient shear rate where it cannot overpass yield shear stress (i.e., the requirement to initiate movement). Given 224 Current Developments in Biotechnology and Bioengineering the particular nature of filamentous fungi cultivation in terms of viscosity contributors, this is certainly an important aspect to consider when selecting a reactor configuration. On an industrial reactor, shear forces are mainly generated by agitation, and while these are much more intense for a mechanical agitation system (i.e., mixing impellers), airbased systems (bubble columns/air-lifts) also generate them to some degree. 3. Traditional bioreactor designs for filamentous fungi cultivation Over the last decades, filamentous fungi have been growingly used in a multitude of biotechnological applications, among which stands out the production of cellulolytic enzymes, antibiotics, chemicals, food additives, but more recently also the bioremediation of different chemical compounds (Gopinath et al., 2016a). Many of these technologies have been implemented for decades with few modifications being introduced in the process. Regarding the bioreactor design, stirred tank reactors, and bubble-column reactors have been largely predominant (Albaek, 2012), while other configurations have been increasingly used such as packed and fluidized-bed reactors, or other variarions of airbased systems (Table 1). More recently, there has been also a significant growth over the technology of SSF, as apposing to submerged fermentation, given raise to a new set of designs which should register a superior demand in the upcoming years. Table 1 Literature reports describing biotechnological applications of filamentous fungi operated under distinct bioreactor configurations (HVC—high-value compounds). Reactor design STR Microorganism Penicillium oxalicum Aspergillus nidulans Penicillium citreonigrum Penicillium oxalicum Penicillium brevicompactum Fusarium equiseti Biotechnological application Substrate Bioremediation Cr(VI) Enzyme production Synthetic media Production of HVC Synthetic media Production of HVC Paneer whey Production of HVC Synthetic media Bioremediation Aspergillus oryzae Tannery wastewater Enzyme production Synthetic media Trichoderma virens Enzyme production Aspergillus niger Production of HVC Aspergillus niger Production of HVC Enzyme production Product(s) Xylanases Fructooligosaccharides Inulinase Mycophenolic acid References Long et al. (2020) Abdella et al. (2020) Nobre et al. (2019) Singh et al. (2019) Patel et al. (2018) Sharma and Malaviya (2016) Polygalacturonases Fontana and Silveira (2012) Modified Kawachi Chitinases Abd-Aziz et al. (2008) medium Waste office Gluconic acid Ikeda et al. (2006) paper Synthetic media Citric acid Papagianni et al. (1998) Cellulases Libardi et al. (2017) Chapter 8 • Bioreactors for filamentous fungi 225 Table 1 Literature reports describing biotechnological applications of filamentous fungi operated under distinct bioreactor configurations (HVC—high-value compounds)—cont’d Reactor design Bubble column Air-lift Microorganism Trichoderma harzianum Penicillium brevicompactum Aspergillus niger Penicillium echinulatum Trichoderma reesei Aspergillus oryzae Air-lift Aspergillus niger Aspergillus japonicas + Aspergillus niger Trichoderma viride Product(s) Domestic wastewater Production of HVC Synthetic media Mycophenolic acid Shu et al. (2010) Production of HVC Beet molasses Enzyme production Synthetic media +cellulose/sorbitol Enzyme production Cellulose-yeast extract medium Enzyme production Synthetic media Bioremediation Tanning effluent Production of HVC Sucrose Bioremediation Cr(VI) Citric acid Cellulases and xylanases Cellulases Production of HVC Synthetic media Bioremediation Cadmium Enzyme production Synthetic media Xylanases Aspergillus terreus Production of HVC Synthetic media Itaconic acid Enzyme production Synthetic media Food industry Soymilk Glucose oxidase Production of HVC Synthetic media Cephalosporin C Bioremediation References Berovic et al. (1993) Ritter et al. (2013) Bannari et al. (2012) Polygalacturonases Fontana and Silveira (2012) Sepehr et al. (2012) FructoLin and Lee (2008) oligosaccharides Morales-Barrera and Cristiani-Urbina (2006) Gluconic acid Klein et al. (2002) Rostami and Joodaki (2002) Aspergillus niger Aspergillus niger + Penicillium austurianum Aspergillus awamori Aspergillus niger Fluidized- Aspergillus oryzae bed Cephalosporium acremonium Packed- Trichoderma bed asperellum Aspergillus awamori Toluene Siedenberg et al. (1997) Lyngstad and Grasdalen (1993) Tr€ager et al. (1992) Kumar and Mulimani (2010) Park et al. (1989) Gopinath et al. (2016a, b) Dı́az et al. (2013) Aspergillus japonicus Enzyme production Grape pomace + orange peels Production of HVC Pearl barley or mannitol Bioremediation Cyanide and formamide Production of HVC Sucrose Aspergillus niger Bioremediation Cephalosporium acremonium Aspergillus nidulans Production of HVC Synthetic media Cephalosporin C Kapoor and Viraraghavan (1998) Kundu et al. (1992) Enzyme production Synthetic media Aspergillus nidulans Enzyme production Synthetic media Aryl alcohol oxidase Xylanases Pardo-Planas et al. (2018) € ller et al. (2015) Mu Penicillium brevicompactum Fusarium oxysporum Tricklebed Biotechnological application Substrate Hydrolytic enzymes Mycophenolic acid Alani et al. (2009) Campos et al. (2006) Fructooligosaccharides Heavy metals Chien et al. (2001) 226 Current Developments in Biotechnology and Bioengineering 3.1 Mechanically agitated stirred tank reactor Stirred-tank reactors (STRs) are possibly the reactor design most commonly used by industry (Wang and Zhong, 2007), being also widely studied by academia. It encompasses a very simple system typically composed by a (vertical) reaction tank with a mechanical agitation apparatus containing a set of specific mixing impellers (Fig. 1A). The impeller selection and the operating velocities enables here a close control not only of the oxygen transfer rate but also of the overall mass and heat transfer, ultimately influencing medium viscosity, nutrients and products diffusion and the shear forces affecting biological elements (cells and/or enzymes). Generally, this reactor configuration enables a high oxygen transfer rate and mixing efficiencies, both particularly positive in filamentous fungi cultivations. From an operational standpoint, it also allows a superior performance under high-viscosity systems, which are very common as hyphal growth proceeds and/or lignocellulosic residues are used. FIG. 1 Schematic representation of bioreactor configurations typically used for filamentous fungi cultivation: STR (A), bubble column (B), concentric-tube air-lift (C), and external circulation air-lift (D). Chapter 8 • Bioreactors for filamentous fungi 227 In relation to the mixing impeller, there is a set of typically employed configurations which are most commonly used by industry, being this selection made according to specific requirements of the process. In this sense, mixing impellers should be considered under the scope of its entire range of functions since agitation aims not only to efficiently disperse substrate components and products, but also to disperse and efficiently break air bubbles. Mixing impellers are typically classified into two main categories according to the type of flows they promote inside the vessel: radial pumping impellers and axial pumping impellers. The first type is characterized by pushing the liquid away in a radial direction and is typically used to disperse air bubbles into the medium. Some examples refer to the Rushton impellers, one of the most common configuration currently employed by industry, but also the Smith impellers. Axial impellers, on the other hand, intend to move the liquid in a direction parallel to the axis (central shaft), promoting a higher mixture of the liquid medium (Doran, 2013). This configuration also enables strong downward flows toward the bottom of the vessel, which became especially important for mediums with high amounts of solids by decreasing the settling of solids and the formation of dead/stagnant zones. Accordingly, STRs for filamentous fungi cultivation typically adopt a combined system of a Rushton impeller at the bottom position and one or more axial impellers in superior positions (Nørregaard et al., 2014). The exact selection of the adopted impellers will always refer to an adequate balance between providing a sufficient degree of shear forces to enable favorable mixing and not achieving excessive levels that could affect cells viability or the integrity of the intended hyphal morphology (Nienow, 1990). Another important aspect in STRs, which also relates to the mixing impellers, is the power consumed for agitation. Although STRs are associated with efficient liquid-liquid and solid-liquid mass transfer, this is achieved at the expense of a significant energy consumption, one of the main drawbacks when compared to non-mechanically agitated systems. On this context, also the impellers selection can have a critical role since different impellers are characterized by a different power number (Hall, 2012), a critical parameter on the estimation of the agitation power, as discussed above. In this sense, it would be highly relevant to find an impeller system with a high discharging number (which directly dictates mixing efficiencies) and a low power number. As an example, Rushton impellers can have an NP around 6 (Derksen and den Akker, 1999) while for a Lightnin A310 this is only 0.3 (Hermajani and Tatterson, 2004), which could mean a drastically lower energy consumption. From a general perspective, STRs are definitely an option to consider for filamentous fungi cultivations. Although presenting a high energy consumption and shear stress, in addition to oxygen transfer rates typically below other systems, their high shear forces also allow to operate under high-viscosity environments, which could be a desirable trait. Furthermore, despite presenting some internal mechanical parts (i.e., mixing impellers), there are no major constraints in relation to operations of downstream processing as one could expect for other configurations such as trickle/packed-bed reactors. Regardless of the final product being a soluble component (e.g., citric acid, penicillin, etc.) or fungal biomass, its final recovery can be conducted efficiently. 228 Current Developments in Biotechnology and Bioengineering 3.2 Bubble columns Right next to the STR, bubble columns represent another frequently used reactor configuration for filamentous fungi cultivation providing two critical features for this type of microorganism: high aeration rates and low shear stress. In general terms, a bubble column reactor is a simple vertical vessel only composed by an air sparger (typically) at the bottom, which injects air bubbles into the liquid broth (Fig. 1B). Depending on the flow of air and the cross-section superficial area, air bubbles will have a given superficial gas velocity (Ugas), which for this system is typically much higher than the superficial velocity of the liquid (Ugas ≫ Uliq). Regarding the liquid phase, it can be operated under a batch or a continuous regime, being the liquid fed either by co- or counter-current. According to superficial velocity of the gas air bubbles will move under distinct flow behaviors: for small values of Ugas (inferior to 1–4 cm s 1), bubbles will move uniformly through the liquid; for higher velocities of the gas, air bubbles will be less uniform and bubble coalescence will occur, leading to the generation of local gradients of medium density, which will result in additional circulatory fluxes (Van’t Riet and Tramper, 1991). Still, bubble columns are commonly associated to considerable mass transfer limitations for liquid-liquid and solid-liquid interfaces. Differently, they are coupled to efficient liquid-gas transfer, which becomes obviously interesting for filamentous fungi. Under this configuration, oxygen transfer is dependent of the superficial velocity of the gas, which is strongly dictated by liquid coalescence, the type of flow and the size of air bubbles (Heijnen and Van’t Riet, 1984). Accordingly, under low viscosity regimes Van’t Riet and Tramper (1991) related the kLa with the gas superficial velocity (Eq. 8). On the other hand, under viscous regimes (commonly found for filamentous fungi cultivations) oxygen transfer could be critically affected by viscosity, as expressed by Eq. (9) early reported by Deckwer et al. (1982). k L a ¼ 0:32U cgas 0:7 k L a ¼ cπ 0:84 (8) (9) c is the superficial velocity of the gas corrected for local pressure and π is liquid where Ugas dynamic viscosity. Despite their mass transfer limitations, and somehow limited application range, bubble columns still represent a very attractive configuration from an industrial perspective. The fact that they almost have no moving parts implies a very simple construction, hence a lower capital cost (Yen et al., 2019). Furthermore, this simpler configuration leads to low maintenance costs and simpler sterilization processes (Wu and Tu, 2016), adding to a reduced contamination risk due to a more closed system. Other important advantage to consider are the reduced shear forces enabled by these systems; although this may constitute an important drawback for an efficient mixing of viscous broths, a lower shear stress can be critical when operating with hyphal structures highly sensitive to these forces. A final aspect to consider, which is probably one of the main advantages in this type of configuration, refers to the drastic reduction of power consumption as energyintensive mechanical agitators are not employed. Chapter 8 • Bioreactors for filamentous fungi 229 3.3 Air-lift reactor In response to some of the most common limitations attributed to bubble columns, alternative configurations emerged derived from this one. Air-lift reactors are a specific type of bubble columns with the particularity that they are divided into distinct liquid circulation zones (Fig. 1C). Hence, an air-lift reactor contains three key areas: a vertical gassed section where air bubbles are injected and flow vertically, commonly known as riser; a liquid descent tube, where the liquid circulates almost/fully degassed, known as downcomer; and the top section, or degassing zone, where air bubbles coalesce resulting in a drop of liquid density (Duan and Shi, 2014). The mechanism behind this design is based on the fact that when air bubbles are injected in the riser, the density of the liquid on this vertical column will decrease, which will cause a vertical ascent of the liquid. As the liquid is degassed on the top section, its density decreases causing its vertical descent in the downcomer (Van’t Riet and van der Lans, 2011). This constant interchange between a gassed (low density) and ungassed state (high density) causes a continuous liquid circulation, and consequently, much better mixing in comparison with bubble columns (Duan and Shi, 2014), especially for liquid-liquid and liquid-solid mass transfer. In fact, for the particular case of the degassing zone (top section) the combined action of liquid circulation and gas release is commonly associated to a highly efficient mixing zone, where the principles of an STR can be considered valid (Verlaan and Tramper, 1987). This is highly relevant given the fact that the limited levels of shear forces generated on this kind of systems may in some cases originate zones with “stagnant” volumes. For this particular reactor, it is still important two distinct two main configurations as air-lifts may either present the riser internally, or as an alternative, separated from the downcomer by a horizontal tube, working as an external circulation loop (Fig. 1D); the later may be advantageous in some cases since the top of the gassed section is more distant from the downcomer, which can reduce the amount of air on this section. Another important distinguishing aspect in relation to bubble columns is that air bubbles on air-lifts have typically a more uniform movement, which reduces bubble coalescence and consequently increases the kLa. On the other hand, it is well established that its improved liquid circulation may in fact mean a lower contact period with the liquid, which may actually reduce the kLa and represent a less attractive option under a given scenario (Stanbury et al., 2017). 3.4 Trickle-bed bioreactor Trickle-bed bioreactors, schematically represented in Fig. 2, resort to the incorporation of solid supports to assemble a packed bed, on the top of which the liquid substrate is fed and trickles downward to the bottom due to different liquid flow mechanisms (overloading, capillary dispersion, and mechanical dispersion) (Lappalainen, 2009). As liquid and gas flow within the interstitial spaces of the packing material, nutrients are provided to the fungus while maintaining moisture and aeration, ultimately promoting growth. 230 Current Developments in Biotechnology and Bioengineering FIG. 2 Schematic representation of a trickle-bed bioreactor. In opposition to a submerged packed bed, trickle-bed bioreactors avoid the complete submersion of the packing, in a distinct approach for the establishment of a three-phase system, promoting a more abundant content of gas within the bioreactor to which the fungus will be exposed. Thus, the constraints of limited gas transfer rates and/or high shear stress imposed in submerged culture systems are smaller in trickled bed bioreactors (Klasson et al., 1992). For a successful establishment of a trickling regime, several hydrodynamic parameters must be taken into account. The central parameter to consider is pressure drop, which is influenced by multiple process variables, namely reactor design (dimension, cross section, particle size, and geometry), operation parameters (gas and liquid flow rates) and properties of the liquid feed (density, surface tension, and viscosity) (Restrepo et al., 2021). One particular aspect in the operation of these reactors is to avoid the formation of preferential flow pathways, which can lead to the appearance of dead volumes and affect the residence time of the fluid in the reactor (Augier et al., 2016). Depending on the intended process, gas can be fed into the reactor in co-current or counter-current regimes (relatively to the liquid feeding), which will impart the concentration of volatile components throughout the packed bed but also the moisture content available, which must also be accounted for when considering fungal metabolism. Besides creating a distinct three-phase system, the solid matrix provides a stable surface for fungal adhesion and growth, enhancing mechanical resistance and stabilizing the mycelium. Different materials can be used for packing trickle-bed bioreactors: inert Chapter 8 • Bioreactors for filamentous fungi 231 materials, which will solely provide the solid support (e.g., polyethylene plastic, lava rocks) € ller et al., 2015) or non-inert materials which can provide (Pardo-Planas et al., 2018; Mu solid support and nutrients for fungal growth (e.g. wheat straw) (Kresinová et al., 2018). As previously referred, the choice of support will impact not only fungal growth but also overall hydrodynamics within the bioreactor, as different packing materials will present distinct porosities, liquid holdups, and pressure drops. Moreover, when considering active solid materials such as lignocellulosic supports, fungal growth and metabolism will modify chemical and structural properties of the solid packing due to hydrolysis of the solid substrate and consumption of nutrients. Thus, hydrodynamic characteristics such as viscosity or pressure drop within the bioreactor will vary throughout the fermentation process, which must be accounted for in the context of fungal growth with especial attention for particular cases, such as the implementation of continuous fermentations. Despite the referred benefits of these solid supports, one must also account for a particular limitation regarding product recovery. Hence, while most fungal products can be easily recovered after fermentation (even enzymes, which are produced extracellularly), with biomass immobilization inclusively facilitating this process, the production of fungal biomass would be unviable under this concept; the strong adhesion to the solid support, previously resembled as an important trait, will now render a very complex recovery process. Accordingly, fungal biomass production is usually conducted on traditional systems such as STRs (Abdullah et al., 2013; Papaspyridi et al., 2010) and air-lifts (Aragão et al., 2020), or SSF systems without an immobilization support. Trickle-bed bioreactors have been applied in combination with filamentous fungi in distinct bioprocess applications, namely bioremediation and production of value added compounds, with examples of filamentous fungi like Aspergillus nidulans and Pleurotus ostreatus being successfully grown immobilized in this bioreactor configuration (Pardo€ ller et al., 2015; Kresinová et al., 2018). Planas et al., 2018; Mu 3.5 Packed-bed bioreactor Packed-bed bioreactors, schematically represented in Fig. 3, also resort to the use of a solid support to assemble a packed matrix. In opposition to trickle-bed bioreactors, packed-bed reactors can also operate under a submerged regime, in which the packing is filled with liquid. Liquid and gas are fed upwards into the reactor, with fungal growth also occurring in the interstitial spaces between packing particles (Zhong, 2011). Hence, while also establishing a three-phase system, the content of liquid within the vessel is higher. Again, control of hydrodynamics is essential, as application of excessive flow rates and high linear velocities in the liquid/gas feed will cause unwanted compression of the packed bed and undesired pressure drops, which will negatively impart mass transfer. As seen for the trickle-bed, a packed-bed configuration also poses risks regarding preferential pathways and dead volumes, especially when compression of the solid matrix occurs (Warnock et al., 2006). Due to the application of low flow rates and the protective effect of the solid support, shear stress imposed in packed reactors is also low, which is beneficial for the growth of filamentous fungi (Cong et al., 2001). 232 Current Developments in Biotechnology and Bioengineering FIG. 3 Schematic representation of a packed-bed bioreactor. The operation of a packed-bed bioreactor allows different strategies for aeration of the medium, as liquid/gas mixing can be performed in a separate vessel, protecting biomass from high shear stress (Warnock et al., 2006). Moreover, even in lower abundancy of gas when compared with the trickle-bed, packed-bed bioreactors promote gas holdup since the particles in the packed bed provide resistance to the rise of gas bubbles, which ultimately depends on pressure drop and rheology characteristics (Taghavi and Balakotaiah, 2019). While similar packing materials can be mentioned for assembling this type of reactors, either inert (e.g., polyurethane foam, alginate, glass beads, polysulfone) (Almyasheva et al., 2018; Campos et al., 2006; Kapoor and Viraraghavan, 1998) or non-inert (pearl barley, grape pomace, orange peels, coir) (Alani et al., 2009; Dı́az et al., 2013; Gopinath et al., 2016b), their intrinsic characteristics must also be taken into account in conjugation with the rheological characteristics of the medium to control process hydrodynamics (Warnock et al., 2006). Packed-bed reactors are quite popular in filamentous fungi fermentations with severable mentionable applications, as for example bioremediation using Aspergillus carbonarius (Arikan et al., 2019), Fusarium oxysporum (Campos et al., 2006), and Aspergillus niger (Gopinath et al., 2016a), the production of value added products such as biodiesel by Aspergillus niger (Almyasheva et al., 2018), mycophenolic acid by Penicillium brevicompactum (Alani et al., 2009), or lovastatin by Aspergillus terreus (Kumar et al., 2014), but also the valorization of lignocellulosic residues such as grape pomace and orange peels (Dı́az et al., 2013). Chapter 8 • Bioreactors for filamentous fungi 233 3.6 Fluidized-bed bioreactor Fluidized-bed bioreactors, schematically represented in Fig. 4, operate under an expanded-bed regime, where control of reactor hydrodynamics targets the enhancement of mass transfer and mixture. To achieve fluidized state, this type of configuration resorts to the application of higher flow rates, sufficient to counteract the sedimentation velocity of the solid and suspend it in the gas/liquid feed, making it behave like a fluid. Thus, mixing properties of fluidized-bed reactors are somewhere between the behavior of packed bed and STR reactors ( Jaibiba et al., 2020). Hydrodynamics in fluidized bed bioreactors must be fine-tuned to allow bed expansion while avoiding wash-out or high pressure drops in the packing material, which will ultimately lead to problems in the bioprocess. Such control within the bioreactor will also depend on some of the previously mentioned parameters, namely flow characteristics (e.g., flow rate and regime, linear velocity), liquid phase characteristics (e.g., density and viscosity), and solid support characteristics (e.g. density, terminal sedimentation velocity) (Li, 2017). By expanding the bed, the occurrence of void volumes or preferential flow pathways within the bioreactor is minimized, therefore enhancing mass transfer when compared with other packed reactor configurations. This is achieved with an overall lower specific power input when compared for example with STR configurations (Porcel et al., 2005). Also, as particles are maintained in suspension and constant motion, the imposed shear FIG. 4 Schematic representation of a fluidized-bed bioreactor. 234 Current Developments in Biotechnology and Bioengineering forces are lower, which has a direct impact on fungal morphology and growth (Kumar and Mulimani, 2010). For instance, when comparing with STR configurations, fluidized bed leads to lesser fragmentation of hyphae and larger mycelium pellets (Porcel et al., 2005). Fluidized-bed reactors can operate without the inclusion of a solid support when targeting solely fluidization of free fungal pellets (Porcel et al., 2005; Sadhasivam et al., 2010). However, fungal biomass can also be immobilized in solid particles (e.g., alginate, celite) (Kumar and Mulimani, 2010; Park et al., 1989). Among a diverse range of applications, these reactors have been applied for bioremediation using Trichoderma harzianum (Sadhasivam et al., 2010), for processes of continuous hydrolysis with Aspergillus oryzae (Kumar and Mulimani, 2010) and for fermentations with Aspergillus terreus (Porcel et al., 2005). 3.7 Rotating-bed bioreactor Rotating-bed bioreactors, schematically represented in Fig. 5, can be described as a modification of the STR configuration where the main difference resides on the assembly of the rotary shaft. In rotating-bed bioreactors, the impellers in the rotating shaft are replaced by a porous bed, on which the fungus adheres and grows (Xu and Yang, 2007). In opposition to the STR, the rotary bed avoids the shear introduced by mixing blades and provides a suitable surface for fungal adhesion and growth. The appearance of smaller pellets or lose mycelia clumps are minimized by promoting the formation of a stabilized biofilm, which presents advantages in terms of downstream processing and biomass reutilization in the case of continuous or successive batches processes (Abdella et al., 2016). However, the creation of a dense immobilized fungal mass can also impose a relevant hurdle in terms of mass transfer. As the biofilm thickens, access of nutrients to the inner section of the fungal biomass will be limited by diffusional constraints, which are only partially counteracted by the centrifugal forces applied in the rotating bed. Nevertheless, immobilization of the filamentous fungus also presents a rheological advantage, FIG. 5 Schematic representation of a rotating-bed bioreactor. Chapter 8 • Bioreactors for filamentous fungi 235 namely the reduction of viscosity associated to biomass growth, which ultimately impairs gas exchange rates throughout the fermentation (Xu and Yang, 2007; Lan et al., 2013). This configuration has been applied in the production of enzymes, namely β-glucosidase using Aspergillus niger (Abdella et al., 2016) and cellulases using Trichoderma viride (Lan et al., 2013), the production of mycophenolic acid by Penicillium brevicompactum (Xu and Yang, 2007), among other cases. 4. Main reactor designs in solid-state fermentation processes An attractive alternative to submerged cultures in the particular case of filamentous fungi refers to SSF. This configuration is characterized by low amounts of free water with the microbial growth essentially occurring on moist particles. Although this may even allow the presence of some droplets of water between solid particles, or the formation of thin layers of water at their surface, there is not a continuous liquid phase and this interparticles space is predominantly filled with air (Mitchell et al., 2006a). These aspects perfectly meet two important characteristics of filamentous fungi, specifically the low requirements of free water and the importance of high aeration rates, which explains being predominantly used by these organisms. On an industrial scale, SSF may enable some important advantages, namely a more concentrated final product, smaller levels of energy consumption and effluents generation, and inferior working volumes (Cerda et al., 2019). There are still, however, important obstacles to an efficient operation: high substrate heterogeneity; inefficient solid/substrate mixing; low heat transfer efficiencies; and a limited level of available technology. Adding to these, also the scale-up to an industrial level still faces important challenges, namely in relation to conducting a homogenous inoculation (Kumar et al., 2021). This may explain why SSF is still a new trend while SmF is already an established technology. In what refers to industrial SSF bioreactors Mitchell et al. (2006b) established a classification into four main classes, according to the level of mixing and aeration, which includes a vast number of different configurations. On the particular case of filamentous fungi, preferential designs include the rotating/stirred drum, tray-like reactors, trickledbed (c.f. Section 3.4) and packed-bed reactors. Packed-bed reactors rely on similar principles as previously discussed in the context of submerged fermentations with some natural differences: the space between bed particles are only filled with air or small films of liquid; the substrate is static, upon which fugal biomass develops. 4.1 Tray-like bioreactors This configuration is one of the most simpler designs in SSF merely consisting in a chamber/vessel with a number of trays positioned in a given conformation. Each tray is filled with solid substrate and inoculated with a specific organism that will grow upon solid degradation. To achieve a maximum superficial area (contact area with air), a large number of trays are typically stacked vertically on the top of each other but still with an adequate 236 Current Developments in Biotechnology and Bioengineering space (gap) for air circulation and heat transference. Aeration is made through the injection of air, which by circulating between the trays can not only control humidity but also temperature. Agitation may occur in some cases but with a low frequency, for instance through a manual operation, which limits its applicability on cases where highly pathogenic fungi are being used (Kumar et al., 2021). Note that, opposing to a packed-bed/trickled-bed, air circulation mostly occurs around the solids bed and not inside it. In this sense, the thickness of the deposited solid represents one of the most critical aspects since an excessive amount of solid may cause overheating or poor aeration (Robinson and Nigam, 2003; Xie et al., 2013). Overall, this design refers to a very simple technology with low capital investment and maintenance costs (Cerda et al., 2019). Still, its scale-up to an industrial level still faces important issues, namely over heat dissipation, mass transfer, and mixing procedures (Benz, 2011). 4.2 Rotating/stirred drum A rotating drum reactor consists on a (horizontal or nearly horizontal) cylindrical drum where the substrate is placed forming a solid bed, and which will be continuously or semi-continuously agitated by rotation of the drum. This configuration allows to address two important limitations found on the previous configuration, specifically the substrate mixing and the dissipation of heat (Cerda et al., 2019). In an alternative configuration known as stirred drum, the solid bed can also be mixed with the utilization of mixing paddles, or similar mechanisms, while the drum structure remains stationary. In both cases, while the solid bed only takes a small part of the drum volume (10–40%), a considerable headspace is provided for efficient solid aeration and/or heat dissipation (Barragán et al., 2016), but which also contributes for a better mixing. By opposition to more static configurations, such as tray reactors, or even packed/trickled-bed reactors, these configurations bring however the issue of shear forces, which in some cases can have a negative effect; it should be noted, however, that shear forces affect fungal biomass in a rather complex way and in some cases can inclusively be beneficial (Stuart et al., 1999). Another additional issue refers to a superior energy consumption, which can be especially relevant if a continuous bed mixing is applied (Yoon et al., 2014). 5. Filamentous fungi morphology and reactor design This section, starting from this life-cycle explanation, briefly highlights the main morphological structures adopted by these microorganisms in SmFs and how they are linked with productivity. Current data available indicates that is very difficult to prescribe generalized solutions for high productivity for different fungal species/strains but also for different products (e.g., proteins, organic acids or secondary metabolites like vitamins) among the same species/strains. To finish, the pros and cons of an upcoming trend of designing the fungal species for the bioreactor instead of designing the bioreactor for the fungal species by engineering approaches is taken into account. Chapter 8 • Bioreactors for filamentous fungi 237 5.1 Life cycle of filamentous fungi Used for the production of energy and high-value chemicals through SmF, the main morphological characteristic that distinguishes filamentous fungi from bacteria and yeast is the complex network of hyphae associated with filamentous growth. The life cycle of filamentous fungi normally starts with the germination of spores (conidia, ascospores, etc.) into hyphae, which in turn extend at their tips in a process denominated polar growth. As growth proceeds, the speed of hyphal extension increases and lateral branching (the formation of new hyphae at intermediate points rather than at the polar axes) starts to appear. Afterward, structures like conidiophore (e.g., Trichoderma) or sporangia (e.g., Eremothecium) are formed in this mature mycelium. The cycle is closed with the formation and release of the spores by these structures (Aguiar et al., 2015; Cairns et al., 2019). This dynamic morphological behavior makes of filamentous fungi very complex microbiological systems to adapt in normalized industrial processes and reactors, mainly during SmFs (Perez-Nadales et al., 2014; Nørregaard et al., 2014). 5.2 Macromorphology of filamentous fungi in submerged fermentations SmFs are the main setup used to cultivate filamentous fungi for industrial purposes. In this fermentation condition, filamentous fungi can adopt three different macrostructures: dispersed mycelia, loose clumps, or compact pellets (Gibbs et al., 2000; Nørregaard et al., 2014; Cairns et al., 2019). The last two are mycelia aggregates that vary in form, size, density, and surface area. Although there is no strict definition to distinguish between clumps and pellets, the latter are recognized to be structures with more defined, spherical, and denser form than the first (Gibbs et al., 2000). For a matter of simplicity, loose clumps or compact pellets will be simply stated as pellets, since they basically give rise to the same fermentations issues unlike what happens with dispersed mycelia. Pellets that are formed in SmFs can be classified as coagulative and non-coagulative (Zhang and Zhang, 2016). Coagulative type of pellets indicates that the spores in a culture agglomerate before germination and polar growth. In non-coagulative pellets, the spores germinate first—which means that a pellet can be formed by a single spore (Veiter et al., 2018). The type of pellet formation can be influenced by several factors, such as: species/ strains, aeration, agitation, pH, electrostatic hydrophobic interactions, among others (Antecka et al., 2016; Veiter et al., 2018). These factors are interdependent, meaning that, for instance, the same species/strain could present different type of pellet formation and characteristics. The main parameters used for pellet monitoring are fullness, circularity, core area, roughness, and diameter (Veiter et al., 2018). The main problem associated with pellets during SmFs is related with the limited or scarce availability of nutrients and oxygen in the core (center) of the pellet (Nørregaard et al., 2014). For this reason, hyphae that are positioned in the core region may suffer from additional stress. There is no simple strategy or factor that can be applied to mitigate this problem. Moreover, when biomass increases in the core region, substrate, and oxygen uptake become increasingly difficult, impairing fungal metabolism, and consequently productivity (Veiter et al., 2018). 238 Current Developments in Biotechnology and Bioengineering Additionally, pellet size can be affected by reactor conditions. For instance, in general terms increased agitation is correlated with low number and smaller pellets. On the other hand, a strong inoculum (high number of spores) can also lead to smaller pellets, at least in Aspergillus species (Veiter et al., 2018). Other factors such as the chemical modulation of the broth viscosity, media constituents and pH have been also studied but no general conclusion can be taken from the data (Antecka et al., 2016). This reinforces the idea stated above that trying to modulate the morphological behavior of filamentous fungi in SmF is a high complex and interdependent mission. Dispersed filamentous growth is another morphological behavior presented by filamentous fungi in SmFs. This type of morphology significantly affects the rheology of the fermentation, since high viscosity of the broth is often observed. In terms of reactor design and manipulation, high viscosity could be counter-balanced with increased shearing force (Gibbs et al., 2000; Nørregaard et al., 2014). However, due to the stronger shearing stress, mainly in the regions near the impellers or air spargers, mycelial breakdown tends to occur. Consequently, increased physiological stress (e.g., oxidative stress) in the fungus may be observed, which could affect metabolite biosynthesis and consequently the productivity of the process. In addition, due to the non-Newtonian nature of this viscous broth, the rheological properties of regions near the shear force (e.g., near the impellers) are different from further regions (Gibbs et al., 2000). The practical consequence of this is related to poor nutrient availability in some regions of the culture broth. Stagnant zones are formed in the vessel where hyphae can have difficult to access to nutrients and oxygen, which rapidly can become depleted. Once again, this effect is particularly relevant when high biomass concentrations are present, since this contribute to even higher broth viscosity. In this situation, the power from the impellers/air spargers to obtain a homogenous virtual mixture also increases, which results in additional costs (Nørregaard et al., 2014). Despite at lesser extent, problems of insufficient mixing can be expanded to temperature and pH control with obvious effects in productivity (Cairns et al., 2019; Gibbs et al., 2000). 5.3 Link between morphology and productivity Productivity could be linked with the morphology of the fungus but it varies according to the species/strain level and the type of product envisaged (Cairns et al., 2019; Pazouki and Panda, 2000; Veiter et al., 2018). Depending on the class of the product that is being produced, a specific macrostructure could be more advantageous. For instance, some trends were observed where proteins seem to be more efficiently produced by dispersed mycelia, whereas organic acid and secondary metabolites (chemicals produced during stationary phases or when the growth rate is near 0) have better results when macrostructures like clumps/pellets are observed (Cairns et al., 2019). However, this is only a generalization since we can find contrary examples such as the production of fumaric acid by dispersed mycelia of Rhizopus arrhizus (Rhodes et al., 1962). Advances in tools to characterize and monitor fungal morphology under production conditions have also the potential to boost the understanding of fungal morphology as Chapter 8 • Bioreactors for filamentous fungi 239 well as its response to different parameters in SmFs (Posch et al., 2013). Such methods go from various microscopic imaging techniques, like confocal laser scanning microscopy, to flow cytometry (Krull et al., 2013). Recently, three dimensional analysis of freeze-dried pellets collected from SmF of Aspergillus niger and Penicillium chrysogenum by X-ray microtomography allowed to quantify several parameters such as hypha length, number of tips and branches, diameter of the pellets, among others (Schmideder et al., 2019). Using these techniques to study high and low producers can give new insights between the link of productivity and morphology or can indicate which species/strain is more suitable for the production of a specific compound. For instance, highly branched hypha of Trichoderma reesei were connected with improved cellulose production (He et al., 2016). Therefore, these imaging techniques could be used as a screening tool of other strains with similar levels of branching. Fragmentation of hypha is another factor that has been studied. For instance, pellet fragmentation in Aspergillus niger, which is directly related to shear conditions and agitation speed, has been connected to the levels of endoglucanase and β-glucosidase production (Buffo et al., 2020). Summarizing, there is no single strategy connecting a specific morphology to better productivities that can be generally applied to different species/strains or to different products. Nonetheless, when there is still no data available about morphology and productivity of a certain product, pellets are normally preferred in production processes due to relatively easier operational conditions (Posch et al., 2013). 5.4 Effect of different reactor designs on morphology Bioreactor design for SmFs of filamentous fungi has mainly taken into account the limitations related with the oxygen transfer rate and the agitation/aeration system used (Musoni et al., 2015). Independently of the morphology of the fungus, one can look to reactor design and check how it can influence macrostructures, adopting culture conditions that are more suitable for a specific bioprocess or a specific fungal morphology. That is, adopt specific systems that provide the conditions for the “best” morphology of the filamentous fungus in order to maximize productivities. However, one must be always present that each fungi is specific as well as each product that they produce, which require different culture and physiological conditions (Musoni et al., 2015). There is scarce evidence that a single bioreactor design or conditions adopted can produce similar responses in different species (Cairns et al., 2019; Gibbs et al., 2000; Nørregaard et al., 2014). Nonetheless, as mentioned above, frequency and type of agitation, and how aeration is supplied on reactors are factors that catch more attention. In brief, increased agitation is usually required for good mass transfer; however, it leads to increased shear stress. In these conditions, mycelia are break more frequently, increasing free hypha in the culture broth and leading to a superior viscosity (Posch et al., 2013). Accordingly, it is important to know the energy generated by impellers and/or aeration systems in order to try to modulate the morphology by adjusting these variables. Beyond the energy generated by these systems, fungal macromorphology is affected by other 240 Current Developments in Biotechnology and Bioengineering factors such as the impact between pellets and impellers (Posch et al., 2013). There are a series of other factors that could affect the morphology of the filamentous fungi in SmFs, namely culture pCO₂ and pO₂, pH, concentration, and type of inoculum (e.g., spores or mycelia), medium composition (Antecka et al., 2016; Gibbs et al., 2000). From these, one of the main factors impaired by high viscosity of the broth (characteristic of fungal fermentations) is oxygen transfer rate; however, insights about the correlation of these parameters with cell morphology are very difficult to include in predictive models (Nørregaard et al., 2014). Several reactor designs are currently used in SmFs of filamentous fungi such as: STR (Abdella et al., 2020; Namboodiri and Pakshirajan, 2018; Ogunyewo et al., 2020; Patel et al., 2018; Saini et al., 2015; Singh et al., 2019), air-lift (Bannari et al., 2012; Sepehr et al., 2012; Fontana and Silveira, 2012), trickle-bed reactor (Pardo-Planas et al., 2018), packed-bed reactor (Arikan et al., 2019), fluidized-bed reactor (Sadhasivam et al., 2010), among others. Next, it will be briefly detailed some specific connections typically made between morphology and specific reactor designs. 5.4.1 Stirred tank reactors Research on the influence of this reactor design on morphology has been focused on the design and the high shear stress generated by the impellers (Nørregaard et al., 2014). This is particularly relevant when pellets are the macromorphology observed, since a high agitation rate usually leads to the fragmenting of hypha from the surface of the pellet or ultimately fully breaks the pellet (Moreira et al., 2003) with obvious effects on growth and productivity (Cairns et al., 2019). When dispersed mycelia is observed, other problems usually arise such as the adherence of the biomass in the impellers. However, this depends on various factors such as the intensity of the agitation, the design of the impellers and the species used (Havlik et al., 2017). Several impeller types are adopted in STRs beyond the Rushton impellers, such as pitched blade, or axial impellers (Gibbs et al., 2000; Nørregaard et al., 2014). These distinct impellers can have different impacts on the fragmentation of € sten et al., 1996), although a different impellers performance may also fungal hyphae ( Ju only occur under given culture conditions since some factors, like the aeration conditions, can mitigate some of these differences (Buffo et al., 2020). Overall, there is no direct relation that can be generally recognized on the effect of the impeller or its speed on fungal morphology. Even the correlation between high shear stress from impellers and decreased productivity is not always verified, as it depends on the filamentous fungus or the growth phase (Cairns et al., 2019). For instance, hypha that are under nutrient-limited conditions are more prone to fragment (Gibbs et al., 2000). Another factor to consider for an STR configuration is the type of inoculum. For instance, a superior enzyme production was reported by Abdella et al. (2016) when pellets were used as inoculum. According to the authors, this can be explained by different morphologies in initial stages since for the fermentations with spores as inoculum (i) the substrate was covered by mycelia, leading to incomplete usage of the substrate and poor mass transfer, and (ii) dispersed hypha was observed, leading to an increased Chapter 8 • Bioreactors for filamentous fungi 241 viscosity. Still, the morphological and productivity problems associated with the inoculum of spores can be solved by immobilization via adsorption, for instance in a rotating fibrous bed reactor (RFBR) (Abdella et al., 2016), counteracting the general trend mentioned above in which superior protein production is associated with growth as dispersed mycelia (Cairns et al., 2019). Other factor that should be taken into account in STRs is the relation between height and diameter of the vessel. Due to the high viscosity of the fluid in filamentous fungi fermentations, especially when dispersed growth is observed, H/D ratios in the range of 2–3 should be considered to help with the mixing of the culture broth (Nørregaard et al., 2014), which is typically lower to those applied for other microorganisms like bacteria or yeast. 5.4.2 Other designs Air-lifts is another type of design frequently utilized. The absence of mechanical agitation for these reactors earlier attracted attention of researchers that wanted to eliminate the effect that mechanical impellers generate in the morphology of filamentous fungi. However, once again, while different productivities between STRs and airlifts were reported, few studies established a relation between this design and specific effects on morphology. Furthermore, one problem that can be linked with airlifts, even more than in STRs, is O2 availability. When viscosity of the broth increases, normally due to high biomass concentrations, satisfactory mixtures are generally more difficult to obtain without the mechanical forces of the impellers in STRs (Gibbs et al., 2000). Fluidized-bed reactors are a type of reactor that when equipped with a pulsed system are able to effectively control pellet size and narrow the dispersion in diameter. This has been a useful strategy to counteract oxygen limitations in the core of large pellets as well as to ease the operation conditions in the production of ligninolytic enzymes (Moreira et al., 2003). Once again, one should be cautious in taking generalizations since what is demonstrated for one fungus cannot be observed in other. In the context of effluents treatment, an interesting work reported the importance of morphology beyond the discussion dispersed mycelia-pellets. The role of aerial mycelia of Fusarium solani in a continuous gas-phase biofilter (to biodegrade n-pentane and other volatile organic compounds) was clearly shown when the bioreactor design promoting aerial mycelia was able to increase more than threefold the elimination capacity of the fungus (Vergara-Fernández et al., 2016). High mycelial growth has been also reported as an important morphological trait for the capacity to biodegrade other pollutant, such as toluene (Gopinath et al., 2016a). 5.5 Tailoring filamentous fungi morphology: Future perspectives The deepening of the knowledge about the morphology of filamentous fungi and its determinants, either genetic or environmental, is of utmost importance to predict the behavior of these microorganisms in submerged cultures and to optimize the bioprocesses where they are employed. However, it can be costlier and laborious to design specific bioreactors 242 Current Developments in Biotechnology and Bioengineering to fit specific bioprocesses than to engineer the fungi morphology to match the required conditions. Moreover, in industrial large-scale production, bioprocesses that more easily adapt to existing infrastructures are more likely to be implemented. There is a clear predominance of STR in industrial processes, due to its versatile and efficient nature (Nørregaard et al., 2014). Therefore, it will be worth to invest in harnessing filamentous fungi with improved growth morphologies for certain products and for certain reactor designs (Cairns et al., 2019). Nowadays, due to great advances in the fields of comparative genomics, transcriptomics, synthetic biology, and genetic/metabolic engineering, it is already increasingly viable to identify several genes that are promising candidates for engineering the morphology of filamentous fungi (Cairns et al., 2019). From these studies, genes belonging to signaling cascades have emerged as the most important target since they can act on several pathways/genes. Fungal morphology is a result of very complex networks that cannot be explained by the absence or presence of a single gene, neither by a single polymorphism. In this regard, a lot of work has been done to understand the genetic basis of filamentous growth, namely in filamentous fungi that are considered of “transition” or “yeast-like”, such as Ashbya gossypii (Aguiar et al., 2015). Ashbya gossypii is a filamentous ascomycete that despite growing exclusively in filamentous form presents a very high degree of genetic similarity with the genome of the budding yeast, Saccharomyces cerevisiae (95% of homology; Wendland and Walther, 2005). By analyzing the genomes of these two microorganisms, there is evidence that the differences between the life cycles of Ashbya gossypii and Saccharomyces cerevisiae do not depend on exclusive molecular factors (genes or proteins) but rather on several sequence modifications (e.g., non-protein-coding DNA), which together lead to different cellular mechanisms controlling cell growth in yeasts and filamentous fungi (Schmitz and Philippsen, 2011). Understanding these differences can pave the way to future genetic strategies that could be applied to modulate the morphology of filamentous fungi. 6. Conclusions and perspectives Filamentous fungi are undeniably in the center of current Industrial Biotechnology and already represent a significant fraction of industrial fermentation processes. Their high robustness and fast growth make this class of organisms a very attractive option for unfavorable environments, such as industrial effluents. On the other hand, their economic impact is clear, being already employed on the production of numerous high value-added compounds. While they can correspond to a vast range of organisms and play an equally vast set of different applications, distinct reactor configurations are normally used intending to meet very specific process and organism requirements. This chapter elucidates how different engineering aspects on bioreactor design are related to some important characteristics of filamentous fungi and how they perform in an industrial context. Special attention was given to the mechanisms of mass transfer and shear stress, but also to the key role of the rheological profile, dictating oxygen availability, nutrients diffusion, cells integrity, among other aspects. Overall, it is clear that the selection of a reactor configuration is Chapter 8 • Bioreactors for filamentous fungi 243 highly specific for each application, being the morphology of the employed organism a decisive aspect to consider. While fungal biotechnology will likely grow noticeably in the upcoming decades, bioreactor design is deemed to play a critical role on this path. In this sense, the ever growing evidences of this close relationship between reactor design, fungal morphology, and cells productivity will drive a higher attention to a more specific process of reactor design. Also, new developments will likely be made to adapt traditional designs, such as the STRs, to particular issues of filamentous fungi such as the shear forces and high viscosities. On the other hand, SSF systems will gradually become more and more present as some of their main bottlenecks are being surpassed, namely temperature control and mass transfer. On this context, it can be expected a growing influence of new technologies, namely in terms of sensors, online control and artificial intelligence, enabling higher levels of process control, which will allow important results not only from an efficiency standpoint but also in terms of energy and materials consumption. 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Academic Press, pp. 165–177. 9 Filamentous fungi processing by solid-state fermentation Marta Cebrián⁎ and Jone Ibarruri⁎ A ZT I, FO OD RE S E AR CH , BAS Q U E R ES E AR CH AND T ECHNOLOGY ALLIANCE ( BRTA) , PARQUE TEC NO LÓ GICO DE BIZKAIA, DERI O, BIZKAI A, SPAIN 1. Introduction Solid-state fermentation (SSF) is a bioprocess where microorganisms grow on moist solid substrates, using these substrates both as inert carriers and as a source of carbon and nutrients. In SSF processes, enough moisture is present to support microbial metabolism, but the free water content is very low and, therefore, the air is in the continuous phase (Webb, 2017). This technology has been known for centuries, being widely used in the production of fermented foods, especially in the East and Asian regions. Some foods that are usually fermented by SSF are rice and soy, but nuts, legumes, fruit, cheese, or grains can also be successfully processed in the production of fermented foods. Examples of products obtained from fermenting rice are koji, tapai, red fermented rice, brewing the Japanese rice wine (sake), while products derived from soybean are tempeh, soy sauce, annatto, and miso (Couto and Sanromán, 2006). Some of these fermented foodstuffs have been used as an important component of the daily diet, providing a valuable source of nutrients for several thousands of years in these regions. Due to its low water activity, SSF can only be carried out by a limited number of microbial species, mainly belonging to the genera of fungi and yeast, although some bacteria have also been successfully used. Filamentous fungi have some unique morphological characteristics that make them an excellent agent for SSF. The tubular hyphae, which emerge from the spore, elongate, and lead to the formation of new branches that are able to colonize and penetrate the solid substrates. This process produces a porous threedimensional structure that is known as mycelium and facilitates the access to nutrients and air. Over the last decades, SSF has become increasingly interesting as a feasible technology for the bioconversion of a wide variety of substrates into valuable products for food, feed, chemical, pharmaceutical, or energetic sectors. The main products that are usually ⁎ Both authors have contributed equally to the chapter. Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00003-X Copyright © 2023 Elsevier Inc. All rights reserved. 251 252 Current Developments in Biotechnology and Bioengineering obtained through SSF are enzymes, organic acids, lipids, bioethanol, biosurfactants, and biopolymers. Apart from previously mentioned foodstuffs, such as grains, wheat bran or legume seeds, other commonly used substrates for SSF are lignocellulose materials, such as sawdust, straws, or wood shavings as well as several plant and animal derived materials (Sadh et al., 2018a, b). SSF presents several advantages in comparison to submerged fermentation (SmF) technology. Some of them can be summarized as follows (Pandey, 2003; Webb, 2017; Srivastava et al., 2019): – – – – – – – – – – – Simple and inexpensive substrates No need for solubilization of nutrients Less strict control of parameters during fermentation Lower energy requirements for stirring and sterilization Lower production of wastewaters No foam generation Reduced downstream process (easy recovery of products or no need for recovery) Higher product yields Less risk of bacterial contamination Whole valorization of wastes Smaller fermenter size It has traditionally been related to the production of enzymes, antibiotics, and organic acids, among others. However, great efforts are being made to replace oil-based refineries with biorefineries based on renewable feedstocks. So, in recent years, remediation objectives are becoming much more important in this field, valorizing unexploited biomass and producing high-value added products with a great variety of applications. Consequently, an economic value is added to these by-products, contributing to the solution of the environmental problems caused by their uncontrolled disposal (Thomas et al., 2013). Therefore, SSF processes have great potential for many emerging applications based on the bioconversion of agroindustrial residues by filamentous fungi, which give a huge number of possibilities to agroindustrial residues, due to the diversity of expanding products, which constitute an important contribution to this development (Lizardi-Jimenez and Hernandez-Martinez, 2017). On the other hand, one of the main problems across the world is food waste. One-third of food produced globally for human consumption (around 1.3 billion tons) is wasted or lost every year (Gustavsson et al., 2011), while an increasing demand for new feed ingredients is expected to occur as world population increases. Thus, SSF can contribute to the production of new and sustainable sources of food and feed ingredients, reducing food, and agricultural waste disposal at the same time (Ibarruri et al., 2021). More recently, SSF has also been studied as a feasible source of microbial secondary metabolites (SMs) that are generated during the fermentation process and can be of interest in sectors such as pharmaceuticals, agronomy, or cosmetics. These compounds Chapter 9 • Filamentous fungi processing by solid-state fermentation 253 (mainly flavoring substances, polyphenols, biopesticides, or biofertilizers), are produced as intermediate compounds during the stationary growth phase, and play an important role as a defense mechanism for the fungi (Kumar et al., 2021). However, operational issues, as well as conditions and process controls, are some aspects of SSF that still need to be solved to allow for a successful scale-up of this technology. Some of the problems that hinder the industrial implementation of SSF are overheating, aeration and agitation optimization, or moisture and effective temperature control, as well as the assurance of an aseptic or hygienic environment (El-Bakry et al., 2015; Marin et al., 2018). These aspects hinder the design of scalable bioreactors and the development of mathematical models that could successfully address material heterogeneity and improve heat transference and control. Nevertheless, over the last few years, several improvements in new reactors and process control knowledge have been achieved, which are expected to overcome these aspects and facilitate its application at industrial scale for a wide range of biotechnological applications. From the microbiological point of view, filamentous fungi growth is also influenced by several factors during SSF. The three major factors are biological factors, including microorganisms and substrates, physicochemical factors (moisture and water activity, temperature, pH, aeration and oxygen requirements, and particle size), and mechanical factors (agitation and mixing) (López-Gómez et al., 2020). All these parameters will be comprehensively examined in this chapter. 2. Main filamentous species and substrates One of the most important elements for the development of a SSF process is a suitable microorganism selection, which will depend on the substrate and the desired endproduct. Filamentous fungi are the most appropriate organisms for this fermentation, due to the similarity of the SSF environment to their natural habitat, where they are able to produce a huge diversity of enzymes and other metabolites, also due to their low water activity requirements (Ugwuanyi et al., 2009; Soccol et al., 2017). In addition, SSF processes enable the fungi to grow on the substrate surface and penetrate through the space between particles, generating in most cases an aerobic environment (Pandey, 2003). There are also other microorganisms that have been used in SSF. Bacteria, being microorganisms with high plasticity, high-growth rate, diverse metabolic fluxes, and in some cases, few nutritional requirements are an interesting option for fermentation processes. Nevertheless, their relatively high minimum water activity (aw) and required selection, adaptation and, in some cases, metabolic modification make them less appropriate for SSF (Ibarruri and Hernández, 2021). Species of Bacillus, Lactobacillus, Pseudomonas, and Streptomyces are the most used since they have some necessary characteristics for this process such as ability to produce a wide range of enzymes, abundant colonization of solid substrates, and adaptability to different conditions (Anupama and Ravindra, 2000; Orozco et al., 2008; Dharmendra, 2012). 254 Current Developments in Biotechnology and Bioengineering Yeasts are another interesting group for SSF where the main genera used are Candida, Kluyveromyces, Saccharomyces, Endomycopsis pichia, and Torulopsis, among others (Anupama and Ravindra, 2000; Gervais and Molin, 2003). The fungi kingdom comprise four phyla: Chytridiomycota, Zygomycota, Ascomycota, and Basidiomycota. As extensively analyzed by Ferreira et al. (2013, 2016), those filamentous fungi belonging to Zygomycota and Ascomycota phyla are the most interesting ones for industry, due to the broad range of possible substrates that can be used and the endproducts produced. Zygomycota is divided into two classes: Trichomycetes and Zygomycetes, which are found worldwide as saprophytes growing on dead organic matter. Zygomycetes are closely related to food applications, the classical example being Rhizopus oligosporus (Nout and Aidoo, 2011), which is used for tempeh preparation. However, they are also known for being pathogens of animals, plants, and of other fungi (Ferreira et al., 2013). Another widely studied Zygomycetes is Mucor indicus, known for its ability to produce γ-linoleic acid (C18:3) (Lennartsson, 2012). The main advantages related to Zygomycota are their high metabolic plasticity and high growth rate, which make them able to grow in a wide range of environments and produce a high variety of hydrolytic enzymes. Major examples of Ascomycetes are the production of antibiotics by Penicillium chrysogenum, the production of a wide range of enzymes by Aspergillus sp. and Trichoderma (Ortiz et al., 2015; Ferreira et al., 2016), and also the production of food, such as soy sauce, miso, sake and rice vinegar by Aspergillus oryzae ( Jin et al., 2019). Another main challenge of modern society is the urgent need for alternative protein sources. Filamentous fungi are rich in vitamin B and provide a high percentage of protein (30%–70%), while nucleic acids are not very high (9.7%) compared to bacteria (Anupama and Ravindra, 2000). Nevertheless, the introduction of new species or processes into the food production system is regulated by the European Food Safety Authority (EFSA) in Europe, which determines that foodstuffs that had been consumed and commercialized within the European Union prior to May 1997, are considered safe and can be marketed. If not, they must undergo the Novel Food Regulation to analyze their safety before being marketed (Fig. 1). Obtaining a safety status requires time and costly safety tests. However, safety regulations in Europe may differ from the regulations in other countries. As an example, Generally Recognized as Safe (GRAS) is a status given by the Food and Drug Administration (FDA) in the United States to any substance or chemical, including sometimes whole organisms, that is considered safe for human consumption. As in Europe, there are two main routes to obtain this status, through years of documented consumption by humans, or through scientific evidence proving that a substance is safe. Several filamentous fungi from Ascomycetes (Aspergillus oryzae) and Zygomycetes (Rhizopus oryzae, Rhizopus oligosporus) have been used as fermentation agents with the main objective of increasing the protein content of several by-products as food or feed ingredients (Dulf et al., 2016; Ibarruri et al., 2021). Fig. 2 shows the main genera and species within Zygomycota and Ascomycota phyla used as biotransformation agents in SSF and their main applications. Chapter 9 • Filamentous fungi processing by solid-state fermentation 255 FIG. 1 Variety of solid by-products from the agroindustrial sector and the variety of value-added products generated by solid-state fermentation. As explained in the above section, in SSF processes, microorganisms grow on solid substrates using them as a source of carbon and nutrients. Therefore, it has been closely related to the valorization of agroindustrial by-products as an eco-friendly process, which mostly uses solid agroindustrial wastes as the source of carbon and other nutrients. All these residues or wastes (agricultural, bakery, winery and brewery by-products, fish and poultry processing by-products, …) are generally rich in different nutrients depending on their origin, mainly composed of starch, lignocellulose, lipids, and proteins. Yet their high water content and their seasonality hinder the implementation of integrated and efficient recovery activities (Ibarruri and Hernández, 2021). The Waste Directive 2018/851 establishes a series of priorities to reduce the environmental impact of waste and promote sustainable bioeconomy. This directive prioritizes reuse and recycling strategies followed by valorization and finally elimination as the last option (Ibarruri and Hernández, 2021). Valorization is defined as the use of a by-product with a useful purpose which replaces other material that would be used to fulfill the FIG. 2 Combinations between main substrates and filamentous fungi applied in SSF process to produce a high variety of products. Chapter 9 • Filamentous fungi processing by solid-state fermentation 257 € et al., 2014). Many sectors are trying to avoid elimination and treatfunction (Ostergren ment costs while applied research related to recovery and conversion processes is growing. This can lead to the generation of financial returns. Indeed, the key to scale-up and establishing valorization processes is technological development. An ideal substrate for SSF processes is the one that gives the required nutrients for the growth of the fungi. In some cases, additional nutrients are needed to achieve better microbial growth. In addition, the substrate can be solid or liquid. In the first case, the solid substrate will be the one that supplies the nutrients and the structure that supports the growth. In the second case, the liquid must be impregnated into an inert solid to support the growth (Krishna, 2005). Carbon and nitrogen are the main nutrients and play a critical role in the growth process. Carbon gives the energy to the fungi and can be obtained from sugars (glucose, fructose) or from polymers (starch, cellulose) (Rodriguez-Leon et al., 2008). Nitrogen also plays a strategic role in biomass formation, its concentration and availability influence the activity and the growth efficiency of the fungi. So, when nitrogen is scarce could lead to lower growth of the fungi. In some cases, the addition of extra nitrogen compounds is necessary to improve the process and protein production (Ibarruri and Hernández, 2018). In addition, as explained by Ibarruri and Hernández (2019), the C/N ratio plays an important role in the production of biomass, and its protein content and organic acid production. A low C/N ratio (10:1 or less) is beneficial for biomass production while a high C/N ratio (from 120:1 to 150:1) promotes the production of organic acids, such as fumaric acid, as the pyruvate flows for fumarate production (Meussen et al., 2012). Before fermentation, the substrate might need to be pretreated such as cut, milled, or granulated to obtain the desired size, be moistened or cooked to increase nutrient availability or be supplemented. Furthermore, the substrate may need to be sterilized or pasteurized outside or inside the bioreactor (Mitchell et al., 2006a). As explained above, agroindustrial wastes are, in general, rich in a variety of nutrients depending on the origin. A simple way to classify them is by their main component where we can find starchy products, proteinaceous products, lignocellulosic products, and substrates rich in soluble sugars. In the case of substrates rich in starch, the most commonly used ones in SSF processes are barley, rice, cassava, wheat, corn, potato by-products, oats, cassava meal, corn meal, sweet potato by-products, and bakery waste, among others (Fig. 2) (Li et al., 2015; Haque et al., 2016; Maryati et al., 2017; Morales et al., 2018; Benabda et al., 2019). They are mainly composed of starch, which is a polymeric carbohydrate comprised glucose units joined by 1,4-alpha glycosidic bonds. It is also the most common carbohydrate in human diets and is used to store energy in plants and animals. Furthermore, starch is easily degradable to simple sugars by amylase enzymes produced by filamentous fungi (Haque et al., 2016; Godoy et al., 2018). In general, agricultural by-products and wastes are rich in lignocellulosic materials, such as lignin, hemicellulose, and cellulose, where SSF can improve their digestibility by reducing fiber fractions (Regalado et al., 2011). During the fermentation of these substrates, fungi improve fermented product digestibility due to their ability to release several 258 Current Developments in Biotechnology and Bioengineering enzymes (hemicellulases, celullases, xylanase, laccase, etc.), which degrade plant cell walls (Lizardi-Jimenez and Hernandez-Martinez, 2017; Sadh et al., 2018a, b; Arredondo-Santoyo et al., 2020). The most commonly studied lignocellulosic substrates are sugarcane bagasse (SCB), soybean hulls, rice hull and stover, wheat bran, corncob, sugar beet pulp, barley husk, wheat and barley straw, wood, brewer spent grain (BSG), coffee husk, spent coffee ground (SCG), and coffee silverskin (Cerda et al., 2017; Cooray and Chen, 2018; Nair et al., 2018; Shin et al., 2019). In these cases, it is necessary to use lignocellulolytic fungi, which are able to produce cellulolytic enzymes to degrade the main polymers in the substrates, such as Trichoderma reesei (Ortiz et al., 2015), Aspergillus niger, Aspergillus awamori, and Aspergillus oryzae (Pensupa et al., 2013; Shin et al., 2019), Rhizopus (Massarolo et al., 2017; Ibarruri et al., 2019), and also ligninolytic white rot fungi (Zuchowski et al., 2013). The most known proteinaceous substrates are oil cakes, which are the solid by-products obtained after the extraction of oil from the seeds or oleaginous fruit. They are an ideal substrate for SSF, due to the carbohydrates, minerals, and remaining lipids on them, apart from their protein content. Therefore, they lead to a wide range of end-products, such as enzymes (e.g., proteases, lipases, etc.) and a wide spectrum of SMs (Dharmendra, 2012; Venkatesagowda et al., 2015; Ahmed et al., 2016; Guneser et al., 2017; Handa et al., 2019; Arsy et al., 2020). As explained by López-Gómez et al. (2020) the most abundant oil cakes are pumpkin, soybean, sesame, groundnut, safflower, rapeseed, cottonseed, mustard, sunflower, canola, linseed, coconut, copra, palm, and olive oil cake. Fruit processing industries are the ones that produce substrates comprised soluble sugars, such as molasses, apple, grape, kiwi and peach pomace, lemon peel and pulp, pineapple and orange wastes, sweet sorghum, sugar beets, carob pods, fruit and vegetable discards, and so on (Fig. 2). They are perfect substrates for organic acid production, phenolic compound release, and enzyme production, among others (Dı́az et al., 2013; Dulf et al., 2016; Kantifedaki et al., 2018; Papadaki et al., 2018; Ibarruri et al., 2021; Zain et al., 2021). The choice of fungi will be dependent on the type/composition of available substrate. Furthermore, different combinations will lead to different end-products. As an example, Rhizopus is a great producer of organic acids (Magnuson and Lasure, 2004), mainly lactic and fumaric acids, but glucose, or another simple sugar content leading to a high C/N ratio (from 120:1 to 150:1) is necessary for a high organic acid production. On the contrary, lower C/N (10:1 or less) ratios are vital to increase protein production (Ibarruri and Hernández, 2018). Similarly, in order to produce specific enzymes, the substrate must be composed of the main substrate for the desired enzyme, for example, if the objective is the production of pectinases, the substrate must have pectin in its composition. 3. Bioreactors and process control parameters: Critical aspects As explained in the introduction of this chapter, the three major biological factors affecting the fermentation process are microorganisms, substrates, and physicochemical factors. Microorganisms and substrates have been described in the previous section, so this Chapter 9 • Filamentous fungi processing by solid-state fermentation 259 section will only discuss these aspects when related to specific information about the fermentation process and focusing solely on the physicochemical and the mechanical factors (agitation and mixing) affecting the process. 3.1 Biological factors Once the microorganism has been selected, the age and quantity of the inoculum are of vital importance, together with the cultivation medium and the physiological state. As explained by Crafack et al. (2014), if the inoculum is not in the correct physiological state, a reduction of the final product will occur, the first hours of fermentation being the most decisive. It has been suggested that the inoculum size is another critical parameter influencing both fungus growth and enzyme secretion during fermentation (Bardiya et al., 1996). A low concentration of inoculum could lead to poor growth of the microorganism. However, it seems that a high inoculum concentration is not always beneficial for the fermentation process which can lead to a limitation in mass transfer (Kumar et al., 2021). As explained by Ezeilo et al. (2019), a low inoculum size leads to low mycelial biomass production, whereas if it is too large, this only leads to an unnecessary increase of fungal growth and nutrient depletion. Thus, a consequence of using a higher inoculum dose than the optimum dose could be lower product generation (Maldonado et al., 2014; Xu et al., 2018). This phenomenon, often seen in mycology, is related to the self-inhibition of spore germination. As an example, Zain et al. (2021) limited the high level of inoculum size (1  108 spores/g of substrate) in a central composite design (CCD) for lactic acid production, using Rhizopus as a biotransformation agent, since a higher spore concentration could cause substrate competition, and could end up reducing the desired product generation (Soccol et al., 1994). Knowing the importance of the effect of inoculum size on the targeted product yield, it is a parameter that must always be included in the optimization trials of SSF, with optimum values between 106 and 108 spores/g of substrate (Ezeilo et al., 2020; Zain et al., 2021). Another important factor related to the inoculum is the growth phase, which is generally started using spore or mycelial inoculum. It is known that spore inoculum adds homogeneity, extends shelf life and facilitates manipulation, while using mycelial inoculum leads to a higher level of proteins due to the instant availability of cellulase and hemicellulase enzymes, when using lignocellulosic materials as substates (Kumar et al., 2021). 3.2 Physicochemical factors 3.2.1 Moisture content and water activity Moisture content and aw are different concepts that must be understood because they are vital factors affecting the SSF process, in addition to being the factors that determine the microorganisms that can grow in the selected substrate. The aw of a food is defined as the ratio between the vapor pressure of the food itself and the vapor pressure of distilled water under identical conditions. Most foodstuffs have a water activity above 0.95, which will provide sufficient moisture to support the growth 260 Current Developments in Biotechnology and Bioengineering of bacteria, yeasts, and fungi (FDA, 2021). As concluded by Raimbault (1998), optimal aw is dependent on the substrate and microorganism selected. Thus, in a study done by Oriol et al. (1988), higher growth and conversion rates were obtained when SCB was added as substrate due to its higher water retention capacity. This can be explained because each substrate has different water binding characteristics, making free water apparent at different moisture levels. This happens when the saturation capacity of the solid is exceeded, which will depend on the substrate. Therefore, moisture content affects microbial growth, but the effect will be dependent on the substrate. In consequence, the optimum moisture level for the cultivation of Aspergillus niger was different on rice (40%) than on coffee pulp (80%), showing the need to determine the optimum levels for each substrate and microorganism. In general, the reduction of aw has a significant effect on fungi growth, extending the lag phase and reducing the specific growth, which results in less biomass production. This free water becomes visible when the substrate saturation capacity is overstepped, which usually matches with 80% of moisture content. The moisture content is the fraction of the total weight that is made up of water, and some studies have revealed that there is an upper and a lower limit for moisture content, above or below which growth is affected (López-Gómez et al., 2020). As SSF is defined as a process that occurs in the absence of free water, microorganisms able to grow at lower aw values are the ones that best fit the process. Most authors consider fungi as the only organisms able to grow below aw of 0.8, although optimal aw is usually higher. In general, bacteria require higher values of aw for growth than fungi. Although the aw is the factor that directly affects the fungi growth, in most studies, the factor measured is the moisture of the substrate with optimal values being around 40%–80%, depending on the substrate and the desired compound (Benabda et al., 2019; Ezeilo et al., 2019, 2020; Naik et al., 2019). As an example, Zain et al. (2021) concluded that, when the moisture of substrates is too high, the lactic acid production can be reduced due to particle agglomeration and indeed, gas diffusion limitation. On the contrary, if moisture is too low, fungal growth will be decreased and lactic acid production will be reduced, mainly due to low oxygen supply, limitation of nutrient transfer, and disruption of enzyme stability. Controlling humidity during SSF is under permanent development, as done by He et al. (2019), where liquid supply was added by successful capillary water control, reducing the heterogeneity of the sample. 3.2.2 Temperature Temperature is one of the most critical factors to be controlled in SSF, thus determining the optimum fungus growth temperature (Arsy et al., 2020). Moreover, temperature changes during the process can have consequences, from enzymatic inhibition to cell death, which can lead to a reduction in biomass and/or certain metabolites (LópezGómez et al., 2020). During SSF processes, fungi metabolism can generate heat that must be removed from the system, otherwise the substrate moisture may be reduced due to evaporation. Chapter 9 • Filamentous fungi processing by solid-state fermentation 261 Simultaneously, condensation drops, generated due to evaporation, can affect the homogeneity of the substrate. So, water must be added in order to replace water evaporation. Nevertheless, water addition also increases the heterogeneity of the process and, therefore, a mixing or homogenization step will be required. So, the main challenge is humidity control to prevent fungal growth reduction, while optimizing the mixing to combat fungal hyphae damage (Khanahmadi et al., 2006). 3.2.3 pH pH is another important factor to take into consideration during fermentation processes and which directly affects the microbial growth. In general, filamentous fungi have the ability to grow in a wide pH range, making them suitable for growing in different agroindustrial substrates. In addition, pH modifications can occur within the fermentation process due to metabolism, which can lead to growth inhibition or metabolism reduction (Ibarruri and Hernández, 2021). Therefore, filamentous fungi are much more adaptable to the SSF process as they can tolerate pH ranges from 2 to 9, with an optimal value of between 3.8 and 6.0 (López-Gómez et al., 2020). Moreover, this ability can also be used to minimize bacterial contamination as they usually need much more restrictive pH control. Measuring the pH during the SSF process is much more complicated than in SmF, mainly due to the substrate particulate structure, which is filled with a continuous gaseous phase. The initial pH is of vital importance and influences the activities of enzymes involved in intracellular metabolism, so it is a critical factor that has been studied in the process optimization of several enzymes production by SSF (Ezeilo et al., 2020; Zain et al., 2021). These studies found that although Rhizopus oryzae NRLL was able to grow at pH 9, the lactic acid concentration was lower than at optimal pH (pH 7), because at pH 9 the fungus produces lactate instead of lactic acid. 3.2.4 Aeration Related to previous parameters, oxygen requirement for SSF processes is a critical factor necessary to control when scaling-up. As explained by Ibarruri and Hernández (2021), filamentous fungi grow in aerobic conditions, so oxygen diffusion must be facilitated from the interparticle space to the biomass and, at the same time, carbon dioxide must be diffused from the biomass to the interparticle space (López-Gómez et al., 2020). As an example, in SSF of fruit and vegetables, the physical structure was critical for Rhizopus growth e, Rhizopus protein content in (Ibarruri et al., 2021). When the substrate was a kind of pure the final fermented product was lower than in particulate substrate. Particulate substrate let the fungus grow between inter- and intraparticles, and enabled higher oxygen diffusion rate, leading to higher biomass production, and consequently, higher protein content. The main problem of the aeration rate is the depth of the substrate bed, which can limit oxygenation when it is not thin enough, with differences between reactor types. Mixing can add extra aeration, but however, this must be controlled to avoid damaging fungal hyphae. Finkler et al. (2017) established that a regimen of three agitations in a total incubation 262 Current Developments in Biotechnology and Bioengineering period of 20 h sufficed to avoid substrate agglomeration and to control the temperature during the SSF of SCB and wheat bran by Aspergillus niger, to produce pectinase in a pilot scale packed-bed bioreactor. Forced aeration is another option for supplying oxygen to the process, with the flow rate and the air quality air being crucial parameters. The air supplied can be dry or humid. If it is dry, it provides better heat removal but reduces the moisture content, while if it is humid, it does not reduce the moisture in the substrate bed. 3.2.5 Particle size Studying the particle size is also a critical aspect because it affects the respiration and nutrient availability in the process. With smaller particle size of the substrate, the specific surface area increases for fungal growth, but consequently, the interparticle space is reduced, decreasing aeration. On the contrary, a bigger particle size enables higher oxygen diffusion but lower surface area for fungi to grow (López-Gómez et al., 2020). Related to particle size, porosity is another important factor in SSF. Porosity represents the intra- and interparticle void spaces in the substrate bed. Karimi et al. (2014) studied the effect of moisture and particle density on porosity, and obtained an experimental relationship for SSF modeling. Changes in porosity can affect gas diffusion as a reduced porosity would hinder gas diffusivity. In addition, these parameters are changing aspects within the fermentation process, affecting other critical aspects for the process success, such as substrate consumption or product concentration, so it is vital to find not only an optimal particle size, but also an optimal surface area and interparticle space. As an example, Rahardjo et al. (2005) concluded that aerial mycelia, related to porosity and surface area, could accelerate enzyme production. 3.3 Mechanical factors 3.3.1 Agitation Agitation is a common solution to avoid oxygen depletion within the different substrate areas, and it can also help to reduce the heterogeneity problems and improve mass transfer and aeration (Vandenberghe et al., 2021). When using filamentous fungi, one of the main concerns is agitation. As explained above, mixing the substrate is important for heat removal. However, agitation of a bed that contains solid substrates with filamentous fungi growing on their surface could damage the fungus hyphae (Mitchell et al., 2006c). So, it is vital to know the sensitivity of the selected fungi to determine the frequency and intensity of the mixing, and, in those cases, to find strategies that improve heat removal from the bed, such as a slower agitation speed and/or using humidified air (Gasiorek, 2008). 3.4 Heat and mass transfer in SSF Mass transfer is not an easy parameter to measure in SSF. Different phenomena take place at the same time and each phenomenon is different depending on the bioreactor type and scale, among other factors (Manan and Webb, 2017). Chapter 9 • Filamentous fungi processing by solid-state fermentation 263 Headspace Substrate bed (a) Particles Biomass Void spaces (c) (b) (d) Aerial hyphae Moist solid particles O2 Gas liquid interface Interparticle gas phase Penetrative hyphae Liquid film with “biofilm hyphae” Solid substrate FIG. 3 Factors that take place in the micro and macroscale level during SSF process. (A) Macroscale system, where the substrate bed, the headspace, and the bioreactor walls can be observed. (B) Substrate at the microscale level. Individual particles and the interparticle space can be observed. (C) Growth of filamentous fungi and the penetration of some hyphae on the surface of the particle. (D) Fungal mat, which includes the aerial layer in contact with the environmental gaseous phase and the wet layer or biofilm, which is the mycelial mat of the fungus where the interstitial pores are filled by liquid. Reprinted by permission from Springer Nature (Mitchell, D.A., Berovic, M., et al., 2006a. The bioreactor step of SSF: a complex interaction of phenomena. In: Mitchell, D.A., Berovic, M., Krieger, N. (Eds.), Solid-State Fermentation Bioreactors: Fundamentals of Design and Operation. Berlin, Heidelberg, Springer Berlin Heidelberg: 13–32). Two main phenomena occur inside the reactor: macro- and microscale phenomena. The macroscale phenomena include the changes that occur within the substrate bed, headspace, and bioreactor walls (Fig. 3A). However, the microscale phenomena are the ones that explain the relationship between solid particles and the environment (Fig. 3B). The space between solid particles is known as void space and it is the fraction that is filled with water and gas. When filamentous fungi grow around the solid particles, the mycelium penetrate deeply and fill the void spaces, with a continuous nutrient and oxygen consumption, and metabolite and enzyme secretion (Fig. 3C). We must bear in mind that the system is highly complex and that it must be described by mathematical modeling. In addition, each process will be different depending on the substrate, the microorganisms, and the type of bioreactor (Mitchell et al., 2006a). Macroscale phenomena include the bioreactor wall, the headspace, and the substrate bed (Mitchell et al., 2006a). In the macroscale phenomena, when the fungus starts growing, heat starts accumulating and transporting by conduction through the substrate bed due to temperature gradients. Using forced aeration, energy can be carried out from the bioreactor, removing O2 and CO2 by connective mass transfer. In addition, if the forced aeration is dry, this can lead to moisture loss. Even though the system is not aerated, water, CO2 and O2 diffusion occur within the interparticle spaces because of concentration gradients. If mixing events occur in the bioreactor, two important aspects must be taken into 264 Current Developments in Biotechnology and Bioengineering account. First, substrate particles must not coagulate after mixing, and second, the fungi must deal with shear stress in order not to be damaged. Likewise, mass and energy transfer can take place from the solids to the headspace, and in the opposite direction. Lastly, heat transfer can occur by conduction from the bioreactor wall to the outside, and also within the walls. As explained above, the microscale phenomena explain the relationship between solid particles and the environment, this being a changing phenomenon during the SSF process. Fig. 3D shows two different layers of the fungal mycelia growing on a solid substrate, the aerial layer which is in contact with the environmental gaseous phase, and the wet layer which is the mycelial mat where the interstitial pores are filled with liquid. Three major phases are specifically distinguished in the particles, the solid, the liquid and the gaseous phases, which cause heat and mass transfer fluctuation within the system (López-Gómez et al., 2020). Fig. 4 shows a complete fermentation process of fruit and vegetable discards fermented with Rhizopus sp., where a complete inclusion of fungal hyphae and solid substrate is illustrated. By means of a schematic perspective, Fig. 3D shows how the fungal mat includes two different layers when grown on a solid substrate, the aerial layer in contact with the environmental gaseous phase, and the wet layer or biofilm which is the mycelial mat of the fungus where the interstitial pores are filled with liquid. The size and the shape of the substrates will determine the void spaces and the degree of continuity between them. The substrate particles are moist and will create a network of fungal hyphae (Mitchell et al., 2006a). Monitoring and controlling microscale phenomena are of vital importance, but however, it is a challenging job due to the complexity of the three phases. The main action to control them is to design operational strategies to control and optimize the transfers between solids and the gas phase. In this situation, fungi would have more access to nutrients in the solid substrate, oxygen, enzymes and water, which will lead to higher productivity and better performance in the SSF process (Manan and Webb, 2017). FIG. 4 Fermentation process of fruit and vegetable discards fermented with Rhizopus sp., where a complete inclusion of fungal hyphae and solid substrate is illustrated. Chapter 9 • Filamentous fungi processing by solid-state fermentation 265 FIG. 5 Events occurring during fungi growth on a solid substrate related to the gas, water, nutrients, and enzyme exchange in the microscale phenomena. Reprinted by permission from Springer Nature (Mitchell, D.A., Berovic, M., et al., 2006a. The bioreactor step of SSF: a complex interaction of phenomena. In: Mitchell, D.A., Berovic, M., Krieger, N. (Eds.), Solid-State Fermentation Bioreactors: Fundamentals of Design and Operation. Berlin, Heidelberg, Springer Berlin Heidelberg: 13–32). An easy way to understand microscale phenomena is described in Fig. 5. It shows all the events happening during the fungi growth on a solid substrate related to the gas, water, nutrients, and enzyme exchange. Generally, most of the oxygen used by the fungi to grow is the oxygen that comes from the gaseous phase (1). This oxygen then passes through the solid particles by diffusion (2). Fungal growth releases hydrolytic enzymes (3), which diffuse across the particle and help to degrade complex polymers, and therefore, soluble nutrients are released (4). The soluble nutrients are now available to be metabolized by the fungus (5), which will need to absorb more oxygen (6) and release it as carbon dioxide (7). The respiration process also releases water, which can be released to the gas phase (8) or be absorbed by the solid and be used by the fungi or other microorganisms (9). Consequently, fungi grow and hypha elongation occurs, and metabolites are released by the fungi (12). As an example, Fig. 6 shows the fermentation process of BSG with Rhizopus sp. (Ibarruri et al., 2019). BSG is an ideal substrate for SSF with filamentous fungi, mainly because of its composition and its particle structure, size, and void space (Xiros and Christakopoulos, 2012). It is composed of small particles with a rigid internal structure, which allows full use of the void spaces and greater fungal growth (Wang et al., 2001), avoiding the presence of unfermented areas. Although it cannot be appreciated, the mycelial structure prevents hyphae from filling more than 34% of the total volume in the void spaces (Auria et al., 1995). 3.5 Bioreactors Scaling-up is one of the main drawbacks of SSF, which must consider all the critical factors explained above: physicochemical, biological, and mechanical factors. In addition, it 266 Current Developments in Biotechnology and Bioengineering FIG. 6 Fermentation process of BSG with Rhizopus sp. plates were photographed during fermentation from time 0 to 192 h. Reprinted by permission from Springer Nature (Ibarruri, J., Cebrián, M., et al., 2019. Solid state fermentation of Brewer’s spent grain using Rhizopus sp. to enhance nutritional value. Waste Biomass Valoriz. 10 (12), 3687–3700). being a process that is highly related to the valorization of agroindustrial wastes makes reaching the homogeneity needed to control the whole process more difficult. As explained by López-Gómez et al. (2020), the disconnection between lab experiments and bioreactor level is one of the most challenging problems to overcome in SSF. In particular, the study of heat and mass transfer are the most critical aspects of the process and raise a challenge when scaling-up solid-state processes (Pandey, 2003; Ali and Zulkali, 2011). The number of types of bioreactors used in the SSF at pilot and/or industrial level is limited due to various reasons, such as the elimination of heat generated in large volumes of substrate, control of process parameters (agitation, oxygen, and temperature), nature of the substrate and the need for pretreatment (sterilization, inoculation), and control of handling factors such as filling, emptying, and cleaning the reactor (Durand, 2003). Bioreactors can be classified taking into account various factors, and it is common to differentiate them by the mixing and aeration systems (Arora et al., 2018). Tray fermentation (Fig. 7) has traditionally been used for the production of fermented foods such as tempeh, miso, koji, or soy sauce in Asian countries (Nout and Aidoo, 2011), it being the oldest type of bioreactor. The trays are stacked on top of each other at controlled temperature and humidity, and scaling-up is achieved by increasing the surface area of the trays. The bottom of the trays is perforated in order to obtain greater aeration. However, only thin layers are suitable for this kind of process, thus limiting the volume of solids that Chapter 9 • Filamentous fungi processing by solid-state fermentation 267 FIG. 7 Diagram of tray bioreactor. Reprinted by permission from Elsevier (Couto, S.R., Sanromán, M.Á., 2006. Application of solid-state fermentation to food industry—a review. J. Food Eng. 76 (3), 291–302). can be fermented (Robinson and Nigam, 2003). Using forced aeration can help to better control temperature and air flow rate, and will improve mass transfer (Ruiz et al., 2012). Many studies have focused on optimizing bed heights, initial moisture content of substrates, and their effect on productivity. Using high substrate loads implies several disadvantages, where mathematical modeling is critical to understand transport and kinetic parameters (Arora et al., 2018). In addition, it requires a large operational area, making it a labor-intensive process. In general, substrate has to be sterilized separately, so it is difficult to apply it to a whole sterile process, except when large aseptic rooms are used, which may be considerably costly (Arora et al., 2018). In packed-bed bioreactors (Fig. 8), commonly known as Raimbault columns, forced aeration helps to replenish oxygen and moisture without the need for mixing. Forced aeration helps to improve the moisture gradients in the substrate, and also to control the temperature within the process. They are generally applied to filamentous fungi when mixing can affect microbial growth. This type of bioreactors allows better control and higher FIG. 8 Diagram of a rotating drum bioreactor. Reprinted by permission from Elsevier (Couto, S.R., Sanromán, M.Á., 2006. Application of solid-state fermentation to food industry—a review. J. Food Eng. 76 (3), 291–302). 268 Current Developments in Biotechnology and Bioengineering substrate load compared to tray bioreactors. They are composed of a cylindrical drum (glass or metal), with jacketed walls when required, and cooling plates improve heat transfer (Arora et al., 2018). However, substrate compaction, air channeling, bed drying, and process heterogeneity can occur during the process. In addition, there are several complex processes, such as in situ sterilization, inoculation, product removal, and downstream processing (Arora et al., 2018). Rotating drum bioreactor (RDB) (Fig. 9) types are classified within reactors by continuous or intermittent mixing. These reactors comprise a drum-shaped container consisting of the drum wall, headspace, and substrate. Air is introduced at one end into the headspace and the bioreactor can be rotated intermittently or continuously to facilitate mixing by the rotating action of the drum itself. A paddle can also be used when the drum is mounted on a central axis, moving this axis and keeping the remaining drum static. The mixing improves mass transfer and heat removal, but however, temperature control is quite complicated. In addition, as explained above, agitation can also damage fungal hyphae, so optimizing mixing events is vital. In drum-type reactors, the useful space is around 30% of the total volume since a high substrate load results in a low product yield. There are useful models for predicting heat transfer in this type of reactors (Stuart and Mitchell, 2003), although they show some difficulties in controlling the temperature at high substrate loads. These bioreactors provide greater control over process factors compared to tray and packed-bed reactors, although they also have difficulties with FIG. 9 Diagram of a packed-bed bioreactor. Reprinted by permission from Elsevier (Couto, S.R., Sanromán, M.Á., 2006. Application of solid-state fermentation to food industry—a review. J. Food Eng. 76 (3), 291–302). Chapter 9 • Filamentous fungi processing by solid-state fermentation 269 Gas exit Culture medium level Solid-substrate Sampling ports Water cooling Air inlet FIG. 10 Diagram of a fluidized-bed bioreactor. Reprinted by permission from Elsevier (Couto, S.R., Sanromán, M.Á., 2006. Application of solid-state fermentation to food industry—a review. J. Food Eng. 76 (3), 291–302). sterilization, product extraction, and postfermentation treatment. The modular designs patented by Biocoon Ltd. (Suryanarayan and Mazumdar, 2001) and Novozyme (Andersen et al., 2015) show promise in terms of obtaining sealed systems for the whole process. Fluidized-bed bioreactors (Fig. 10) are composed of a vertical chamber with a perforated base plate. The substrate and the microorganism are constantly kept in a fluid state by the action of the upward flow of air, which generates mixing. It is important to analyze the type of substrate. A sticky substrate can form agglomerates, which cannot be fluidized, and heterogeneity in the size can hinder the complete fluidization of the solid particles. Furthermore, its application is limited, and it is also highly energetic, so hardly any studies have been carried out to date. Spouted-bed bioreactors are similar to fluidized bed bioreactors. However, the air flow is only through the central axis of the bed, and therefore, only part of the bed is fluidized (Mitchell et al., 2006b). It is the most suitable bioreactor for sticky and irregular substrates as the constant impacts through the center of the substrate hinder agglomeration. As a 270 Current Developments in Biotechnology and Bioengineering result, effective heat and mass transfer is provided, which in turn, can lead to greater performance (Cerda et al., 2017). For better understanding of bioreactors in SSF, we recommend the further reading of Arora et al. (2018) and López-Gómez et al. (2020). Table 1 summarizes the characteristics and limitations of the above-described bioreactors. The selection of the most suitable reactor is a difficult decision, and depends mainly on the substrate and microorganism. In the last decade, bioreactors in SSF have undergone continuous research, but however, there are still many challenges to be overcome (Arora et al., 2018; Vandenberghe et al., 2021). Thus, when designing a bioreactor, the main efforts should be aimed at creating a balance between optimal fungi growth conditions and optimal operation conditions, minimizing temperature deviation, microorganism damage and substrate bed water activity, and maximizing oxygen supply (Mitchell et al., 2006c). 4. Products and current industrial applications As previously commented, SSF presents a high potential for the production of several ingredients for the feed, food, chemical, and pharmaceutical sectors. SSF has traditionally been used to produce metabolites such as organic acids, enzymes, biosurfactants, antibiotics, and aroma compounds. Over the last decades, SSF has also been attracting new interest in the mitigation of environmental impacts and biofuel production, because of its wide variety of applications in valorizing unexploited biomass. Regarding biofuel production, SSF is a suitable technology for biodiesel, bioethanol, biobutanol, or biohydrogen production. More recently, SSF has also been extensively studied as a suitable technology to obtain biologically active SMs with increased added value in several industrial sectors. For example, a variety of applications and products can be achieved, from aroma and flavors to agronomic molecules such as plant growth hormones and biopesticides, as well as antioxidant and antimicrobial compounds, amino acids, vitamins, biosurfactants, pigments, or bioplastic promoters. The rising demand of eco-friendly products and processes has led to a stronger development of “bio-based” manufacturing industries. In this context, SSF has attained much relevance over the last decades as it offers several environmental advantages as well as some benefits from the economic point of view, in comparison to SmF processes. The use of low cost agroindustrial substrates as fermentation materials has transformed SSF into an attractive technology for bioprocesses, like bioremediation, bioleaching, biopulping, biobeneficiation, or biological delignification (Thomas et al., 2013). Another advantage of SSF is the high resistance of microorganisms (fungal, bacterial or yeast species) to catabolic repression (inhibition of enzyme synthesis) in the presence of several substrates, such as glucose, glycerol, or other carbon sources, thus producing a high yield of targeted compounds (Lizardi-Jimenez and Hernandez-Martinez, 2017). Some of the main products obtained by SSF and their applications are described in more detail in the following sections. Chapter 9 • Filamentous fungi processing by solid-state fermentation Table 1 271 Characteristics and limitations in SSF bioreactors. Type of bioreactor Characteristics—Limitation Tray bioreactors • • • • • • • • • • • • • • • Packed-bed bioreactor • Rotating drum bioreactor Fluidized-bed bioreactor Spouted-bed bioreactor • • • • • • • • • • • • • • • • • • • • • • • • • Simple structure, easy to operate Temperature and moisture gradients generated Bed caking Larger area required Labor intensive Forced-aeration, nonmixed Axial temperature and gas concentration gradients exist Nonuniform growth Heat accumulation Difficult to obtain a whole sterile process Difficulties to harvest the final product Difficult to scale-up Bed caking Labor intensive The spacing between the internal cooling plates and the temperature of the cooling water Cooling water temperature is varied during fermentation in response to bed temperature Forced-aeration, nonmixed Continuously or intermittently mixed Improved mass and heat transfer Shear effect may cause damage to microorganisms Slumping flow may cause a little mixing Complicated reactor construction Difficult operation for large-scale fermentation Good moisture and temperature control due to good mixing Considered better and more uniform Removal of metabolic heat is efficient Operating in continuous or semi-continuous mode Good moisture and temperature control due to good mixing Removal of metabolic heat is efficient Excellent growth of aerobic microorganisms due to efficient aeration No bed caking High shear damage to microorganisms Difficult to fluidize large, coarse, and sticky particles High cost Continuously or intermittently mixed High mass and heat transfer rate High mass and heat transfer rate Continuously or intermittently mixed Lower power requirements than fluidized-bed systems Good in handling large, coarse, nonuniformly sized, and sticky particles Need further investigation on characterization and scale-up From Manan, M., Webb, C., 2017. Design aspects of solid state fermentation as applied to microbial bioprocessing. J. Appl. Biotechnol. Bioeng. 4, 1–25; López-Gómez, J.P., Manan, M.A., et al., 2020. Solid-state fermentation of food industry wastes. In: M.R. Kosseva, C. Webb (Eds.), Food Industry Wastes (second ed.). Academic Press, 135–161 (Chapter 7). 272 Current Developments in Biotechnology and Bioengineering 4.1 Enzymes The global market for industrial enzymes was estimated at USD 5.6 billion in 2019, and it is scheduled to reach USD 8.7 billion by 2026, recording a CAGR of 6.5%, in terms of value. The highest sales of enzymes are located in the leather market, followed by the bioethanol market. In addition, the food and beverage enzyme segment is expected to reach about USD 3.2 billion by 2023 (MarketsandMarkets, 2019). Of the various products produced by SSF, industrial enzymes have been the most successful at a commercial level, because of the wide variety of enzymes that can be produced, as well as the wide range of fungi and substrates that can be used. These enzymes include amylase, L-asparraginase, cellulase, including glucosidase, chitin deacetylase, chitosanase, exo- and endo-glucanases, fibrinolytic enzymes L-glutaminase, inulinase, invertase, laccase, levansucrase, lipase, homocysteine-lyase, mannanase, pectate lyase, phytase, lichenase, alkaline protease, acid protease, tannase, xylanases, and xylosidase, among others (Table 2). Enzyme production by SSF presents several advantages compared to SmF, such as high-volumetric productivity, low cost of the equipment involved, better product yield, less waste generation, and less time-consuming processes (Bhargav et al., 2008). It is also noteworthy that enzymatic activities in SSF are higher than those in SmF (Lizardi-Jimenez and Hernandez-Martinez, 2017). Biofuel production is one of the most interesting applications for enzymes produced by SSF, because of the increasing growth of cellulases to saccharify lignocellulosic raw materials. Xylanolytic enzymes and laccases for xylose and lignin degradation, respectively, are another important group of enzymes for the biofuel program. Substrates used for production of these enzymes can be rice husk and rice bran, wheat, soybean, and SCB with Aspergillus spp. as the main fermentative fungi (Thomas et al., 2013). Enzymes for food and feed applications are also successfully produced by fungal SSF to improve their nutritional quality. Some of the most interesting ones are α-amylase, α-galactosidase, glucoamylase, pectinase, inulinase, invertase, protease, lipase, and phytase, which can be produced by Aspergillus spp., Geotrichum spp., or Tricoderma spp., among others. The detergent production and laundry sector is another field of application for enzymes produced by fungal species in SSF. It is estimated that 29% of the global enzyme market corresponds to these enzymes, including protease, xylanase, alkaline amylase, lipase, etc. One example is the production of lipase by Tricoderma harzianum using agroindustrial residues like SCB and castor oil cake supplemented with olive oil (Thomas et al., 2013). Large-scale fermentation for enzyme production has been carried out in drum-, tray-, or deep-trough type fermenters, although much attention has to be paid to avoid disruption of fungal mycelia during agitation and rotation in some types of reactors (Chen and Qiu, 2010). One of the successful technologies for large-scale enzyme production is the PlaFractor process developed by Biocon (India) and acquired by the Novozyme company. Chapter 9 • Filamentous fungi processing by solid-state fermentation 273 Table 2 Enzyme production by solid-state fermentation using by-products as substrate and filamentous fungi as fermentation agent. Substrates Products Microorganisms References Rice bran, wheat bran, soybean meal, and wheat flour Wheat bran Wheat bran, orange peel, and lemon peel Wheat bran, soybean hulls, and rapeseed meal Wheat bran and sugarcane bagasse Soybean bran Alkaline protease Aspergillus oryzae Fath and Fazaelipoor (2015) Polygalacturonase Polygalacturonase A. oryzae A. giganteus Demir and Tari (2016a, b) Ortiz et al. (2017) Glucoamylase, protease, cellulase, and xylanase Pectinase A. awamori and A. oryzae Musaalbakri Abdul and Webb (2018) Finkler et al. (2017) Babassu cake Cellulolytic enzymes A. niger Exoamylase, endoamylase, protease, xylanases, and cellulases Pectinase Trichoderma reesei NRRL6156 A. awamori Gasparotto et al. (2015) de Castro and Sato (2015) A. oryzae Biz et al. (2016) Pectinase A. niger Pitol et al. (2016) Citrus pulp and sugarcane bagasse Wheat bran and sugarcane bagasse Waste bread Soybean meal Wheat bran and citric pectin Defatted rice bran Orange and grapefruit rind Glucoamylase, protease Phytase Pectinase A. awamori A. niger A. niger LB-02-SF Melikoglu et al. (2015) Saithi and Tongta (2016) Poletto et al. (2015) Amyloglucosidase, exo-PG Naringinase Colla et al. (2017) Mendoza-Cal et al. (2010) Wheat bran Sorghum straw Amylase Xylanase Wheat bran Sugarcane bagasse, corn cobs, coconut husk, and candelilla stalks Castor bean waste Sugarcane bagasse and soybean oil Palm kernel cake and palm pressed fiber Chickling vetch peels and grasspea peels Xylanase Ellagitannase A. niger Aspergillus foetidus Aspergillus niger Aspergillus niger HPD-2 Aspergillus oryzae Aspergillus tubingensis FDHN1 Aspergillus oryzae Aspergillus niger GH1 Lipase Lipase Protease Protease Chen et al. (2014) Adhyaru et al. (2016) Pirota et al. (2013) Buenrostro-Figueroa et al. (2014) Penicillium simplicissimun Thermomucor indicaeseudaticae M31S A. oryzae Godoy et al. (2011) Ferrarezi et al. (2014) Aspergillus terreus NCFT4269.10 Sethi et al. (2016) Tsouko et al. (2017) Continued 274 Current Developments in Biotechnology and Bioengineering Table 2 Enzyme production by solid-state fermentation using by-products as substrate and filamentous fungi as fermentation agent—cont’d Substrates Products Microorganisms References Wheat bran, soybean meal, cotton seed and orange peel Jatropha seed cake Protease Aspergillus niger LBA 02 de Castro et al. (2015) Protease Aspergillus versicolor CJS-98 Canola cake Protease Aspergillus oryzae CCBP001 Veerabhadrappa et al. (2014) Freitas et al. (2015) From López-Gómez, J.P., Manan, M.A., et al., 2020. Solid-state fermentation of food industry wastes. In: M.R. Kosseva, C. Webb (Eds.), Food Industry Wastes (second ed.). Academic Press, 135–161 (Chapter 7); Soccol, C.R., Costa, E.S.F.d., et al., 2017. Recent developments and innovations in solid state fermentation. Biotechnol. Res. Innov. 1 (1), 52–71; Lizardi-Jimenez, M.A., Hernandez-Martinez, R., 2017. Solid state fermentation (SSF): diversity of applications to valorize waste and biomass. 3Biotech 7 (1), 44. In Japan, enzymes are also produced at industrial scale using SSF (Suryanarayan, 2003). Some examples of enzymatic products developed by SSF are Synergen or Allzyme produced by the company Alltech (USA) through SSF of Aspergillus niger, with applications in feed formulation (Bowyer et al., 2020). 4.2 Organic acids Organic acids have been extensively used by the food industry as food additives and preservatives. SSF presents a clear advantage for the production of organic acids, due to the feasible and efficient extraction of acids from the fermented matter (Lizardi-Jimenez and Hernandez-Martinez, 2017). Table 3 summarizes different SSF processes to produce organic acids. Table 3 Organic acid production by solid-state fermentation using by-products as substrate and filamentous fungi as fermentation agent. Substrates Products Microorganisms References Apple pomace Pineapple waste Orange peel Sugarcane bagasse Tea waste and molasses Pomegranate seeds and husk Cranberry, creosote bush, and pomegranate Citrus processing wastes Fumaric acid Citric acid Citric acid Citric acid Gluconic acid Ellagic acid Rhizopus oryzae 1526 Aspergillus niger DS I Aspergillus niger CECT-2029 Aspergillus niger PTCC-5010 Aspergillus niger ARNU-4 Aspergillus niger Das et al. (2015) Kumar et al. (2010) Torrado et al. (2011) Yadegary et al. (2013) Sharma et al. (2008) Robledo et al. (2008) Ellagic acid Aspergillus niger GH1 Sepúlveda et al. (2016) Aspergillus niger ATCC 1015 Kuivanen et al. (2014) L-galacturonic acid From Lizardi-Jimenez, M.A., Hernandez-Martinez, R., 2017. Solid state fermentation (SSF): diversity of applications to valorize waste and biomass. 3Biotech 7 (1), 44. Chapter 9 • Filamentous fungi processing by solid-state fermentation 275 4.2.1 Citric acid Citric acid is commonly used in the food and pharmaceutical industries. and it can be also obtained by chemical synthesis, although the cost is much higher than by using fermentation. This acid is mainly produced in SmF system by Aspergillus niger, but SSF is also proposed by several authors as a suitable technology, as different agroindustrial by-products can be used as substrate (apple pomace, coffee husk, wheat straw, mixed fruit, cassava bagasse, etc.) (Couto and Sanromán, 2006). 4.2.2 Lactic acid Lactic acid has a broad scope in the food, pharmaceutical, leather and textile industries, and more recently, it has garnered more attention as a starting material to produce biodegradable polymers used in medical and other industrial products. Unlike in chemical synthesis, which generates a racemic mixture of L- and D-lactic acid, several microbes can produce only L-lactic acid. Various studies have been developed that focus on the production of L(+)-lactic acid by Rhizopus oryzae in solid-state conditions operating with substrates such as SCB or paper sludges, with similar or even better results than those obtained with SmF (Couto and Sanromán, 2006; López-Gómez et al., 2020). 4.2.3 Succinic acid Succinic acid is a bulk chemical that can be used as a de-icer in the food and beverage industry, or as a building block for various commodity chemicals and biodegradable polymers. It is currently produced from fossil fuels, but fermentative processes have also been suggested for its production. Du et al. (2008) presented a biorefinery process for fractionated wheat to produce glucoamylase and protease enzymes by Aspergillus awamori and Aspergillus oryzae. The resulting product was hydrolysed and used as substrate to produce succinic acid with Actinobacillus succinogenes. Other organic acids that can be produced by filamentous fungi through SSF are gallic acid, gluconic acid, or ellagic acid (Thomas et al., 2013). 4.3 Bioactive compounds/secondary metabolites SMs are the intermediate products of metabolism, which are produced in the course of a fermentation process. SMs are produced during the stationary phase and their main roles are related to competition, antagonism, and self-defense mechanisms. The pathways of microbial SMs are polyketone synthesis, mevalonate synthesis, shikimic acid, and sugar derivative pathways (Zhang et al., 2018). They do not play any role in the growth and reproduction activities, so they are only produced under specific conditions and by some species, depending on several factors like medium composition and nutrient concentration, temperature, pH, light, etc. These metabolites can be applied in different sectors, such as the pharmaceutical, food, cosmetics sector, etc. (Kumar et al., 2021). 276 Current Developments in Biotechnology and Bioengineering Bioactive compounds are defined as SMs triggering pharmacological or toxicological effects in humans and animals. They are highly heterogeneous, including alkaloids, mycotoxins, plant growth factors, pigments, flavonoids, antibiotics, and phenolic acids, among others, with varying chemical structures (hydrophilic or lipophilic) (Sadh et al., 2018a, b). SSF is a suitable alternative for the release of bioactive compounds due to the production of enzymes required for the breakage of cell walls of plants and biotic materials. In recent years, SSF has been applied to the production or the extraction of different antioxidant molecules (Lizardi-Jimenez and Hernandez-Martinez, 2017). Table 4 summarizes different SSF processes to produce pigments, antibiotics, and phenolic compounds. Table 4 Secondary metabolites, pigments, phenolic compounds, and antibiotics production by solid-state fermentation using by-products as substrate and filamentous fungi as fermentation agent. Substrates Products Microorganisms References Jackfruit seed Corn cob Pigments Pigments Babitha et al. (2007) Velmurugan et al. (2011) Sugarcane bagasse Cephalosporin C (antibiotic) Lovastatin (antibiotic) Monascus purpureus Monascus purpureus KACC 42430 Acremonium chrysogenum C10 Penicilium funiculosum NCIM 1174 Green gran husk, black gram husk, wheat bran, and orange peel Potato Rice bran Wheat Cuadra et al. (2008) Reddy et al. (2011) Sambacide (antibiotic) Phenolic compounds Phenolic compounds Fusarium sambucinum B10.2 Rhizopus oryzae Rhizopus oryzae RCK2012 Chickpeas Maize Phenolic compounds Phenolic compounds Apple pomace Phenolic compounds Grapefruit by-products Antioxidant compounds Gallic acid Antioxidant, antimicrobial, anticancer, anti-HIV, antihyperuricuric, antitubercular Cholesterol lowering agent (statin) Cordyceps militaris SN-18 Thamnidium elegans CCF 1456 Phanerochaete chrysosporium Aspergillus niger GH1 Larios-Cruz et al. (2018) Aspergillus niger Aspergillus niger G131 Saeed et al. (2020) Carboue et al. (2018) Monascus ruber Zhang et al. (2018) Fruit peels and seeds Vine shoors, pine saw dust Rice, miller, corn, barley, and wheat Dong et al. (2016) Schmidt et al. (2014) Bhanja Dey and Kuhad (2014) Xiao et al. (2014) Salar et al. (2012) Ajila et al. (2011) From Lizardi-Jimenez, M.A., Hernandez-Martinez, R., 2017. Solid state fermentation (SSF): diversity of applications to valorize waste and biomass. 3Biotech 7 (1), 44; Kumar, V., Ahluwalia, V., et al., 2021. Recent developments on solid-state fermentation for production of microbial secondary metabolites: challenges and solutions. Bioresour. Technol. 323, 124566. Chapter 9 • Filamentous fungi processing by solid-state fermentation 277 4.3.1 Pigments Some examples of bioactive compounds produced by SSF are lycopene from tomato waste that can be used as an antioxidant agent, as well as a coloring agent in the cosmetics, pharmaceutical, or food industries (Sadh et al., 2018a, b). Pigments are produced by fungi as a defensive and survival mechanism. Pigment production largely depends upon the medium composition, aldohexoses, such as glucose and dextrose, being the best carbon sources. Other factors affecting pigment production are nitrogen concentration and source, temperature, and pH. These pigments have been used as pharmaceuticals or food additives. Monascus sp. has been reported to produce at least six different pigments (yellow pigments—monascin, ankaflavin, orange pigments—moascorubrin and rubropunctatin, and red pigments—monascorubramine and rubropuntamine). For example, M. purpureus CMU001 produced the highest pigment yield (129.63 U/g dry weight) when corn meal was supplemented with 8% glucose (Thomas et al., 2013). 4.3.2 Phenolic compounds Phenolic compounds present diverse biological activities including antioxidant properties. SSF has been exploited to enhance the phenolic compounds fraction in selective food products to improve their antioxidative attributes, such as black beans. Several studies also revealed that phenols can be successfully released into the media during fermentation processes by fungal species in solid-state. This is due to the fungal extracellular enzymatic systems that produce hydrolases to degrade polysaccharides and ligninolytic enzymes that open phenyl rings, increasing phenol content in the media (Martins et al., 2011). Two different fungi, Rhizopus oligosporus and Aspergillus niger, were investigated for phenolic compound release and antioxidant activity during SSF of apricot pomaces, producing an increase of phenolic content of 70% and 30%, respectively (Kumar et al., 2021). Rhizopus oryzae also produced an increase of phenols after growing on rice bran or on fruit and vegetable by-products (Schmidt et al., 2014; Ibarruri et al., 2021). 4.3.3 Antibiotics Antibiotics are SMs that are produced by microorganisms in stress conditions. For example, penicillin is generated by Penicillium chrysgenum when the culture medium has a very low glucose concentration and starts to consume lactose (Kumar et al., 2021). Antibiotics were traditionally produced by SmF, but however, some studies indicate that production by SSF is better because the solid substrates are natural habitats for fungi and in addition, because it is possible to use agroindustrial by-products. Some examples are cephalosporin C produced using SCB by Acremonium chrysogenum C10, and lovastatin by Penicillium funiculosum NCIM 1174, using different agroindustrial by-products, such as black gram husk, green gram husk, orange peel, and wheat bran (Lizardi-Jimenez and Hernandez-Martinez, 2017). Sambacide is a novel tetracyclic triterpene sulphate, which has been successfully produced using potato as fermentation substrate by Fusarium sambucinum (up to 278 Current Developments in Biotechnology and Bioengineering 1.9  0.08 mg/g of substrate dry weight). This compound also exhibited remarkable antimicrobial activity against S. aureus and E. coli (Dong et al., 2016). 4.4 Biological control agents/biopesticides Replacement of pesticides for the control of plant pests and diseases is of great interest due to the environmental impact this can cause. Entomopathogenic fungi are able to infect insects and arachnids directly through penetration of the host cuticle (Sala et al., 2020). Therefore, the production of fungal spores of these species grown on solid substrates can be used as alternative biocontrol agents. Some of the more studied species are Beauveria bassiana grown on rice husk (Sala et al., 2020), or brewer’s spent grain (Qiu et al., 2019) or Trichoderma harzianum grown on several agroindustrial wastes, such as vinegar production by-products, cattle dung, and rice straw, used for the biocontrol of Fusarium wilt in cucumber (Chen et al., 2011) (Table 5). 4.5 Biofuels Biofuels for renewable energy production has been garnering great attention in recent decades, to replace fossil fuels and reduce the resulting environmental impact. SSF can be used as a feasible technology to produce both biodiesel and bioethanol, using renewable raw materials, and therefore, contributing to reduce the greenhouse gas (GHG) emissions. Table 6 summarizes different SSF processes to produce biofuels. 4.5.1 Biodiesel Biodiesel production by oleaginous microorganisms is highly appealing for the biofuel industry. In this process biodiesel can be produced using lipids and lipases produced by SSF. Lipids are generally produced when the nitrogen source is limited while carbon source is in excess. Some of the fungal species involved in lipid production belong to Table 5 Biopesticide production by solid-state fermentation using by-products as substrate and filamentous fungi as fermentation agent. Substrates Products Microorganisms References Refused potatoes Parboiled rice Winery wastes Corn wastes, wheat bran Biological control agent Biological control agent Biological control agent Biological control agent Santa et al. (2005) Tarocco et al. (2005) Bai et al. (2008) Cavalcanti et al. (2008) Millet grain Sweet potato Parboiled rice texturized with water hyacinth Biological control agent Biological control agent Biological control agent Beauveria bassiana Beauveria bassiana Tricoderma viride Tricoderma harzianum sp., T viride sp., T. koningii sp., T. polisporum sp. Beauveria bassiana Isaria javanica Isaria fumosorosea Kim et al. (2011) Kim et al. (2014) Angel-Cuapio et al. (2015) From Lizardi-Jimenez, M.A., Hernandez-Martinez, R., 2017. Solid state fermentation (SSF): diversity of applications to valorize waste and biomass. 3Biotech 7 (1), 44. Chapter 9 • Filamentous fungi processing by solid-state fermentation 279 Table 6 Biofuel production by solid-state fermentation using by-products as substrate and filamentous fungi as fermentation agent. Substrates Products Microorganisms References Rice straw Ethanol Takano and Hoshino (2018) Sweet sorghum Pear pomace Rice bran, soybean meal Wheat straw bran mixture Rice hulls Ethanol c-linolenic acid Gamma-linolenic acid Lipids Oleic acid, palmitic acid, linoleic acid Lipids Mucos sp. and cellulolytic enzymes Mucor indicus Mortierella isabellina Mucor rouxii Aspergillus oryzae Mortierella isabellina Aspergillus tubingensis TSIP9 Cheirsilp and Kitcha (2015) Palm empty fruit bunches Molaverdi et al. (2013) Fakas et al. (2009) Jangbua et al. (2009) Hui et al. (2010) Economou et al. (2011) From Takano, M., Hoshino, K., 2018. Bioethanol production from rice straw by simultaneous saccharification and fermentation with statistical optimized cellulase cocktail and fermenting fungus. Bioresour. Bioprocess. 5 (1); Lizardi-Jimenez, M.A., Hernandez-Martinez, R., 2017. Solid state fermentation (SSF): diversity of applications to valorize waste and biomass. 3Biotech 7 (1), 44. Mucor, Mortierella, and Aspergillus genera, and can produce several lipids such as γ-Linolenic, gamma linolenic, linoleic, oleic, palmitic acids from rice bran, soybean, sorghum, wheat straw, rice hulls, or pear pomace (Table 6) (Lizardi-Jimenez and HernandezMartinez, 2017). On the other hand, lipases are also important enzymes for biodiesel production. Their main advantages are that the process is less polluting than in chemical catalysts and less energy consuming, and that they allow the catalysis reactions to develop in non-aqueous media. Nevertheless, biodiesel production using lipases is more expensive due to the cost of commercial enzymes (Liu et al., 2013). 4.5.2 Bioethanol Bioethanol has traditionally been produced by SmF. However, in recent years, some researchers have shown the viability of using SSF processes for the production of hydrolysates from several agroindustrial lignocellulosic wastes, which can be used as a source of sugar for bioethanol production, e.g., wheat straw by Aspergillus niger or sweet sorghum using Mucor indicus. Bioethanol is produced through a two-step process where a first hydrolysis phase of lignocellulosic substrates is needed to produce enough sugars, which are then used in the second fermentation phase. These two processes can be developed separately or simultaneously (simultaneous saccharification and fermentation). SSF presents some advantages in comparison to SmF for ethanol production, e.g., energy and time saving, easy operation, cheaper feedstocks, and environmentally friendlier processes (Lizardi-Jimenez and Hernandez-Martinez, 2017). 4.6 Biosurfactants Biological surfactants (BSs) are amphipathic molecules that are involved in cell development, microbial survival under disadvantageous conditions, and the regulation of osmotic pressure (Banat et al., 2021). BSs present lower toxicity and higher biodegradability in 280 Current Developments in Biotechnology and Bioengineering comparison to synthetic surfactants, and can also be used in food emulsification, bioremediation, new polymer synthesis, and cosmetic applications. They have a stable activity at extreme temperature, pH, and salinity conditions, and they also have applications in oil industries. Although BS produced in SmF are extensively used in bioremediation purposes, the main drawback is the high production cost. SSF allows the use of agroindustrial by-products as substrates, which reduces costs, and it avoids the typical foaming problems during SmF production (Krieger et al., 2010). BS production is also affected by growing conditions (pH, water content, temperature, or carbon source). Generally, hydrophobic carbon sources are reported to be better than hydrophilic ones in promoting BS production. Sophorolipids and rhamnolipids are two groups of extracellular biosurfactants that can be produced by SSF. Although productivities are still lower than those obtained by SmF, it is expected that, by devoting more effort to SSF optimization, both in terms of the reactor design and control parameters, further improvements can be reached (Cerda et al., 2019). 4.7 Biopolymers Biopolymers are produced by microorganisms as a reserve energy source, and they are stored in the cells as intracellular granules when nitrogen, phosphorus, sulfur, or magnesium sources are limited in the medium (Sindhu et al., 2015). SSF has also been explored to produce several biopolymers such as exopolysaccharides (EPS), polyhydroxyalkanoates (PHA), poly-3-hydroxybutyrate (PHB), dextran, etc. EPS are of great industrial significance as they can be produced by a large number of microbial species, and some of them show some biological effects such as antitumor, hypoglycemic, and immunostimulant activities. Several studies have been conducted to produce these EPS in SmF although the expensive media components have led to searching for alternative systems like SSF (Thomas et al., 2013). Nevertheless, there are a few reports about higher medicinal fungi cultivated on agroindustrial residues for polysaccharides production. Two polysaccharides, DGS1 and DGS2, were obtained by SSF of Fusarium solani DO7 with immunomodulatory activity (Zeng et al., 2019). Rapeseed meal was also used for the growth of Aspergillus sojae at pilot scale, to produce β-glucan enriched water extracts with an immunomodulatory effect in human peripheral blood mononuclear cells (PBMCs), and mouse bone marrow-derived macrophages (BMDM). Many other researchers have analyzed chitosan and chitosan-glucan complex production in different filamentous fungi (Aspergillus, Mucor, Penicillium, Rhizopus), concluding that SSF is the process with the highest production yields for the production of these polysaccharides under optimized conditions to reduce fermentation costs (Sutter et al., 2017). PHA and PHB are biodegradable polymers used for bioplastic production, and their production by fermentative processes has been extensively studied in recent decades. SmF is the main process proposed for PHA production, but however, the process has been economically unfavorable, and SSF has recently been considered as a potential option to reduce the production cost of PHA (Thomas et al., 2013). Chapter 9 • Filamentous fungi processing by solid-state fermentation 281 First reports on the use of agroindustrial wastes for the production of PHA by Cupriavidus necator were developed by Oliveira et al. (2004) with soy cake alone, or supplemented with sugarcane molasses producing up to 4.9 mg PHA/g (dry weight) in 60 h. They also reported the production of PHB by SSF process with Ralstonia eutropha, grown on babassu and soy cake, obtaining productions of about 1.2 mg/g on soy cake in 36 h. This process provides a biopolymer that is identical to a commercial PHB produced by SmF, the only difference being a higher molar mass and a lower degree of crystallinity, which represent an advantage for a broader range of applications (Oliveira et al., 2004). 4.8 Other secondary metabolites 4.8.1 Phytostimulatory hormones Phytohormones are metabolites that influence a wide range of development processes in plants, which include dormancy, germination, stem elongation, sex expression, flowering, induction of enzymes, and leaf and fruit senescence. Many fungal strains, such as Aspergillus niger, Fusarium oxysporum, Penicillium cyclopium, A. flavus, P. funiculosum, P. corylophilum, and Rhizopus stolonifer are capable of producing phytostimulatory hormones like indole acetic acid (IAA) and gibberellic acid (GA) (Waqas et al., 2012). Less information is available on the production of phytohormones by SSF although GA production has also been reported by strains of Gibberella fujikuoroi and Fusarium moniliforme grown on industrial by-products like soy bean, citric pulp, soy husk, SCB, coffee husk and cassava bagasse. Results indicated that the best medium was citric pulp with a production of 5.9 g/kg of dry pulp after 3 days’ fermentation (Rodrigues et al., 2009). 4.8.2 Aroma Flavor and fragrance compounds play a vital role in the commercialization of food, feed, cosmetic products, and also for pharmaceutical industries. Interest in the use of SSF to produce aroma compounds is also increasing after the successful use of this bioprocess in the production of various enzymes and metabolites (Soccol et al., 2017). Different studies have suggested that a greater yield of aroma compound can be obtained by using SSF in comparison to SmF. Filamentous fungi like Trichoderma viride, Rhizopus oryzae, and Aspergillus niger are capable of producing fruit flavors or coconut aromas by SSF processes on SCB and sugar beet molasses, jatropha cake or olive pomace (Kumar et al., 2021). Vanillic acid, which is a flavoring agent and also a precursor for vanillin production, has also been produced by Phanerochaete chrysosporium in lignocellulosic wastes at a concentration of 73.69 mg vanillic acid/g of dried wastes (Thomas et al., 2013). 4.8.3 Polyunsaturated fatty acids Polyunsaturated fatty acids (PUFAs) provide several health benefits in the diet, such as a reduction in low-density lipoprotein and an increase in high-density lipoproteins. Some of the PUFA (omega-3 and omega-6) are essential fatty acids that the body needs but 282 Current Developments in Biotechnology and Bioengineering humans are not able to synthesize. So, PUFAs are of great interest in food, nutraceutical, pharmaceutical and feed applications, a growing market estimated to reach USD 48.9 billion by 2025, and growing at a CAGR of 11.7% (Industryarc, 2019). Morteriella species are the main fungal microorganisms involved in PUFA production by SSF with production yields from 1.7% to 15.8% of oil per fermented mass. In order to reduce costs, agroindustrial by-products such as wheat straw, cereal-based products (bran, straw), wastes (orange peel, pear pomace, press cake, brewer’s spent grain), are mainly used. Depending on the substrate a pre-treatment is sometimes needed to break down insoluble materials (Ochsenreither et al., 2016). Soybean meal and distillers’ dried grains were also used as substrates for PUFA production by Mortierella alpine. These fermented products enriched in PUFA were used for feeding laying hens, resulting in an increase of ω-6 and ω-3 proportions in chicken breasts and liver (Yang and Zhang, 2016). 5. Conclusions and perspectives SSF by filamentous fungi has experienced important developments over the last decades (especially during 1980–90), because of its environmental and economic advantages in the valorization of agroindustrial by-products, as well as in the production of valuable compounds with applications in a wide range of industrial sectors. This has led to the development of new bioreactors and mathematical modeling of the processes as well as to an increase in the number of patents (from less than a hundred until 1980 to around 10,000 in the last decade 2010–20). Several fungal species, and especially filamentous fungi, have demonstrated clear advantages as fermentation agents in SSF. In fact, they are the most widely used microorganisms for these processes due to their ability to grow on a large variety of substrates like food and agricultural wastes with a low water activity, reducing water and energy consumption for fermentation in comparison to SmF. Nevertheless, some of these advantages become disadvantages when trying to scale-up and make the fermentation process reproducible. As described in this chapter, inoculation of the specific fungal strain, or consortia, adapted to the substrate and to growth conditions, is a key factor for the successful production of targeted bioproducts. This, in some cases, requires a previous sanitization step to remove or reduce native microbial population from the substrate, which could interfere in the fermentation or even in the hygienic quality of the final product. Fungal biomass quantification is also difficult to determine and control due to the type of growth where the hyphae are closely linked to the substrate. Advances in the understanding of fungal microbiology will also be key for future developments of this technology. The use of mixed culture strains is also a promising opportunity to couple several biochemical processes because of the synergistic effect between several species, which could lead to more effective fermentation, and to the production of new valuable compounds. However, there are still limited studies on mixed fermentation optimization, and this field needs further development. Chapter 9 • Filamentous fungi processing by solid-state fermentation 283 Another important challenge is the variability of the substrate composition in relation to its chemical nature, particle size, water content, or surface area, which can lead to low reproducibility of the process. Heterogeneity of raw materials causes problems in microbial population evolution and metabolism. This is especially important when using industrial waste mixtures that can vary seasonally in generation and composition. As previously set out, there is a huge number of products that can be produced by SSF (from enzymes and organic acids to biopesticides, biosurfactants, biofuels, aromas, and bioactive compounds or bioplastics), and this number is expected to increase considerably in the future due to the intensive research in this field. Nevertheless, optimization of the downstream process is also an important factor in the development of fungal SSF processes. Although there are remarkable advances in biochemical engineering, the separation of the desired compound from the solid matrix is technically and economically a challenging process. Therefore, SSF has been mainly used in the production of metabolites with a low degree of purity. However, the most important aspect that hinders fungal SSF is the development of specific and feasible reactor designs. The objectives in this field are focused on the reduction of operational constraints, like heat removal or adequate mixing strategy, adequate monitoring and control, but also on the continuous and standardized production of compounds to guarantee their quality. The control of parameters is a key point that needs to be solved at an industrial scale because of the difficulties in obtaining representative samples. So, the slow development of automation and control has become an important handicap for industrialization. Further engineering developments are needed to provide effective technologies for forced ventilation, temperature control, effective mixing, and easy operation. These technologies must solve urgent problems like low oxygen diffusion in the solid matrix, temperature and concentration gradients, heat accumulation, modeling of growth kinetics, and transport phenomena. These aspects need to be developed in a multidisciplinary way, including fluid dynamics, computational mathematics and modeling, or porous medium theory. As a result of the lack of scalable full-scale reactors, and effective controlling and operating technologies adapted to each specific application, the level of industrial implementation of SSF technology is still low in comparison to SmF. Despite these technical limitations for scaling-up, many industrial SSF facilities operate worldwide. Currently the market is dominated by large companies (Novozyme, Dupont, DMS, Monsanto, …), although productions are still at a relatively low scale, and mainly focused on enzymes and organic acid production. Nevertheless, taking into account that climate change mitigation is one of the most challenging and urgent objectives worldwide, and that biorefinery and circular economy strategies are contributing to the development of new industrial activities, fungal SSF experiences are expected to develop considerably in the near future. This could lead to the implementation of new production companies, including small and medium enterprises, and new bioproducts with a reduced environmental impact that substitute more contaminant chemicals in fields like biofuels, biopesticides, biosurfactants, or biopolymers. 284 Current Developments in Biotechnology and Bioengineering References Adhyaru, D.N., Bhatt, N.S., et al., 2016. Insight on xylanase from Aspergillus tubingensis FDHN1: production, high yielding recovery optimization through statistical approach and application. Biocatal. Agric. Biotechnol. 6, 51–57. Ahmed, A., Zulkifli, I., et al., 2016. 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Biotechnol. 171, 2075–2081. 10 Production of industrial enzymes by filamentous fungi Zohresadat Shahryaria,b and Seyyed Vahid Niknezhadc,d a SWE DISH C ENTRE FOR RE SOUR CE R ECOVE RY, UNIVERS ITY O F BORÅ S, BOR ÅS , SW EDEN AV IDZYME COMP ANY, SHIRAZ, IRAN c BURN AND WOUND HEALI NG RESE ARCH CE NTER , S H I R A Z U N I V E R S I T Y O F ME DI CAL SCIENCES, SHIRAZ, IRAN d PHARMACEUTICAL SCIENCES RE SEARC H CENTE R, SHIRAZ UNI VE RSI T Y OF MEDIC AL SCI ENCES , SHI RAZ, IRAN b 1. Introduction Enzymes are used today to make several commercial products. Enzyme-based products and solutions are used in a broad variety of industry sectors from food (Collados et al., 2020; Li and Lu, 2020; Raveendran et al., 2018) and cosmetic (Khan and Rathod, 2015; Sunar et al., 2016) to biofuel (Lv et al., 2021; Srivastava et al., 2020). Interestingly, filamentous fungi are among the most widely used microorganisms in industrial enzyme production. Filamentous fungi, as a member of a large group of eukaryotes, are heterotrophic and obtain nutrition by hydrolyzing complex material into simple molecules for uptake and use in biosynthesis and energy production (Lv et al., 2021). Regarding the primary demand of filamentous fungi to process diverse and complex substrates, this kind of microorganism can produce a vast variety and a large number of enzymes such as amylase, xylanase, pectinase, protease, etc. In addition to their natural ability in enzyme production, remarkable properties make them attractive host for recombinant enzyme production as well. Fungal enzymes, both natural and recombinant form, have been utilized in the industrialization of detergents, starch, drinks, food, textile, animal feed, baking, pulp and paper, leather, chemical, and biomedical products in recent decades (Lv et al., 2021). Although filamentous fungi have contributed greatly to enzyme production, implementation of their industrial cultivation encounters challenges considering their pathogenesis effects, bi-products production such as toxin and protease and high broth viscosity. The aim of this chapter is to describe the fermentation process and critical challenges in the implementation of enzyme production by filamentous fungi. The current bioprocess strategies and various approaches and techniques in fungal enzyme production have been highlighted and discussed to underline the most important research achievements in this field. Besides natural enzyme production, recombinant enzyme production using filamentous fungi, as a candidate host, has been investigated as well. Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00004-1 Copyright © 2023 Elsevier Inc. All rights reserved. 293 294 Current Developments in Biotechnology and Bioengineering 2. Applications of industrial enzymes The use of enzymes in industrial applications has been increased because of their several distinct advantages such as operation in milder reaction conditions, exceptional product selectivity, and lower environmental and physiological toxicity (Al-Ghanayem and Joseph, 2020; Chapman et al., 2018). Today, the quality of food supply issue is one of most concerns in the food industry. In this regard, enzymes such as amylase, glucose isomerase, and pectinase are professionally applied for the purification of the food production and improvement of the food components such as flavor, aroma, color, texture structure, appearance, and nutritive value (Raveendran et al., 2018; Singh et al., 2016). Enzymes are also applied in new parts of food industry like fat modification, sweetener technology, and beverages (Li et al., 2012). In dairy industries, enzymes like lactose are used as a digestive aid to improve factors such as the solubility and sweetness in milk products (Khan and Selamoglu, 2020). After a nonstop increasing universal request for milk and meat consumption, the quality and quantity of animal diet formulation have been taken into consideration. In feed industries, the addition of feed enzymes such as phytases, xylanases, glucanases, α-galactosidases, α-amylases, proteases, and polygalacturonases to the animal diet causes a rise in the digestibility of nutrients and decrease in the antinutritional factors (ANFs) content (Singh et al., 2016). In textile industries, enzymes allowed the improvement of environmentally friendly technologies in fiber processing to reduce the responsibility of waste generation from desizing of fabrics, bleaching chemicals, and dye. Additionally, enzymes make strategies to enhance the final product quality (Choi et al., 2015). Enzymes have also contributed expressively to the growth and improvement of detergent industries. The enzyme-based cleaning agents are less expensive, environmentally friendly, biodegradable, and work at low temperatures as compared to caustic or acid € rko € k, 2019). Lipases, cellulases, proteases, and amylases are imporcleaning regimes (Gu tant groups of hydrolytic valuable enzymes utilized in detergent industries. 3. Advantages of enzymes production by filamentous fungi Enzyme production by filamentous fungi is interesting due to their easy cultivation, highvalue protein secretion, simultaneous biomass production, and stress resistance (Shahryari et al., 2019). Their filamentous mode of growth enables effective colonization of substrates and provides a large surface to volume ratio facilitating the uptake of nutri€ sten, 2019). It has been reported that filamentous fungi such as Trichoderma reeents (Wo sei and Phanerochaete chrysosporium, can effectively degrade different types of lignocellulosic materials by secretion of specific enzymes (Troiano et al., 2020). In addition, filamentous fungi can perform post-translational modifications including glycosylation, protease cleavage, and disulfide bond formation, which are necessary for enzyme function and activity (Wang et al., 2020). Furthermore, despite industrially preferred yeast, Saccharomyces cerevisiae, filamentous fungi can utilize pentose sugars, mainly xylose and Chapter 10 • Industrial enzymes by filamentous fungi 295 arabinose, which are the main sugars in agricultural residues such as sugarcane bagasse, straw, and corn stover. The prominent potential of filamentous fungi in the utilization of agro-industrial waste as inexpensive feed-stocks for the production of valuable bio-chemicals such as enzymes can lead to a dramatic decrease in the enzyme production cost ( Jun et al., 2011; Troiano et al., 2020). Filamentous fungi also have an interesting ability to convert commonly available substrates such as cellulose into organic acids. This ability has at least two major evolutionary advantages for the organism, but it is also a factor that explains the frequent utilization of fungi as a production organism in industry. By turning sugars into organic acids, the fungi make sure that few other organisms have the metabolic capability to digest carbon and energy of this form. In an acidic environment, other non-acidophilic organisms, especially pathogens, cannot survive and fungal products such as enzymes can be produced without this type of contaminants (Nørregaard et al., 2014). In addition, enzyme production by filamentous fungi brings several benefits related to downstream processes. For example, in solid-state fermentation (SSF), extensive growth of the fungi on the solid substrate could bring more damage to the crystalline structure of the substrate, which may finally result in easier enzyme extraction (Shahryari et al., 2020). Due to the large size of filaments, purification of the produced enzyme and filamentous fungi biomass separation from cultivation media is applicable using simple filtration. The preference for filamentous fungi comes from superior enzyme productivity compared to yeast and bacteria, high enzyme activity at neutral pH values, and enzymatic thermal stability (Troiano et al., 2020). Fungal α-amylase is more accepted, Generally Recognized as Safe (GRAS), than yeast and bacterial α-amylase (Gupta et al., 2003). Fungal xylanase is also reported to be more active than yeast and bacterial xylanase (Kulkarni et al., 1999). 4. Filamentous fungi in enzymes production 4.1 Aspergillus Aspergillus is one of the most prominent and commercially utilized filamentous fungi for the production of industrial enzymes (Hu et al., 2011). It is a common and widespread genus distinguishable by the morphology of the conidiophore, the structure that bears the asexual spores (Troiano et al., 2020). Aspergillus produces a broad range of enzymes related to the degradation of plant polysaccharides, such as cellulose, xylan (Li et al., 2018), xyloglucan, galactomannan and pectin (Li et al., 2018), and several enzymes used in food and feed production such as glucoamylases (Pasin et al., 2017; Xian and Feng, 2018), proteases (Osmolovskiy et al., 2021; Tang et al., 2020), and phytases (Neira-Vielma et al., 2018; Sanni et al., 2019). In fact, AB Enzymes, BASF, Chr. Hansen, DuPont, and Novozymes are only a few examples of companies that are still using Aspergillus sp. in the large-scale manufacturing of commercial products such as enzymes (Ntana et al., 2020). 296 Current Developments in Biotechnology and Bioengineering 4.2 Fusarium Fusarium is filamentous fungi belonging to the Ascomycota phylum. The application of Fusarium sp. as enzyme producers in the most diverse fields have been extensively ^ a et al., reported in the last years (Li et al., 2020; Martı́nez-Pacheco et al., 2020; Pesso 2017). This fungus is a potential commercial producer of extracellular laccase (Abd El-Rahim et al., 2020), chitin deacetylase (Suresh et al., 2014), chitinase (Laien and Mohammadi, 2021), and β-glucosidase (Gao et al., 2012; Olajuyigbe et al., 2016). Fusarium strains have also been extensively studied for the production of proteases. Currently, some companies are employing proteases and alpha-amylases from Fusarium sp. for cleaning purposes. The patent previously assigned to Novo Nordisk (US5288627A, nowadays assigned to Novozymes) claims the application of endo-proteases produced by Fusarium oxysporum DSM 2672 with specific characteristics for use in detergent formulations (Nielsen et al., 1994). 4.3 Penicillium The filamentous fungal genus, Penicillium, comprises more than 300 species, many of which are common soil inhabitants (Kumar et al., 2018). Many Penicillium species have great potential in the secretion of lignocellulose hydrolysis enzymes. The P. citrinum MTCC 6489 strain produces high xylanase and cellulases content (Dutta et al., 2007). The wide numbers of extracellular enzymes produced by Penicillium species have an important role in the food and feed industries. Remarkable examples of hydrolases of Penicillium include various cellulolytic enzymes and other polysaccharases, such as thermoactive polygalacturonase used in citrus juice clarification, Nuclease P1, used as a flavor enhancer in the food industry (Meena et al., 2018), xylanase (Meshram et al., 2008), together with a variety of lipases and proteolytic enzymes (Hamlyn et al., 1987; Sahay and Chouhan, 2018). 4.4 Rhizopous Rhizopus is one of the most economically important members of filamentous fungi. Different characteristics make this genus interesting for considerable application in enzyme production. Rhizopus has a worldwide distribution with a high prevalence in tropical and subtropical regions. It has been isolated from many substrates, including a wide variety of soils, decaying vegetation, fruit, vegetables, and seeds. Rhizopus requires a simple ecosystem to survive and can vigorously grow between 25°C and 45°C (Cantabrana et al., 2015). A large number of carbohydrate digesting enzymes have been reported to produce from Rhizopus filamentous fungi. Apart from the cellulases and hemicellulases, several other enzymes such as protease, urease, ribonuclease, glucoamylase, lipase, and polygalacturonase are also found to be secreted by Rhizopus from pentose sugars and agricultural wastes (Battaglia et al., 2011). Chapter 10 • Industrial enzymes by filamentous fungi 297 4.5 Trichoderma Trichoderma is characterized by rapid growth and successful colonization in diverse environments, from rich and complex soil to sterile biological fermenters (Schuster and Schmoll, 2010). It is a successful genus due to a high capacity for secreting lytic and proteolytic enzymes (Gajera and Vakharia, 2012). Enzymes from Trichoderma are used in wide-ranging applications such as brewing processes (β-glucanases), fruit juice production (pectinases, cellulases, hemicellulases), and feed additive (xylanases). Enzyme production in Trichoderma can be induced by substrates including lactose, sophorose, xylobiose, D-xylose, and L-sorbose ( Jun et al., 2011). Trichoderma reesei, which represents the primary industrially employed species of the Trichoderma genus, has also shown the ability to use lignocellulosic carbon sources for the production of cellulases, amylases, hemicellulases, lignin-degrading enzymes, peptidases, proteinases, and transport proteins (Cologna et al., 2018). Generally, different filamentous fungi species that are utilized in enzyme production processes are listed in Table 1. Table 1 Filamentous fungi species that are utilized in enzyme production. Filamentous fungi Substrate Enzyme Production References Starch Corn flour Starch Wheat bran α-Amylase Glucoamylase Amyloglucosidase Endoglucanase 22,100 U/g 346 U/mL 886 U/g 79,566.77 U/mL Sahnoun et al. (2015) Jain and Katyal (2018) Colla et al. (2017) Sukumaran et al. (2009) Sukumaran et al. (2009) Hemansi et al. (2018) da Silva Menezes et al. (2017) Wang et al. (2010) Aspergillus A. oryzae S2 A. niger A. niger NRRL 3122 A. niger MTCC 7956 A. niger MTCC 7956 A. niger RCKH-3 A. brasiliensis BLf1 Wheat bran β-Xylosidase 2.84 U/mL Wheat bran Rice husk β-Xylosidase β-Xylosidase 87.6 IU/g 28.1 U/g A. oryzae Rapeseed meal Protease 728 U/g F. verticillioides Gamba grass Endoglucanase 6.5 U/mL F. verticillioides Gamba grass Cellobiase 6.8 U/mL F. oxysporum F. oxysporum Rice chaff Corn stover Fibrinolytic enzyme Endoglucanase 154,500 U/L 304 U/g Fusarium de Almeida et al. (2019) de Almeida et al. (2019) Tao et al. (1998) Panagiotou et al. (2003) Continued 298 Current Developments in Biotechnology and Bioengineering Table 1 Filamentous fungi species that are utilized in enzyme production—cont’d Filamentous fungi Substrate Enzyme Production References F. oxysporum Corn stover Cellobiohydrolase 4.1 U/g F. oxysporum Corn stover β-Glucosidase 0.140 U/g F. oxysporum Corn stover Xylanase 1840 U/g F. oxysporum Corn stover β-Xylosidase 0.041 U/g Panagiotou et al. (2003) Panagiotou et al. (2003) Panagiotou et al. (2003) Panagiotou et al. (2003) Corn stover β-Xylosidase 15.1 U/mL Ye et al. (2017) Brewer’s spent grain Olive oil Xylanase Lipase 15.19 U/mL 1.62 U/mL Terrasan et al. (2010) Turati et al. (2019) Wheat straw Wheat straw Rice straw Rice straw Rice straw Rice straw Xylanase β-Xylosidase FPase CMCase Cellobiohydrolase β-Glucosidase 115.2 U/mL 89 mU/mL 1.4 U/mL 2.0 U/mL 0.6 U/mL 2.7 U/mL Liao et al. (2012) Liao et al. (2012) Liao et al. (2015) Liao et al. (2015) Liao et al. (2015) Liao et al. (2015) Penicillium P. oxalicum RGXyl1 P. janczewskii P. gracilenta CBMAI 1583 P. oxalicum GZ-2 P. oxalicum GZ-2 P. oxalicum GZ-2 P. oxalicum GZ-2 P. oxalicum GZ-2 P. oxalicum GZ-2 Rhizopus R. oryzae R. oryzae R. microsporus IBBL-2 R. oryzae 262 Bread waste Bread waste Wheat bran Amylase Protease L-Asparaginase 100 U/g 2400 U/g 17.18 U/mL Waste cooking oil Lipase 22 IU/mg R. microsporus Wheat bran Amylase 358 U/g R. chinensis Wheat flour with wheat bran Lipase 24,447 U/kg Benabda et al. (2019) Benabda et al. (2019) Ashok and Kumar (2021) Balasubramaniam et al. (2012) de Barros Ranke et al. (2020) Sun and Xu (2008) Horticultural waste powder Wheat straw β-Xylosidase 10.4 U/g Xin and Geng (2010) Cellulase Chahal (1985) Wheat straw Horticultural waste powder Wheat bran Xylanase CMCase (carboxy methyl cellulase) Endoglucanase 17.2 IU/mL; 430 IU/g 540 IU/mL 90.5 U/g Trichoderma T. reesei RUT C30 T. reesei QMY-1 T. reesei QMY-1 T. reesei RUT C30 T. reesei RUT C30 15.0 U/mL Chahal (1985) Xin and Geng (2010) Sukumaran et al. (2009) Chapter 10 • Industrial enzymes by filamentous fungi 299 5. Platforms for enzymes production by filamentous fungi Cultivation of filamentous fungi to produce enzymes and other bio-products can occur in solid-state and submerged fermentation (SmF) systems. 5.1 Solid-state fermentation for enzymes production by filamentous fungi In SSF, microorganisms are cultivated on the surface of the solid support in the absence or near absence of free water. These conditions are suitable for the growth of filamentous fungi, which typically grow in nature on solid substrates. The solid substrate is a main element in SSF. It provides nutrient sources such as carbon and nitrogen for microorganisms and plays the role of physical support for the growth of microorganisms (Webb and Manan, 2017). Agro-industrial residues are generally considered as the potential substrate to provide vital nutrients in SSF, which can decrease the cost of enzyme production significantly. Economical and effective enzyme complexes, containing cellulases, hemicellulases, phytase, etc. can be prepared by SSF using different agro-industrial residues (Pandey et al., 1999). Table 2 shows the list of agro-industrial residue that is utilized in filamentous fungi cultivation and enzyme production. The main applicable SSF platforms are the tray (Fig. 1A) and the packed-bed (Fig. 1B) bioreactors. The most important differences between these platforms are the solid substrate thickness. In the tray bioreactor, the substrate thickness is less than that in the packed bed bioreactor. While forced aeration is arbitrary in the tray bioreactor, it should be used in the packed bed bioreactor during fermentation. 5.2 Submerged fermentation for enzymes production by filamentous fungi SmF is the other fermentation system that is usually applied for enzyme production by filamentous fungi. SmF is carried out in liquid media consisting of water and specific nutrients for the cultivation of microorganisms. Fungal species such as Aspergillus (Amorim et al., 2019; Florencio et al., 2016; Regner et al., 2019), Trichoderma (Baskaran and Krishnan, 2020; Delabona Pda et al., 2013; Li et al., 2019), Fusarium (de Almeida et al., 2019; Kamble et al., 2019), and Penicillium (Dwivedi et al., 2009; Turati et al., 2019) are reported for enzyme production by SmF. Several SmF platforms for the industrial cultivation of filamentous fungi exist. The stirred-tank bioreactor (STR) (Fig. 2A) and the bubble column bioreactor (BCR) (Fig. 2B) are the most promising platforms of SmF. The primary difference between them is the presence of spinning impellers in the STR, while the BCR operates without moving parts. The result of this is a higher degree of mixing and oxygen mass transfer in the STR, which gives higher productivity, but at the cost of increased power consumption (Nørregaard et al., 2014). 300 Current Developments in Biotechnology and Bioengineering Table 2 Agro-industrial residues utilized in SSF to produce enzyme. Agro-industrial residues Filamentous fungi Enzyme References Sugarcane bagasse Brewery spent grain Wheat bran Aspergillus niger Cellulases and xylanases Moran-Aguilar et al. (2021) Trichoderma reesei NCIM 1186 and Penicillium citrinum NCIM 768 Aspergillus ficuum Aspergillus niger Aspergillus ficuum Aspergillus niger Aspergillus niger Cellulases Lodha et al. (2020) Phytase Phytase Phytase Phytase Invertase Shahryari et al. (2018) Vassilev et al. (2007) Tian and Yuan (2016) Buddhiwant et al. (2016) Ohara et al. (2015) Aspergillus niger PN1 Tannase Mansor et al. (2019) Aspergillus niger Cellulases and xylanases Moran-Aguilar et al. (2021) Aspergillus caespitosus CCDCA 11593 Trichoderma spp. and Aspergillus niger Aspergillus tubingensis TSIP9 Trichoderma reesei QM Aspergillus niger CECT2088 Aspergillus niger CCUG33991 L-Asparaginase Fernandes et al. (2021) Cellulase, xylanase, amylase and β-glucosidase Cellulase and xylanase Grujic et al. (2015) Intasit et al. (2021) β-Glucosidase β-Glucosidase Intasit et al. (2021) Leite et al. (2019) Xylanase Khanahmadi et al. (2018) Cellulase, hemicellulase and pectinase Lipase de Siqueira et al. (2010) Wheat straw Olive waste Potato waste Groundnut oil cake Wheat bran, soybean meal, cottonseed meal and orange peel Rice bran, brewer’s rice, spent coffee ground and desiccated coconut residue Sugarcane bagasse and brewery spent grain Pereskia aculeata miller Spent mushroom compost Palm empty fruit bunches Palm empty fruit bunches Brewer’s spent grain Wheat bran, sorghum stover, corn cob and soybean meal Cotton residue Palm kernel cake, soybean meal, and coir pith Aspergillus oryzae and Aspergillus terreus Aspergillus niger Prabaningtyas et al. (2018) 5.3 Comparison between solid-state and submerged fermentation in enzymes production by filamentous fungi SSF has certain advantages such as low production cost, less energy and space, easy downstream process, high concentration products, low contamination risk, and high productivity (Webb and Manan, 2017). Despite these advantages, due to limited water content and low thermal conductivity of solid substrates, SSF suffers from poor heat and oxygen mass transfer. Chapter 10 • Industrial enzymes by filamentous fungi 301 (A) Silica gel Air out Co2 /O2 Analyzer E-data logger Temperature data logger Computer Fermented substrate Air filter Thermocouples type K Circular mesh tray Air in FI Rotameter Air nozzle Humidifier tank Compressed air Water Bath (B) Co2 O2 Silica gel Co2/O2 Analyzer Air out Thermocouples type K E-data logger Computer Temperature data logger Fermented substrate FI Rotameter Air in Air filter Pump Water reservoir 2 Compressed air Bioreactor Water reservoir 1 FIG. 1 Solid-state fermentation platforms, the tray bioreactor (A) and the packed-bed bioreactor (B) (Manan and Webb, 2020). In recent years, some efforts have been made to design innovative SSF bioreactors. In the so-called Zymotis design, vertical heat exchanger plates are embedded in a packed bed bioreactor to minimize the problem of temperature rise in the bed (Hejazi et al., 2010). In another attempt, the trickling of water during fermentation along with a helical perforated bed is utilized to facilitate the heat and mass transfer in SSF (Shahryari et al., 2020). However, scale-up of laboratory-scale processes to industrial-scale has shown to be principally challenging. In contrast, in SmF, water is abundantly present, and variations in temperature, oxygen concentration, and nutrients are insignificant (de Souza et al., 2015). This allows 302 Current Developments in Biotechnology and Bioengineering (A) Co2 Seed fermenter O2 Carbone source Nutrient Motor Co2/O2 Analyzer Filter E-data logger Computer Temperature data logger Fermentation process Baffle Cooling water Separatore Separated enzyme Sparger Fermented broth including enzyme Air Compressed air Sterile filter (B) Co2 Co2/O2 Analyzer Filter E-data logger Computer Temperature data logger Fermentation process Seed fermenter O2 Carbone source Nutrient Cooling water Separatore Sparger Separated enzyme Fermented broth including enzyme Air Compressed air Sterile filter FIG. 2 Submerged platforms, the stirred-tank bioreactor (STR) (A) and the bubble column bioreactor (BCR) (B). Chapter 10 • Industrial enzymes by filamentous fungi 303 installation of online sensors on the fermenter, as well as the development of control strategies on the basis of such measurements (pH, temperature, and oxygenation of the culture medium) that would not be possible in SSF (da Silva Menezes et al., 2017). Due to the ease of automation and better mass and heat transfer, SmF can be scaled-up more easily than SSF. However, the major disadvantages associated with the SmF processes are the low productivity and dilute product, less product stability, high production cost, and complexity of the medium (de Souza et al., 2015). 6. Challenges for the development of industrial enzymes production by filamentous fungi Despite the filamentous fungi great potential in industrial enzyme production, a number of process engineering challenges such as high viscosity broth, toxin production, and lower enzyme stability as a consequence of protease secretion, are associated with the development of filamentous fungi industrial cultivation, especially in submerged platforms. Some of these challenges are discussed below. 6.1 Troubles in oxygen supply Production processes involving filamentous fungi are often highly aerobic. In the culture of aerobic microorganisms, oxygen serves as a substrate for energy generation, and it affects cell growth and metabolite production directly or through changes in morphology (Gibbs et al., 2000). The viscous characteristic of some filamentous fungi fermentation broths, during their submerged cultivation, can result in poor mixing and insufficient momentum and mass transfer. Low solubility and inadequate mass transfer introduce oxygen as a limiting component in such fermentation systems (Gibbs et al., 2000). Insufficient dissolved oxygen (DO) during fungal fermentation arise undesirable morphological changes, mycotoxin production, fungal autolysis, and enzyme production suppression (Sharma et al., 2009). Providing sufficient DO and avoid the detrimental effects of its deficiency, some techniques have been utilized to maintain the DO at a higher level, such as increase the aeration, headspace pressure, and agitation rate and replace air with pure oxygen (Gibbs et al., 2000). 6.2 Toxin production Filamentous fungi can produce a wide range of secondary metabolites which show antimicrobial activity. These types of fungal secondary metabolites are used as fungi defence against other organisms in nature and called mycotoxin. Mycotoxins are low-molecularweight compounds that are naturally produced after the stop of the fungi growth phase (Bhatnagar et al., 2002; Calvo et al., 2002). Mycotoxins represent serious toxicity and pose health threats in humans and animals depend on the type of toxin, the extent of exposure (based on duration period and dose), age, nutritional status, and health of the affected 304 Current Developments in Biotechnology and Bioengineering organism (Arnau et al., 2020). Generally, the major factors that affect mycotoxin production are temperature, water activity, relative humidity, pH, fungal strain, and substrate (Daou et al., 2021; Mannaa and Kim, 2017). Over 300 mycotoxins have been identified from different species of filamentous fungi. The common filamentous fungi producing mycotoxins are dominated by three genera, Aspergillus, Penicillium, and Fusarium (Greeff-Laubscher et al., 2020). These filamentous fungi can produce a variety of mycotoxins when fermentation is extended beyond the usual time needed for the production of primary metabolites. Table 3 (Agriopoulou et al., 2020) provides a list of ordinary mycotoxins produced by filamentous fungi. Table 3 Mycotoxin can be produced from filamentous fungi during the fermentation process (Agriopoulou et al., 2020). Mycotoxin Acronym Fungal species Aflatoxins B1, B2, G1, G2 AFB1 AFB2 AFG1 AFG2 OTA Aspergillus flavus Aspergillus parasiticus Ochratoxin A Fumonisins B1, B2, B3 Zearalenone FB1 FB2 FB3 ZEN Trichothecenes (type B: deoxynivalenol) DON Patulin PAT Trichothecenes (type A: HT-2) Trichothecenes (type A: T-2 toxin) Enniatins HT2 ENNs Ergot alkaloids EAs Alternariol AOH T-2 Aspergillus ochraceus Aspergillus carbonarius Penicillium verrucosum Penicillium nordicum Fusarium verticillioides Fusarium proliferatum Fusarium graminearum Fusarium culmorum Fusarium equiseti Fusarium cerealis Fusarium verticillioides Fusarium incarnatum Fusarium graminearum Fusarium culmorum Fusarium cerealis Penicillium expansum Bysochlamis nı́vea Aspergillus clavatus Penicillium patulum Penicillium crustosum Fusarium langsethiae Fusarium sporotrichioides Fusarium langsethiae Fusarium sporotrichioides Fusarium tricinctum Fusarium avenaceum Claviceps purpurea Claviceps fusiformis Claviceps africana Alternaria alternata Chapter 10 • Industrial enzymes by filamentous fungi 305 Several strategies have been evaluated to control mycotoxin production and/or decrease mycotoxin effects including microbial detoxification (Nešic et al., 2021), binder agent (Nazarizadeh and Pourreza, 2019), and bio-protection (Murugesan et al., 2015). However, there is no single technique that has proved effective against the wide array of mycotoxins. 6.3 Production and secretion of endogenous proteases and peptidases Filamentous fungi have the potential to produce several protein-degrading enzymes. For example, in A. oryzae, more than 130 peptidase genes have been identified (Arnau et al., 2020). The activity of endogenous proteases can be responsible whenever degradation of the enzyme of interest is observed during fermentation or during storage. However, an investigation of the various proteins secreted into the medium can be performed to identify the protease’s contribution to product degradation. Numerous research studies have reported protease involved in the regulation of enzyme activity during filamentous fungi fermentation (Arnau et al., 2020; Papagianni and Moo-Young, 2002; White et al., 2002). Protease production and activity varies depending on the strain, the medium, and the culture conditions. For example, it has been reported that protease activity in Aspergillus nidulans is generally repressed in the presence of low-molecular-weight forms of C, N, S, and P (White et al., 2002). Lower protease production has been seen under conditions of nutrient excess, while starvation as carbon and nitrogen-limited cultures can cause higher protease production (White et al., 2002). It can be concluded that besides the fungal source and its responsible genes, protease levels production and degradation effects vary depending on growth and storage conditions. 6.4 Lack of sufficient information Despite numerous researches on enzyme production by filamentous fungi, in some cases still, sufficient information is on demand. The absence of reliable fungal metabolite reference libraries, methodology variation, and the absence of a proper public source of these data are major deficiencies in the field of filamentous fungi enzyme production. In addition, the exact structure behind protein secretion from filamentous fungi is still poorly understood. Knowledge on improved protein secretion for one protein may be limited to the specific protein and strain and is not necessarily transferable to other proteins or fungal systems (Meyer et al., 2016). 7. Strategies to improve enzymes production by filamentous fungi Enzyme production in SmF of filamentous fungi, the prevalent industrial platform, is influenced by different parameters. Based on the effect of each parameter, bioprocess strategies can be considered to improve enzyme production as discussed below. 306 Current Developments in Biotechnology and Bioengineering Disperse morphology Pellet morphology Developing hyphae Spores FIG. 3 Filamentous fungi different morphologies (Posch et al., 2013). 7.1 Filamentous fungi morphology in enzymes production In filamentous fungi, new cells form at the tip of the existing hyphae and, these hyphae subdivide and grow into different structures or mycelia (Nørregaard et al., 2014). As shown in Fig. 3, two extreme types of morphology are generally known for filamentous fungi, pellets, and free filaments. Moreover, an intermediate aggregated morphology named clumps is observed between these extreme forms. Filaments growth forms, as well as the shape and size of pelleted growth forms, are influenced by many factors included specific strain properties, environmental factors such as medium composition, DO concentration, spore numbers in the inoculum, type of inoculum, pH, temperature, cultivation conditions, reactor type and scale, and agitation intensity (El-Enshasy et al., 2006). Generally, electrostatics, hydrophobicity, and interactions between spore wall components are the main triggers for pellet formation. The determinative parameter is different for different fungal species. For instance, in strains of Penicillium, electrostatics, and hydrophobicity play a dominant role, and pellet forms as a result of high pH. While the pellet formation of Aspergillus terreus depends on the type of carbon sources (Nair et al., 2016) which can be the result of interactions between spore wall components, notably salt bridging between polysaccharides (Veiter et al., 2018). Filamentous fungi morphology has a strong influence on enzyme production and excretion. During the production of fungal metabolite, the desired morphology varies from one product to another. For example, free mycelia are required for the production of glucose oxidase from Aspergillus niger (El-Enshasy et al., 2006) whereas, the higher level of fructofuranosidase and glucoamylase is produced by the pellet form (Driouch et al., 2012). Filamentous fungi morphology may cause a significant difference in final enzyme productivity by changing the number of active tips of the mycelia. On the other hand, Chapter 10 • Industrial enzymes by filamentous fungi 307 different morphological forms of filamentous fungi result in different types of broth rheology. Filamentous growth in culture usually causes a high apparent viscosity and nonNewtonian rheology, which leads to several undesirable results, such as inadequate heat and mass transfer followed by media heterogeneity and reduced productivity (Antecka et al., 2016). In contrast, pelleted growth usually demonstrates a low apparent viscosity and more or less Newtonian rheology. Subsequently, cultures with pelleted growth are often assumed to be well mixed, which can result in an appropriate heat and mass transfer (Grimm et al., 2005). However, the size of the pellet plays an important role in productivity yields. For large pellets, production occurs only within a thin layer at the pellet surface and, the inner pellet can suffer from oxygen starvation due to a mass transfer limitation and do not contribute to the production (Driouch et al., 2010). Generally, the reduction of pellet size during fermentation is a significant issue for more product formation (Germec et al., 2017). These morphological complications and their subsequent influences on fungal enzyme production play a significant role, especially in industrial implementation. 7.2 Agitation In filamentous fungi fermentation, agitation intensity has been identified as an important variable influencing enzyme production process productivity. Agitation intensity affects both fungal morphology and mass and heat transfer in the broth (Gibbs et al., 2000; Wang et al., 2005). In general, increasing agitation intensity reduces the pellet size and even changes the growth morphology from pelleted to filamentous growth forms (Cui et al., 1997). It can also result in shorter mycelial hyphae and larger numbers of tips (Ahamed and Vermette, 2010). Additionally, in a fungal fermentation broth, a high agitation rate plays a crucial role in the provision of sufficient mixing and mass transfer, especially when the fungal cells grow in a freely dispersed form and subsequently results in high apparent viscosity. On the other hand, mechanical forces can impose serious damages to fungi filaments and mycelia (Wang et al., 2005). Concerning enzyme production, to experience the benefits of agitation, its intensity should be designed in a range to meet process requirements in terms of the DO level and bulk mixing and avoid exerting high shear stresses on fungal mycelia. 7.3 Fungal mycelia immobilization Fungal mycelia immobilization, called immobilized fermentation, involves cells that are immobilized on a solid surface via physical methods, including physical adsorption and entrapment methods (Fig. 4). Immobilization of fungal mycelia is a suitable technique to overcome the free mycelia-related difficulties such as fungi shear damage by impellers, high viscosity and subsequently inadequate heat and mass transfer, and insufficient oxygen provision. This technique has shown substantial improvement in the production of the relevant bio-products (Zhu, 2007). Immobilized cell systems make it easy to separate cells from the liquid medium, which makes repeated batch culture possible and simplifies the operation of both the 308 Current Developments in Biotechnology and Bioengineering FIG. 4 Different immobilization strategies for filamentous fungi (Sen et al., 2021). continuous culture and subsequent downstream processes. Fungal mycelia immobilization can also eliminate the cellular lag phase, thus, accelerate the fermentation rate in repeated batch fermentation, which could substantially reduce the fermentation period. Pelleted fungal growth can also be viewed as a self-immobilization system (Prabaningtyas et al., 2018). Recently, the potential of immobilized fungal cells has been investigated for the production of various enzymes such as laccase (Couto and Toca-Herrera, 2007), xylarez et al., 2011), chitinase (Halder et al., 2014), nase (Garai and Kumar, 2013; Peralta-Pe amylase (Roble et al., 2020), etc. 7.4 Feeding strategies and cultivation mode Batch, fed-batch, and continuous fermentation systems can be utilized in SmF to produce enzymes from filamentous fungi (Fig. 5). For the closed state of the batch system, this type of fermentation is better for non-growth associate product formation (Ali et al., 2018). However, the batch processes have some disadvantages such as time loss to cleaning (CIP) and sterilization (SIP) for each fermentation, limitation of operating time, re-inoculation requirement for each fermentation, and substrate or product inhibition (Berenjian, 2019). Continuous fermentation systems benefit from long runs, steady-state conditions resulting in easier process control and consistency of product quality, which results in high productivity (Li et al., 2014; Nieto-Taype et al., 2020). Apart from batch and continuous systems, fed-batch fermentation is applied to overcome substrate inhibition or catabolite repression by intermittent feeding of the substrate. Besides, fed-batch fermentation ensures higher cell concentration and product formation in a bioreactor in comparison to batch fermentation (Zhu et al., 2019). Many enzymes are Chapter 10 • Industrial enzymes by filamentous fungi 309 Fermentation Fed-batch Batch pH controller Acid Air Continuous pH controller Pump Base Air Fresh medium pH controller Pump Acid Base Acid Air Base Fresh medium Medium out FIG. 5 Batch, fed-batch and continuous fermentation systems (Kushwaha et al., 2019). exposed to catabolite repression, where enzyme synthesis is prevented by the presence of a rapidly utilized carbon source (Zhang et al., 2020). In this case, fed-batch fermentation, a process strategy using the pulsed addition of a carbon source, is appropriate to keep the substrate away from the critical concentration for catabolite repression. Moreover, in the cultivation of filamentous microorganisms, a fed-batch strategy is used to control the fungal morphology, reduce fungal broth viscosity, thus, enhance enzyme productivity (Haack et al., 2006; Prasad Uday et al., 2017). In addition, during fed-batch cultivation of filamentous fungi, DO content, as a critical parameter in enzyme production, can be controlled by manipulating the substrate feed rate and, subsequently, the rate of oxygen consumption (Roque et al., 2021). In pectinase production by Aspergillus oryzae, fed-batch cultivation and addition of pectin 24 h after inoculation has been performed to reduce viscosity caused by pectin, facilitate heat and mass transfer mechanisms during the initial hours of the process, and control the DO concentration (Meneghel et al., 2014). Generally, supe ska et al., 2017), xylanase rior production of various enzymes such as amylase (Celin (Prasad Uday et al., 2017), cellulase (da Silva Delabona et al., 2021; Roque et al., 2021), and lignin peroxidase (Liu et al., 2021) from filamentous fungi has been reported as a result of fed-batch cultivation implementation. 7.5 Fermentation pH Fermentation medium pH is of prominent importance during filamentous fungi cultivation and enzyme production. Many genes in filamentous fungi are regulated by ambient pH (Peñalva and Arst, 2002). Generally, fungal spores exhibit negative surface charges 310 Current Developments in Biotechnology and Bioengineering (Douglas et al., 1959) that are affected by pH and ionic strength. Therefore, apart from the regulatory effect on gene expression, cultivation pH also affects fungal morphology (Veiter et al., 2018). Ambient pH also heavily influences the hydrophobicity of proteins and produced enzyme structure. Consequently, the development of an optimal pH control strategy is useful in obtaining higher enzyme productivity. Filamentous fungi have different optimal pH conditions for producing different enzymes. It has been reported that cellulase production by Aspergillus niger occurs optimum at pH 6, while optimum xylanase production pH is 5.5 (Li et al., 2018). Implementation of a pH control strategy could be a viable method to reduce extracellular protease activity, thereby increasing desired enzyme yields. Moreover, pH value has also been shown to affect the biosynthesis of other fungal metabolites such as mycotoxins. For example, aflatoxin production needs a pH value of 4.0, and the lower the pH, the higher its synthesis. Fumonisin B1 also needs a pH of 4.0 to 5.0 to be synthesized and is not stable in an alkaline medium (Daou et al., 2021). 7.6 Cultivation media Generally, carbon and nitrogen sources are two essential factors affecting cell growth and product formation in filamentous fungi. Besides their direct effect on filamentous fungi growth, these vital sources may either repress or induce enzyme expression genes. For example, glucoamylase, amylase, and a-glucosidase production are induced by starch; however, the presence of glucose represses the production of these enzymes (Murakoshi et al., 2012). Not only desired enzymes but also undesired extracellular proteases can be affected by cultivation media. Most extracellular proteases are repressed under the conditions of high glucose and ammonium levels in the medium (Sharma and Singh, 2016). Therefore, for the degradation effects of proteases on produced enzymes, the regulation of proteases by carbon and nitrogen sources would benefit enzyme production. 7.7 Engineering of filamentous fungi Mutagenesis is a widespread method for generating microbial strains with improved production characteristics (Meyer et al., 2016). Recent advances in molecular genetics, marker recycling, and genome editing could be used to alter transformation and metabolism based on optimized design implemented with computer knowledge (Wakai et al., 2017). Bioinformatic analyses of the many fungal genomes result in the identification of a wide range of dispensable genes that can be removed using Cas9 multiplexing gene deletion strategies. This strategy can be utilized as the clean background for the production of enzymes, reducing the risk of pathogenesis effects, co-production of undesirable mycotoxins or undesired cross-chemistry and peptidases that makes pathway analysis difficult and reduces yields (Meyer et al., 2016). For example, the deletion of proteases in T. reesei improves the production level of alkaline endoglucanase (Wakai et al., 2017). Chapter 10 • Industrial enzymes by filamentous fungi 311 Table 4 Examples of filamentous fungi engineering along with its effect on enzyme production improvement. Filamentous fungi Approaches Improvements Aspergillus niger A single amino acid mutation in XlnR (XlnRV756F) by a forward genetic screen A single point mutation in Xyr1 (Xyr1A824V) by UV mutagenesis The point mutation in AraR (AraRN806I) by UV mutagenesis A mutation of AraR (AraRA731V) expressed under control of the promoter gpdA The DNA-binding domain of ClrB was combined with the C-terminal sequence of XlnRA871V forming an artificially designed chimeric TF Overexpression of amyR gene (multicopy) Overexpression of gaaR gene under the control of the strong constitutive gpdA promoter Overexpression of manR using the constitutive tef1 promoter Deletion of cre-1 Constitutive expression of xylanases under repressing conditions Strong constitutive expression of xylanase and cellulase encoding genes Inducer-independent expression of the genes of fibrinolytic enzymes Constitutive production of α-Larabinofuranosidase 7.3-fold increase in cellulase production Trichoderma reesei Aspergillus niger Penicillium oxalicum Penicillium oxalicum Aspergillus niger Aspergillus niger Aspergillus oryzae Neurospora crassa 2- to 15-fold increase in amylolytic activities Strongly elevated production of pectinases Increased mannanolytic (1.5- to 5.5-fold) and cellulolytic (1.5-fold) activities Increased amylase and β-galactosidase secretion as well as cellulolytic activity and expression level of cellulolytic genes Replacing the original signal peptide with a more efficient one, a fusion of heterologous protein to a naturally secreted one, regulation of UPR and ERAD, optimization of the intracellular transport process, regulation of mycelium morphology, and optimization of the sterol regulatory element-binding protein (SREBP) are the other genetic engineering strategies can be used to improve enzyme production characteristics (Wang et al., 2020). Examples of filamentous fungi engineering along with its effect on enzyme production improvement have been shown in Table 4 (Meng et al., 2021). 8. Heterologous protein expression in filamentous fungi There are several kinds of hosts for protein expression, including both prokaryotic and eukaryotic protein expression systems. Different protein expression systems have been compared in Table 5 (Zhang et al., 2020). The ability of filamentous fungi in high-level protein secretion was one of the main features in considering them as potential hosts for producing high-value recombinant proteins. Besides the elevated protein production, protein secretion into the medium results in the simple recovery of a recombinant protein (Peberdy, 1994). Along with the ability to 312 Current Developments in Biotechnology and Bioengineering Table 5 Comparison of protein expression in different systems (Zhang et al., 2020). Organisms Growth and culture conditions Genetic transformation Posttranslational modification Expression efficiency Cost Prokaryote E. coli Bacillus subtilis Fast and high efficiency, simple media requirement Fast, high efficiency, and safe Well-defined, simple, and high efficiency No posttranslational High without efficient secretion Low cost Convenient for gene modification Almost none High yield with secretory expression and produces no lipopolysaccharide Low cost Fast and high efficiency, easy scale-up High cell density, easy scale-up Fast and high efficiency Well-established manipulation Yes, but hyperglycosylation Low cost Well-established manipulation Complex manipulation and lower transformation efficiency Complex manipulation, long period, and lower transformation efficiency Excellent tool for recombinant glycoprotein production Yes, but hypermannosylation Typical eukaryotic posttranslational modifications Moderate and mannosylation of secreted proteins Moderate of secreted proteins High and efficiency secretion Tailor-made glycans High expressing Cost and potential contamination with microorganisms Glycosylation of protein terminal with mannose glycans High expressing but cannot be expressed continuously High cost Complicated technology Proper protein folding, posttranslational modifications Moderate High cost and potential contamination with animal viruses Eukaryote S. cerevisiae Komagataella pastoris Filamentous fungi Plant cell Safe and efficacious Insects Safe for vertebrates, more demanding culture conditions Slow growth and expensive nutrient requirement, limited largescale industrial production Mammal cells Low cost Low cost Chapter 10 • Industrial enzymes by filamentous fungi 313 secrete high-level proteins, filamentous fungi can perform post-translational modification such as glycosylation, peptide chain shearing, and disulfidation (Wang et al., 2020). This group of microorganisms has a powerful secretory pathway, which gives them the ability to produce eukaryotic proteins correctly (Zhang et al., 2020). Thus filamentous fungi have great potential to be considered as hosts for recombinant enzyme production. Aspergillus species, Trichoderma species, and Penicillium species are the common filamentous fungi applied as a protein expression host (Singhania et al., 2017). Even though filamentous fungi are the proper host for protein expression, their genetic manipulation is a complex and time-consuming process, thus they are not suggested for the expression of proteins that are easily produced by other hosts with a notable yield (Zhang et al., 2020). 9. Economic aspects and filamentous fungal enzymes market The yield coefficient is the most important economic parameter for any fermentation, mostly related to the carbon substrate as the main cost in the fermentation systems. Filamentous fungi fermentation systems represent a high yield coefficient that gives some economic space for energy and downstream processing costs (Nørregaard et al., 2014). During fermentation, the rheological characteristic of the broth causes increasing energy input to provide better oxygen transfer and, the energy cost is high. However, costs of the downstream process are relatively low associated with the ability of filamentous fungi to secrete products. In addition, the filamentous fungi can perform the enzyme posttranslational modification before its secretion. This process also reduces the cost of the associated downstream process (Ntana et al., 2020). Considering the potential and economic aspect of enzyme production by filamentous fungi, several companies are interested in filamentous fungi to manufacture industrial enzymes. Companies such as AB Enzymes, BASF, Bayer, DSM, DuPont, and Novozymes are leaders in using filamentous fungi as cell factories in enzyme production. Filamentous fungi enzymes have a growing market. For instance, the production of plant biomassdegrading enzymes by filamentous fungi alone was a €4.7 billion market in 2016, which is expected to double by 2026 (Meyer et al., 2016). 10. A roadmap toward future research on filamentous fungal enzymes production Enzyme secretion in filamentous fungi is very complicated and highly controlled, and in spite of several types of research, current knowledge in this field remains limited. Different topics can be a roadmap toward future research: • • Understanding fungal pathogenesis in different strains and strategies to control this side-effect. Methods for filamentous fungi morphology adaptation to different substrates and environmental conditions. 314 • • Current Developments in Biotechnology and Bioengineering Bioprocessing strategies can be utilized in solid-state fermentation to overcome its current challenges. Definition of optimal gene structure for enzyme expression. 11. Conclusions and perspectives Filamentous fungi represent an enormously important platform for the industrial production of enzymes. More than their inherent capacity for the secretion of enzymes, they have underlying features such as appropriate stress tolerance, high yield coefficients, and the simplified downstream process. Moreover, filamentous fungi ability to grow on inexpensive media and perform post-translational modifications to produce complex proteins makes them a promising source of enzyme production. Despite their undeniable potential, filamentous fungi industrial cultivation encounters several challenges such as mycotoxin production, proteases and peptidases secretion, troubles in mixing, and oxygen supply as a result of viscose medium, which can confine the utilization of this potent enzyme source. The research on bioprocessing technologies may have an enhancing role for large-scale filamentous fungi fermentation process and enzyme production. 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Laurent Dufosse CHEMISTRY AND BIOTECHNOLOGY OF NATURAL PRODUCTS (CHEMB IOPRO), UNIVERSITY O F REUNION I SLAND, E SIROI FOO D SCIENCE, SAINT-DENIS CEDEX 9 , R EUNI ON I S L AND, F RANCE 1. Introduction Penetration of the fermentation-derived ingredients into the industries is increasing year after year. Examples could be taken from the following fields: thickening or gelling agents (xanthan, curdlan, gellan), flavor enhancers (yeast hydrolysate, monosodium glutamate), flavor compounds (gamma-decalactone, diacetyl, methyl-ketones), acidulants (lactic acid, citric acid), etc. Efforts have been made in order to reduce the production costs of pigments produced by microbial fermentation compared to those of synthetic pigments or pigments extracted from natural sources such as plants. The successful marketing of natural pigments derived from algae (non-conventional sources) or extracted from flowering plants (conventional sources), both as food colorants and nutritional bioactive supplements, reflects the presence, and importance of niche markets in which consumers are willing to pay a premium for “natural healthy ingredients.” Among non-conventional sources, filamentous fungi are known to produce an extraordinary range of pigments (Kalra et al., 2020) that include several chemical classes such as carotenoids (Gmoser et al., 2018), melanins, flavins, phenazines, quinones, and more specifically monascins, violacein or indigo. The success of any class of pigment produced by fermentation depends upon its acceptability by the consumers, regulatory approval, and the capital investment required to bring the product to the market. Thirty years ago, personnel from food industries expressed doubts about the successful commercialization of fermentation-derived food grade pigments (Fig. 1) because of the high capital investment requirements for fermentation facilities, and the extensive and lengthy toxicity studies required by the regulatory agencies. Public perception of fungal-derived products for food use also had to be taken into account. Nowadays some fermentative food grade pigments from filamentous fungi are existing in the market: Monascus pigments, Arpink red™ from Penicillium oxalicum, riboflavin from Ashbya gossypii, lycopene and β-carotene from Blakeslea trispora. The production Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00011-9 Copyright © 2023 Elsevier Inc. All rights reserved. 325 326 Current Developments in Biotechnology and Bioengineering FIG. 1 Using the filamentous fungi as microbial cell factories for the production of bioactive pigments (description in three steps, from left to right: (i) filamentous fungi are used as pigment producers—biodiversity, versatility, (ii) fermentors allow the easy scale-up of the production, (iii) two examples of pigments produced). yield in the case of β-carotene as an example, could be as high as 17 g/L of the Blakeslea trispora culture medium (Park et al., 2000). Thus, the present chapter emphasizes the crucial role those filamentous fungi are currently playing and are likely to continue to play in the future. These microbial cell factories are and will continue to be effective for the production of food grade pigments because of the versatility in their pigment color and chemical profile, amenability for easy large-scale cultivation, and a long-term history of well-studied production strains. In this review, we draw the attention of both the academia and the food industry to some stimulating research findings in the area of fungal pigments. We illustrate this by polyketide-Monascus-like pigments from the new non-mycotoxigenic, and thus potentially safe fungal production strains, and the promising and yet unexplored hydroxy-anthraquinoid pigments with an amenability to be cultivated for the large-scale industrial production as natural food colorants of microbial origin. Additionally, fungi from marine ecological niches are discussed as potential producers of novel color hues and structures, and the ways to explore the metabolic potential of these pigment producing marine fungi. Chapter 11 • Production of bioactive pigmented compounds 327 2. New non-mycotoxigenic fungal work horses for the production of polyketide pigments as food colorants The red and the yellow polyketide pigments—from the well-known fungus Monascus sp. have been commercially produced and legally used as food colorants in the form of pigment extracts and/or as traditionally used dried fermented red rice powder in South East Asia for more than thousand years (Feng et al., 2012). However, Monascus pigments are not approved as food colorants both in the European Union and the United States, mainly due to the risk of the possible contamination by the nephro-, and hepato-, toxic metabolite citrinin. There have been controversial views over the safety of these pigments though co-production of a toxic metabolite together with the main target compound by a fungal host is not alien and one of the examples is the case of a mycelial fungal food product Quorn™ by Fusarium venenatum whereby a potent cytotoxic metabolite 4, 15-diacetoxyscirpenol could be produced [fortunately the industrial conditions used for mycoprotein production (with excess nutrients present) do not induce mycotoxin production. Nevertheless, samples of Quorn™ mycoprotein are taken regularly from the production line to test for the presence of trichothecenes as well as other mycotoxins] (Whittaker et al., 2020). Another example is the production of fumonisins by one of the well-known industrial workhorse Aspergillus niger. The point is that the fungal producer is designated Generally Recognized as Safe (GRAS) for the processes involved for the food use and not the organism as such. It implies that the safe production of Monascus pigments can be designed keeping the hazard analysis and critical control points in mind. Nonetheless, the presence of the mycotoxin citrinin issue has triggered investigations to find possibilities for minimizing citrinin accumulation by (i) manipulating culturing conditions (e.g. effect of dissolved oxygen) (Pereira et al., 2008), (ii) developing strains incapable of synthesizing citrinin by metabolic engineering ( Jia et al., 2010), and (iii) simply screening for genera other than Monascus that produce polyketide pigments (Mapari et al., 2005). Some strains among Talaromyces species (formerly Penicillium sp.) viz. Talaromyces aculeatus, T. funiculosus, T. pinophilus, and T. purpurogenus have been discovered to produce Monascus-like polyketide azaphilone (MPA) pigments without co-producing citrinin or any other known mycotoxins using chemotaxonomic rationale (Mapari et al., 2008a). Epicoccum nigrum is also shown to be non-mycotoxigenic fungal producer (Mapari et al., 2009a) of polyketide pigment orevactaene with antioxidant property (Mapari et al., 2008b). The red and the yellow pigment extracts from T. aculeatus and E. nigrum, respectively, have been shown to exhibit enhanced photostability over the commercially available red Monascus colorant and turmeric in liquid food model systems (Mapari et al., 2009b). Recently, Talaromyces purpurogenus (formerly Penicillium purpurogenum) and related species such as Talaromyces amestolkiae, Talaromyces ruber, Talaromyces stollii, have also been reported to produce MPA pigments (Yilmaz et al., 2012). Strains of these species are likely to be non-mycotoxigenic and non-pathogenic to humans, however, their 328 Current Developments in Biotechnology and Bioengineering individual mycotoxin profiles and pigment producing abilities have yet to be explored. Currently, the pigment production potential and pigment producing cellular mechanisms of strains of T. purpurogenus are being investigated (Mendez et al., 2011; Arai et al., 2013; Santos-Ebinuma et al., 2013). More recently, a patent has been granted for a submerged cultivation method for some of the non-mycotoxigenic strains of Talaromyces sp. whereby, the concentration of pigments was significantly enhanced and the number of pigment constituents was significantly reduced with MPA pigment PP-V [(10Z)-12carboxyl-monascorubramine] being the major compound (Mapari et al., 2012). Thus, recent research activities indicate that the above-mentioned new fungal cell factories have advantages in (i) potentially eliminating the risk of contamination by mycotoxins especially citrinin, (ii) reducing the pigment constituents and thereby easier characterization and quality control, (iii) providing the colorants with improved stability. In addition, the new fungal production strains have a potential to be engineered to produce a new generation of natural functional food ingredients with bioactive properties. 3. Focus on azaphilones 3.1 Toward mycotoxin-free Monascus red Monascus has been used to produce natural colorants and food supplements for more than 1000 years in Asia, and approximately more than 1 billion Asian people consume Monascus-fermented products as part of their daily diet. The first known source reporting the use of these red colorants was a recipe for the preparation of red pot-roast lamb, in which meat was simmered with hong qu (red rice koji, made with Monascus purpureus), as handed down in the Qing Yilu in AD 965. Monascus species are known to produce six major azaphilone pigments, namely the yellow monascin and ankaflavin, the orange monascorubrin and rubropunctatin, and the red monascorubramine and rubropunctamine. To date, more than 50 different chemical structures have been identified (Yang et al., 2015), as azaphilones easily combine with nucleophilic nitrogen containing compounds. Using next-generation sequencing and optical mapping approaches, a 24.1-Mb complete genome of a Monascus purpureus YY-1 industrial strain has been described for the first time and this will allow major improvements in the process in the coming years (Yang et al., 2015). It consists of eight chromosomes and 7491 genes. M. purpureus belongs to the Aspergillaceae, mainly comprising the genera Monascus, Penicillium, and Aspergillus. Phylogenetic analysis at the genome level provides the first comprehensive prediction of the biosynthetic pathway for Monascus pigments. Comparative genomic analyses demonstrated that the genome of M. purpureus is 13.6–40% smaller than that of closely related filamentous fungi and has undergone significant gene losses, most of which likely occurred during its specialized adaptation to starch-based foods. Some polyketide synthases (PKSs) are expressed at high levels under high pigment yielding conditions. The citrinin PKS C6.123 has also been found in the genome (Yang et al., 2015), paving the way for research aiming at non-mycotoxin producing strains, if Chapter 11 • Production of bioactive pigmented compounds 329 suppression of the citrinin gene does not change the ability of the strain to produce pigments, which seems to be feasible, as described by Fu et al. (2007). The latter group has shown that monascorubrin and citrinin are synthesized by two separate pathways. When the PKS gene responsible for synthesis of citrinin was disrupted, red pigment production from the fungus was not affected. Comparative transcriptome analysis revealed that carbon starvation stress, resulting from the use of relatively low-quality carbon sources, contributed to the high yield of pigments by suppressing central carbon metabolism and augmenting the acetyl-CoA pool. As for other pigments produced by biotechnology, the problem is to have enough carbon oriented in the correct pathway, i.e., the pigment pathway (Wang et al., 2021). Woo et al. (2014) investigated another filamentous fungus, Penicillium marneffei, for production of azaphilones exhibiting black, yellow and red hues. The polyketide gene cluster and biosynthetic pathway were reported for monascorubrin in this red pigment-producing, thermal dimorphic fungus, taking advantage of available genome sequence and faster growth rate compared to Monascus species (Woo et al., 2014). The red pigment of P. marneffei has been shown to consist of a mixture of more than 16 chemical compounds, which are amino acid conjugates of monascorubrin and rubropunctatin. This occurs as amino acids can be conjugated under specific conditions without enzymatic catalysis, i.e., by Schiff base formation (Fig. 2; Woo et al., 2014). The aforementioned polyketide gene cluster and pathway have been shown to be also responsible for the biosynthesis of ankaflavin and citrinin, the latter being a mycotoxin exerting nephrotoxic activity in mammals (Kumar et al., 2014). Twenty-three putative PKS genes and two putative PKS-non-ribosomal peptide synthase hybrid genes were identified in the P. marneffei genome (Woo et al., 2014). Woo et al. (2014) systematically knocked out all 25 PKS genes of P. marneffei. They also knocked out genes located up and downstream of the PKS gene responsible for red pigment production and characterized the pathway for biosynthesis of the red pigment. However, it is still questionable whether it will be possible to produce mevinolin/lovastatin-free (a cholesterol-lowering drug that is undesired in normal foods) and citrinin-free red pigments from P. marneffei, as the latter, a mycotoxin, appears to be an early byproduct of the biosynthetic pathway. 3.2 Monascus-like pigments from non-toxigenic fungal strains Some species of Talaromyces secrete large amounts of red pigments. In literature, this biosynthetic potential has been linked to species such as Talaromyces purpurogenus, T. marneffei, and T. minioluteus often known under their previous Penicillium names. However, since some of them do not exert sufficient stability for pigment production, then such species should be avoided for scale-up production. Isolates identified as T. purpurogenus have been reported to be of industrial interest as they can produce extracellular enzymes and red pigments. Some of these isolates may also produce unwanted mycotoxins such as rubratoxin A and B and luteoskyrin in addition to FIG. 2 Hypothetical pathway of monascorubrin, ankaflavin, and citrinin biosynthesis in P. marneffei. Adapted from Woo, P.C., Lam, C.W., Tam, E.W., Lee, K.C., Yung, K.K., Leung, C.K., Sze, K.H., Lau, S.K., Yuen K.Y., 2014. The biosynthetic pathway for a thousand-year-old natural food colorant and citrinin in Penicillium marneffei. Sci. Rep. 4, 6728. https://doi.org/10.1038/srep06728. Chapter 11 • Production of bioactive pigmented compounds 331 RED RED C21H18O7 C18H20O7 COLOURLESS ORANGE C23H26O5 C25H32NO5 C22H33NO5 COLOURLESS COLOURLESS C32H43NO4 FIG. 3 Structures of some of the most characteristic compounds produced by Talaromyces atroroseus. Adapted from Frisvad, J.C., Yilmaz, N., Thrane, U., Rasmussen, K.B., Houbraken, J., Samson, R.A., 2013. Talaromyces atroroseus, a new species efficiently producing industrially relevant red pigments. PLoS One 8(12), e84102. other undesirable extrolites that may be toxic following intraperitoneal (spiculisporic acid) and intravenal (rugulovasine A and B) injections in cats (Frisvad et al., 2004). Consequently, mycotoxin production may limit the use of isolates of a particular species in biotechnology, and Frisvad et al. (2013) concluded that Talaromyces purpurogenus may thus not be recommended for industrial production of red pigments. Talaromyces atroroseus sp. nov., described by the same group (Isbrandt et al., 2020), produces the azaphilone biosynthetic families mitorubrins and Monascus pigments without being accompanied by mycotoxin synthesis (patent applications WO2012022765, US 20110250656) (Fig. 3).
union island lagoon, Indian Talaromyces albobiverticillius strain 30,548 isolated from Re Ocean, is also under investigation by our group from the Reunion island university based on this tropical volcano, located between Madagascar and Mauritius. As it has been found for Monascus, these azaphilone pigments may react with amino groups containing compounds, to which reaction they owe their name, providing intense dark red colors (Mapari et al., 2012; Gao et al., 2013). 4. Fungal hydroxyanthraquinoid (HAQN) pigments as potential food colorants HAQN pigments are widespread in nature (plants, insects, lichens) and have also been found abundantly in microorganisms, particularly in filamentous fungi belonging to the genera Penicillium sp. and Aspergillus sp., with different color hues. For example, the pigment emodin is isolated from the strains of Penicillium citrinum and P. islandicum. 332 Current Developments in Biotechnology and Bioengineering As a first commercial product within this chemical family, the natural food colorant Arpink red™ (now Natural Red™) is manufactured by the Czech company (Ascolor Biotech followed by Natural Red) and has been claimed to be produced by fermentation and bioprocess engineering using the fungal strain Penicillium oxalicum var. Armeniaca CCM 8242, a soil isolate. Some strains of Aspergillus sp. (A. glaucus, A. cristatus, and A. repens) were found to produce known yellow and red HAQN compounds such as emodin (yellow), physcion (yellow), questin (yellow to orange-brown), erythroglaucin (red), catenarin (red), and rubrocristin (red) (Caro et al., 2012). However, using strains of Aspergillus and Penicillium sp., several known mycotoxins are co-produced in the medium, e.g., secalonic acid D, oxaline, citrinin, tanzawaic acid A, cyclochlorotine, islanditoxin, luteoskyrin, erythroskyrin, rugulosin, or aspergiolide A. Many of these mycotoxins are pigmented and are naphtoquinones by chemical nature. All these fungal secondary metabolites (the yellow and the red HAQN pigments that show substitution on both aromatic rings as well as the naphtoquinone-type mycotoxins) biosynthetically arise by the same polyketide pathway. This infers that these fungal strains are not safe, and therefore, cannot be used for the production strains of hydroxyanthraquinoid pigments as potential natural food grade colorants. Traditional mutagenesis and/or metabolic engineering methods to eliminate the production of mycotoxin(s) should be investigated as an alternative strategy. Species of Eurotium sp. (E. amstelodami, E. chevalieri, and E. herbariorum) have been found to produce the yellow pigment physcion and the red pigment erythroglaucin; however, they have been reported to produce, in addition, the mycotoxin echinulin and two benzaldehyde coloring compounds: flavoglaucin (yellow) and auroglaucin (red) (Gessler et al., 2013). Along similar lines, co-production of the red hydroxyanthraquinoid pigments and mycotoxins such as fusaric acid, nectriafurone, monoliformin, and gibepyrones, has been shown in strains of Fusarium oxysporum isolated from roots of diseased citrus trees. Apart from those mycotoxigenic fungi, other filamentous fungi have the ability to produce known HAQN pigments which arise biosynthetically through the polyketide pathway, without co-production of mycotoxins. As an example, a strain of Dermocybe sanguinea (¼ Cortinarius sanguineus) has been identified as a producer of the red HAQN glycoside dermocybin-1-β-D-glycopyranoside, giving the typical red color of the fruiting body and the spores, together with the pigments emodin and physcion (Bechtold, 2009). As of now, hydroxyanthraquinoid pigments used in food are from plants (European madder root color, i.e., Rubia tinctoria, sold in Japan up to 2004) or insects (carminic acid extracted from cochineal insects; Dactylopius coccus), however there is an increasing interest both from the academia and the industry about the readily available microbial sources. 5. Focus on anthraquinones 5.1 Fungal natural red™ As the first fungal commercial product claimed as a member of the anthraquinone pigment class, the natural red food colorant Arpink red™ (now Natural Red™) has been Chapter 11 • Production of bioactive pigmented compounds 333 manufactured by two Czech companies. Initially by Ascolor Biotech s.r.o., then followed by Natural Red™, who both produced the pigment by fermentation and bioprocess engineering using the fungal strain Penicillium oxalicum var. Armeniaca CCM 8242, a soil isolate. Numerous patents have been filed by Ascolor, e.g., WO 9,950,434; CZ 285,721; EP 1,070,136; US 6,340,586 cited in Sardaryan et al. (2004). The cultivation of the fungus in liquid broth requires carbohydrates (such as sucrose and molasses), nitrogen (corn extract, yeast autolysate, or extract), zinc sulfate, and magnesium sulfate. The optimum conditions for performing the microbiological synthesis are pH value in the range of 5.6 to 6.2, and temperature between 27°C and 29°C. On the second day of incubation, the red colorant is released into the broth, increasing up to 1.5–2.0 g/L of broth after 3 to 4 days [WO 9,950,434; CZ 285,721; EP 1,070,136; US 6,340,586 cited in Sardaryan et al. (2004)]. After biosynthesis of the red colorant is completed, the liquid phase is filtered or centrifuged, and separated from the biomass. The liquid is then acidified to pH 3.0–2.5 to precipitate the colorant. The precipitate is dissolved in ethyl alcohol, and filtered. Following removal of alcohol from the filtrate, the microcrystalline form of the colorant is obtained, i.e., a dark red powder. The colorant produces a raspberry-red color in aqueous solution, stable at pH > 3.5 (4–60°C). Solutions at pH 7.0 are even stable after 30 min of boiling. Toxicological data are widely available about this red pigment. This includes patents containing information about acute oral toxicity in mice 90-day sub-chronical toxicological study, acute dermal irritation, acute eye irritation, anti-tumor activity, micronucleus test in mice, AMES test (Salmonella typhimurium reverse mutation assay), estimation of antibiotic activity, and test results of estimation of five mycotoxins [WO 9,950,434; CZ 285,721; EP 1,070,136; US 6,340,586 cited in Sardaryan et al. (2004)]. After evaluating all the documents provided by the company, the Codex Alimentarius Commission made the following statement on the occasion of its Rotterdam meeting on March 11–15, 2002: “… there will not be any objections to use the red coloring matter Arpink Red” in – – – – – meat products and meat product analogs in the amount up to 100 mg/kg non-alcoholic drinks in the amount up to 100 mg/kg alcoholic drinks in the amount up to 200 mg/kg milk products and ice creams in the amount up to 150 mg/kg confectionery in the amount up to 300 mg/kg Subsequently, this biotechnologically produced anthraquinone was sold and used in Czech Republic for several years. The joint FAO/WHO Expert Committee on Food Additives (JECFA) evaluation process made some progress, and the legal situation concerning Arpink Red™ was discussed during the 63rd Annual JECFA meeting in Geneva, June 8–17, 2004. Additional data were requested, however, the company Ascolor appeared to stop its activities, and a new company, named Natural Red™, was established in 2012. 334 Current Developments in Biotechnology and Bioengineering Pros and cons are quite difficult to judge in this case, as private companies using a fungal strain that is not publicly available, have conducted the whole development. No academic paper has been published, and much information, in particular, confirmation of genus/species of the fungal strain, chemical structure of the anthraquinone pigment(s), and absence of mycotoxins (e.g., secalonic acid D) is lacking. 5.2 Other fungal anthraquinones Anthraquinones are widely spread throughout the kingdom of fungi (For example, in Aspergillus sp., Eurotium sp., Fusarium sp., Dreschlera sp., Penicillium sp., Emericella purpurea, Curvularia lunata, Mycosphaerella rubella, Microsporum sp., etc.), and thus might serve as alternative sources independent of agro-climatic conditions (Caro et al., 2012; Gessler et al., 2013). This is in contrast to plant- and animal-derived sources. Anthraquinones exhibit a broad range of biological activities, including bacteriostatic, fungicidal, antiviral, herbicidal, and insecticidal effects (Gessler et al., 2013). Presumably, in fungi, these compounds are involved in interspecific interactions. For example, anthraquinones synthesized by endophytic fungi protect the host plant from insects or other microorganisms (Gessler et al., 2013). The present picture of fungal anthraquinones is quite complex, with a great variety of chemical structures (Fig. 4), a huge number of factors or parameters which may have impact on the composition of quinoidal pigments biosynthesized by a particular species. Among them, e.g., habitat, light, pH, temperature, O2 transfer, liquid/solid media, culture medium, C and N sources, C:N ratio, presence of organic acids, mineral salts, and inoculum have been considered (Caro et al., 2012). Today, research priority is focused on a small number of fungal anthraquinoneproducing species meeting the following profile of requirements established by Mapari et al. (2009a) during the identification of potentially safe fungal cell factories for the production of polyketide natural food colorants using chemotaxonomic rationale: – – – fungus shall be non-pathogenic to humans fungus shall be non-toxigenic under a broad range of production conditions fungus shall be able to produce in liquid media 6. Fungi from marine ecological niches as novel sources of chemically diverse pigments Recent literature abundantly reports the interest for marine organisms with respect to the production of new molecules and, among them, new pigments. Indeed, many marine ecological niches are still unexplored and it seems plausible that unique features of marine environments such as high salinity, low temperature, lack of light, and high pressure can be the inducers of unique substances synthesized by marine microorganisms. The potential of marine microorganisms to produce unique and original molecules could therefore Chapter 11 • Production of bioactive pigmented compounds Altersolanol A, Alternaria sp. 335 no common name, Fusarium oxysporum NaO3 S Cynodontin, Dreshslera avenae 3-O-Methyl alaternin sulfate, Ampelomyces sp. FIG. 4 Some anthraquinones from fungal origin (color of the box reflects color of the main pigment produced by the fungus). come from specific metabolic or genetic adaptation processes to meet very specific combinations of physico-chemical parameters. For now, the highest diversity of marine fungi seems to be found in tropical regions, mainly in tropical mangroves which are extensively studied because of their high richness in organic matter favorable to the development of these heterotrophic microorganisms. Anyway, it seems obvious that in extreme 336 Current Developments in Biotechnology and Bioengineering environments the fungal species with pigmented cell walls in the spores and mycelium, can tolerate dehydration-hydration cycles and high solar radiation better than the moniliaceous fungi, whose cells are devoid of pigments. For example, melanin, a black phenolic pigment which has a significant antioxidant activity and sporopollenin (brown product of oxidative polymerization of β-carotene) are very common in many fungi (dematiaceous hyphomycetes) and may protect the biological structures, giving them an excellent durability and a high potential for survival in hostile environments. Another example is the deep green pigment cycloleucomelone (terphenylquinone) from an A. niger strain isolated from the mediterranean sponge Axinella damicornis, as well as from its terrestrial counterparts (Hiort et al., 2004). Marine fungi are also able to produce bright colors, from yellow to red, mainly belonging to polyketides. Indeed, several review papers illustrate that polyketides seem to dominate marine natural products of fungal origin (Ebel, 2010). As examples, yellow pigments physcion and macrosporin, have been reported to be extracted from the endophytic Alternaria sp., isolated from the fruit of the marine mangrove tree Aegiceras corniculatum in Zhanjiang, Guangdong (South China Sea) (Huang et al., 2011). The orange questin, the yellow asperflavin, and the brown 2-O-methyleurotinone have been reported to be produced by Eurotium rubrum from the inner tissue of the stem of the marine mangrove plant Hibiscus tiliaceus near Hainan Island (China) (Li et al., 2009). Also, the yellow oils citromycetin and 2,3-dihydrocitromycetin have been isolated from a marine derived Penicillium bilaii (Capon et al., 2007). The examples of other molecules that can also color fungal structures or be excreted as secondary metabolites include tetrahydroauroglaucin (yellow) and isodihydroauroglaucin (orange) which have been extracted from Eurotium sp., an isolate from leaves of the mangrove plant Porteresia coarctata (Dnyaneshwar et al., 2002). The yellow compounds flavoglaucin including the mycotoxin citrinin have been shown to be produced by the marine-derived fungus Microsporum sp. in Korea (Li et al., 2006). A fungus of the genus Periconia isolated from hypersaline environment (solar saline in Puerto Rico) subjected to high solar radiations has been shown to produce a still unidentified, and unusual blue pigment (Cantrell et al., 2006). To date, most of the studies on the marine fungi have highlighted that these fungal genera and species are facultative and not obligate microorganisms. With regards to the industrial production of dyes, this may be considered as an advantage because strictly marine fungal species (able to grow only in the marine environment) are often difficult to culture at a large scale (Ebel, 2010). The feature to culture at a large scale is highly required for the industrial production of biochemicals including the pigments. Ubiquitous strains including the members of the genera Aspergillus and Penicillium are frequently encountered in marine habitats and usually produce enough biomass for chemical studies or industrial exploitations. These two genera have been intensively investigated for decades in the quest for interesting secondary metabolites both from the terrestrial and the marine origin. Among these secondary metabolites, some are considered as new, although in many cases, they are biogenetically closely related to natural products described previously from their terrestrial counterparts. For example, the so Chapter 11 • Production of bioactive pigmented compounds 337 called novel yellow 2,3-dihydrocitromycetin from the marine-derived isolate of Penicillium bilaii, collected from the Australian Huon estuary (Port Huon, Tasmania) has also been identified from a soil isolate of Penicillium striatisporum (Capon et al., 2007). Up-to-now a few unique colored compounds have not yet been found their counterparts produced from the terrestrial isolates. The examples are the brown bisdihydroanthracenone derivative, eurorubrin or the new orange anthraquinone glycoside [3-O-(α-D-ribofuranosyl)-questin] from Eurotium rubrum, isolated from the inner tissue of the stem of the mangrove plant H. tiliaceus around Hainan Island (China). Seemingly, no terrestrial counterpart has been discovered yet for the new yellow compound dimethoxy-1methyl-2-(3-oxobutyl) anthrakunthone produced by the mangrove’s endophytic Fusarium sp. ZZF60 isolated from the South China Sea (Huang et al., 2010), or for the red alterporriols: K, L & M from the endophytic Alternaria sp. found in the fruit of the mangrove’s shrub Aegiceras corniculatum (Zhanjiang Guangdong, South China Sea) (Huang et al., 2011). Penicillium commune G2M isolated from the mangrove plant H. tiliaceus (Hainan Island, China) has also been reported to synthesize a pale-yellow oil characterized as 1-O-(2,4-dihydroxy-6 methylbenzoyl)-glycerol (Yan et al., 2010), and Penicillium sp. JP-1 from the inner bark of an Aegiceras corniculatum tree collected in Fujian (China) has been claimed to produce a red pigment named penicillenone (Lin et al., 2008). Finally, the yellow anthracene-glycoside asperflavin-ribofuranoside from the marine-derived fungus Microsporum sp. (Korea) (Li et al., 2006) appears only to be produced by marine fungi. Many marine fungi have been reported to be endophytes and to make associations with higher life forms (plants, algae, corals). Examples have shown that under these conditions the fungi may proceed to biochemically mimic the host organism (Strobel et al., 1996). This is not surprising since the fungi have to deal with the marine environment and the biological context of the host. Algae can then be considered as valuable sources for the isolation of pigment producing marine fungi to the extent that many algae are pigmented. Therefore, the algicolous fungi may produce unusual and novel dyeing molecules. In addition, co-cultivation of marine fungi with other microorganisms from the same ecosystem has been proved to be successful in activating silent gene clusters to produce bioactive secondary metabolites (Brakhage and Schroeckh, 2011). Even if the microorganism can be easily genetically manipulated and simply scaled-up for metabolite production, the modification of cultivation parameters such as media composition can also possibly induce and regulate secondary metabolite biosynthesis (Calvo et al., 2002). Inactivation or enhancement of selected steps of a biosynthetic pathway by a chemical approach can then be an alternative tool to metabolic engineering, using mutations or genetic transformation techniques. One of the main advantages of using inhibitor and precursor feeding is that the genetic and epigenetic background of the cell remains unchanged. However, the overexpression of a transcriptional factor controlling a metabolic pathway can affect the expression of certain genes or modify some cellular processes. The use of such techniques requires a thorough knowledge of the biosynthetic pathways and the enzymes involved. 338 Current Developments in Biotechnology and Bioengineering FIG. 5 Products from filamentous fungi and their various applications. Thus, filamentous fungi seem very versatile and could be used for various applications (Fig. 5). 7. Conclusions and perspectives The current use and the potential of using filamentous fungi as pigment and natural colorant sources for food applications is promising considering the ever-rising demand by the consumers to replace their synthetic counterparts. Filamentous fungi are readily available raw materials that can be tailored to make microbial cell factories for the production of food grade pigments because of their chemical and color versatility in their pigment profile, easier large-scale-controlled cultivation, growth on agro-industrial residues and Chapter 11 • Production of bioactive pigmented compounds 339 a long-term history of well-known production strains for the production of a variety of other biochemicals including colorants. Emphasis has been put on the screening for specific pigments such as natural blue, or red colorant for cochineal extract/carminic acid/ carmine partial replacement by the food colorant industries. In this regard the hydroxyanthraquinoid pigments and/or novel chemical classes from marine pigment producing fungi could be an interesting avenue to be explored further. As in the case of other food additives and/or ingredients, all sources of natural pigments and colorants (plants, minerals, insects, microalgae, microorganisms) will coexist in the market, with market shares depending on consumer’s expectations, industrial prices, and availability. Acknowledgments
would like to thank the Conseil Re
gional de La Re
union, Re
union island, France, and the Laurent Dufosse
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Taherzadehc a DEPARTMENT OF FOOD AND AGRICULTURAL PRODUCT T ECHNOLOGY, UNI VERSITAS GADJ AH M A D A , Y OG Y A K A R T A, I NDO N E S I A b DEPARTME NT OF FOOD SCIENCE AND TECHNOLOGY, IPB U N I VE R S I T Y , B OGOR, INDONESIA c SW EDISH C ENTR E FOR R ESOUR CE RECOVE RY, UNIVER SIT Y OF BORÅS, BOR ÅS , SW EDEN 1. Introduction The United Nations (UN) figures the global population to reach 9.7 billions by 2050, which consequently increases the food demand, particularly protein-rich diets (United Nations, 2019). Animal protein is an important protein source, hence the world demand for animal protein is predicted to be doubled in 2050 (FAO, 2019), which results in raising concerns for sustainability and food security. It is generally accepted that animal-based foods give higher negative impact on the environment since they are more greenhouse gases (GHGs) intense than plant-based foods (Tilman and Clark, 2014). The rise of animal-based food demand puts more pressure on land and water usage as well as losses of biodiversity and other essential ecosystem services (Van Zanten et al., 2016). In terms of health, overconsumption of processed meat is linked to a high intake of saturated fatty acids, which harms our health. Besides environmental and health concerns, ethical issues related to animal production drives a dietary shift toward reduction of meat consumption. Therefore, an alternative source of protein that provides high protein quality with a low environmental impact is desired. Filamentous fungi have been consumed for centuries in several countries in the form of fermented foods. For instance, tempe, a fermented soybean by filamentous fungi is the staple source of protein in Indonesia. Pure biomass of filamentous fungi was introduced to the market 3.5 decades ago and obtained a positive response as a promising meat replacer, and now there are several companies with their products on the market. Filamentous fungi have similar quality of protein to several animal-based foods with a significantly lower requirement of land and water usage; thus, it can be considered as a more environmentally friendly protein. In addition, filamentous fungi can grow in a wide range of substrates, thus enabling them to convert organic wastes into a rich and diverse set of valuable products. This is in accordance with the concept of a sustainable bio-circular economy which has gained interest in very recent years. This chapter presents the application of filamentous fungi as food, the protein quality, the environmental impact, the safety issue, the production of filamentous fungi-based food both in traditional and in modern Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00007-7 Copyright © 2023 Elsevier Inc. All rights reserved. 343 344 Current Developments in Biotechnology and Bioengineering FIG. 1 Various filamentous fungi-based food products (the fungal-based sausage was provided by Swedish Company Millow). industry, as well as the prospects and the challenges in developing filamentous fungibased food. The applications of filamentous fungi for food are summarized in Fig. 1. Filamentous fungi can be consumed as sole biomass in the form of mycoprotein, in which Quorn is one successful example. They can be consumed with their growing substrates in the form of fermented food such as tempe, soy sauce, miso, oncom, etc. Furthermore, filamentous fungi are also used for making food ingredients including coloring agents, flavor enhancers, and enzymes. 2. Filamentous fungi as protein source 2.1 Protein quality of various protein sources Proteins are among the essential nutrients in the human diet. Proteins are available in a variety of foods, which are mainly classified into animal- and plant-based proteins. Several important animal protein sources include cattle, poultry, goat, pig, seafood, egg, and milk. Meanwhile, plant-based protein sources are dominated by soybean, bean, Chapter 12 • Filamentous fungi for food 345 nuts, and pulses (Hertzler et al., 2020; Vogelsang-O’Dwyer et al., 2021). Despite the aforementioned protein sources, there are several emerging protein source alternatives such as insects, cultured meat, and fungal protein (Hadi and Brightwell, 2021). There are several factors to be considered when selecting protein sources, including nutritional quality, price, safety, as well as health and environmental impacts. The nutritional quality of protein is evaluated according to protein content and quality. The protein quality is related to protein digestibility and bioavailability (Schoenlechner et al., 2008). The digestibility of protein is a crucial factor as it describes how the protein is best utilized in the human body, which can be presented in the value of net protein utilization (NPU) and protein digestibility-corrected amino acid score (PDCAAS). NPU measures protein quality by calculating the nitrogen used for tissue formation divided by the nitrogen ingested from food (Hoffman and Falvo, 2004); thus, it provides information on how efficiently the body utilizes protein consumed in the diet. Besides the protein content, the essential amino acid (EAA) content in a protein source is also necessary to be considered. EAAs are amino acids that cannot be produced by the human body and must be ingested from our diet. The PDCAAS compares the EAA content of a test protein with that of a reference EAA pattern and a correction for differences in protein digestibility (Schaafsma, 2005). The bioavailability of the protein is affected by unfavorable compounds which inhibit nutrient absorption, which is called antinutritional factors (Ram et al., 2020). The safety of protein is related to the presence of toxic and allergenic compounds. An example of an antinutritional factor is phytic acid, whereas the toxic compound com^mara and Madruga, 2001). The nutritional quality monly found in plants is cyanic acid (Ca and the safety of various protein sources are presented in Table 1. Protein digestibility shown by NPU and PDCAAS is relatively higher in animal protein than in plant-based protein (Table 1). However, animal proteins are usually high in saturated fat and cholesterol, which have a negative impact on human health. Animal proteins may pose food safety risks, including heavy metals in wild fish (Hayes et al., 2016; MoraEscobedo et al., 2018) and environmental contaminants frequently found in meat as the contaminants are highly soluble in fat (González et al., 2020). Besides, it has been reported that high animal protein intake could increase the risk of cardiovascular disease, cancer (De Souza et al., 2015; Song et al., 2016), kidney stones (Tracy et al., 2014), and mortality (Virtanen et al., 2019). In addition, consuming <70% of animal protein in the diet leads to a higher healthy eating index score (Sokolowski et al., 2020). Consumption of red meat, excluding poultry, has been reported to correlate with the occurrence of diabetes in China (Du et al., 2020). In contrast, replacing red meat with white meat can lead to a lower risk of type-2 diabetes, according to a Danish study (Ibsen et al., 2019). Furthermore, animal proteins are relatively more expensive, and there is a growing concern for animal welfare (de Gavelle et al., 2017). Plant proteins show a comparable amount of protein content with those of animal proteins (Table 1). However, they contain phytic acid and cyanic acid as antinutritional and toxic compounds, respectively. The PDCAAS value of plant protein is also relatively lower than that of animal proteins. Nevertheless, there is a challenge in developing plant-based Table 1 Protein content, protein quality, fat, and antinutrient compounds from various protein sources. Protein type Protein source Protein total Net protein (% wb) utilization (%) Cattle Beef 22.7–23.7 (USDA, 2021) 11–12.3 (USDA, 2021) 17.4–22.5 (USDA, 2021) 16.9–21.1 (USDA, 2021) 3.33–3.75 (USDA, 2021) 22–24.4 (USDA, 2021) 13.6–20.35 (USDA, 2021) 38.55 (USDA, 2021) Egg Chicken Pig Dairy Milk Aquatic animals Tuna Shrimp Plant Insect Fungi Soy Protein digestibility corrected amino acid score (%) 73 (Lagrange, 2004) 0.92 (van Vliet et al., 2015) 12.5 (F) (USDA, 2021) 94 (Lagrange, 2004) 1 (van Vliet et al., 2015) 11.36 (F) (USDA, 2021) 8.93 (F) (USDA, 2021) 83 (Lin and Huang, 1986) n.a 0.92 (Boye et al., 2012; Negrão et al., 2005) 1 (Burd et al., 2019) 82 (Lagrange, 2004) 1 (van Vliet et al., 2015) 70.1 (Castrillón et al., 1 (Gilani, 2012; Mitchell et al., 1996) 1989) n.a 1 (Dayal et al., 2013) 61 (Lagrange, 2004) 0.9 (van Vliet et al., 2015) Wheat 13.7 (USDA, 2021) Pea 2.8–8.8 44.7–60.7 (Savage (USDA, 2021) and Deo, 1989) Peanuts 40.5 (Lin and Huang, 0.52 (Hoffman and Falvo, 25–25.2 2004; US Dairy Export Council, (USDA, 2021) 1986) 1999) Seaweed 0.54–5.92 52.7–67 (Garcı́a0.43 (Cian et al., 2014) (USDA, 2021) Garibay et al., 2014) Cricket 44.41 (Tinarwo et al., 2021) Oyster 2.9–3.31 mushroom (USDA, 2021) Quorn Tempe Fat (F) Phytic acid (PA) Cyanic acid (CA) (%) 61.9 (Homolková et al., 2019) 0.45 (van Vliet et al., 2015) 0.67 (van Vliet et al., 2015) 75.2 (Oibiokpa et al., 0.65 (Stone et al., 2019) 2018) 83.9–86.9 (Valencia 0.45 (Dabbour and Takruri, del Toro et al., 2004) 2002) 11–15 (Sadler, 75 (Trinci, 1991) 2003) 20.8 50–60 (Gandjar, (KEMENKES, 1999) 2017) 0.99 (Quorn, n.d.) 0.8–0.9 (Bintanah et al., 2019; Hermana et al., 1999) 21.19 (F) (USDA, 2021) 3.33 (F) (USDA, 2021) 0.49 (F) (USDA, 2021) 1.18 (F) (USDA, 2021) 2.3 (PA) (Karkle and Beleia, 2010) 0.03 (CA) (Adejoh et al., 2020) 0.77 (PA) (Oatway et al., 2001) 0.0007 (CA) (Iwe et al., 2017) 0.112 (PA) (Warkentin et al., 2012) 0.811 (PA) (Yagoub and Abdalla, 2007) 0.0048 (CA) (Ingale and Shrivastava, 2011) 0.025 (PA) (SánchezMoya et al., 2019) 0.0003 (CA) (Marasabessy and Sudirjo, 2017) 10.40 (F) (Udomsil et al., 2019) 0.41 (F) (USDA, 2021) 0.0009 (PA) (Majesty et al., 2019) 3.2 (F) (Sharp, 1993) 8.8 (F) (KEMENKES, 2017) 0.255 (PA) Chapter 12 • Filamentous fungi for food 347 meat alternatives due to technological hurdles to mimic the texture and flavor of the meat. Soybean contains eight allergenic proteins; two of these, namely β-conglycinin and glycinin, represent 70% of soybean proteins (Helm et al., 2000; Nadathur et al., 2016). Furthermore, the taste and flavor of plant-based food are not favorable for part of consumers, which might be the reason for low intake of legumes in several Organization € sler et al., 2012; for Economic Co-operation and Development (OECD) countries (Scho Tobler et al., 2011). Cultured meat has been proposed to meet the requirement of high-quality protein with low impact on the environment. However, several challenges to produce this meat included high-cost production and the availability of scaling-up technology (Post and Hocquette, 2017). In addition, a consumer study on artificial meat reported that only 5%–11% of the respondents were willing to eat the meat while 49%–64% would not accept the artificial meat in the future (Hocquette et al., 2015). The low consumer acceptance was related to perceiving culture meat as not natural, healthy, and safe (Post and Hocquette, 2017). More recently, insects have emerged as an alternative protein source. There are more than 2000 different species of insects eaten worldwide. Beetles (31%) and caterpillars (17%) are the dominant species, followed by wasps, bees, and ants (15%), crickets, grasshoppers, and locusts (14%), and true bugs (11%) (Jongema, 2015). As described in Table 1, cricket has higher protein content than animal proteins with comparable value of NPU. From an environmental perspective, it offers several advantages as it produces less GHG and uses less land and water than conventional livestock (Oonincx et al., 2010). However, there are several obstacles in promoting insects as a new alternative protein source which can be classified as nutrient, safety, sensory, and cultural issues. In terms of nutrients, the PDCAAS is lower than that of animal proteins (Table 1). In addition, many insects lack tryptophan and lysin and have poor digestibility due to the chitin exoskeletons (Henchion et al., 2017). Several insects also pose allergenic proteins as part of their defensive system. Various insects contain allergens and the incidence of allergic reactions due to the consumption of silkworm pupae, cicadas, and crickets have been reported in China (Feng et al., 2018). Similarly, caterpillars, sago worms, locusts, grasshopper, and bees have also been associated with insect allergies (de Gier and Verhoeckx, 2018). Unfortunately, the allergens are very stable, which remain present even after thermal processing and digestion (de Gier and Verhoeckx, 2018). From the sensory point of view, the appearance and the strong odor of the insect often evoke rejection. Moreover, eating insects is considered culturally inappropriate and disgusting as they usually inhabit dirty places, making neophobia to insects more severe. Fungal protein could be one alternative protein source. Fungi with a fruiting body (macrofungi) known as mushrooms have been consumed for centuries. Besides mushrooms, a filamentous fungus (microfungus) has been commercialized since 1985 under the brand of Quorn, and several new companies have entered this market in the last few years using filamentous fungi as protein source in their food products. Table 1 shows that the protein content of Quorn is similar to egg and the NPU is similar to beef, the PDCAAS is higher than beef and chicken, and the fat is four times lower than beef. 348 Current Developments in Biotechnology and Bioengineering In addition, fungal protein is high in fiber and has texture similar to meat (Sadler, 1994). Furthermore, fungal protein is high in lysine, threonine, and zinc, nutrients that are present in low amounts in cereal and vegetables (Sadler, 1994). However, the protein content of mycoprotein is relatively lower than animal protein. 2.2 Proteins and environmental concerns Nowadays, as there has been growing concern on environmental aspects, people’s awareness of the impact of food production on the environment has increased. Hence, current food preference is not only based on its nutrient, but also on the carbon footprint as well as water and land use. Agricultural sector is one of the highest contributors to GHG production, in which livestock industry becomes the key player contributing 12%–18% to global GHG production (Allen and Hof, 2019). Global production of major animal and plant protein sources in 2014–2018 is presented in Fig. 2. There are several reasons why animal protein is not favorable from environmental point of view: (a) less efficiency of energy since it requires two times conversion including growing a feed then raising cattle instead of growing food crops that can be directly consumed by human, (b) requires higher land and water use than plant-based protein, (c) intensified land use for feed production, in turn, will increase conversion of forests, wetlands, and natural grasslands into agriculture lands which endanger the biodiversity and ecosystem, and (d) GHG emission from enteric fermentation and manure (Mogensen et al., 2020; Van Zanten et al., 2016). For instance, the production of 1 kg of beef protein requires 18 times more land, 10 times more water, 9 times more fuel, 12 times more fertilizer, and 10 times more pesticides compared to the produc et al., 2015). tion of the same amount of kidney beans (Sabate The GHG emissions of various animal protein sources are presented in Table 2, showing that beef produces the highest GHG emissions. Red meat shows significantly FIG. 2 Production quantity of selected crops and livestock products in 2014–2018 (FAOSTAT, 2021). Chapter 12 • Filamentous fungi for food Table 2 349 Greenhouse gases from various protein sources. No. Protein source GHG (kg CO2 eq/kg) Major contribution References 1 Beef 23.4–27.2 Enteric fermentation (70%–75%) 2 Lamb 6.1 Enteric fermentation (70%–75%) 3 4 5 6 Rabbit Pork Poultry Milk 3.86 4.6 5.52 2.05 7 8 9 10 Egg Insect Fish Soy 1.6 2.57 2.12 0.19–0.32 kg 11 12 Quorn Tempe 1.23–1.63 0.323 n.a. Feed production n.a. Feed production, enteric methane, and manure management Poultry feed (63%) Feed production Feed production Diesel combustion (machinery) and the production of fertilizer n.a. LPG gas (0.12315 kg CO2-eq/kg) Wiedemann et al. (2015) Wiedemann et al. (2015) Cesari et al. (2018) Six et al. (2017) Cesari et al. (2017) Thoma et al. (2013) Taylor et al. (2014) Halloran et al. (2017) FAO (2017) Castanheira and Freire (2013) Kazer et al. (2021) Dunuwila et al. (2019) higher GHG emissions than white meat. The major contributors to GHG emissions are feed production and enteric fermentation. Therefore, reducing meat consumption would give a significant contribution to mitigating climate change. Insects lead to lower GHG emissions, particularly nitrous oxide and methane, than conventional livestock (Oonincx et al., 2010). Similarly, substitution of animal base protein with oat protein both partially and fully would decline GHG emissions by 8% and 13%, respectively, as well as land use by 14% and 26% (Mogensen et al., 2020). Although plant protein is desirable in terms of land use and GHG emission, industrialization of farming has a negative impact on water use, soil degradation, pollution, deforestation, and habitat loss. Meanwhile, filamentous fungi offer more advantages from an environmental point of view due to the following reasons: (a) the conversion rate of protein production is five times higher than animal (Sadler, 1994); (b) the land usage is low since fungi are usually grown in a bioreactor with high metabolic rates (Schweiggert-Weisz et al., 2020); (c) fungi could grow in a wide range of substrates from glucose, simple sugars, protein, starch, lipid, lignin, cellulose, as well as in organic waste stream and agricultural residues, hence they play an important role in biorefinery; and (d) the GHG emission of fungal production is 10–20 times lower than beef (Kazer et al., 2021; Wiedemann et al., 2015). 2.3 Smart proteins As aforementioned, fungi are desirable protein sources with regards to nutrient and environmental points of view. Fungi could convert various substrates into protein-rich biomass and metabolites. Since they have a bigger particle size, the harvesting, separation, 350 Current Developments in Biotechnology and Bioengineering and recovery of filamentous fungi are easier than using other microorganisms. Mycoprotein poses an outstanding nutrient, with almost a half and a quarter of the dry weight are protein and fiber, respectively (Finnigan, 2011). In addition, several health benefits of fungi consumption have been reported in correlation with antidiabetes and antihypertension potential. The antidiabetes activity might be related to poor digestibility of fiber content in the cell wall, which slows down the absorption process in the intestine and raises the nutrient in the plasma (Warrilow et al., 2019). The PDCAAS of fungi is very close to the maximum score (0.996) and higher than beef and chicken (Edward and Cumming, 2010). Several toxicity testing of mycoprotein on gastrointestinal, oestrogenic, and exposure effects on the eyes and skin have been conducted. The findings of all studies showed that there was no sign of toxicity. In addition, an allergenicity study of mycoprotein which involved 400 subjects reported that only 1%–4% of the population exhibited adverse reactions (Bender and Matthews, 1981). Besides its nutritional value and safety, several factors need to be considered prior to consumer acceptance, including palatability, convenience, availability, and price. Several strategies are applied to address these issues. To produce palatable products, the fungi are selected based on the taste. The texture of some microfungi is identical to that of meat fibrils in terms of filament diameter and presence of longitudinal alignment; hence they provide similar eating quality to meat (Finnigan, 2011). 3. Filamentous fungi in food applications 3.1 Fungal filaments vs mushrooms Edible fungi are an important part of culinary and have been consumed for 1000 years in ancient civilizations such as Greek and Chinese (Valverde et al., 2015). In the past, edible fungi were not only valued as nutritious and tasty food but also as traditional medicine. There are two types of edible fungi, including macrofungi and microfungi. Macrofungi is a group of fungi with large and visible fruiting body and produce spores. Macrofungi belong to family of Ascomycota and Basidiomycota, with mushroom as the most well-known example of macrofungi. Several genera of macrofungi are Agaricus, Lentinula, Pleurotus, Auricularia, Volvariella, Amanita, Boletus, Cantharellus, Cordyceps, Cortinarius, Laccaria, Lactarius, Leccinum, Lentinus, Lycoperdon, Macrolepiota, Morchella, Polyporus, Ramaria, Russula, Suillus, Terfezia, Termitomyces, Tricholoma, and Tuber. In contrast, microfungi can only be observed with the aid of microscope and are commonly known as molds. The edible microfungi mostly form hypha, which is long-like filaments; thus they are called filamentous fungi. Several genera belong to this category include Rhizopus, Aspergillus, Mucor, Monascus, Neurospora, Penicillium, Fusarium, and Trichoderma. According to FAO, there are 1154 species from 85 countries that are edible and are eaten by people around the world (Halling, 2006). The world production of mushrooms and truffles in 2019 was 11.9 million tons corresponding to an increase of 36% compared to the production in 2009. The highest mushroom producers were Asia contributing 78% to the global production, followed by Europe, America, and Australia (FAOSTAT, 2020). The highly consumed mushrooms include champignon/button (Agaricus bisporus), shitake Chapter 12 • Filamentous fungi for food 351 (Lentinula edodes), oyster (Pleurotus ostreatus), paddy straw (Volvariella volvaceae), jelly ear (Auricularia auricula-judae), and enoki (Flammulina velutipes). Among them, button, shitake, and oyster mushroom dominate the market and account for three-quarters of mushroom cultivated globally, whereas Pleurotus spp., represent three-quarters of mushrooms cultivated globally. The high consumption of mushrooms is related to their excellent properties such as palatability, low lipid, high fiber, and high protein contents, sufficient amino acid, and vitamin content (Boonsong et al., 2016). In addition, they contain a number of nutraceutical compounds including terpenes, polysaccharides, bioactive proteins, and antioxidants which promote their health effect against degenerative disease, tumors, cancers, cardiovascular disease, diabetes, and osteoporosis (Papoutsis et al., 2020). Furthermore, mushrooms exhibit antioxidant, antibacterial, antifungal, antiinflammatory, immune-modulatory, antitumor, and antiviral properties. Besides the nutritional and health effects, the highly accessible product is also the driving force for mushroom consumption (Martinez-Medina et al., 2021). The safety aspect of mushroom consumption is still under discussion. The number of toxic mushrooms is considered low. For instance, of the 14,000 species of mushrooms that are edible, only 100 species are considered as toxic (Kamal et al., 2009; Tran and Juergens, 2020). Several toxic compounds were detected in mushrooms including amatoxin, psilocybin, muscarine, coprine, allenic norleucine, and gyromitrin (Tran and Juergens, 2020). Ingestion of toxic mushrooms leads to several clinical symptoms depending on the type of mushroom. For instance, acute gastroenteritis is caused by backyard mushrooms, hallucinations are caused by Psilocybe, liver toxicity is caused by Galerina, Lepiota, and Amanita, and seizures are caused by Gyromitra, Paxina, and Cyathipodia micropus species (Tran and Juergens, 2020). Mushroom poisoning occurs mainly due to eating raw mushrooms, misidentification of the mushroom, food shortages, and different physiological response to edible fungus (Boa, 2007). Filamentous fungi could be consumed either as a pure fungal biomass (mycoprotein) or as fermented food products. The hyphal structure of mycoprotein could imitate protein fibril in meat and thus promotes a similar texture of meat tissue. This property is not owned by plant-based meat analogous, hence it becomes an excellent candidate as meat replacer. Several examples of fermented products using filamentous fungi are tempe, soy sauce, oncom, and miso. In fermented food, the filamentous fungi are consumed together with substrate residue. For instance, in tempe, the fungi are consumed together with the bean as the substrate. Tempe is a fermented soybean by Rhizopus oligosporus which is currently gaining worldwide interest outside of its original country, Indonesia. Tempe is among the top three protein sources with consumption rate of 0.146 g/per capita in Indonesia (Badan Pusat Statistik, 2021). Tempe supplies 10% of protein consumption which is significantly higher than chicken egg (1.25%) and meat (3.15%) (Forum Tempe Indonesia, 2017). 3.2 Fermented food using filamentous fungi Fermentation using filamentous fungi has been employed for a long time and known to extend shelf life of food such as legumes, milk, and cereals. The most commonly used strains belong to genus Aspergillus (A. oryzae, A. sojae, A. flavus), Rhizopus (R. oryzae, 352 Current Developments in Biotechnology and Bioengineering Ranunculus stolonifer, R. oligosporus), or Penicillium (P. camemberti, P. requeforti), and Mucor (Mucor mucedo). There are over 20 types of fermented foods using filamentous fungi in the world (Table 3). To date, the application of filamentous fungi in food fermentation is intentionally conducted to impart desirable biochemical changes. These fermented foods also remain an integral part of people’s diet in fulfilling their daily nutritional requirements, especially in some developing countries in Africa and Asia. Tempe in Indonesia and miso in Japan are examples of food fermented by filamentous fungi that hold important role in the diet, especially regarding nutritional value (El Sheikha and Montet, 2016). Fermentation using filamentous fungi offers advantages both in developing and developed countries. In developing countries with higher prevalence of protein deficiencies, fermented foods provide high protein and vitamin nourishment. For instance, vitamin B12, B6, and B2 significantly increased up to 400 folds, hence fermentation can be considered as biological enrichment for vitamins (Keuth and Bisping, 1993). In addition, from the environmental point of view, fermented foods which are commonly produced from agricultural waste or food by-products, are beneficial for sustainable production. For example, oncom is a nutritious and tasty delicacy made from low-cost peanut press cake, cassava waste, and okara which is consumed by millions of people in Indonesia. In this section, the three most consumed fermented foods will be discussed, namely tempe, soy sauce, and oncom. Tempe is one of the most popular dishes made from fermented soybean, soy sauce is widely used as condiments, whereas oncom is a representative example of fermented food made from food by-products. 3.2.1 Tempe Tempe (or tempeh) is a traditional fermented soybean food product originally from Indonesia. Although tempe is mainly produced from soybean, this fermented food can also be made from other raw materials, such as legumes, cereals, and food by-products. The legumes include black gram (Phaseolus mungo), broad bean, bakla, horse bean, field bean, fava beans (Vicia faba), chickpea, garbanzo bean (Cicer arietinum), common bean, red kidney bean (Phaseolus vulgaris), cow pea (Vigna unguiculata), horse (wild) tamarind (Leucaena leucocephala), jack bean (Canavalia ensiformis), lablab bean, Hyacinth bean (Lablab purpureus), lima bean (Phaseolus lunatus), mungbean or green gram (Vigna radiata), Vigna pigeon pea or red gram (Cajanus cajan), sesban bean (Sesbania grandipora), sweet lupine, lupine bean (Lupinus albus), velvet bean (Mucuna pruriens), winged bean (Psophocarpus tetragonolobus), yellow pea (Pisum sativum), grass pea (Lathyrus sativus), Bambara groundnut (Voandzeaia subterranea), ground bean (Macrotyloma geocarpa Harms), velvet bean (Mucuna pruriens var. utilis). Meanwhile, cereals that can be made for tempe are barley (Hordeum vulgare), wheat (Triticum vulgare), oat (Avena sativa), red sorghum (Sorghum bicolor). Food by-products include soy cake from soymilk, coconut residue after coconut-oil pressing (Cocos nucifera), ground peanut (Arachis hypogaea) press cake, cassava (Manihot esculenta Crantz) fibers, and soybean hulls (Yoneya, 2003). Tempe is made by dehulling, soaking, boiling, cooling of soybean followed by inoculation with mold which is then allowed to ferment for 48–72 h at room temperature. Table 3 Fermented food from filamentous fungi. Fermented food (other No. names) Description (incl. ingredients) 1 Tempe Whitish fermented soybean (or Rhizopus oligosporus, other ingredients as listed R. oryzae, Ranunculus above) cake bound together stolonifer, and R. Arrhizus by the fungal mycelia 2 Hamanatto (toushih, tao-si, tao-tjo) Fermented soybean with dark Pediococcus halophilus, color applied as condiment Lactobacillus delbrueckii, Torulopsis versatilus Microorganisms Processing step References Dried soybeans are cleaned and partially cooked then dehulled. The soybeans are soaked overnight in water to allow natural fermentation so lactic acid bacteria could lower the pH (around 3.5–5.2). The soybean is then washed and cooked. After adequately cooled to room temperature, the cooked soybean is inoculated with the starter. The mixture is then packed to allow the second fermentation to occur. This step would take time for 1–3 days Soybeans are cleaned, soaked for about 3 h and cooked. After cooked soybeans are cooled to 30°C, they are carefully mixed with roasted barley flour and Aspergillus oryzae (in tane koji) and left to ferment in room temperature for 3–4 days. Then the mixture is sun-dried for a week. After drying, 15% saltwater is added to the mixture. Ginger, shiso leaves, and pepper are used as an additional seasoning. The process is continued by second fermentation for 3–6 months or longer Ashu et al. (2015), Hesseltine and Wang (1980), and Yoneya (2003) Hesseltine and Wang (1980) and Yoneya (2003) Continued Table 3 Fermented food from filamentous fungi—cont’d Fermented food (other No. names) Description (incl. ingredients) Microorganisms Processing step References A. oryzae, A. soyae (Koji) Zygosaccharomyces rouxii, Candida versatilis, C. etchellsii (Moromi) The koji is produced by inoculating cooked and mashed soybean or wheat with A. sojae or A. oryzae conidia. The process continued with 3 days of incubation. The koji is then mixed with soybean or wheat mash in the fermentation tanks, including water and salt. The mixture is left to ferment which results in brown semiliquid product. The fermentation is halted after approximately 6 months; the liquid is separated from the mash by pressing through several filtration cloths. The resultant liquid is then pasteurized Soybeans, rice or barley are soaked, cooked, then cooled before being mixed with prepared starter (koji) and salt. After mixing the soybeans are crushed and transferred to a fermentation chamber with a lid. The duration of fermentation is about 10–15 days for sweet miso and 2–6 months for salty miso. Sometimes fermentation can be carried out for up to 2 years or longer Ashu et al. (2015) and Hesseltine and Wang (1980) 3 Dark reddish liquid with salty Soy sauce, (shoyu, chiang-yu, shi-tche, toyo, kanjang, kecap, and umami taste from soybean, and wheat see-ieu) 4 Miso (tauco, chiang, doenjang, Yellow to brownish paste used A.oryzae, Sepia rouxii, Torulopsis etchellsii, soybean paste) as flavoring and soup base P. halophilus, R. oligosporus from soy bean, rice, barley Ebine (1986), Hesseltine and Wang (1980), and Yoneya (2003) 5 6 Fermented black soybean used Aspergillus aegyptiacus, Mucor Black soybeans are cleaned, sp., Rhizopus sp., and bacteria soaked in water for for condiments and creates approximately 3–4 h, cooked, soy paste and soy sauce and then cooled until the temperature reaches 30°C. The cooked soybean is then inoculated with the starter. The process continues with fermentation at 30°C for 3–4 days, which results in called douchi qu. The douchi qu is rinsed with water and mixed with 16% salt solution, ginger, and a mix of powdered spices Several fermentation methods Rhizopus chinensis., Mucor Fermented tofu (sufu, fu-ru, fu- Creamy chunks made of have been developed to yield dispersus, M. flavus, soybean curd and served as ju, tou-fu-ju, bean cake, different fermented tofu M. racemosus, M. praini, M, condiment Chinese cheese, soy cheese, substilissimus, M. circinelloides, variations, but basically, there Chinese soybean cheese, are two steps in making M. hiemalis, Antimucor tofuyo, toufu-ru, toufu-ju, rufu, fermented tofu, i.e., the taiwanensis, A. elegans, funan, fuyu, touyu, tau-zu, preparation of tofu and Aspergillus spp. tafuri, tau ju, tau-fu yee, chao, dehydrated tofu (pehtze), and dau-phu-nyu) the fermentation process including soaking and ripening. The tofu is cut into smaller cubes and dehydrated at room temperature for 24–48 h in which prefermentation might occurs (some procedures introduce Mucor flavus or Bacillus sp.) then washed or sprinkled with alcoholic beverages (awamori), salt and seasonings. The production of red sufu uses red koji containing Douchi Mora-Escobedo et al. (2018) and Zhang et al. (2007) Ashu et al. (2015), Cheng et al. (2009), Hesseltine and Wang (1980), Nout and Aidoo (2002), and Yasuda (2011) Continued Table 3 Fermented food from filamentous fungi—cont’d Fermented food (other No. names) 7 8 9 Description (incl. ingredients) Microorganisms Processing step M. angka or M. purpureus, while the production of pale cream-yellow tofu uses yellow koji that does not contain any Monascus culture. The koji is inoculated into the mixture (moromi), allowed to ferment at 25–30°C, and ripened after around 5 months Meju Fermented soybean A. oryzae, A. sojae, Bacillus Cooked soybeans are crushed condiment subtilis and into cubes and then left to dry by hanging them outdoors to let the natural fermentation occurs. Typically bacterial fermentation precedes fungal fermentation Peanut press-cakes, which oil Oncom Peanut press-cake Rhizopus oligosporus has been extracted, are soaked Neurospora sitophila, in water for about 1 day, N. intermedia drained, (sometimes added with other side products from cassava and soybean), and cooked. The starter is inoculated after the mixture has been cooled off and then left to ferment for about 1–2 days in a bamboo case or in banana wrap at room temperature (25–30°C) White-mold cheese, Camember Soft cheese from goat or cow, Lactococcus lactis ssp. cremoris, Raw or pasteurized milk is ripened by surface mold and L. lactis ssp. lactis, Lactobacillus inoculated with lactic bacteria cheese, Brie cheese, and prematured for about 24 h delbruckii ssp. bulgaricus, Coulommier, and Carre de l’est characterized by the (10–12°C). After the pH appearance of white mycelia L. helveticus, Streptococcus References Ashu et al. (2015) Beuchat (2018) and Hesseltine and Wang (1980) Ashu et al. (2015), Hesseltine and Wang (1980), and Spinnler and Leclercq-Perlat (2007) decrease to 6.45–6.5, rennet is added to start coagulation process. After the soft curd has been formed, approximately 1 h, obtained curd is cut into smaller cubes and then left to settle for about 15 min. The cubes are transferred to cheese container and left overnight (about 8 h) to drain, and then the soft curd is flipped over. The draining continues for another 8 h. Then the curd is taken out from the container and sprinkled with salt or brine. The curd is then allowed to mature in a low humidity chamber (85% RH). Once the mold starts to grow, the curd is flipped once a day until the mold growth has covered all the surface, which is the beginning of the aging process for about 2–3 weeks. The obtained cheese can then be wrapped and stored for another 4–6 weeks or longer for final aging Ardo (2007), Cantor et al. Soft cheese, from goat or cow, L. lactis ssp. cremoris, L. lactis Pasteurized raw milk is Blue cheese, bleu cheese, (2017), and Hesseltine and introduced with the starter ssp. lactis, Leuconostoc spp., Requefort cheese, Gorgonzola, characterized gr by the culture, and acidification Wang (1980) greenish blue appearance of yeasts (e.g., Debaromyces Stilton, Danablue, Cabrales, hansenii, Kluyveromyces lactis, occurs. When the desired pH Penicillium requerforti Chetwynd, Gamonedo, has been achieved, rennet is and K. marxianus) Kopanisti, Adel Picon, BejesS. thermophilus, L. delbrueckii, added to further coagulate the Tresviso, Rokpol, Civil milk. The P. requeforti inoculum Brevibacterium linens, is introduced on the top of the Penicillium roqueforti curd. Then the thick curd is cut thermophilus, Debaromyces hansenii, Kluyveromyces lactis, Geotrichum candidum, Penicillum camemberti 10 Continued Table 3 Fermented food from filamentous fungi—cont’d Fermented food (other No. names) 11 Gamalost cheese Description (incl. ingredients) Microorganisms Processing step References into relatively large chunks and rested for few minutes to produce moist and porous texture (small internal openings to allow aerobic fermentation of P. requeforti). The curds are transferred to a perforated container for draining and stirred intermittently to prevent hardened texture. The curd is flipped frequently to accelerate the process. After draining, the curd is sprinkled with salt or brine. Then the ripening of the cheese is started. The last step is the ripening or aging process for about 60–90 days or longer, in which blue cheese flavor starts to appear. To inhibit further fermentation and inactivate the P. requeforti, heat treatment is applied after aging step Norwegian indigenous cheese L. lactis ssp. lactis, L. lactis ssp. To pasteurized skimmed milk is Qureshi et al. (2013) inoculated with lactic acid ripened with mold cremoris, L. lactis ssp. lactis bacteria starter, which causing biovar diacetylactis, the pH to decrease. After Leuconostoc cremoris several days of fermentation, Mucor mucedo the milk is deliberately warmed up. The curd is then removed from the mixture through precipitation and then transferred to cheese mold. After enough draining, the curd is taken out and then mold is inoculated and left to ripen for about 4–5 weeks 12 13 14 Black or white sticky rice is cleaned, steamed, and then cooled on a tray. A little sugar solution is sprinkled on the cooked rice. Starter is carefully inoculated for even distribution before the mixture is transferred to closed fermentation chamber or banana wrap to allow fermentation for approximately 3 days Peeled cassava is washed, Tapai singkong, tape singkong Sweet and slightly sour Rhizopus oryzae, Aspergillus steamed, and cooled off. The fermented cassava spp., Acetobacter and cooked cassava is inoculated Saccharomyces cerevisiae. Lactic acid bacteria would also with the starter, mixed grow in fermented cassava with carefully, and transferred in a dark chamber for 1–5 days or sour flavor longer in a room temperature for fermentation process to occur. However, a variation of cassava pretreatment would result in a different flavor and different fermented microbes. Substrate with higher Aw will result in the possibility of lactic acid bacteria growth imparting sour flavor The conventional process of Ang-kak (red rice, anka, red qu, Dark red rice used for its color Monascus purpureus Chinese red kojic rice) due to the presence of orange M. ruber, M. anka, M. pilosus making angkak is started by soaking the polished rice pigments (rubropunctatin and overnight then cooking it. After monascorubrin), purple cooled down, the cooked rice is pigments (rubropunctamin inoculated with Monascus spp. and monascorubramin), and then followed by solid-state the yellow pigments fermentation. For about 1 week (ankaflavin and monascin) the mold will grow and produce the red pigments Tape ketan (Lao-chao, chiuniang) Sweet and slightly alcoholic soft rice cake from glutinous rice Amylomyces rouxii, Rhizopus chinensis, Saccharomyces fibuligera, S. manlange, Endomyces fibuliger, Hyphopichia burtonii Berlian and Aini (2016) and Hesseltine and Wang (1980) Nurjannah and Nurhikmah (2020) Hesseltine and Wang (1980) and Nout and Aidoo (2002) Continued Table 3 Fermented food from filamentous fungi—cont’d Fermented food (other No. names) Description (incl. ingredients) 15 Katsuobushi Shimmered Smoked Skipjack Tuna (Katsuwonus pelamis) 16 Gari (garry, garri) Fermented cassava products 17 Fufu Fermented cassava products Microorganisms Processing step Aspergillus repens, A. candidus, The head and the fish’s internal P. glaucum organs are excluded, and the fish is chopped into fillets and cooked in hot water. The process continues with removing rib bones and smoking fillets until the golden brown color is achieved. In the following 1–2 days, the smoking process is repeated for 6–8 h, about 12–13 times. Aspergillus spp. is then inoculated on the fillets through spraying, and the fillet is left to sun-dry Aspergillus flavus, A. niger Peeled cassava was immersed in water and grated. Then the process continues by draining in a cloth bag and allowing the natural fermentation and drying to occur for 3–7 days. Frying process follows after the pulp has already dried Aspergillus flavus, A. niger Natural fermentation of peeled cassava is conducted for 72 h in water immersion. It is further processed into a mash and drained in a cloth bag. The pulp is then sieved and cooked with occasional stirring to get a thick paste References Ashu et al. (2015) and Nout and Aidoo (2002) Ashu et al. (2015) Ashu et al. (2015) 18 Kum-kum 19 Khamir bread 20 Koji 21 Jiuqiu (Chinese koji) Fermented cassava products Aspergillus flavus, A. niger The fermented cassava pulp is crushed and formed into round pellets which are then smoked. If the pellets are wrapped with banana leaves or plantains, it is called bobolo and meyondo Bread from fermented Aspergillus niger Sorghum flour, garlic, onion, sorghum grain lemon juice, and water are mixed and left to ferment for 24 h. The mixture is then heated Rice is steamed and then Starter culture from soybean, Aspergillus sojae, A. oryzae allowed to cool. The inoculum rice, or wheat used for (A. sojae or A. oryzae) is production of other fermented introduced, and the substrate is foods thoroughly mixed to facilitate the homogenous spreading of the inoculum. The mixture is stored in heaps at a warm temperature by insulating it with a cloth. After several hours, before mycelium’s appearance, the mixture is split into smaller portions in cases. The environment is set at warm and humid conditions to encourage fermentation, in which the fungi will grow white and fluffy. In the case of soybean and wheat substrate, the cooked and crushed soybean or wheat is left to cool and then incubated within 3 days Starter mix of mold, yeast and mix of mold (commonly found Traditionally, the cooked is A. oryzae), yeast and bacteria subtrate is left to cool and bacteria from wheat, rice, ferments naturally barley or pea for production of other fermented foods Ashu et al. (2015) Ashu et al. (2015) Ashu et al. (2015) Ashu et al. (2015) Continued Table 3 Fermented food from filamentous fungi—cont’d Fermented food (other No. names) Description (incl. ingredients) 22 Gochujang, Kochujang 23 Sake Microorganisms Processing step References Spicy condiments made of chili, meju, rice, wheat powder, and salt Aspergillus oryzae, A. sojae, B. subtilis Alcoholic drink from rice S. cerevisiae, S. rouxii, Aspergillus oryzae Ebine (1986) and Nout and Meju, steamed cereal flour (rice, barley or sometimes with Aidoo (2002) added wheat flour), fine red chili powder, salt, and water are mixed and allowed to ferment for around 2–6 months in a fermentation chamber or pot. The ingredients and method of making gochujang might differ depending on the regions and available raw materials Conventionally polished rice is Bokulich et al. (2014) cooked and cooled down to reach room temperature. The A. oryzae culture is introduced to the cooked rice and thoroughly mixed. This mixture is allowed to ferment for 2 days and the resulting product called koji. Koji will later be added to other mashed cooked rice of larger quantity and mixed evenly. The process continues with fermentation stage for 10–25 days to obtain fermentation starter called moto (or shubo) before the mixture is transferred to larger vessed and mixed with the larger quantity of rice, water, and koji to obtain the main mash or moromi, in which the main fermentation or sake brewing will be conducted for 20–30 days. The separation of sake and the solid substrate is conducted by pressing and filtration. The last step is pasteurization Chapter 12 • Filamentous fungi for food 363 The soaking process is aimed to increase the moisture of the bean, soften the bean, remove antinutrient compounds soluble in water, and acidifying the bean to prevent the growth of pathogens. Sometimes, to acidify the system, lactic acid bacteria or lactic acid can be used. As some unwanted components dissolve in water, the water is replaced with fresh water prior to cooking. The dehulling process is aimed to remove the barrier facilitating the penetration of the mycelium on the bean. Cooking is applied to soften the bean and kill the pathogens. After cooking, the water is discharged immediately, and the cooked bean is spread out on a bamboo tray to remove the excess water which could trigger bacterial spoilage. The cooled beans are then inoculated with 104 colony-forming units per gram of bean using tempe starter containing fungal sporangiospores of mainly Rhizopus oligosporus (Ahnan-Winarno et al., 2021). It is followed by packing the inoculated soybean in banana leave or perforated plastic bags (100–300 g) and stored at room temperature (25–30°C) for 24–48 h. The resulting tempe is a compact soybean tied to each other by the whitish mycelium of the fungi. The number of spores influences the characteristics of the tempe. If the spores are less than 102 CFU/mL of spore, the growth of the fungi becomes irregular, the fermentation process is too slow and very susceptible to bacterial spoilage as it does not dominate the microflora of the tempe. In contrast, if the spores are too high , the fermentation process occurs rapidly resulted in fast increase in temperature leading to the death of the fungi. It takes shorter fermentation time; however, it has shorter shelf-life and is tasteless since the flavor compound is generated. In addition, the starter culture also affects folate and vitamin B12 (Mo et al., 2013). The steps of the tempe making also affect the microflora. For instance, in a process that applies two times boiling the LAB is lower than that of applying one time boiling and resulting in different composition of tempe, particularly on carbohydrate and protein (Nurdini et al., 2015). The fungus primarily used for tempe fermentation is Rhizopus oryzae, although Rhizopus oligosporus and Aspergillus oryzae can also be used. Several studies have also reported microorganisms other than molds such as lactic acid bacteria, Acetobacter indonesiensis, Klebsiella pneumoniae, Bacillus subtilis, Flavobacterium sp., Brevundimonas sp., Pseudomonas putida, and Acinetobacter sp. which involve in tempe production (Barus et al., 2008; Nurdini et al., 2015). Lactic acid bacteria acidify the soybean during soaking which inhibits the growth of pathogenic bacteria (Nurdini et al., 2015). Generally, the fungus inoculum is available as a starter made from dried ground cooked rice inoculated with R. oligosporus (laru), dried ground tempe, or fungus grown on oak leaves (usar). Tempe has been known to be consumed by Javanese people in Indonesia since 17th century (Astuti et al., 2000). Tempe is used to be consumed after addition of spices and deep-fried or made into various dishes with vegetables or made into chips. However, currently, tempe has also been made into burger, meat loaf, etc. As with other cereal fermentation, the main purpose of fermentation is to mainly improve the texture, appearance, and acceptance, and not for improving the shelf life. Tempe can generally be stored at room temperature for 2–3 days and 5–7 days at refrigerated temperature. During storage, the fermentation progresses resulting in a bitter taste and spoilage. Traditionally, in Java, 364 Current Developments in Biotechnology and Bioengineering this spoiled tempe (tempe bosok in Javanese language) is made into sambal tumpang and enjoyed as a local delicacy in certain community in Central Java. In addition to having improved sensory quality, tempe fermentation also produces bioactive compounds such as isoflavon aglycone, gamma amino butyric acid (GABA), superoxide dismutase (SOD), peptides, and other antimicrobial agents. Isoflavone has been reported to have similar function as estrogen which could prevent osteoporosis in menopausal women and reduce the risk of arteriosclerosis (Yoneya, 2003). 3.2.2 Soy sauce Soy sauce is a dark brown liquid condiment made by soybean fermentation. Soy sauce was originated in China over 2500 years ago and spread out to Japan, Korea, and several Southeast Asia countries. Soy sauce has a different name in each country, in China it is called Jiàngyóu, in Japan the name is shoyu, in Korea the name is Ganjang, while in Indonesia it is known as Kecap. The taste of soy sauce is mainly salty, except in Indonesia where sweety soy sauce is more popular and called “kecap manis.” Soy sauce is made by two states of fermentation, i.e., solid-state koji fermentation followed by a liquid state of moromi fermentation. During koji fermentation, several microorganisms are detected including Aspergillus oryzae, A. sojae, A. niger, A. flavus, A. parasiticus, and Rhizopus species. Among the fungi, Aspergillus oryzae and Aspergillus sojae are the most common. A. oryzae secretes proteolytic and amylolytic enzymes, while A. sojae releases various enzymes related to amino acid production and flavor fermentation. Among these enzymes, glutaminase is one of importance as it converts amino acid and glutamine into glutamic acid, one of the key flavors for umami taste. After koji fermentation, the koji product is added with brine solution and fermented in a vessel for months or years which is known as moromi fermentation. In this fermentation, Pediococcus halophilus and Zygosaccharomyces rouxii are the dominant microbes. P. halophilus firstly grow in the substrate due to its high salinity tolerance. During its growth, P. halophilus produces lactic acid and acetic acid which reduce the pH from 6 to 7 to 5.5 enabling the growth of Z. rouxii. At the end of fermentation, the pH is 4.6–4.9 and the product contains lactate, acetate, ethanol, glycerol, reducing sugars, and formol nitro€ ling et al., 1994). In addition, various flavors are also produced in this stage. The gen (Ro alcohols and esters are related to the type of yeast involved in the fermentation. Besides Z. rouxii, several yeasts are involved in moromi fermentation such as Candida etchellsii, C. versatilis, Zygosaccharomyces cryptococcus, and various genera belong to Candida, Kluyveromyces, Millerozyma, Saccharomycopsis, Peronospora, Pichia, Trichosporon, Wickerhamomyces, Rhodotorula sp., Debaryomyces, Torulaspora, Microbotryum, and Tetrapisispora (Song et al., 2015). 3.2.3 Oncom Oncom is a traditional fermented food in the form of mycelium-bound cake. It is made from a mixture of peanut press cake, cassava waste, or tofu waste (okara) inoculated with Neurospora fungi. Oncom is originated from West Java, Indonesia, and consumed by Chapter 12 • Filamentous fungi for food 365 millions of people with consumption per capita of 0.96 kg per year in 2018 (Hakim and Sumantri, 2018). More than 20 dishes can be prepared from oncom. Oncom is characterized by its unique color and taste. The color of oncom varies from yellow, orange, red, to black. The color is affected by the medium and the fungi used. Red and black oncom are the most common oncom which is produced using Neurospora sitophila and Rhizopus oligosporus, respectively. Besides those fungi there are several fungi that play an important role in oncom fermentation including Neurospora crassa, Neurospora intermedia, Mucor javanicus, M. circinolloides, and M. rouxii. Oncom is characterized by fruit-like, alcoholic, mincemeat, and almond character. Oncom made from food by-products is a successful example of producing nutritious and sensory acceptable products from low-cost material through fermentation. This concept is important particularly due to the growing global population demanding a sustainable food supply. There are several steps for making oncom including soaking, water removal, rinsing, mixing, steaming, molding, cooling, inoculation, packaging, and incubation. The first step is soaking the raw material separately. The duration of soaking varies between oncom producers from 1 to 12 h. The rinsing is aimed to remove the remaining oil in the peanut cake. The water is drained using a sieve. The ratio of peanut cake with cassava waste or oncom varies between oncom producers and this determines the quality of the oncom. The higher the peanut press cake used, the higher the quality of oncom. The steaming is conducted for 30 min to 2 h. After steaming, without waiting for it to cool, the mixture is inoculated into a cubic form with a size of 9  12 cm and a depth of 3 cm. The cake is then placed on a bamboo shelf. After cooling, the cake is inoculated by sprinkling using inoculum or milled semidried oncom from a previous batch. A small number of oncom producers do not use inoculum and rely on spontaneous fermentation from the surrounding air which has been contaminated with the spores of the mold. This is done by putting the cake in one room without ventilation. The incubation takes place for 18–48 h depending on the raw material. Okara requires longer incubation than that of peanut press cake and cassava waste (Beuchat, 2018). The fungus degrades starch, lipid as well as protein and converts their macromolecules into alcohol and ester which are responsible for oncom aroma. The lipase hydrolyzes triglycerides into free fatty acids, whereas protease is responsible for free amino acids and peptide production which is associated with meaty flavor characteristics. Fermentation also increases protein solubility. The fungi used greatly affect the sensory of the oncom. For instance, oncom made from Mucor has better taste, aroma, and texture as well as higher soluble nitrogen content (Sastraatmaja et al., 2002). Oncom has similar nutrient to tempe. A 100 g of oncom contains approximately 14.9 g protein, 6 g lipid, 12.3 g fiber, 0.2 g calcium, 0.11 g phosphor, 0.01 iron, 0.2 g total carotene, and 0.03 vitamin B1 (Departemen Kesehatan Republik Indonesia, 1995). According to its composition, oncom can be considered as a nutritious food with high protein, fiber, minerals, and vitamins. Several studies explored the potential of oncom to be developed as a functional food. Oncom contains phytase, a phytate degrading enzyme. Phytate is considered as antinutrient compound that inhibits the adsorption of mineral from our diet. 366 Current Developments in Biotechnology and Bioengineering A study by Kanti and Sudiana (2016) proves that oncom fungi, Neurospora crassa, and Neurospora sitophila can be used to produce phytase to improve poultry nutrient quality. In addition, red oncom has a fibrinogenolytic activity, a protease that is able to degrade fibrin or fibrinogen. A lack or disruption of the fibrinolytic system on a regular basis triggers several thrombosis-related diseases such as stroke, atherosclerosis, hypertension, and diabetes (Afifah et al., 2015). Furthermore, it has been reported that defatted soy oncom is able to decrease the plasma cholesterol level significantly (Matsuo, 2000). 3.3 Natural food additives and metabolites from filamentous fungi Food additive is defined as a component added to food for a specific purpose and remains in the food but it does not characterize the product. The specific purpose of food additives includes manufacture, processing, coloring, and preservation. Filamentous fungi play an important role in commercial production of diverse food additives such as organic acids, enzymes, vitamins, polyunsaturated fatty acids (PUFAs), and flavor compounds. Filamentous fungi transform a wide variety of substrates into various products; hence the product is regarded as natural product. Recently, there has been a growing interest in natural products due to high consumer awareness toward clean label products and sustainability. This is further supported by the technological advancement to produce higher yields of safe food additives such as modern techniques for choosing nontoxic fungal strains, advanced knowledge on biosynthesis regulation, and potential of genetic engineering (Copetti, 2019). Filamentous fungi produce a number of organic acids that are used as food additives. The organic acids include citric acid, gluconic acid, itaconic acid, malic acid, lactic acid, fumaric acid, succinic acid, tartaric acid, isoascorbic acid, kojic acid, ɣ-linolenic acid, and arachidonic acid (ARA). In the food industry, organic acids can be used as pH regulator, acidulant, chelating agent, emulsifier, preservative, flavoring agent, antibrowning agent, nutritional supplement (Archer et al., 2008). The organic acids produced by filamentous fungi play a major role in food industry. For instance, 95% of the citric acid in global market obtained from fungi (Copetti, 2019), in which citric acid represents 80% of food acidulant (Moresi and Parente, 2014). Citric acid is applied as pH control in jams, jellies, juices, softdrink, and wine. Several fungi that are able to produce citric acids include Aspergillus niger, A. wentii, and Penicillium citrinum (Copetti, 2019). They are also primary flavor producers such as 2-methoxy-3-isopropyl pyrazine, 2-hydroxy-3-6-substituted pyrazines, acyclic terpenoids: geraniol and citronellol, methyl ketones. These flavors give earthy, nutty, roasted, potato, soy sauce, essential oil, blue cheese, etc. In addition, they also produce PUFAs including arachidonic acid (Ω-6; ARA) and docosahexaenoic acid (Ω-3; DHA). Among them, A. niger is the most frequently used in the food industry. 3.3.1 Natural colorant from filamentous fungi Among metabolites that can be produced by filamentous fungi, pigments are among the most interesting products since this production capability is not widespread among microorganisms. Various pigments produced by filamentous fungi are presented in Table 4. Filamentous fungi are a promising source of natural pigments such as lycopene, Chapter 12 • Filamentous fungi for food Table 4 367 Pigments produced by filamentous fungi. Filamentous fungi Pigments Color Monascus purpureus, Monascus ruber, and Monascus pilosus (Patakova, 2013) Monascus purpureus, Monascus ruber and Monascus pilosus (Patakova, 2013) Monascus purpureus, Monascus ruber, and Monascus pilosus (Patakova, 2013) Blakeslea trispora (Copetti, 2019) Monascorubramine and rubropunctamine Red Monascorubrin and rubropunctatin B. trispora (Copetti, 2019) Ashbya gosyppi (Copetti, 2019) Monascus pilosus (Akihisa et al., 2005) Monascus purpureus (Knecht and Humpf, 2006) Mutant of Monascus purpureus (Hsu et al., 2010) Mutant of M. kaoliang (Jongrungruangchok et al., 2004) Monascus ruber (Loret and Morel, 2010) T. aureoviride, T. harzianum, and T. polysporum (Caro et al., 2015) Fusarium graminearum (Frandsen et al., 2006) T. atroroseus sp. nov (Mapari et al., n.d.) Talaromyces spp. (Lebeau et al., 2017) Neurospora intermedia (Gmoser et al., 2018) Status of industrial scale code Use and restriction N.A. Food Orange Allowed in southeast Asia but restricted in Europe and USA N.A. N.A. Food Monascin and ankaflavin Yellow N.A. N.A. Food β-carotene Yellow to yellow-orange Yes E160a Lycopene Yellow to red Yes E160d Food, drug, cosmetics Food Riboflavin Bright yellow Yes E101 Food Xanthomonasin A&B Yellow N.A. N.A. N.A. N.A. N.A. N.A. Yellow N.A. N.A. N.A. Yellow N.A. N.A. N.A. N.A. N.A. N.A. Chrysophanol Yellow, blue fluorescent Red N.A. N.A. N.A. Aurofusarin Red N.A. N.A. N.A. Azaphilone Red N.A. N.A. N.A. N-threoninerubropunctamine Red N.A. N.A. N.A. β-carotene Yellow to orange N.A. N.A. Food Monascopyridine A-D Monaphilone A & B, purpureusone, monashexenone Monascusone A & B Monarubrin, rubropunctin 368 Current Developments in Biotechnology and Bioengineering natural red, riboflavin, Monascus red, and β-carotene. Several of these pigments are regarded as food-grade and have been produced on industrial scale. They include natural red from Penicillium oxalicum, riboflavin from Ashbya gossypii, lycopene, and β-carotene from Blakeslea trispora as well as Monascus red. All these compounds, with the exception  et al., 2014; Mapari et al., of Monascus red, are permitted to be used in Europe (Dufosse 2010). Another potential pigment from filamentous fungi is a yellow to orange β-carotene extracted from Neurospora intermedia (Gmoser et al., 2018). This fungus is used for making a traditional dish in Indonesia, namely Oncom. Although oncom has been consumed by Indonesian people for centuries, however, the natural pigment from Neurospora intermedia has not been commercialized yet. Several criteria for a good food colorant include nontoxicity, high stability toward temperature, pH and light, ability to improve the appearance and organoleptic properties, composition and coloring, compatibility to a wide variety of matrices, and solubility in water (Ogbodo and Ugwuanyi, 2017). Considering these factors, several fungal pigments meet these criteria, hence they have been produced commercially such as β-carotene from B. trispora and riboflavin from Ashbya gosyppi. The β-carotene has been produced , 2018). In addiin large-scale in the USA, Canada, Australia, and New Zealand (Dufosse tion, fungal pigments offer a number of advantages that are not owned by artificial or other pigments such as seasonal independence compared to plant-based pigment, sustainability, natural character, functional properties, and nutritional value. Fungal pigments that are used as food colorants for humans are actually produced to support physiological needs for the fungi. The fungi secrete the pigments in order to do photosynthesis and to maintain membrane integrity and stability, to survive under nutrient and other stresses, and as defense systems against pathogens. Fungal pigments can be produced either in submerged fermentation or solid-state fermentation. Each mode of fermentation has pros and cons. Solid-state offers higher yield, lower water usage, and lower risk of contamination, however, nutrient transfer and pigment extraction are the challenges of using this mode. On the other hand, the submerged fermentation offers higher efficiency of carbon consumption and better transfer of nutrients by mixing. The main drawbacks of this mode include high energy and advanced technology requirement, enhanced risk of contamination, and slow growth of the fungi (Joshi et al., 2003). The fungal pigments can be classified into predominantly cell-bound known as intracellular or secreted outside the cell thus accumulating in the fermentation broth known as extracellular. The selection of the mode of fermentation and downstream processing could consider the type of the pigment. For instance, submerged cultivation is preferable for extracellular pigment production as it makes the downstream process easier. However, for intracellular pigment, it is recommended to use solid-state fermentation, in which the fermented solid is directly used as colorant (Ogbodo and Ugwuanyi, 2017). The typical downstream process for fungal pigment is extraction using solvents or adsorption using resins. The solvents used for extraction are methanol and ethanol (Lebeau et al., 2017). Following the extraction, the pigment is further concentrated using a rotary evaporator or subjected to spray drying to remove the solvent. Chapter 12 • Filamentous fungi for food 369 Several factors affecting pigment production include growth medium, pH, light, aeration, temperature, and incubation time. The growth medium that gives significant effect is carbon source and nitrogen source. For the carbon source, glucose is the most preferable substrate. The type of carbon not only influences the yield but also the color shade of the pigment and incubation time. For instance, replacing glucose with sucrose in fermentation using M. purpureus, resulted in changing the color from very dark liver pigment into a light and uneven red pigment (Joshi et al., 2003). In addition, Monascus sp. required 3 days longer incubation time to produce pigments when using cassava bagasse compare to using sugar medium (Tallapragada and Dikshit, 2017). The type and concentration of nitrogen affect pigmentation. Organic nitrogen is generally considered better than inorganic nitrogen for pigmentation and concentration of nitrogen above 10% is detrimental for pigmentation. The recommended organic nitrogen was sodium caseinate, peptone, and yeast extract, whereas the suggested inorganic nitrogen sources include ammonium chloride, ammonium nitrate, and glutamate (Joshi et al., 2003). The pH influences the shade of the color. For instance, M. purpureus secreted yellow pigment at pH 4.5 and red pigment at pH 6.5 (Gunasekaran and Poorniammal, 2008). In general pH of 6 is favorable for the growth and pigment production of fungi, although the optimum pH for Penicillium sp. to produce pigment is reported at pH 9. Light is another factor affecting pigment production. In general, fungi produce higher pigments in the dark (Velmurugan et al., 2010). Fungi require oxygen to produce biomass as well as pigment. The higher the aeration rate, the higher the pigment yield. However, an agitation rate above 200 revolutions/min decreases the yield (Gunasekaran and Poorniammal, 2008). The temperature required for pigmentation in several reported fungi is in the range of 25–30°C (Joshi et al., 2003). The optimum incubation time for pigmentation varies from 8 to 12 days depending on the species of the fungi (Tallapragada and Dikshit, 2017). Although the advantages, there are several challenges and limitations in developing fungal pigment including (i) high manufacturing cost and price; (ii) limited scale-up technology and downstream processing; (iii) safety issues related to citrinin, a toxin produced by Monascus; (iv) requirement for toxicological studies; (v) strict food legislation and regulatory process; and (vi) technical aspects to optimize the yield of fungal pigments as , 2018; pigment production is negatively correlated with biomass production (Dufosse Morales-Oyervides et al., 2020). Once these challenges can be addressed, the market of fungal pigments can be expanded since currently, 90% of pigments are of synthetic origin. 3.3.2 Flavor enhancer from filamentous fungi Conventionally, fermented foods from filamentous fungi have been used to improve sensory quality of food, for example, soy sauce (shoyu), miso, tempe bosok (overripe tempe), katsuobushi, and cheese (Table 3), especially umami and kokumi taste (Diez-Simon et al., 2020). The liquid by-product separated from, e.g., the fungal biomass Quorn, could also be further processed into flavoring product, Quessent (Wiebe, 2004). The exceptional flavor from the fermented food is assumed to be due to the lipolytic and proteolytic action of the fungi on the substrate, for instance, development of flavor during maturation in 370 Current Developments in Biotechnology and Bioengineering Camembert cheese (Galli et al., 2016). Both volatile aroma active compounds and nonvolatile taste active compounds from fungal metabolites are found to enrich the flavor characteristics of the supplemented dishes. Soy sauce, fermented food with strong savory taste, has been one of the main seasonings for Japanese dishes, beside sugar (satou), vinegar (suu), and salt (shio). Different kinds of soy sauces, imparting different kinds of sensory characteristics, especially umami and kokumi, have been reviewed (Diez-Simon et al., 2020). Umami compounds naturally available in soy sauce are the amino acid aspartate, and the nucleotides 50 -IMP and 50 -GMP (Kong et al., 2018). However, other low molecular peptides as well as several water-soluble compounds from Maillard reaction, even with low concentration, might contribute to augment the savory taste in soy sauce (Lioe et al., 2010). Similar findings were also reported in miso, where peptides resulted from the Maillard reaction formed a characteristic of mouthfulness in ripened miso (Ogasawara et al., 2006). Water soluble taste active compounds in over fermented tempe (tempe semangit) that complements Indonesian dishes as condiments, have been characterized by Utami et al. (2016). It was reported that over fermented tempe contained a high free amino acids which are responsible for umami taste activity. While kokumi-active gamma-glutamyl peptides in blue cheese have been characterized by Toelstede and Hofmann (2009). A group of γ-glutamyl dipeptides were found to induce the attractive kokumi flavor of matured Gouda cheese. 3.3.3 Enzymes from fungi Filamentous fungi are key players in industrial enzyme production. In the food industry, enzymes are applied in order to improve efficiency, ease the manufacturing process, and increase quality (Zhang et al., 2018). Filamentous fungi are vastly exploited in industrial enzyme production due to their versatility and high productivity, especially in the food industry. Some enzymes that are approved for use in food are presented in Table 5. Filamentous fungi produce a wide variety of enzymes on commercial scale such as amylases, cellulases, proteases, xylanases, glucoamylases, lipases, and pectinases. Xylanases and pectinases secreted by Trichoderma spp., Penicillium spp., Rhizopus spp., and Aspergillus spp. are used in juice clarification, bread improvement, and production of monomers. Fungal amylase is applied in manufacturing of glucose syrup, whereas proteases mainly generated by Rhizopus spp. and Aspergillus spp. are needed for meat tenderization (Copetti, 2019). Submerged fermentation technique for filamentous fungi remains dominant in industrial enzyme production. Major species that were commonly used in enzyme production are from the genus Aspergillus, i.e., A. niger and A. oryzae that have been recorded as generally recognized as safe (GRAS) followed by A. awamori. Trichoderma reesei has also gained interest due to its capability in producing high amount of cellulase and hemicellulase (Hjort, 2006; Leisegang et al., 2006). The major advancements were obtained through classical strain enhancement and optimization of fermentation conditions, especially combined with the breakthrough in recombinant DNA technology. Gene disruption technique has solved the major shortcoming in the stability and purity of enzymes produced by Aspergillus oryzae, A. niger, and A. awamori, in which the fungi produced both amylase and protease (Hjort, 2006; Leisegang et al., 2006). Chapter 12 • Filamentous fungi for food 371 Table 5 Commonly used recombinant enzymes from filamentous fungi and their application in the food industry. Enzymes Host Donor Application References Aminoacylase Aminopeptidase Aspergillus sp. Aspergillus sp. Rhizopus sp. L-amino Catalase Aspergillus niger Aspergillus sp. Cellulase Trichoderma reesei, Aspergillus oryzae Humicola sp., Trichoderma sp. acids synthesis Hydrolysis protein, remove bitter taste protein, modify flavor food preservation (with glucose oxidase), removal of hydrogen peroxide from milk prior to cheese production Animal feed, clarification of fruit juice Archer et al. (2008) Archer et al. (2008) Chymosin Calf Cheese making α-Galactosidase Aspergillus niger var awamori Aspergillus oryzae Aspergillus sp., Trichoderma sp., Penicillium sp. Glucoamylase Rhizopus oryzae Aspergillus awamori, A. niger β-Glucanase Trichoderma reesei Trichoderma sp. Glucose oxidase Aspergillus niger Aspergillus sp. Lipase Aspergillus oryzae Rhizopus niveus Penicillium requeforti Candida sp., Rhizomucor sp., Thermomyces sp. Phytase Aspergillus niger Aspergillus sp. Hydrolize raffinose and stachyose in processing soybean milk and other legume based products Beer production, bread quality improvement, high glucose, and high fructose syrups To remove haze resulting from Botrytis growth on the grapes Food shelf life improvement, food flavor improvement Encourage formation of emulsion in situ formation of emulsifiers in baking, to modify the compositions of fatty acids from food substrate, texture, and flavor development in cheese, preventing rancidity in butter To increase mineral digestibility of processed food Archer (2000), Ough (1975), and Sirbu (2011) Grassin and Fauquembergue (1996) and Sukumaran et al. (2005) Archer (2000) Archer (2000) Archer (2000), Blanco et al. (2014), and James et al. (1996) Archer (2000) Archer (2000), Hanft and Koehler (2006), and Zhu et al. (2006) Archer (2000), Hjort (2006), and Leisegang et al. (2006) Archer (2000) Continued 372 Current Developments in Biotechnology and Bioengineering Table 5 Commonly used recombinant enzymes from filamentous fungi and their application in the food industry—cont’d Enzymes Host Donor Application References Protease Aspergillus oryzae, A. niger Rhizomucor sp. Archer (2000), Aruna et al. (2014), Hjort (2006), Leisegang et al. (2006), and Miguel et al. (2013) Xylanase Aspergillus niger, A. oryzae, Trichoderma reesei (longibranchiatum), Penicillium sp., and Humicola insolens Aspergillus sp., Trichoderma sp., Thermomyces sp. Brewing, meat tenderization, coagulation of milk, bread quality improvement, prevent acrylamide formation in food processing, energy drink production Ethanol and xylitol production, improve bakery products, aid the processing of the juice and wine industry, beer quality improvement Archer (2000), Camacho and Aguilar (2003), and Dervilly et al. (2002) The capacity of fungi for large-scale production and purification gave birth to the growing interest of research and commercial utilization (Archer, 2000). Some enzymes from filamentous fungi have advantages compared to other sources, for example, xylanase used in bread making from filamentous fungi (commonly A. niger, Trichoderma spp., and Humicola insolens) has an acidic optimum pH, high stability, and higher amount than those produced by yeast and bacteria (Polizeli et al., 2005). 3.3.4 Other food ingredients A sweetener and flavor modifier, thaumatin, from the fruit of Thaumatococcus danielii is considered as food-grade ingredient being approximately 3000 folds more potent than sucrose. Due to limited amounts in plants, the microbial production from bacteria, yeast, and fungi were attempted (Hjort, 2006; Leisegang et al., 2006). Successful efforts in increasing thaumatin production were achieved with P. roquefortii and A. niger var. awamori. More than two folds increased in thaumatin production with A. awamori were reported (Lombrana et al., 2004). Nonvolatile flavor compounds industrially produced by filamentous fungi such as citric acid, and a plethora of volatile compounds, namely vanillin, linalool, jasmonate, lactones, and methyl ketones have synthesized via de novo or through biotransformation and bioconversion processes (Archer et al., 2008). Another filamentous fungi product as food ingredients is fat mimetics, which act as processing aid. The fat mimetics is produced by dispersing mycoprotein treated with high-pressure homogenization (Finnigan et al., 2017). 3.4 Fungal mycelium-based food In Section 2.3, the usage of filamentous fungi in fermented food production or as food ingredients has been discussed. In this chapter, the usage of whole fungal biomass as food is addressed. The first fungal biomass that has been produced commercially and marketed is Fusarium biomass with the brand of Quorn and manufactured by Marlow Foods, Ltd. Chapter 12 • Filamentous fungi for food 373 Quorn is the first mycoprotein approved for human consumption available in the market. However, it took 20 years of work before its commercialization as it is considered as a novel food so safety approval by comprehensive clinical studies is required. It has been consumed for 36 years in its origin country and spread to other countries such as Belgium, Holland, Switzerland, Sweden, Denmark, Norway, Australia, and USA. Research to find a fungal candidate for mycoprotein production began in 1967 with over 3000 soil taken samples from around the world. The selected fungus was Fusarium graminearum code A3/5, which was later identified by O’Donnell et al. (1998) to be Fusarium venenatum (PTA 2684). The fungus was then used for commercial production and the originated fungal biomass was developed into various products such as fillet, nuggets, pieces, patties, meatballs, steak, slices, etc. In addition to Quorn, there are other companies now in the market such as Swedish Svampsson that are introducing now other foods based on fungal biomass to the market. Among the several factors behind the success of commercialization of the mycoprotein, a similar eating quality of Quorn with that of meat is one of the most important properties. This property is formed during freezing and frozen storage by the bundle of mycelium of mycoprotein which creates a fibrous meat-like texture. Quorn both in pieces or in mince obtained the highest liking score compared to other meat-free proteins including tofu, tivall-stir fry pieces, good bite chicken style, vivera-vega stir fry, and cultured meat (Hashempour-Baltork et al., 2020). Mycoprotein generally contains 45% protein on dry basis or 11%–15% on wet basis. This amount is similar to the protein content in egg, higher than milk and lower than meat. However, it contains all of the EAAs and the PDCAAS is 0.996 which is near to perfect protein (Edward and Cumming, 2010). This score is higher than that achieved by beef or chicken. The fat of mycoprotein is ca. four times lower than meat, while the essential PUFA content of mycoprotein is eight times higher than meat (Hashempour-Baltork et al., 2020). This indicates that mycoprotein can be used as healthy fat diet. In addition, it contains high fiber up to 6% (Finnigan, 2011), and higher vitamin (39.4 mg/kg) compared to meat (0.61 mg/kg) (Hashempour-Baltork et al., 2020). Mycoprotein has been suggested to have several health benefits such as increasing satiety, lowering cholesterol in serum, improved glycemic response, and increased insulin sensitivity. Regarding the safety of Quorn, mycoprotein holds a “GRAS” status. In addition, the result of toxicology tests of the Quorn Strain A 3/5 has shown that no toxic metabolites were produced by this strain (Solomons, 1986). However, Jacobson and DePorter (2018) reported that there are allergenic reactions related to Quorn consumption. The reactions include urticaria and anaphylaxis, emesis, diarrhea, nausea. 4. Nutritional consideration In several developing countries, there are many traditional fermented foods that involve filamentous fungi. Several fermented foods have higher vitamins and other nutrients compared to raw materials. While the industrialized countries can afford to enrich its foods with synthetic vitamins or other nutrients, many of the developing countries must rely upon biological enrichment to fulfill their essential nutrient requirements. The ability 374 Current Developments in Biotechnology and Bioengineering of filamentous fungi to utilize complex polymers enzymatically and degrade antinutrients, for example, phytate breakdown to increase mineral bioavailability in cereal and legume-based foods (Greiner and Konietzny, 2006), or cyanogenic glucosides breakdown in cassava. This could lead to improvement in the digestibility of the substrate (El Sheikha and Montet, 2016). Filamentous fungi can enrich fermented foods through a synthesis of EAAs, fatty acids, and vitamins. Therefore, in addition to traditional food fermentation, currently, the production of nutritional supplements through filamentous fungi flourished. The actions of the fungi on the substrate also create impacts on the sensory characteristics of the fermented food, such as texture, color, and flavor. The microorganisms would produce aldehydes, esters, ketones, acids, and sulfur compounds that influence the flavor profile. The metabolites that are excreted by the fungi might also contain bioactive compounds. Though there is a possibility that the fungi produce toxic compounds, the metabolic activity of the fungi also could degrade the harmful compounds in foodstuffs minimizing the level of toxins (Hjort, 2006; Leisegang et al., 2006). 4.1 Proteins Protein is one of the most important nutritional contents in filamentous fungi. The criteria for determining the protein quality are already discussed in Section 2.1. However, this section is more focused on amino acid profile, more specifically the EAA content. As EAAs could only be obtained from the diet, food containing a complete and sufficient level of EAA is of interest. The EAA of mycoprotein, tempe, and miso are presented in Table 6, showing consumption of 100 g of mycoprotein, soybean tempe, and soybean miso Table 6 Essential amino acid contents in filamentous fungi-based foods. Parameter/ component Histidin (g/100 g) Isoleucine (g/100 g) Leucine (g/ 100 g) Lysine (g/ 100 g) Methionine (g/100 g) Phenylalanine (g/100 g) Threonine (g/ 100 g) Valine (g/ 100 g) Soybean tempe (Bujang and Taib, 2014) Soybean miso (Zarkadas et al., 1997) Requirements mg/kg body weight (Palmer, 1989) 1.42 3 8–12 5.2 2.90 4.865 10 8.6 4.75 7.736 14 8.3 3.29 6.483 12 2.1 1.04 1.943 13 (plus cystine) 4.9 3.54 5.379 14 (plus tyrosine) 5.5 0.87 4.315 9 6.2 3.33 5.688 13 Mycoprotein (Finnigan et al., 2017) Chapter 12 • Filamentous fungi for food 375 could fulfill the daily requirement of EAA for an adult person. For instance, the requirement of methionine, one of the most limiting amino acids in legumes, for a 60 kg person is 0.78 g and this can be fulfilled by ingesting 100 g of all filamentous fungi-based food, which is equal to, e.g., three serving slices of tempe. 4.2 Vitamins Filamentous fungi are capable to produce several vitamins include riboflavin (vitamin B2) pantothenic acid (vitamin B5), β-carotene (provitamin-A). Riboflavin has been industrially produced via de novo biosynthetic pathway involving Ashbya gossypii and Eremothecium ashbyii. The metabolic flow of vitamin B5 has been synthesized via biotransformation with a lactonohydrolase from the genera Fusarium, Gibberella, and Cylindrocarpon. Industrial production of β-carotene has been conducted involving the fungi B. trispora (Archer et al., 2008). Fermentation has been reported to increase vitamins and nutrients in the final products. For instance, tempe, a fermented soybean cake by Rhizopus sp. has a tremendously higher vitamin B12, vitamin B6, riboflavin, thiamine, nicotinic acid, and nicotinamide than soybean (Keuth and Bisping, 1993). 4.3 Lipid Some fungi-producing oils are Fusarium equiseti, F. oxysporum, and Aspergillus sydowii (Azeem et al., 1999). Commercially, lipid extracted from plants is still preferable compared to those of filamentous fungi. However, the market needs supplemental PUFAs, such as docosahexaenoic acid (DHA) and ARA. These two PUFAs, essential for an infant’s neural and retinal development, are available in mother’s milk but absent in cow milk and infant formula. In that case, the demand for long-chain unsaturated oil can not be satisfied by plants that have not been yet reported to produce lipid longer than C18, but the PUFA might be obtainable through fish oil, algae lipid, or microbial oil from filamentous fungi. While fish oil also remained an option, the possibility of heavy metal pollution in seafood inhibits further application of fish oil in infant formula. Therefore, oil from filamentous fungi would be an alternative solution (Ratledge, 2004). Organisms involved in the industrial microbial oil or single cell oil production should generate high amount of the desired lipid as the main PUFA, namely Crypthecodinium cohnii which produces triaglycerols with the content of DHA of up to 40%–50% of total fatty acids (Ratledge, 2004). Mucor circinelloides and Mortierella isabellina has been cultured to generate linoleic acid. Mucor circinelloides has also been used in the production of fat substitutes for cocoa butter (Sancholle and Losel, 1996) while for the commercial production of ARA, Mortierella alpina was preferred by the industry (Hjort, 2006; Leisegang et al., 2006). Fungal biomass, mycoprotein (e.g., Quorn), has been reported to contain has an ideally healthy fatty acid profile due to lower content of saturated fatty acid (40% PUFA, 11% MUFA, and 11% saturated fatty acid). PUFA in 100 g mycoprotein consists of 4.3 g linoleic acid and 6.9 g linolenic acid (Finnigan et al., 2017). Tempe before frying and miso also have been claimed to have low content of saturated fatty acid and no cholesterol (Dinesh Babu et al., 2009; Okouchi et al., 2019). 376 Current Developments in Biotechnology and Bioengineering 4.4 Other health aspects/functional properties The high fiber content of mycoprotein has been shown to lower total and low-density lipoprotein (LDL) cholesterol, promote satiety, and attenuate the glycemic response when eaten together with carbohydrate-rich foods (Finnigan et al., 2017). Soy sauce could increase gastric juice production, lower blood pressure, exhibit antimicrobial activity against several pathogens, and show antioxidant, anticarcinogenic, and anticataract activities (Kataoka, 2005). Several reports advocated functional properties of tempe, namely lowering the risk of strokes and heart disease, preventing osteoporosis, decreasing the risk of cancer and digestion problems, including decreased risk of heart disease and strokes, osteoporosis, cancer, and digestive disorders, and lessening the symptoms of menopause (Dinesh Babu et al., 2009). Miso has reportedly been linked to prevention of metabolic disease, possessing antioxidant activity, and suppression of visceral lipid accumulation (Okouchi et al., 2019). Gamalost cheese was reported to possess angiotensin I converting enzyme (ACE) inhibitor activity (Qureshi et al., 2013). 5. Safety of fermented foods using filamentous fungi 5.1 Mycotoxins Section 2.2 has shown the utilization of filamentous fungi in food fermentation. Most of the foods have been consumed for decades and historically assumed as safe. However, the fermentation conditions which tend to support microbial growth (room temperature, high aw, neutral pH, long incubation time) have prompted several studies to evaluate their safety, i.e., from the toxigenic molds and fungi similar to the ones used and from the potential bacterial contamination that may render the foods unsafe. Several fungi belonging to the genera of Aspergillus, Penicillium, and Fusarium have been reported to produce toxic secondary metabolites known as mycotoxins. Mycotoxins are generally heat stable thus capable of withstanding heat commonly applied in food processing and they have been implicated with hepatocellular carcinoma, esophageal cancer, human nephropathies, liver cancers, increased mortality and morbidity, child undernutrition, and stunting. Production of mycotoxins is supported by warm temperature and high humidity. Control of the molds in agricultural commodities has to be carried out through Good Practices since they are in the field, during storage, processing, and their transport or distribution. Aspergillus flavus, A. parasiticus, and A. nomius have been reported to produce aflatoxins B1, B2, G1, and G2 (Payne and Brown, 1998). Recently, aflatoxins were also reported to be produced by other aspergilli such as A. bombycis, A. ochraceoroseus, A. pseudotamari, A. tamari, as well as Emerciella astellata, dan Emerciella venezuelensis (Kumar, 2018). Aflatoxin B1 is the most toxic (Luning et al., 2007) of the four toxins and is classified as class 1 carcinogen (IARC, 1993). Aflatoxin B1 has been linked to hepatocellular carcinoma especially in the community with endemic hepatitis B virus (Kew, 2013). These fungi can grow Chapter 12 • Filamentous fungi for food 377 on ground nuts and cereals thus there is a concern for mycotoxin production in the foods fermented by filamentous fungi. At present, total aflatoxins in cereals and nuts is regulated at maximum of 15 ppb (Codex Alimentarius Commission, 2019). Several countries in Europe apply more stringent limits while other countries such as the USA, Indonesia, or India has more lenient limits at 20–30 ppb. Other toxigenic aspergilli, such as Aspergillus ochraceus, A. carbonarius, A. niger, along with Penicillium verrucosum have been reported to produce ochratoxins. Ochratoxin A, the most toxic member of ochratoxins, is reported to cause renal diseases in humans and is classified as a possible human carcinogen based on animal studies (Reddy et al., 2010). The toxins have been found in various foods such as cereal grains, grape, wine, salted fish, coffee beans, etc. (Bui-Klimke and Wu, 2015). Fungi from the genus Fusarium have been reported to produce various mycotoxins. Fumonisins are produced by Fusarium verticillioides, F. proliferatum, F. oxysporum, F. globosum, and other Fusarium spp., as well as by Alternaria alternata f. sp. lycopersici (Scott, 2012). Of the many fumonisins, fumonisin B1 (FB1) is the most frequently found and has been reported in various foods such as corn, rice, cowpea seeds, triticale, sorghum, beans, soybeans, asparagus, and beer. FB1 is categorized as possible carcinogenic to humans (IARC, 1993) and is linked to esophageal cancer (Marasas, 2001) as well as neural tube defect (Missmer et al., 2006). Trichothecenes are groups of mycotoxins produced by Fusarium, Myrothecium, Stachybotrys, Trichothecium (Polak-Sliwiniska and Paszczyk, 2021). Trichothecenes are a family of more than 200 toxins and classified into type A (T-2 toxin, HT-2 toxins), type B (e.g., deoxynivalenol or vomitoxin), type C (e.g., crotocin), and type D (e.g., verucarrin) (McCormick et al., 2011). Fusarium is found especially in wheat, barley, oats and maize, Myrothecium species in muskmelon and tomato and Stachybotrys sp. has been correlated with damp building-related illnesses. Meanwhile, Trichoderma species have been associated with mushrooms and grapes diseases and Trichothecium species are commonly found in the soil and on decaying organic materials. Fusarium graminearum, F. culmorum, F. cerealis, F. equiseti, and F. verticillioides also produce zearalenone (ZEN), mycotoxins with potent estrogenic activity in animals. In a study simulating tempe production using soybean and maize contaminated with ZEN, ZEN was metabolized by Rhizopus and Aspergillus into a-zearalenol (a-ZEL) and ZEN-14-sulfate, respectively. These metabolites are significantly more toxic than ZEN, thus increase the risk of intoxication from fermented foods (Borzekowski et al., 2019). On the contrary, during soaking of aflatoxin-contaminated maize in doklu fermentation, ^ te d’Ivoire dish, the number of aflatoxins was reduced by 72% (Assohoun et al., a local Co 2013). The author suggested possible aflatoxin removal by lactic acid bacteria involved in the fermentation; thus, resulting in lower health risk. Similarly, Adebiyi (2019) proposed that microorganisms involved in fermentation could decrease the level of mycotoxins through physical binding, noncovalent binding, adsorption/absorption, attacking the functional group responsible for toxicity, biotransformation (e.g., glycosylation), degradation, reduction by fermentation metabolites. On the other hand, biogenic amines (BAs) and aflatoxin have been detected in soy sauce. As the koji fermentation occurs spontaneously in traditional soy sauce, the fungi 378 Current Developments in Biotechnology and Bioengineering involved in this process are not controlled, hence some of them are mycotoxin producers. Aspergillus parasiticus is reported to produce aflatoxin during soy sauce fermentation (Maing et al., 1973). In addition, Sukhla and Kim reported the occurrence of BA and aflatoxin in soy sauce in the range of 2.80–20.42 mg/L and 0.00–4.80 μg/kg, respectively (Shukla and Kim, 2016). Similarly, it has been reported that 188 out of 209 (89.95%) of soy sauces in China were contaminated by aflatoxin B1, in which only two samples exceeded the maximum permitted limit of European Union and all of them were below the Chinese regulation level (Xu et al., 2012). A safety issue related to oncom is the presence of aflatoxin which might be present in the raw material or during fermentation. The maximum limit of aflatoxin in oncom is 20 ppb (INSC, 2009), while the oncom made from peanut press cake, okara, cassava waste contain 5.14–6.77 ppb aflatoxin, hence they are safe for consumption (Wikanta, 2019). 5.2 Other harmful contaminants Several safety issues related to soy sauce are the presence of 3-chloropropane-1,2-diol (3-MCPD), aflatoxins, and BAs. There are two types of soy sauce namely biological soy sauce which is made by fermentation as explained above and chemical soy sauce which is produced using acid hydrolysis. The chemical soy sauce can be made only in few days as it employs acid hydrolysis conducted at high temperatures to degrade the protein. This kind of soy sauce is reported to contain 3-MCPD, a by-product during hydrolysis (Lee and Khor, 2015). 3-MCPD is classified as carcinogenic by International Agency for Research on Cancer (IARC) which is probably carcinogenic to human and cause cancer, birth defects, and other reproductive harm. 5.3 Pathogenic bacteria in fermented food A challenging study suggested the safety risk of tempe when contamination occurs during production (Tanaka et al., 1985). Tempe made from unacidified soybeans artificially inoculated with different Clostridium botulinum posed the risks of toxins formation when incubated in vacuum packages after fermentation and steaming. Inoculation of Staphylococcus aureus prior to fermentation resulted in >106 CFU/g increase while inoculation after fermentation and steaming led to an increase in the bacterial counts up to 108 CFU/g and detection of S. aureus enterotoxins in some samples. Additionally, Salmonella typhimurium also grew well during the fermentation (>106 CFU/g increase in 1 d), although it grew relatively slowly at 25 and 15°C in tempe inoculated after fermentation and steaming. The study recommends that high level of hygiene during tempe fermentation and refrigeration (5°C) of the product following fermentation are to be applied to prevent potential outbreaks (Tanaka et al., 1985). In the USA, an outbreak (n ¼ 8) due to Salmonella paratyphi linked to tempe consumption was reported in 2012 (Griese et al., 2013). The S. paratyphi was also found in the starter culture and this poses a risk of contamination. Concerns pertaining food safety hazard of tempe have been reported in the USA. It is suggested that the starter culture was contaminated with S. paratyphi (Marasas, 2001), and prompted the authority to recall the contaminated batch of tempe. Chapter 12 • Filamentous fungi for food 379 Tempe with the most food safety concerns is tempe bongkrek, i.e., tempe made from coconut press cake (instead of soybean) which is fermented using Rhizopus oligosporus. Tempe bongkrek poisoning is caused by bongkrekic acid produced by contaminating bacterium Burkholderia gladioli (previously known as Burkholderia cocovenenans). An outbreak due to consumption of tempe bongkrek has caused death of nearly 2000 people since 1951. In addition, the endosymbiont bacterium of Rhizopus microsporus used for making tempe such as Burkholderia produces a toxin, namely rhizonin which is a hepatotoxic cycloprotein (Partida-Martinez et al., 2007). 6. Industrial production of filamentous fungi-based food Filamentous fungi have been applied as food source worldwide. In the Western world, the fungal biomass is mainly used to produce meat-free protein such as Quorn. Meanwhile in Asia, several filamentous fungi play an important role in production of fermented food. In this section, industrial production is divided into two groups, namely traditional and modern processes. 6.1 Traditional processes of food from filamentous fungi 6.1.1 Tempe Tempe is one example of filamentous fungi-based product which is mainly produced by traditional methods. In Indonesia, there are over 100,000 tempe producers representing mostly small and medium enterprises with a capacity of 10–2000 kg soybean per day. Tempe has a significant contribution to economic growth of Indonesia as it creates millions of direct and indirect jobs with business turnover of billions USD. Several businesses are related to tempe including supplier, distributor, transporter, and retailer. Approximately half of soybean usage in Indonesia is for tempe productions (PUSIDO Badan Standardisasi Nasional, 2012). Kopti is an association of tempe enterprises in Indonesia that established in 1975 to assure the quality of tempe material and provide training for its members on how to produce tempe. Tempe is one of the staple proteins for Indonesia mainly due to its affordable price. The raising of global attention on tempe is mainly driven by its nutritional values as plantbased protein. Tempe is introduced in other countries mainly by Indonesian immigrants although in some cases by their citizens who visited or lived in Indonesia. Tempe was firstly available in the European Market in 1946, since then the number of tempe factories increased and in 1984, 18 tempe factories were reported. Similarly, in America, the number of tempe companies raised by more than four times, from 13 to 53 during 5 years (1979–84) (Shurtleff and Aoyagi, 1984). This fact makes tempe to be the fastest-growing soyfood market in the USA. Tempe and its derived products that dominate the US sales include regular soy tempe (33%), tempe burgers and other second-generation tempe products (48%), and soy and grain tempe (19%) (Shurtleff and Aoyagi, 1984). In 1984, the eight largest companies outside its origin country were located in Japan, The Netherlands, and the USS with average weekly production between 2100 and 6885 kg/week (Shurtleff and Aoyagi, 1984). 380 Current Developments in Biotechnology and Bioengineering In general, the process of making tempe consists of soaking, boiling, dehulling, cooling, inoculation, packaging, and incubation. In traditional methods, dehulling is commonly done using wet method, i.e., by soaking the beans to loosen the hull and let the hull floating or with dry method by mechanical abrasion. Meanwhile at the industrial scale, dehulling is done using motor-driven disc impactor and fermentation is done in a tray (Nout, 2005). In traditional production, two different starter cultures are generally used: laru, i.e., fungi grown in cooked rice then dried and usar, i.e., fungi grown on certain leaves and let dry (Fig. 3). The diversity of tempe microflora leads to inconsistency of tempe quality which becomes a problem in large-scale production. 6.1.2 Soy sauce The top producer of soy sauce is China which contributes to more than 60% of the global € kenberg, 2017). Soy sauce is made by traditional small-scale industry and in production (Ho a large-scale modern factory. Aroma is a crucial property in soy sauce affecting consumer selection and distinguishes soy sauce from different origins. Aroma is generated during fermentation and it is influenced by raw materials, processing methods, and microorganism. In traditional soy sauce production, the koji and moromi fermentation occur spontaneously using an indigenous microflora as the processes are performed in nonsterile conditions. The microbial community in traditional soy sauce may be more complex than in the controlled industrialized process, thus they produce distinctive flavors which makes traditional soy sauce to be sold at a higher price (Yang et al., 2017). However, as the microflora is poorly controlled, the quality of the product is inconsistent and consequently, the market share of traditional products has been constantly diminishing. Soy sauce is mainly made from yellow or black soybean. The process and equipment used for traditional soy sauce are presented below: a) Sortation and boiling The sortation is conducted manually mainly to remove gravel and other contaminants. The soybean is then soaked overnight. The soaked soybean is then boiled for 1–5 h. The aim of this step is to soften the beans; hence, the mycelium of the fungi can be easily penetrated into the bean and hydrolyze the protein into amino acids and flavoring compounds that contribute to the taste of the soy sauce. This step is also essential to reduce the pathogens in the soybean and to increase the digestibility of the protein. After boiling, the softened bean is rapidly spread in a bamboo tray to remove the excess water and heat to prevent spoilage (Sardjono, 2016). b) Koji fermentation After the temperature of the bean reaches room temperature, sometimes it is mixed with wheat or rice flour. The bamboo tray is then covered by other bamboo trays, gunny sacks, or rice straw for approximately a week. This step is known as koji fermentation which is aimed to form the flavor compound. In traditional soy sauce production, the koji fermentation occurs spontaneously by microbes living in the air, from previously used trays and Chapter 12 • Filamentous fungi for food 381 FIG. 3 Production of traditional tempe culture “Usar”: (A) Hibiscus tiliaceus leaves; (B) spreading the boiled soybean on the leaves; (C) covering the soybean with the leaves; (D) incubation at room temperature; (E) sporulation; and (F) ready to use Usar (personal documentation taken by Anang Juni Yastanto). covered materials. The water activity (aw) is set at 0.95–0.97 to obtain optimum condition of koji fermentation. If the aw is lower, the fungi start to form spores which cease the enzyme production. During koji fermentation, the temperature increased, thus it needs agitation. During fermentation, the oxygen diffuses slowly to grow and prevent sporulation. After sufficient growth of the molds, the koji product is crumbled to remove the 382 Current Developments in Biotechnology and Bioengineering mycelium followed by sun drying. The dried beans are then put in plastic, ceramic, and wooden vessels (Sardjono, 2016). c) Moromi fermentation In this stage, a brine solution is added to the dried koji product in the vessel with a ratio of 1 part of koji product to 4–6 parts of brine solution. The brine solution is made by dissolving crude sea salt in water. The vessel is put outside under the sun and stirred regularly. It is covered during rainfall and at night to maintain the temperature. This process aims to enhance the flavor formation, prolong the shelf-life, and select appropriate microorganisms. The brine solution varies from 18% to 24%. The fermentation duration varies from months to years. It has been suggested that a period of 150 days is optimum for flavor formation in traditional Chinese-type of soy sauce (Gao et al., 2010). d) Filtration and cooking The brine is then filtered using a cloth and subjected to cooking. Cooking is aimed to kill pathogens and improve the shelf life. In traditional soy sauce fermentation, cooking is commonly conducted at 60–80°C for 30 min. However, it is recommended to cook at 80°C for 30 min to obtain the optimum flavors (Gao et al., 2010). In Indonesian soy sauce, caramelized palm or cane sugar is added during cooking to make a sweet soy sauce which is a more common type of soy sauce. Besides sugars, sometimes spices, monosodium glutamate, and thickening agent are also added at this stage. After cooking, the solution is filtered again and subjected to bottling. 1 kg of soybean could produce approximately 10 L of soy sauce. 6.2 Modern processes 6.2.1 Modern process of tempe production The number of modern tempe factories has been raising consistently. As the largest tempe producer in the world, approximately 2.4 million tons of tempe were produced per day in Indonesia in 2012 (PUSIDO Badan Standardisasi Nasional, 2012). There are several methods for tempe productions in modern tempe factories, which are mainly classified into dry and wet method depending on the method of dehulling. In the wet method, the soybean is firstly soaked followed by dehulling manually or using mechanical rubber, hull removal, and soaking, while in the dry process the soybean is dehulled by mechanical abrasion, followed by hull removal and soaking. The remaining process for wet and dry processes are similar. They include cooking, inoculation, packaging, and incubation. Wet process offers an advantage as it is conducted manually by workers so it does not require a major equipment and the mechanical damage of the soybean can be minimized. However, this process is considered less hygienic and significantly higher water usage compared to the dry method. Generally, the step of making tempe in modern process is similar to that of traditional one. However, in modern processes, some factories use dry method, inoculum powder, Chapter 12 • Filamentous fungi for food 383 and higher sanitary level of the production process. Modern tempe factories use a standardized inoculum in the form of powder which contains a single culture of a certain strain of Rhizopus, thus the quality is more homogenous. In addition, the higher sanitary level in modern tempe production is applied by using stainless-steel food-grade equipment and practicing personal hygiene for the workers. The different hygienic and sanitary level of tempe production is related to the presence of biologically active compound in tempe. Tamam et al. (2019) reported that tempe produced in a good sanitation level has more bioactive peptides (with antihypertensive, antidiabetic, antioxidative, and antitumor properties) than that of tempe produced using moderate or poor sanitation level. This might be due to that the metabolite profiles are mostly influenced by the starter culture (Kadar et al., 2020), hence the direct physical contact of the worker’s hand with the soybean which introduces the contamination should be avoided. In brief, several steps of tempe production in modern factories include soaking, boiling, dehulling, cooling, inoculating, packaging, and incubation (Fig. 4). The soybean is soaked overnight in a steel vessel and it is followed by boiling for 30 min using large pressure cooker and continuous soybean cooking machine, dehulling using a grinder followed by soaking for 24 h. Subsequently, the bean is boiled for 1 h and drained in a perforated steel table. After cooling, 1 kg of inoculum is added to 1500 kg soybeans. The inoculated soybeans are then transported on a conveyor to a package machine that divided the soybeans by weight into perforated plastic bags. Incubation takes place for 24 h. Soaking 1 Boiling 1 Incubation Packaging Dehulling Inoculation Soaking 2 Boiling 2 FIG. 4 Modern tempe processing (pictures taken from tempe factory UD Super Dangsul, Yogyakarta, Indonesia). 384 Current Developments in Biotechnology and Bioengineering 6.2.2 Production of Quorn Large-scale production of Quorn is started by growing F. venenatum on media containing glucose, vitamins, trace minerals, and ammonia. The fermentation takes place in an 14,000 L airlift fermenter operated in a continuous mode. The freeze-dried culture of Fusarium venenatum is grown in a lab-scale fermenter prior to transfer to the main airlift fermentor. Several fermentation parameters are strictly controlled including pH, temperature, dissolved oxygen, nutrient concentration, and growth rate (Trinci, 1991). The fermentation temperature is kept at 28–30°C and the pH is maintained at 6.0. These conditions resulted in a specific growth rate of 0.17–0.20 h 1 and yielded 300–350 kg biomass h 1 (Wiebe, 2002). The fermentation usually occurs for 6 weeks. The fermentation broth is heated at optimum temperature of 72–74°C for 30–45 min to reduce the RNA content from 10% to 1%, which is below the limit required by the World Health Organization (2%). Subsequently, the mycelial biomass is heated at 90°C for preservation. It is followed by centrifugation to collect the biomass and further concentrated by vacuum chilling to obtain 24% of total solid (Finnigan, 2011). At this point, the mycoprotein is ready to be further processed into Quorn food. To create Quorn piece and mince, the processing steps are similar, however, the ingredient is slightly different. The processing steps include mixing, forming, cooking, and freezing. The mycoprotein is firstly mixed with egg albumin, malt extract, and water for making Quorn mince, however, the malt extract is replaced by natural flavor in Quorn piece. The egg albumin is needed to form fibrous bundles. Subsequently, the temperature is increased to 90°C in order to denature the egg, which is then fixed the texture after forming. It is followed by freezing at 10°C for 30 min to settle the fibrous bundle. The ice crystal growth during the freezing force the mycelium together and create the fibrous bundle. This fibrous bundle creates an eating quality of meat which differentiate Quorn from other meat-free protein. After freezing, the product is packed and kept in cold storage. The production scheme is illustrated in Fig. 5. FIG. 5 Production of mycoprotein. Adapted from Finnigan, T.J.A., 2011. Mycoprotein: origins, production and properties. In: Handbook of Food Proteins. Woodhead Publishing Limited. https://doi.org/10.1533/9780857093639.335. Chapter 12 • Filamentous fungi for food 385 7. Conclusions and perspectives Filamentous fungi holds a wide applications in food due to its high nutritional quality with low environmental impact. Although their excellent properties, development of filamentous fungi-based food faces some challenges including the mycotoxin issue, allergen, neophobia, and high production cost. Several attempts hence are needed to address those challenges such as avoiding mycotoxin-producing fungi, conducting comprehensive allergenic studies, formulating filamentous fungi into tasty familiar products, involving an influencer, role model, or officials in the health and food sector for promotion by explaining the nutritional value, safety, and sustainability. The introduction of filamentous fungi in the market as a nutritious family member of mushroom and truffle could eliminate the new phobia. Advanced technologies which could decrease the production cost are needed to increase the acceptance of this product in the market. In addition, filamentous fungi have been shown to give a positive impact on human health thus it becomes a good candidate as a functional food. Furthermore, filamentous fungi could grow in agricultural waste and food by-products which convert low-cost material into nutritious, healthy, and tasty products. The excellent nutritional value together with its environmentally friendly protein would drive the demand for this protein, particularly if it is supported by policies and regulations. Acknowledgment This work was supported by the Ministry of Research and Technology of Indonesia through the grant of PDUPT with the contract number of 2847/UN1.DITLIT/DIT-LIT/PT/2020. References Adebiyi, J.A., 2019. 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Introduction There is no doubt that providing food for the population is one of the biggest challenges concerning the governments for the future. This issue will be more critical when reports show that population will grow beyond 9 billion people by 2050 (FAO, 2018). Supplying huge amount of food and energy resources for this growing population from one side and generation of giant amount of waste from other side, make the situation even more complicated. Farm products, including animal-based protein producing sector is cooperated as a significant part of protein supplying for human. However, putting pressure on domestic animal farms in order to increase production, concomitantly rises the need for high quality feed ingredient. Among feed ingredients, protein source ingredients are the most valuable component. Utilization of food-grade protein source ingredient such as fishmeal and other agricultural products, e.g., soybean meal, sunflower meal, etc., for animal feed is not sustainable. Fishmeal, the most common protein source ingredient, is commonly produce from pelagic fish species such as anchovy, sardine, jack mackerel, or capelin, which their stock resources declined since last decades (FAO, 2018). Limitations of supply, rapid expansion in different farming sectors and increase in the demand has resulted in incredible increase in fishmeal price. Even though, different alternative protein sources such as soybean meal have introduced as new feed ingredient, these feedstuff do not meet the nutritional requirements of farmed animals. On the other hand, competition with their application as food, limited land, and water resources for agricultural purposes are still obstacles in the way of these alternatives as suitable resources for animal feed (Karimi et al., 2018). Therefore, a great effort has been given to find an appropriate alternative protein source ingredient for animal feed. Various commercial feed recipes assigned to different animals, contain protein, fat, minerals, vitamins, etc. Feed composition and formulation is greatly affected by several biotic factors, e.g., animal species, life stage, health status, etc. (NRC, 2011). Feed ingredient, however, generally vary from simple elements to complex compounds and chemicals. However, as the basic classification, they grouped in three macronutrient groups, protein, fat, and carbohydrates. It must be taken into account that a variety of additives Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00002-8 Copyright © 2023 Elsevier Inc. All rights reserved. 399 400 Current Developments in Biotechnology and Bioengineering such as vitamins, minerals, antioxidant agents, etc., need to be added to the recipes to meet all nutritional requirement. From both animal health and growth standing points, protein source ingredient is the most important part in the feed. In addition, since protein source ingredient is the most expensive component composing animal feed, it can be as key factor determining final feed production cost (Sanchez-Muros et al., 2014). Inclusion of ingredients entitle additives can enhance feed quality and lower feed production cost. Recently, filamentous fungal biomass has proposed as one of promising renewable protein sources with the application in animal feed (Karimi et al., 2018). In this chapter different aspects of its feed application will be discussed. 2. Animal compound feed The term “compound feed” refers to the feed type that formulated based on the nutritional requirements of each individual animal (Hardy, 2010). Nutritional quality of feed attributed to the specific animal can be evaluated previously in the experiments and accordingly a range of different raw material of varied sources are mixed and processed by processing equipment. Development in the knowledge of animal specific requirements in the terms of amino acids, fatty acids, minerals, and vitamins, resulted in formulating more accurate diet for each animal species. There is no doubt that protein supply is the most important part in animal feed production. Currently, fishmeal is the preferred protein supplement to the animal diet and therefore, its nutritional properties is described in next section. 2.1 Fishmeal Fishmeal is a nutrient-rich feed ingredient with high digestibility that is the preferred animal protein supplement in the diets of domestic animals, especially fish and shrimp (Bimbo and Crowther, 1992). It contains high quantities of energy per kilogram and is a unique source of protein, lipids (oils), minerals, and vitamins. Fishmeal can be manufactured from wide range of aquatic animals but is generally made from wild catches, small marine fish that contain a high percentage of bones and oil, and therefore, they not consider for direct human consumption. These fishes can be termed as “industrial fish” since most of them are caught with the only purpose of fishmeal and fish oil production. Highquality fishmeal normally contains between 60% and 72% crude protein by weight. Typical diets for fish may contain from 32% to 45% total protein by weight, and diets for shrimp may contain 25%–42% total protein. The percentages of inclusion rate of fishmeal in diets for carp and tilapia may be from 5% to 7%, and up to 40% to 55% in trout, salmon, and some marine fishes. A typical inclusion rate of fishmeal in terrestrial livestock diets is usually 5% or less on a dry matter basis (FAO, 2018). Any complete diet must contain some protein, but the nutritional value of the protein relates directly to its amino acid composition and digestibility. The amino acid profile of fishmeal is what makes this feed ingredient attractive as a protein supplement. Although Chapter 13 • Filamentous fungi as animal and fish feed ingredients 401 most of the oil usually gets extracted during processing of the fishmeal, the remaining lipid typically represents between 6% and 10% by weight but can range from 4% to 20% (Olsen and Hasan, 2012). Fishmeal is an excellent source of the essential polyunsaturated fatty acids (PUFAs) in both the omega-3 and omega-6 families of fatty acids. Fishmeal and oil contain more omega-3, than omega-6 fatty acids. In contrast, most plant lipids contain higher concentrations of omega-6 fatty acids. Fishmeal also contains valuable phospholipids, fat-soluble vitamins, and steroid hormones (Kris-Etherton et al., 2002). 2.2 Economic and environmental aspects of fishmeal as feed ingredient The fishmeal and fish oil industries are among few major animal industries existing nowadays that still relies greatly on a wild catches. The supply is currently stable at 6.0–6.5 million tons yearly. Roughly, 4–5 tons of whole fish are required to produce 1 ton of dry fishmeal (Olsen and Hasan, 2012). The majority of fishmeal produced is incorporated into commercial diets fed to fish, shrimp, swine, poultry, dairy cattle, and other animals such as mink (Fig. 1). It is unlikely that supplies of commercially available fishmeal and oil will be able to keep pace with the projected increase in worldwide production of aquaculture and terrestrial animal feeds. In most recent years, aquaculture has used approximately 46% of the total annual fishmeal production, a figure that is expected to rise as demand for aquaculture products increases in the next decade (FAO, 2018). The best approach in feed formulation is to use high-quality feedstuffs to manufacture a diet that meets the nutritional and energy requirements of the aquaculture species in question. If a portion or all of the fishmeal in a diet can be replaced successfully with other high-quality protein sources, doing so will contribute greatly toward protecting the surrounding environment and promoting a sustainable aquaculture industry. New information on nutrient requirements of aquatic organisms coupled with advances in feed technology indicates that species-specific fish diets can be made by partial or total replacement of fishmeal with other plant and animal proteins. FIG. 1 Comparison of the status of fishmeal consumption in different sectors of animal farming (between 2002 and 2010). Data collected from Olsen, R., Hasan, M., 2012. A limited supply of fishmeal: impact on future increases in global aquaculture production. Trends Food Sci. Technol. 27, 120–128. 402 Current Developments in Biotechnology and Bioengineering Balancing nutrients in diets by using the minimum amount of fishmeal to meet specific amino acid requirements for fast growth and reproduction and reducing feed costs constitute one of the principal objectives in formulation of fish feeds. The animal feed production industry, such as aquaculture industry, must continue to seek out alternative sources of high-quality plant and animal-based protein ingredients for their feedstuffs. Presently, this is an active area of research in aquaculture nutrition. If supplies of fishmeal do not increase, the “fishmeal trap” will start to constrain producers of shrimp and carnivorous fish as the world market price of fishmeal increases in response to increasing demand. As presented in Table 1, average inclusion of fishmeal in commercial compounded feed from 1995 to 2010 in major groups of farmed species had an impressive reduction. The increased knowledge has also resulted in improved feed conversion ratios (FCR). For example, the reported FCR of fed carps dependent upon industrial compounded aquafeed was 2.0 in 1995 and is predicted to be reduced to 1.7 in 2015, for marine shrimps from 2.0 to 1.5 and salmon from 1.5 to 1.3 (Olsen and Hasan, 2012). FAO (2018) reported that the inclusion of fishmeal would continue to decrease in compounded feed for farmed fish and shrimps in the future. From 2010 to 2020, the average levels of fishmeal in the feed are projected to be reduced further from 16% to 8% for shrimps, from 26% to 12% for marine fish, from 22% to 12% for salmon and from 2% to 3% to 1% in carps and tilapias. 2.3 Alternatives to the fishmeal Plant proteins have been and will probably continue to be the main choice as replacement of fishmeal in aquaculture diets. However, plant protein meals have several nutritional drawbacks compared to fishmeal particularly in diets prepared for the carnivorous species which are not adapted to plant feed. In addition to a relatively low content of proteins, the presence of antinutritional components will reduce the digestion or absorption of nutrients, counteract the function of vitamins and may even induce toxicity (Francis et al., 2001; Krogdahl et al., 2010; Tacon and Hasan, 2007). Soybean meal is a by-product of the soybean oil industry. Soybean meal is used as a source of protein by the animal feed industry, either for direct use at farm level, or blended in the mixed feeds. Soybean meal protein levels generally reach around 44%–48% of the total dry matter as compared to 62%–70% in the case of fishmeal (the richest source of protein available for feeding animals). Soybean and other legume meals, which are widely used in the diets of most farm animals such as pigs and chickens, are a good source of lysine and tryptophan but are limiting in the sulfur-containing amino acids methionine and cysteine (Bakke-McKellep et al., 2007). Plant-based proteins, even when properly processed, are usually not as digestible as fishmeal; and their inclusion rate into the diet is often limited as it results in depressed growth rates and feed intake (Karimi et al., 2019). Proteins in cereal grains and other plant concentrates do not contain complete amino acid profiles and usually are deficient in the essential amino acids (EAAs) lysine and methionine. Overall protein digestibility values for fishmeal are consistently above Chapter 13 • Filamentous fungi as animal and fish feed ingredients 403 Table 1 Production of main farmed fish in the world, commercial feed consumption, FCRa during the years 1995–2020. Species group Total production % on commercial feed Average FCRa % of fishmeal in feed Total feed usedb Total fishmeal usedb 1995 2005 2010 2015 2020 925 2664 4113 6043 8087 75 89 95 97 100 2.0 1.8 1.6 1.5 1.4 28 24 16 12 8 1387 4268 6251 8793 11,322 388 1024 1000 1055 906 1995 2005 2010 2015 2020 533 1402 2137 3140 4613 50 70 73 75 80 2.0 1.9 1.9 1.8 1.8 50 38 26 18 12 533 2050 2964 4239 6643 267 779 771 763 797 1995 2005 2010 2015 2020 537 1382 1734 2213 2825 100 100 100 100 100 1.5 1.3 1.3 1.3 1.3 45 35 22 16 12 806 1796 2255 2877 3672 363 629 496 460 441 1995 2005 2010 2015 2020 5154 9100 11,670 14,190 16,459 20 45 50 55 60 2.0 1.8 1.8 1.7 1.6 10 8 2 1 1 2062 7371 10,503 13,275 15,801 206 590 210 133 158 1995 2005 2010 2015 2020 704 1980 3386 5453 8012 70 80 85 90 95 2.0 1.8 1.7 1.6 1.6 10 8 3 2 1 985 2852 4893 7852 12,178 99 228 147 157 122 5773 18,337 26,866 37,036 49,613 1323 3250 2624 2568 2424 Year Marine shrimp Marine fish Salmon Fed carpc Tilapia Sum of all five groups 1995 2005 2010 2015 2020 a 7853 16,528 23,040 31,047 39,996 FCR: feed conversion ratio (total feed intake/total increase in biomass). In 1000 tons. c Excluding silver carp, bighead carp, and Indian major carp. Data adapted from Tacon, A., Hasan, M., 2007. Global synthesis of feeds and nutrients for sustainable aquaculture development. Study and Analysis of Feeds and Fertilizers for Sustainable Aquaculture Development, vol. 497. FAO, pp. 3–17. b 404 Current Developments in Biotechnology and Bioengineering 95%. In comparison protein digestibility for many plant-based proteins varies greatly, for example, from 77% to 96%, depending on the species of plant (Tacon, 2004). The structural nature of plants is totally different from that of animals. Proteins isolated from plants are associated with indigestible nonstructural carbohydrates (oligosaccharides) and structural fiber components (cellulose), which are not associated with animal proteins. Presence of these components is thought to be contributing obstacles to efficient utilization of proteins in many economically plant-based feedstuffs. The lack of nutritional inhibitors or antinutritional factors in fishmeal also makes this meal more attractive than plant proteins for use in aquaculture diets. Antinutritional factors are compounds that interfere with nutrient digestion, uptake, or metabolism and can also be toxic. For example, a naturally occurring antinutritional factor in uncooked soybeans is the Kunitz trypsin-inhibitor that prevents the enzyme trypsin from breaking down dietary proteins in the intestine of animals. Lathyrogens in chickpeas also disrupt collagen formation. Collagen is the most abundant protein present in animals, making up most of connective tissue and providing structural support. Another very important problem with plant-based protein alternatives for fishmeal is low acceptability (palatability) by animal resulted in nutrient leaching and low feed intake (Francis et al., 2001; Krogdahl et al., 2010). Apart from plant proteins, there are other protein sources which may substitute for fishmeal in aquaculture feed. Terrestrial animal by-product meals such as meat and bone meal, blood meal, and poultry by-product meals are considered feed ingredients of good nutritional quality. It has been estimated that potential quantities of these by-products are two to three times higher than that of fishmeal (Tacon and Metian, 2015). For safety reasons, it is important animal feeds of the same animal species. The ban on using by-products from warm-blooded animals in fish feed in many countries stems from the fear of transmissible spongiform encephalopathies (TSEs) disease transmission (Tacon and Hasan, 2007). Varieties of animal by-products have been used as protein sources for livestock. As high-quality protein sources, blood meal, and plasma protein (spray dried plasma) have been used successfully in nursery diets, and have also been used to stimulate feed consumption in early weaned pigs (Hansen et al., 1993; Kats et al., 1994). However, blood products, and also milk products, are expensive and more likely existence of pathogens limited their application as animal feed ingredient. Recently, single-cell proteins (SCPs), a novel protein source, has introduced as promising alternative ingredient for fishmeal. Different nutritional characteristics of this valuable protein source is discussed in next sections. 3. Single-cell proteins (SCPs) The term SCP is referred to the bulk of dead, dried biomass of single-cell organisms (Nalage et al., 2016). SCP can be prepared from different organisms, e.g., microalgae, bacteria, and fungi. SCPs enclose many positive nutritional characteristics such as high-quality proteins, vitamins, pigments, structural polysaccharides which make it potential to be used in the animal feed as sustainable and renewable feed component Chapter 13 • Filamentous fungi as animal and fish feed ingredients 405 ( Jones et al., 2020). Inclusion of SCP can address the nutritional shortcoming of inclusion of plant-based meals and reduce the need for fishmeal in the feed. In addition to the nutritional properties, SCP is considered as promising pre/probiotics (Glencross et al., 2020). General properties for the organisms to be considered as suitable SCP is listed below (Anupama and Ravindra, 2000): – – – – – No infectious to the plants and animals Capable to include in feed/food Containing of high nutritional value components No toxic compounds Low production cost Recently developed SCP-grade food product, Mycoprotein, is an alternative product for meat which is produced from filamentous fungi, Fusarium venenatum. A well-known market brand Quorn is commercially approved to supply as food since 1983 and nowadays is accepted by costumers as high quality food. Introduction of SCP-based animal feed ingredient in the same concept can aid animal farming industry and indirectly improve human food security by producing higher quality meat. Filamentous fungi are a protein-rich and fast growing microorganism. Its application as SCP has investigated by a few studies. For example, nutritional value of Rhizopus oryzae has explored and as a conclusion, researchers reported that dietary filamentous fungi can have positive effects on fish health (Abro et al., 2014; Bankefors et al., 2011). 4. Fungi kingdom The Kingdom fungi comprised by eukaryotic and heterotroph organisms accounting to around 1.5 million species. Based on the last taxonomical classification, in the Kingdom of fungi is divided into eight main phyla, Chytridiomycota, Zygomycota, Glomeomycota, Ascomycota, Basidiomycota, Blastocladiomycota, Microsporidia, and Neocallimastigomycota (Kendrick, 2017). According to the lifecycle, fungi can be categorized in three distinct groups, namely, unicellular, macrofilamentous and multicellular filamentous fungi. From the ecological standpoint, they play a very significant role in the nature, where they contribute to the recycling of nutrients (Wakai et al., 2017). Macrofilamentous fungi refer mainly to mushrooms and truffles. Many of them are commonly included in human diet due to beneficial nutritional composition and favorable taste (Boland et al., 2012). Multicellular filamentous fungi (often referred as molds) include some species industrially used for production of a wide range of products such as organic acids, enzymes, and antibiotics. Furthermore, some species have been used for production of food products. For instance, Aspergillus spp., Neurospora spp., Rhizopus spp., Fusarium spp., and Monascus spp. are categorized as generally recognized as safe (GRAS) and have food applications. For instance Sake, Shoyu, and Miso are the fermented products using Aspergillus spp. and Neurospora spp. have been used traditionally in some indigenous East Asian food, Oncom. Fusarium venenatum is the well-famous strain of edible filamentous fungi 406 Current Developments in Biotechnology and Bioengineering involved in mycoprotein production known as Quorn. During growth, microscopic spores elongate and branch out creating a 3D macroscopic structure easily recovered from the medium. The use of filamentous fungal biomass for food applications is related to the extended group of nutrients present. Those include high protein content, good PUFAs, vitamins, minerals, and antioxidant and immune stimulant components. Filamentous fungi are enabled to degrade complex substrates due to the production of various enzymes such as amylases, lipases, proteases, pectinases, phytases, etc. Considering their nutritional composition and range of substrates that can be used for growth, filamentous fungi have extensively been investigated for valorization of low-value substrates through their conversion into valuable products. One of the products produced, the filamentous fungal biomass, has increasingly been considered a potential alternative protein source. Filamentous fungi can be cultivated either in submerged cultivation of solid-state fermentation. Food products such as Quorn are produced through submerged fermentation, while tempeh, tofu, and oncom are produced via solid-state fermentation. The product obtained via solid-state fermentation is normally a mixture of fungal filaments and remaining substrate, whereas in submerged fermentation a purer filamentous fungal biomass can be obtained via submerged fermentation via close control of cultivations conditions and content of suspended solids. High content of the latter normally leads to entanglement with fungal filaments; hence influencing the final composition of the fungal biomass. This can have both positive and negative impacts depending on the final applications and composition of the suspended solids. 5. Fungal biomass composition as animal feed nutrient 5.1 Protein and amino acid profile Filamentous fungal biomass carry valuable amount of protein. As a general fact, protein level in highly related to the species as well as cultivation factors, e.g., substrate composition, oxygen availability, pH, etc., but 30%–50% (w/w) dry biomass of most of filamentous fungi is crude protein (Table 2) (Karimi et al., 2019). Therefore, fungal biomass is evaluated as suitable protein source ingredient for animal feed. From nutritional stand point, they are considered as most important constituent in all feed types both since proteins are included in many important biological and economic processes. Proteins connect to the wide range of functions within organisms, such as catalyzing metabolic reactions, deoxyribonucleic acid (DNA) replication, stimuli response, cell shape and structure and transporting molecules from one location to another. Hence, proteins are among essential macronutrient for every living organisms to maintain growth and health ( Joint FAO WHO UNU Expert Consultation on Protein and Amino Acid Requirements in Human Nutrition. Food and Agriculture Organization of the United Nations, World Health Oorganization, and United Nations University, 2007). Apart from protein concentration, protein quality is mainly related to the amino acid composition. In fact, true protein concentration is directly attributed to the amino acid profile in the biomass. In the other word, proteins are carriers for amino acids. The main Chapter 13 • Filamentous fungi as animal and fish feed ingredients 407 Table 2 Protein content present in the biomass originated from cultivation of filamentous fungi in various substrates. Protein content (% of dry weight) References 39 46 Barker et al. (1981) Jin et al. (1999) 50–60 30.4 28 47–63 Ferreira et al. (2012) Mitra et al. (2012) Liang et al. (2012) Wikandari et al. (2012) Spent sulfite liquor 30–50 Lennartsson (2012) Vinasse 49.7 Wheat ethanol stillage Corn ethanol stillage Wheat ethanol stillage 56 48 55 43 43 Nitayavardhana et al. (2013) Ferreira et al. (2014) Wheat bran Dairy waste 40 40 Yunus et al. (2015) Mahboubi et al. (2017) Wheat lignocellulosic residues 50 Nair (2017) Fungi Substrate Aspergillus oryzae Rhizopus oligosporus Palm oil waste Starch processing wastewater Spent sulfite liquor Corn ethanol stillage Corn ethanol stillage Tempeh Rhizopus sp. Mucor circinelloides Pythium irregulare Rhizopus, Mucor, Rhizomucor Mucor indicus Rhizopus sp. Rhizopus oryzae Neurospora intermedia Aspergillus oryzae Rhizopus sp. Rhizopus oligosporus Aspergillus oryzae Neurospora intermedia Rhizopus oligosporus Neurospora intermedia Aspergillus oryzae Neurospora intermedia Rasmussen et al. (2014) Bátori et al. (2015) functions of amino acids in the organism are (a) protein synthesis, (b) being substrate for necessary metabolite synthesis, e.g., peptides (glutathione, carnosine, etc.), neurotransmitters, NO, CO, H2S, and (c) energy supplier throughout amino acid oxidation (Deutz et al., 2014). However, they also incorporate in rebuilding of various type of proteins (Wu, 2016). Even though amino acids, indirectly have impact on animal health by providing metabolic compounds, it is confirmed that large number of amino acids can manipulate animal immunity system through different mechanisms. EAAs and conditionally EAAs must be provided by the diet because the organisms have not capacity to produce them in the body. In opposite, a number of amino acids can be synthesized in the body de novo (non-EAAs; NEAAs). These amino acids is more or less similar in animals including humans, while there is small differences between then regarding being essential or not (Table 3). Several parameters can affect amino acid synthesize in animal bodies, e.g., availability of substrate, animal species, life stage, physiological condition, gut microbiome, environmental factors, and health status of animal (NRC, 2011). The term “functional amino acids” (FAAs) refer to the given amino acids (arginine, glutamine, glycine, BCAA, proline, etc.) which are included in the regulation of significant physiological reactions can affect and enhance growth and health, effective reproduction success and tissue development. In this concept, even NEAA may consider as required compound to supply in the feed (Wu, 2010). 408 Current Developments in Biotechnology and Bioengineering Table 3 Profile of different groups of amino acids in different animals, namely in mammals, poultry, and fish (Wu, 2010; Halver, 2013). Mammals Poultry Fish EAA NEAA CEAA EAA NEAA CEAA EAA NEAA CEAA Arg Cys His Ile Leu Lys Met Phe Thr Trp Tyr Val Ala Asn Asp Ser Gln Glu Gly Pro Tau Arg Cys Gly His Ile Leu Lys Met Phe Pro Thr Trp Tyr Val Ala Asn Asp Ser Gln Glu Tau Arg Cys His Ile Leu Lys Met Phe Pro Thr Trp Tyr Val Ala Asn Asp Ser Gln Glu Gly Tau Beyond amino acid composition, balance in amino acid composition is another issue in protein suitability evaluation. Imbalanced feed in regard to amino acid profile may result in amino acid antagonism and toxicity, which in long-time period consumption can reduce feed intake, induce aggressive behavior and lower growth (Dwyer, 2003). Karimi et al. (2019) have studied amino acid composition of three filamentous fungi. As it is presented in Fig. 2 fungal biomass is rich in arginine, methionine, phenylalanine, threonine, glutamine, branched-chain amino acids (BCAAs: leucine, isoleucine, and valine), alanine, asparagine, cysteine, and tyrosine. In addition, the concentrations for proline and glycine, which are categorized as functional amino acids, are in higher level comparing with plant-based protein sources including soybean meal. Fishmeal and soybean meal, the two most usual protein supplementation sources in aqua-feed, contain 62%–70% and 46%–50% crude protein, respectively. Considering this, new protein sources, such as fungal biomass, with comparatively high-protein content (see Fig. 2 for a comparison), have the potential to be used as an alternative protein source for fishmeal and soya. 5.2 Fat and fatty acid content Filamentous fungal biomass, in similar to other fungi, contain fat which is accumulated intracellularly (Passoth, 2017). Different filamentous fungi species are capable to store various range of fats. For example, oleaginous fungi are capable to produce and store high concentrations of lipids, up to 80% of cell dry biomass. This characteristic, existence of lipids in this high level, makes them attractive source for broad spectrum of applications such as biofuel, chemical, and food/feed additives production. For instance, fatty acids Chapter 13 • Filamentous fungi as animal and fish feed ingredients 409 100% 90% 80% 70% 60% a 50% 40% 30% 20% 10% 0% AO NI RO FM Arginine Histidine Isoleucine Leucine Methionine Phenylalanine Threonine Valine SBM Lysine 100% 90% 80% 70% 60% 50% b 40% 30% 20% 10% 0% AO NI RO FM SBM Alanine Aspargine Cysteine Glutamine Glycin Proline Serine Tyrosine FIG. 2 Comparison of profiles of EAAs (A) and NEAAs (B) found in filamentous fungi (Aspergillus oryzae (AO), Neurospora intermedia (NI), Rhizopus oryzae (RO)), fishmeal (FM), and soybean meal (SBM) (Karimi et al., 2019). including omega-3 and -6, fatty alcohols, alkanes, and carotenes can be isolated from fungal biomass (Passoth, 2017). Lipids are among macronutrients together with carbohydrates and proteins. In general, they are energy sources for living cells, but since they are compartment of cell membrane, they play several vital roles in cell integrity and transportations (FAO, 2010). Furthermore, fatty acids are essential substances for many important processes in living organisms, which directly affect growth, heath, and performance. Lipids exist in the complex with other biocompounds and form different biomolecules such as lipoproteins and glycolipids which are critical in the cell functionality (Burlingame et al., 2009). 410 Current Developments in Biotechnology and Bioengineering Different types of fat, triglycerides, phospholipids, and cholesteryl esters, contain fatty acids. Fatty acids are important to keep cell functionality; hence, they are dietary necessary substances. Basically, fatty acids are formed in either saturated (saturated fatty acids; SFAs) or unsaturated fatty acids. Those of unsaturated fatty acids with more than one double bond PUFAs are valuable molecules in every organisms (Kim et al., 2016). Among PUFAs, the 18–20 or more carbon fatty acids are of particular importance (since plants and some organisms are not capable to synthesize them) and called long-chain polyunsaturated fatty acids (LC-PUFAs) which are nutritionally significant and this group have key roles in animal health maintenance (Blondeau et al., 2014). Filamentous fungi contain high value PUFAs; and therefore, regarded as suitable alternative source providing PUFAs in feed (Asadollahzadeh et al., 2018). Basically, animals including human are able to synthesize LC-PUFAs such as arachidonic acid (20:4n-6; AA), eicosapentaenoic acid (20:5n-3; EPA), and docosahexaenoic acid (22:6n-3; DHA) from C18 fatty acids. However, production rate is not sufficient, particularly in the case of incidence of disease (Bhardwaj et al., 2011). Therefore, either consumption of PUFAs as dietary supplement in the form of pure PUFA or PUFAs-rich products can boost health status. Filamentous fungal biomass, e.g., oleaginous fungi are reported to synthesize omega-3 FAs including EPA and DHA. For example, the genus Mortierella has found to synthesize wide range of PUFAs and gamma-linolenic acid (18:3n-6) was commercially produced using them. Arachidonic acid is commercially produced by M. alpine (Passoth, 2017). Karimi et al. (2019) studied ascomycetes and zygomycetes fatty acid content. Collected data from lipid analysis is presented in Fig. 3. Oleic acid, linoleic acid, and palmitic acid were exist in considerable concentrations and arachidonic acid, alpha linolenic acid (ALA) also were detected in lower amount. Optimization of cultivation conditions such as pH, temperature, aeration rate, etc., and utilization of suitable substrate for cultivation as well as selection of high potential fungi species in lipid synthesize and storage, can improve lipid and fatty acid production by filamentous fungi. Linoleic acid can bioconvert to the arachidonic acid and ALA is the precursor substrate for biosynthesis of EPA and DHA, most important derivatives of fatty acids in disease responses. Aforementioned LC-PUFAs, have several roles, which are mainly important that is described in the next section. 5.2.1 Arachidonic acid (AA) As a fundamental component of cell membrane, it is necessary element in all stages of cell development and growth specifically in bad cell conditions (Das, 2018). AA effect is mainly on membrane fluidity and permeability and therefore can control cell signaling which is done by control of in/out flux of protein to the cell. In addition, it can enhance ion entrance and exit to the cell throughout regulation of sodium-potassium channels (Seah et al., 2017). AA positively affect immune system by activation of immune cells such as eosinophils, neutrophils, macrophages, and consequently respiratory burst mechanism is approved as an impact of exogenous and endogenous supplementation of AA. Chapter 13 • Filamentous fungi as animal and fish feed ingredients 411 100% 90% 80% 70% 60% a 50% 40% 30% 20% 10% 0% AO NI RO FM SBM C14:0 (Myristic acid) C16:0 (Palmitic acid) C16:1 (Palmitoleic acid) C18:0 (Stearic acid) C18:1 (Oleic acid) C18:2 (Linolelaidic acid) C18:3 (Linolenic acid) C20:0 (Arachidic acid) 100% 90% 80% 70% b 60% 50% 40% 30% 20% 10% 0% AO % SFA NI % MUFA RO % PUFA FIG. 3 (A) Comparison of the profile of fatty acids found in filamentous fungi (Aspergillus oryzae (AO), Neurospora intermedia (NI), Rhizopus oryzae (RO)), fishmeal (FM), and soybean meal (SBM). (B) The fraction of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) is presented in the lower figure (Karimi et al., 2019). AA together with other three types of oxygenases: cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome p450 can react with molecular oxygen, which led to eicosanoids and other inflammatory compounds synthesize. AA and its derivatives such as prostaglandins (PGs) also are included in muscle cells generation (Gomez Candela et al., 2011). Bae et al. (2010) evaluated the impact of adding different levels of ARA in the diet of juvenile eel, Anguilla japonica, to investigate growth rates and carcass quality. After 12 weeks feeding trial, growth promoting (both specific growth rate (SGR) and weight gain (WG)) have been reported as result. In addition to growth promoting effect, it has been found that dietary ARA, improve reproduction success in fish. Viability of larvae, higher 412 Current Developments in Biotechnology and Bioengineering hatching success and increase in egg size are the main improved features in yellow tail, Seriola dorsalis fed with ARA in the diet (Stuart et al., 2017). Turbot juvenile fish fed with the diet containing ARA as a single HUFA (0.78% of diet dry weight) showed better growth rate and survival when these parameters compared with the fish fed with reference diet (Castell et al., 1994). In the other study, gilthead sea bream, it has been confirmed that, feed containing higher levels of ARA can enhance fish growth and survival (Fountoulaki et al., 2003) and immunity system (Koven et al., 2001). Fountoulaki et al. (2003) stated that 1% inclusion of ARA can improve fish growth significantly, while fish fed with 1.8% extra ARA had higher survival rates. Koven et al. (2001) found that when fish (gilthead sea bream) larvae fed with the ARA-enriched rotifers, they were more resistant against acute stresses such as handling, etc. 5.2.2 Linoleic acid (LA) In parallel to the elucidation of relationship between diet and health, a new idea has emerged as functional lipids. LA is 18-carbon chain FA that contains two-double bounds and it is classified as n-6 FAs. The first and main health beneficial of LA in the literature is obesity prevention. Reduced energy intake, enhancement in fat metabolism, changes in skeletal metabolism have reported as the major effect, which lead to antiobesity effects of LA (Bhardwaj et al., 2011). Although there is still doubt on the positive impact of LA on prevention of cardiovascular disease (CVD) but its consumption has confirmed in a number of studies to reduce CVD risk (Kim et al., 2016; Kus-Yamashita et al., 2016; Morenga and Montez, 2017). Anticancer effects of LA consumption also have been investigated. While McGowan et al. (2013) have reported positive effect of LA consumption 10 days prior to the surgery in breast cancer, however, there is other studies, which are in contrast with this findings. LA has showed to reduce undesired effects of immune activation and inflammatory responses. Replacement of soybean oil with commercially enriched LA oil in growing finishing pigs resulted in higher quality in pig bacon fattening and meat quality (Marijana et al., 2012). Ebeid et al. (2011) found that chickens fed with the diet consist of various levels of LA, had increased amount of omega-3 and -6 PUFAs in egg yolk. Paulino et al. (2018) fed the juvenile tambaquic (Colossoma macropomam) with the higher rate of LA/ALA, ranging from 3.1 to 26.9. The researchers reported that higher rates of LA/ALA significantly improve fillet quality indices such as EPA, DHA, and ARA content which is suitable for human as costumers. 5.2.3 Alpha linolenic acid (ALA) As another member of functional FAs, ALA, which is belonging to the n-3 FAs, is the precursor for other most important LC-PUFAs, EPA, and DHA. It is highly documented that
rrez et al., 2019). ALA has neuroprotective and antiinflammatory properties (Gutie Omega-3 FAs are essential to promote brain development and maintain normal health
rrez et al., 2019). Like other of mammalian, human are not capable to synthelevel (Gutie size ALA de novo and therefore it should obtain from the diet. Consumption of ALA-rich Chapter 13 • Filamentous fungi as animal and fish feed ingredients 413 diet has been shown to have reduction impact on low-density lipoprotein (LDL) which is the main cause of atherosclerosis and coronary heart disease (CHD). In addition, it is demonstrated that it has antiarrhythmic properties and ALA consumption can protect against CVD (Blondeau et al., 2014). Increasing levels of ALA in the diet resulted in higher concentrations of ALA in the liver and fillet of baramondi, Lates calcarifer (Tu et al., 2012). Higher growth rate and lower mortality is reported in postpartum piglets by consumption of ALA (Roszkos et al., 2020). In another study, it is found that dietary ALA can improve reproduction success. In this work, feeding pigs with the diet containing ALA resulted in enhancement in oocyte maturation and embryo development. Total cell count of blastocyst improved by consumption of ALA (Sampels et al., 2010). 5.2.4 Oleic acid Oleic acid (18:1n-9) has a double bound on carbon 9 position. A number of studies showed that oleic acid is efficient bioactive compound in prevention of ischemic heart disease. In addition, it is stated that consumption of oleic acid in the diet has inhibitory effect on platelet aggregation. The people consumed oleic acid had lower total plasma cholesterol as well as immunomodulatory effect via affecting neutrophil function and production of reactive oxygen species and T-cell proliferation promoting effect (Lopez-Huertas, 2010). It is mentioned in the literature that balanced ratio of PUFA to SFA in food/feed must be supplied. In this regard, the ratio of 0.45 of PUFA/SFA has reported to be safe and below that is considered as nonhealthy (Gomez Candela et al., 2011). Considering high concentrations of PUFA in filamentous fungi, for example in Karimi et al. is more than 1, filamentous fungal biomass could be an interesting source to meet the requirements. 5.3 Cell wall components Filamentous fungi similar to plants, some of algae and bacteria consist a functional rigid cell wall. Although the general functions that described for filamentous fungi cell wall is highly similar to that of described for other organisms, its composition may be different with other organisms. Furthermore, cell wall composition become different among various species in the kingdom fungi during the evolution process (Quintin, 2018). In addition, there is evidence regarding cell wall structure and composition alteration in respect to fungi ontogeny stages such as sporulation or development (aging of fungal culture) and environmental condition, e.g., nutritious circumstances (Snarr et al., 2017). To emphasize on the importance of cell wall of filamentous fungi, it is interesting to mention that cell wall comprise around 30% of cell dry weight and roughly, 20% of total genetic material is to regulate cell wall composition and structure. In general, the cell wall is largely composed of polysaccharides. However, small proportions of proteins and lipids also are present. In this section, functional compound present in filamentous cell wall will discuss (Bowman and Free, 2006). 414 Current Developments in Biotechnology and Bioengineering 5.3.1 Glucans Glucan is a polymer made of glucose building blocks. Glucans are acting like a flexible cement to maintain cell wall flexibility. Glucans are the most containing polysaccharides in the fungal cell walls. There are various types of glucans, e.g., β-1,3, β-1,6, and α-1,3 glucans. In α-glucans, sugars building blocks are linked together via α-1,3-bond while β-1,3 and β-1,6 bond are used to make β-glucans. Although in some cases small amount of β-1,4glucan bonds also have been detected in a number of fungi species. The most common glucans among the fungi cell wall are β-glucans where β-1,3-glucans are present in zygomycetes, basidiomycetes, and ascomycetes, whereas, β-1,6-glucans are founded in ascomycetes and basidiomycetes (Bowman and Free, 2006). Glucans are arranged generally in large linear molecules in the form of microfibrils contributing to the cell wall strength (Cabib et al., 1988). Biological response modulators including immunostimulants have been intensively explored in animal production industries, recently. Glucans are among the most studied immunostimulants in aquaculture. In Channel catfish, Ictalurus punctatus, applying 0.05% of beta-glucan showed effective immunostimulant properties. In this study, phagocytic activity, reactive cells to nitroblue tetrazolium (NbT) were significantly stimulated. The author concluded that adding 0.05% beta-glucan to the commercial feed can enhance innate immune system in the channel catfish (Sánchez-Martı́nez et al., 2017). In several other studies in fish, beta-glucan has to confirmed to stimulate nonspecific immune system by improving immune factors such as phagocytic activity, respiratory burst activity, nitric oxide, complement, and lysozyme activity and white blood cell count (Bridle et al., 2005; Zaragoza et al., 2011; Jaafar et al., 2011). Application of beta-glucan in chicks also showed that dietary beta-glucan can actively aid immune system against A. salmonella thyphimurium by increase in villus height and gablet cell numbers ( Jacob and Pescatore, 2017). Paul et al. (2012) reported that glucans extracted from the fungi pleuratusflorida can stimulate immune system and protect broiler chickens against Newcastle disease. 5.3.2 Chitin Chitin is homopolymer of β-1,4-N-acetylglucosamine which is the second most abundant biopolymer after cellulose in the nature. It is connected to the rigidity and cell shape. Chitin basically composes lower amount of cell wall composition comparing with glucans. However, almost in many of fungi species, chitin is present. Considering the chain arrangement of chitin into the microfibrils, three types of chitin exist in the nature, β, α, and γ which only α-chitin is occurred in fungal cell wall. Chitin is present in variable concentrations in fungi cell wall ranging from 2% in Saccharomyces cerevisiae to 60% in Allomyces macrogynus. However, average amount of 20% is considered in fungal cell wall (Lopez-Romero and Ruiz-Herrera, 1986). Application of chitin as immunostimulant has investigated in a number of studies. Various parameters of nonspecific immune system such as super oxidase anion production, myeloperoxidase activity, nitric oxide production were elevated in different fish species such as catla, Catla catla (Sangma and Kamilya, Chapter 13 • Filamentous fungi as animal and fish feed ingredients 415 2015), gilthead sea bream, Sparus auratus (Esteban et al., 2000), and rainbow trout, Oncorhynchus mykiss (Vahedi and Ghodratizadeh, 2011). 5.3.3 Chitosan Chitosan is a polymer of glucosamine and diacetyl chitobiose which are bound via β-1,4bonds. Mainly, chitosan is founded in zygomycetes, ascomycetes, and basidiomycetes species. Chitosan can be synthesized throughout the deacetylation of acyl glucosamine of chitin (Cabib et al., 1988). Chitosan functions is assumed to be enhancing the accumulation processes of negatively charged molecules in the cell wall and protecting chitin against enzymatic hydrolyses of chitinases due to elastic features of chitosan (Beauvais
, 2018). and Latge Chitosan has been confirmed to improve immune function in animal feeding studies. It has been reported that, relative weight of thymus, serum level of IGF-1, INS, GH, T3, T4, IgM, IgA, complement system were significantly improved by supplementation of chitosan in the Huoyan geese diet (Miao et al., 2019). Chitosan also in approved to actively stimulate immune system in fish and shell fish. Injection of 2 and 4 μg/g of chitosan significantly increased hemocyte cells and respiratory burst activity after 2 days and phagocytic activity after 1 day (Wang and Chen, 2005). In another study, 1% supplementation of chitosan in the feed, improved superoxidase anion production, and elevate lysozyme activity in common carp (Gopalakannan and Arul, 2006). 5.3.4 Mannose Mannose is another component of filamentous fungi cell wall. Mannan polysaccharides are frequently found in almost all of fungi species. They are commonly associated with wall proteins. One of greatly confirmed application of filamentous fungal cell wall polysaccharides is use as immunostimulants. Interestingly, cell wall biopolymers such as chitin, chitosan, glucans, etc., can improve immunity status of animals (Hernandez Chavez et al., 2017; Karimi et al., 2018; Sakai, 1999). Immunostimulants are the substances that improve resistance of organism against infectious substances and organisms. They boost innate immunity responses to protect the organism against stressful condition and disease incidence. Generally, immune mechanisms are common in higher vertebrates including mammals, aquatic animals, etc. (Wei et al., 2016) and consist of a group of cellular and humoral-based factors that can act to protect the organism against invading pathogens such as microorganisms; toxins, etc. Almost in large number of organisms, immune system comprises innate and adaptive responses, which both of these systems can react via cellular and humoral mechanisms. Innate immune system is the most general reactions and consist of physical barriers such as skin and mucus, other chemical factors like complement systems, antimicrobial enzymes, e.g., lysozyme, interleukins as well as cellular mechanisms such as granulocytes, monocytes, macrophages, and natural killer cells ( Jin et al., 2018). Often the bioactive compounds found in fungal cell wall, which discussed earlier, can trigger immune system particularly as antigen-like substances to immune responses 416 Current Developments in Biotechnology and Bioengineering induction by production of some specific antibody. The other mechanism that the immunostimulants are involved in is without any antigenic property and instead they enhance immune response of other antigens nonspecifically. 5.4 Minerals It is greatly approved that filamentous fungi similar to other organisms such as algae and bacteria are able to achieve different concentrations of various elements from the surrounding substrate (Beever and Burns, 1981). Different species of filamentous fungi contain valuable minerals. Although the concentrations are variable due to several parameters such as species, type of living (wild or cultured), cultivation conditions, life stage, living environment, and substrate, however, considerable amount of macro and microminerals composition have reported in the literature in filamentous fungal biomass (Siddiquee et al., 2015). Karimi et al. (2019) reported that filamentous fungi species, Aspergillus oryzae, Neurospora intermedia, and Rhizopus oryzae, contain Ca, K, P, and S were in considerable level while Mg and Na content also were detected in lower amount. In addition to the mentioned elements, iron, zinc, magnesium, and selenium, which are categorized as microminerals, reported to be included in fungal biomass. Their availability in the fungal biomass is directly related to the growth stages and its uptake level decrease over the cultivation time (Boriová et al., 2014). Generally, minerals such as P, K, Ca, and S are involved in very wide range of functions in organisms. Instance, P is a part of nucleotide, skeleton, cell membrane phospholipids, coenzymes, DNA, ribonucleic acid (RNA) and has buffering effect (Takeda et al., 2012). In addition, P is a component of nucleotides, skeletal tissues, phospholipids, coenzymes, DNA, RNA, and special enzymes involved in energy production (Bloomfield, 1997). Moreover, P has a buffering effect and helps an organism maintain a normal pH (Fairweather-Tait and Cashman, 2015). Ca is involved in blood clotting (vertebrates), muscle functions (such as contractions), nerve impulse transmission, osmoregulation, membrane permeability, hormone, and enzyme secretions, and acts as a structural component of teeth and bones. K is an essential macromineral used particularly to balance the acid-base equilibrium, as well as for osmoregulation and maintaining muscle and nerve activity (He and MacGregor, 2008). If these minerals are not supplied at sufficient levels in the diet, the organism will become susceptible to different pathological problems. For example, a deficiency in P will lead to a reduction in growth capacity and feed conversion, skeletal malformation, intermediary metabolism impairment, a reduction in tissue hardness, a reduction in antibody production, and reduced weight gain (Fairweather-Tait and Cashman, 2015; Yamauchi et al., 1996). Other minerals and particularly microminerals such as selenium, zinc, iron, and magnesium are considered as essential microminerals and must be included in animal diet. For example, selenium has significant roles in seleno-proteins, which are effective in cell signaling regulation, intracellular hemostasis, and several other important physiological processes (Fairweather-Tait and Cashman, 2015). As mentioned before, filamentous fungi are capable to absorb chemicals from surrounding substrate. These elements could be heavy metals also, however, there is evidence Chapter 13 • Filamentous fungi as animal and fish feed ingredients 417 that describe such chemical elements such as arsenic, tellurium, etc., could not store in high concentrations in filamentous fungal cells, because living filamentous fungal cells transform them to alkylated or methylated compound effectively. Throughout such biotransformation, generated methylated compounds are in the gas form and highly volatile. Therefore, they release from the cell to the substrate under the definition of bio volatilization (Boriová et al., 2014). 5.5 Pigments Natural pigment are known as the colorant substances, which are produced by living organisms while it can be stored in the cell and/or excreted to the substrate. Plants, a number of animals, bacteria, and filamentous fungi are capable to store pigment. Higher water solubility, possibility of production over the whole year regardless of seasonal variation and higher efficiency in production during the time are the advantages of microbial including filamentous fungi pigment production over other methods. Moreover, independency of environmental condition and capability of production under controlled cultivation circumstances are added to the advantages of natural pigment production by microbial sources like filamentous fungi (Siqueira, 2015). Filamentous fungi are known as pigment producers. Considering GRAS nature of edible filamentous fungi, they are accounted to be a promising feed grade pigment producers. Filamentous fungal pigments are grouped in two major classes, carotenoids and polyketides, including carotenoids, melanines, azaphilones, anthraquinones, flavin, phenazine, quinones, etc. Polyketide pathway is employed by Monascus spp. whereas other type of filamentous fungi such as N. intermedia produces carotenoids instead. Pigments can be synthesized nonbiologically, however, due to the problems arising from their toxicity and not environment friendly being, natural pigments are growing in acceptance to introduce in the market (Gmoser et al., 2017). Pigments produced via fermentation process by various fungi such as Monascus, Penicillium oxalicum, Ashbya gossypii, Blakeselea trispora are already available in the market (Esteves Torres et al., 2016). Monascus is well famous genus of filamentous fungi for its natural pigment production. Major pigments which are produced by Monascus, including M. purpureus and M. ruber, they mostly belong to azaphione pigments which give the color ranging from yellow, orange to red. Pigments aid filamentous fungi to overcome the problems arising from exposure to the high intensity light and
, 2017). ultraviolet radiations, invading organisms such as bacteria and insects (Dufosse Animals have lack of the genetic information that code pigment synthesize; and therefore, it is necessary to add the pigments in their diet. Therefore, pigments and in particular natural types of pigments are becoming one of most desired additives to include in animal feed. Pigments serve as several important functions in organisms. In plant, they attract the light to make the photosynthesis possible. In addition, they are precursors for retinoid and retinoic acid which they are required for vision and cell signaling in the animals body. Adding color to egg yolk and fish flesh has led to increase in customer preferences (Karimi et al., 2018). In addition to these applications, antioxidant activity of pigment and mainly carotenoids is greatly accepted, and it is suggested that its consumption in diet can reduce 418 Current Developments in Biotechnology and Bioengineering the risk of CVD, cancer, eye disease, and health disorders (Fiedor and Burda, 2014). Several potential applications have been described for filamentous fungal pigments including addition in animal feed, e.g., fish and poultry feed purpose to improve sensory attributes in flesh and egg yolk and skin. Filamentous fungi pigments as other pigments, which are already used in the industry, can use as colorant in leather, textile, drug, cosmetic, food, and feed packaging industries (Gmoser et al., 2017). 5.6 Antioxidant agents Filamentous fungi possess different molecules such as phenolic acids, phenyl propanoids, and flavonoids as well as other polymeric compounds such as lignin, melanin, and tannins, which are demonstrated as antioxidant compounds. Polyphenolic substances have been demonstrated to aid cell to protect against oxidative stresses by different mechanisms including inhibiting and/or scavengering free radicals and reactive oxygen species. Reactive oxygen species are generated normally because of living cellular metabolism. Although they can improve immune defense, cellular functionality, and metabolic pathways in lower concentrations, its high concentration can be deteriolous to the cell and living organism. Every living organism can protect against the damages caused by reactive oxygen species in normal condition using endogenous antioxidants system but higher concentrations of accumulated reactive oxygen species in different physiological body condition can oats be covered by endogenous protection system. Filamentous fungi have been demonstrated to be valuable sources of natural antioxidant. Generally, it has been shown that fermentation can increase antioxidant activity of feed. For example, fermented rice bran with Rhizopus oryzae increase free phenolic compound by 100%; and therefore, antioxidant activity improved. Arora and Chandra (2010) have been reported that Aspergillus sp. and Monascus purpureus can be considered as valuable sources for phenolic compounds. Antioxidant capacity of phenolic compounds is mostly dependent on redox ability of the phenolic compounds feed phenolic hydroxyl group and the capacity to the localized electron from chemical structure. Oxidative stresses are related to wide range of disease particularly neurodegenerative disease. Oxidative stress induced by excess reactive oxidative species in vivo has reported to damage carbohydrates, lipids, proteins, and nucleic acid (NA) and cause different disease such as cancer, arteriosclerosis, and complicating disorders of diabetic peoples. Taste, color, and flavor of feed also changes by oxidative damage, in particular in lipid peroxidation during preservation time. Thus, affectively decrease feed quality, nutrient value, and biofunctionality. Basically, synthetic antioxidant compounds such as α-tocopherol, ascorbic acid, and BHA are added to the feed products to stop or at least reduce oxidative reactions. Antioxidants have been confirmed to negatively affect oxidative stress and therefore prevent or decrease the incidence of aforementioned disease. Filamentous fungi contain antioxidant substances that can suppress reactive oxygen species generation and prevent relevant disease incidence and improve health status in animals. Chapter 13 • Filamentous fungi as animal and fish feed ingredients 419 5.7 Vitamins Vitamins are essential elements which animals cannot synthesize them de novo; therefore, it is needed to be supplemented by the feed (NRC, 2011). Filamentous fungi require vitamins for metabolism and they are able to synthesize a number of vitamins. Fungi including mushrooms are a good source of vitamins. They are rich in a number of vitamins such as B-complex and vitamin D reported that fungal biomass contain considerable contents of vitamin C, folic acid, B1 (thiamine), B2 (Riboflavin), and niacin larırmak, 2007). (Çag Filamentous fungi Ashbya gossypii have utilized for commercial production of B2 (Riboflavin) (Aguiar et al., 2015). Riboflavin is a precursor for flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) which are essential cofactors for numerous enzymes such as dehydrogenases, oxidases, oxidoreductases that participate in a range of redox reactions critical for major biological process. This vitamin is commercially used as a yellow colorant and to make favorable aroma and taste used as animal feed additive. Riboflavin was produced synthetically for many years but its production shifted toward the microbial synthesis nowadays. According to the studies, A. gossypii can produce 5 g/L while 14–20 g/L production also reported for B2 production by A. gossypii in the literature (Aguiar et al., 2015). Vitamin B6 is the term that refers to all biological forms of pyridoxine including pyridoxine, pyridoxal, pyridoxal 5-phosphate, and pyridoxamine. It is required co-factor in very important enzymatic functions mainly in amino acid metabolism. It is reported that B6 may have antioxidant properties as well. Variety of animals including most of fish species are not capable to synthesize vitamin C because of not having the enzyme: L-gulonolactone oxidase required in the vitamin C synthesize pathway (Lee, 2015; Lee et al., 2015). Major symptoms of ascorbate deficiency in fish include reduced growth, scoliosis, lordosis, internal and fin hemorrhage, distorted gill filaments, fin erosion, anorexia, and increased mortality (Lee et al., 2015). Vitamin C is necessary in broad spectrum of physiological functions including growth, development, reproduction, wound healing, response to stressors, and nutrient metabolism such as lipid through its function in carnitine biosynthesis. Because of antioxidant activity, vitamin C plays important role in the immune system and in combating with to infectious diseases of fish (Halver and Hardy, 2002). Vitamin C also is involved in the production of catecholamine in fish which control stress responses trough endocrine. Therefore, in situation of exposure to stressful condition, ascorbic acid requirement is needed in higher concentrations and if required concentrations be provided stress-induced downregulation of the immune system will compensate (NRC, 2011). 5.8 Nucleotides Nucleotides exert numerous important physiological and biochemical functions including incorporation into genetic material, mediating energy metabolism, and cell signaling. In addition, they are necessary components of coenzymes, allosteric receptors, and 420 Current Developments in Biotechnology and Bioengineering agonists in the cell level (Carver and Allan Walker, 1995). Therefore, while they can be synthesized in the body, their inclusion in the daily diet has health beneficial to the animal and numerous studies have shown evidences that dietary nucleotide deficiency may hurt liver, heart, intestine, and impair immune responses. Accordingly it seems that dietary supplemented nucleotides improve lymphocyte maturation, activation and proliferation, macrophage phagocytosis, immunoglobulin responses, and expression of kind of cytokines either in human and variety of animal (Grimble and Westwood, 2001). Importance of supplementation of dietary nucleotides in fish diet has highlighted since Burrells et al. (2001) reported described how its dietary inclusion can have positive impacts on various salmonid species. In a number of studies with fish it has confirmed that dietary nucleotides can enhance innate immunity system via improve on the immune responses at humoral and cellular levels. Sakai et al. (2008) noticed that feeding the fish, Cyprinus carpio, with the diet containing commercial nucleotides in the increases in serum complement (alternative pathway), lysozyme activity, phagocytosis, and superoxide anion production of head kidney phagocytes. In addition, Li et al. (2004) and Cheng et al. (2010) have shown that blood neutrophil oxidative radical production and head kidney macrophage superoxide anion production were in higher level comparing with the fish fed with common feed. Supplementation of nucleotides increases head kidney leucocyte super oxidase anion production and plasma lysozyme activity. The authors concluded that inclusion of 120–140 mg nucleotide/kg in the diet improve immune system in tilapia (Shiau et al., 2015). In addition to boosting immunity system, several health beneficial reported by adding nucleotides to the fish diet. Enhanced resistance against pathogens, better growth rates and integrity in gut-intestinal tract, boosted and quicker response to the vaccination, increasing capacity of osmoregulation and overall, enhancement in fish performance are among reported consequences of supplementation of nucleotides in the fish diet (Li and Gatlin, 2006). 5.9 Prebiotics Prebiotics are known as nondigestible substances that can positively influence the host by improve growth and/or promoting the metabolism of one or a group bacteria present in the intestine microbiome that promote organisms immunity, and therefore, keep balance in the host’s intestine (Gibson and Roberfroid, 1995). The definition of prebiotics term was modified by Gibson et al. (2004) to “selectively fermented ingredients that allow specific changes, both in the composition and/or activity in the gastrointestinal microbiota that confers benefits upon host well-being and health.” Thus mentioned dietary additive compounds indirectly and gradually change the microbiota in the host gastrointestinal (GI) tract. As discussed earlier in the section for immunostimulants, fungal cell wall composed of oligosaccharide biopolymers such mannan-oligosaccharides and fructooligosaccharides which their prebiotic effects are confirmed (Davani-Davari et al., 2019). Application of prebiotics in aquaculture gained great attention recently to improve Chapter 13 • Filamentous fungi as animal and fish feed ingredients 421 production capacity, increased nutrient utilization efficiency, and increase infectious disease resistance. Prebiotics seems to represent a high potent alternative to the traditional approaches used to treat the disease. 6. Animal feed Filamentous fungal biomass as feedstuff to be used in chicken diet has investigated in a few researches. Rezaei et al. (2019) fed 70 broiler chickens with Neurospora intermedia to evaluate its digestibility, they used fungal biomass as 30% of total diet which was composed of wheat-soybean meal-based control diet. According to their reported results, there were no significant differences in feed intake and body weight gain at the end of experiment. Based on their result digestibility values for crude and amino acids, cysteine, methionine, and threonine were as the same level as other protein sources commonly used in animal feed. In another research, broiler chicken has fed with tea fungus (consortium of two yeasts, Pichia sp., Zygosaccharomyces sp., and bacterium Acetobacter sp.). Different levels of inclusion (0, 50, 100, 150, 200 and 250 g/kg) were applied to evaluate chicken performance when tea fungus utilized as feed ingredient. Authors reported that, 150 g/kg inclusion in poultry feed enhanced feed intake, weight gain, and performance efficiency factor and carcass quality. As the control test, they reported that there was no abnormalities in histopathological examinations of liver and moreover, 100% of chickens survived during the trial (Murugesan et al., 2005). There are only a few studies described fungal fermented feed application in ruminants. Fungal treated rice straw with Comprinus fimetarius resulted in improved digestibility of cell wall and liquor feed intake in goats (Cone et al., 1996). In addition, Shrivastava et al. (2011) fed goats with fungal treated substrate and as a result, higher digestibility values in dry matter, organic matter. Crude protein, hemicellulose and cellulose were reported. In another study, sheep has fed with C. subvermispora treated bamboo in the compound feed containing Alfa alfa hay, wheat bran, and soy bean meal. In this study, higher digestibility of fungal treated bamboo in the organic matter, NDF and ADF digestion measured when it is compared with untreated bamboo. Fermented soybean and soybean meal have been studies in poultry nutrition. Hirabayashi et al. (1998) reported that fermented soybean meal with Aspergillus improves weight gain and phosphorous retention in chicks. Likewise, it was reported that feeding fermented soybean meal with Aspergillus to broilers enhances the daily feed intake, daily body weight gain, the activity of the enzymes trypsin, lipase, and protease, and increases the villus height (Feng et al., 2007a, b). In addition, Aspergillus oryzae can be used to lessen the antinutritional effects of potential feedstuffs. Fermentation using Aspergillus oryzae and Neurospora sitophila of both Jatropha seed meal and jatropha seed cake could be one way to enhance their nutritional properties and to reduce the amount of toxins and antinutritive compounds (Wina et al., 2010). Yang et al. (2007) fed piglets with different types of soybean products. According to their results, piglets fed with fungal (A. oryzae) fermented soy protein grow as well as other 422 Current Developments in Biotechnology and Bioengineering treatments including soy protein concentrate which is more expensive than soybean. Moreover, authors noticed that fermented product of soy protein-enhanced piglet performance comparing with those fed unfermented soy proteins. Similar result found when weanling pigs fed with fermented soybean. They reported that digestibility was higher in fermented soy bean than common soybean. Standard ideal digestibility for most of indispensable amino acids was greater in fermented soybean meal than fishmeal-based diet and only Lys, Thr, and Trp were not significantly greater. Overall, they conclude that inclusion of 10% fermented soybean meal can replace fishmeal, chicken meal, or poultry by-product meal without any adverse effect in digestibility and final weight. More digestibility of phosphorous has been reported by the authors comparing with soybean meal that diminish need of adding inorganic phosphorous in the diet. A. awamori, isolated from Japanese fermented food, Koji, has been evaluated in broilers chicken (Saleh et al., 2011). In this study, 15-day (365  3 g) broiler chicken was fed with the diet supplemented with A. awamori at various levels of 0.01%, 0.05%, and 0.1% of feed. Feeding with mentioned diets containing fungi, resulted in higher weight gain, decrease in saturated fatty acids, and increase in unsaturated fatty acids in the muscle. Overall, enhanced growth performance observed when fungal biomass added to the diet. Authors noticed that A. awamori can be utilized as a useful probiotic agent in the broiler chicken farms. Application of A. oryzae has been investigated in order to use as probiotic compound against salmonella contamination in poultry (KyungWoo et al., 2006). They found that, when diet supplemented with A. oryzae, it had inhibitory effect on colonization of salmonella and E. coli in the chickens gut. The author stated that, since, A. oryzae is a favorable substrate for different positive-effect bacterial strains, such as lactobacillus, they can support higher concentrations of such bacterial community in the gut microbiome. Effect of feeding of fungal biomass to the fish has received great attention recently. Wagner et al. (2019) replaced 40% of fishmeal with Zygomycetes (R. oryzae) to investigate its influence on fat and fatty acids content and composition of arctic charr (Salvelinus alpinus). They found that when fungal biomass supplemented in the feed, positively lowered liver lipid content and higher concentrations of DHA in the liver observed comparing with the reference diet. Nile tilapia have fed with two diets containing different concentrations (106 and 8 10 CFUg 1) of A. oryzae to assess probiotic effects of filamentous fungi (Dawood et al., 2019). After 60 days feeding trial, significant improvement in weight gain and feed efficiency reported. When fish fed A. oryzae, challenged with hypoxia stress, higher activity of antioxidant enzymes (SOD and GPX) and higher blood antimicrobial capacity (bactericidal and phagocytosis activity) against A. hydrophila observed. Also elevated serum protein, nitro bluetetrazolium, immunoglobulin, and lysozyme activity were observed as beneficial health effect of A. oryzae in the feed. The authors concluded that A. oryzae supplementation in fish feed can significantly boost immunity status of Nile tilapia against hypoxia stress. Apparent digestibility of R. oryzae investigated in Perca fluvitialis (Eurasian perch) and S. alpinus (Arctic charr) (Langeland et al., 2014). Digestibility coefficient for crude protein, Chapter 13 • Filamentous fungi as animal and fish feed ingredients 423 amino acid, gross energy was not significantly different with the reference diet in Arctic charr. Same result found regarding the Eurasian perch. Penaeus vannamei fed with defatted groundnut oil cake fermented by filamentous fungi, A. niger as a replacement of fishmeal ( Jannathulla et al., 2018). After the 45-day growth trial, data showed that when cake added to the feed was not fermented by A. niger, in the level of 50 g/kg had no significant adverse effect on growth, while fermented cake by filamentous fungi allowed the researchers to add 100 g/kg in the diet. FCR, protein efficiency ratio, and apparent protein utilization were improved by inclusion of fermented cake. Growth performance, feed intake, carcass composition of rainbow trout have monitored when fish fed fermented wheat grains (Pascual et al., 2018). Pleurotus ostreatus (PWD) and Lentinus edodes (LWD) were filamentous fungi species used in order to perform fermentation. Fish growth, feed intake and efficiency, nutrient retention efficiency for crude protein, crude fat and phosphorus, body lipid content were increased using feed contain fermented materials. 7. Applications of fungal biomass as feed In addition to the specific cell structure differences of various fungal species, other factors, such as culture conditions, cultivation media, etc., contribute to the final composition of fungal biomass (Gopalakrishnan et al., 2012). These differences in the protein, lipid, vitamin, pigment, etc., of fungal biomass challenge the introduction of a standardized method for their application in fish feed. As a number of filamentous fungi strains in genera such as Aspergillus, Fusarium, Monascus, Neurospora, and Rhizopus are categorized as GRAS by the United States Food and Drug Administration (USFDA), their application as animal feed and even human food is allowed (Ferreira et al., 2013). However, different measures should be taken into consideration when applying them as a fish feed supplement. One of the factors that may limit the application of fungal biomass as mammalian food is its high content of NAs, which may cause an increase in plasma uric acid in the long-term, leading to gout and kidney stone formation (Rumsey et al., 1992). However, fish species such as salmonids have the ability to produce high levels of active liver uricase that enables them to metabolize NA without health risks (Kinsella et al., 1985). Filamentous fungi may produce mycotoxins such as aflatoxin, ochratoxin, citrinin, and fusarin (Bennett and Klich, 2003). Therefore, special attention must be paid to prevent the inclusion of mycotoxins as an ingredient in fish feed. In addition, a number of fungal species, including Fusarium, Aspergillus, Exophiala, Scytalidium, and Mucor have been isolated as opportunistic filamentous fungal pathogens from various fish and shellfish species such as Atlantic salmon (Salmo salar) and rainbow trout (Onchorhynchus mykiss) (Ramaiah, 2006). Considering the problems pointed out related to fungal mycotoxins and pathogenicity, it is critical that the fungal biomass that is to be used in fish feed is properly dried and treated prior to application as a fish feed supplement. Another issue with the application of fungal biomass cultivated on waste streams is that filamentous fungi are highly tolerant to xenobiotic compounds present in waste streams. The high efficiency 424 Current Developments in Biotechnology and Bioengineering of filamentous fungi in absorption and adsorption of various environmental pollutants has introduced them as a low cost bioremediation solution (Tišma et al., 2010). Different species of white rot fungi have been used for removal of various types of pollutants such as phenols ( Justino et al., 2009), polycyclic aromatic hydrocarbons (Quintero et al., 2007), dyes (Faraco et al., 2009), and heavy metals such as lead and cadmium (Sankaran et al., 2010). Kapoor et al. (1999) successfully removed different heavy metal ions including lead, cadmium, copper, and nickel from the culture media using A. niger. In another study, Delgado et al. (1998) used Fusarium sp. for the removal of nickel, cadmium, and copper from wastewater. However, there is evidence that describe such chemical elements such as arsenic, tellurium, etc., could not store in high concentrations in filamentous fungal cells, because living filamentous fungal cells transform them to alkylated or methylated compound effectively. Throughout such biotransformation, generated methylated compounds are in the gas form and highly volatile. Therefore, they release from the cell to the substrate under the definition of biovolatilization (Boriová et al., 2014). In order to prevent xenobiotic compounds such as heavy metals from reaching our dining tables through fish fed with filamentous fungi, it is imperative to assure that fungi has been cultivated on by-product and waste streams with no health threatening compounds. 8. Economic and environmental aspects Animal proteins such as cattle, pork, and fish meat are incomparable constituents of almost human daily diet. Therefore, demands for such protein products are growing in parallel with the ever growing population of the world and force meat production industries to provide more products in the market. Scaling-up in production and supply of meat and seafood needs to be accomplished with expansion in providing high-quality feed ingredient. Currently, fishmeal and soybean meal are the common sources for protein utilized in animal feed industries. Natural resources to provide fishmeal are limited and according to the current situation, other agricultural products, such as soybean meal, etc., must compensate the shortage of fishmeal. However, high amount of water and land consumption to farm, competition with human feed and biologic problems with the use of such product as feed ingredient, e.g., low digestibility, health problems, and antinutrient content have limited their application as animal feed ingredient. Filamentous fungi have been proposed as alternative proteins sources. Fungal biomass contains a range of other compounds with nutritional relevance including EAAs, fat, PUFAs, vitamins, minerals, β-glucans, and chitosan. Presence of various kinds of bioactive compounds in fungal biomass, not only can supply essential proteins for farming animals, it can promote animal health by providing essential component involving in animal health and immunity. Increase in production, product quality and food security and enhancement in economic aspects is considered as a step forward toward animal protein production in sustainable way which is consequence expected from the application of high quality feed ingredient. On the other hand, Chapter 13 • Filamentous fungi as animal and fish feed ingredients 425 potential to grow filamentous fungi on industrial and municipal side streams, residuals, and wastes tackle down the production cost issue arising from high cost of synthetic substrate and also can contribute positively to waste management and to the environmental footprint of the food production chain. 9. Conclusions and perspectives Inclusion of filamentous fungal biomass in the animal feed can aid to improve nutritional properties of animal compound feed. Production of such valuable nutrient source by utilization of low-value streams generated by industrial sector as substrate can address their environmental footprint and give the opportunity of supplying a renewable high quality protein source ingredient in feed production sector. The application of filamentous fungal biomass as a potential alternative to fishmeal can remediate some of the challenges obstacle aquaculture industry expansion. From the other stand point, production of valueadded bioproducts via fungal bioconversion of organic-rich waste streams opens new windows for circular economy. Presence of considerable concentrations of protein, fatty acids, pigments, and immunostimulants in the fungal biomass can contribute to the nutrient quality of fish feed. However, it is noteworthy that as the biomass obtained from the different fungal strains differs in amount and type of constituents, standardization of the application of fungal biomass in fish feed still requires extensive research work. Even though, the research field can benefit from a broader investigation of animal species in order to evaluate the potential of application of filamentous fungal biomass as additive in common feed diets. This would strengthen fungal biomass potential as animal feed ingredient and screen its application versatility. In addition to this, in the future, technoeconomic and life-cycle assessment investigations is highly required in regard to evaluating the application of filamentous fungal biomass as animal feed source. This will emphasize on the potential impact of filamentous fungi on the valorization of low-value residues and on the compound feed industry sector. Acknowledgments €xtverket through a European Regional Development Fund. This work was supported by the Tillva References Abro, R., Moazzami, A.A., Lindberg, J.E., Lundh, T., 2014. Metabolic insights in Arctic charr (Salvelinus alpinus) fed with zygomycetes and fish meal diets as assessed in liver using nuclear magnetic resonance (NMR) spectroscopy. Int. Aquat. Res. 6, 63. Aguiar, T., Silva, R., Domingues, L., 2015. Ashbya gossypii beyond industrial riboflavin production: a historical perspective and emerging biotechnological applications. Biotechnol. Adv. 33, 1774–1786. Anupama, Ravindra, P., 2000. Value-added food: single cell protein. 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Wu, G., 2016. Dietary protein intake and human health. Food Funct. 7, 1251–1265. Yamauchi, M., Anderson, J.J.B., Garner, S.C., 1996. Calcium and Phosphorus in Health and Disease. CRC Press, New York. Yang, Y.X., Kim, Y.G., Lohakare, J., Yun, J.H., Lee, J.-K., Kwon, M.S., Park, J.I., Choi, J., Chae, B.J., 2007. Comparative efficacy of different soy protein sources on growth performance, nutrient digestibility and intestinal morphology in weaned pigs. Asian Australas. J. Anim. Sci. 20, 775–783. Yunus, F.U.N., Nadeem, M., Rashid, F., 2015. Single-cell protein production through microbial conversion of lignocellulosic residue (wheat bran) for animal feed. J. Inst. Brew. 121, 553–557. Zaragoza, O., Fajer-Avila, E., Almazán-Rueda, P., 2011. Influence of β-glucan on innate immunity and resistance of Lutjanus guttatus to an experimental infection of dactylogyrid monogeneans. Parasite Immunol. 33, 483–494. This page intentionally left blank 14 Production of alcohols by filamentous fungi Behzad Sataria and Hamid Amirib,c a DEPARTMENT OF FOOD TECHNOLOGY, COLLEGE OF ABURAIHAN, UNIVERSITY O F T EHRAN, TEHRAN, IRAN b DEPARTMENT OF BIOTECHNOLOGY, FACULTY OF BIOL OGICAL SCIENCE AND TECHNOLOGY, UNIVERSITY O F I SFAHAN, ISFAHAN, IRAN c E NV IR ONMENT AL RES EARCH I N S T I T U T E , UNI VERSITY OF ISFAHAN, I SFAHAN, IRAN 1. Introduction Ethanol is a colorless, flammable, and volatile alcohol with the chemical formula CH3CH2OH, often abbreviated as EtOH. Being the only nontoxic alcohol, ethanol has been used in alcoholic drinks for a long time. With the spread of coronavirus in early 2020, the use of ethanol as antiseptic agent and hand sanitizer gels becomes overspreading. A solution of 70% ethanol (in water) has the highest ability in dissolving the membrane lipid layer of the virus and deactivates it. Ethanol is used in different industries as a solvent because of its unique structure, capable of dissolving both hydrophilic and hydrophobic compounds. As a solvent, it is used in paints, tinctures, markers, perfumes, and deodorants. Many water-insoluble medications, e.g., cough and cold medications, uses ethanol as solvent media with concentrations of up to 25%. Besides, ethanol is used as an antimicrobial preservative in the preparation of many liquid medicines. Having a high vapor pressure, ethanol can be easily moved away from solutions, making it a suitable solvent for extraction purposes. Because of low toxicity and low freezing point ( 114.14°C), it is used as a coolant to keep the temperature below the freezing point of water, when needed (Rosillo-Calle and Walter, 2006). However, the major application of ethanol is for transportation fuel. Ethanol is blended with gasoline with different volumes to use in spark-ignition engines. Depending on the percentage of ethanol and gasoline, the obtained fuel is coded as E(n) (n is the percentage of ethanol in the blend) (Fig. 1). To address the weakness of ethanol, e.g., relatively high volatility, low energy density, and high solubility in water, the production of n-butanol fuel by reviving the old acetone-butanol-ethanol (ABE) fermentation has been suggested by different researchers. The market for biofuels, especially ethanol as the major contributing fuel in transportation, is highly influenced by the oil market. The Brent crude oil price was $22.58 (£18.19) per barrel on March 30, 2020, which reached to its lowest price since November 2002. Declining stock market index and global economy in the long term and plummeting Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00016-8 Copyright © 2023 Elsevier Inc. All rights reserved. 435 436 Current Developments in Biotechnology and Bioengineering E5 5% anhydrous ethanol 95% gasoline E10 10% anhydrous ethanol 90% gasoline E15 15% anhydrous ethanol 85% gasoline E25 E85 E100 25% anhydrous ethanol 75% gasoline 85% anhydrous ethanol Brazilian hydrous ethanol (contains ca. 5% water) 15% gasoline FIG. 1 Ethanol blends for use is spark-ignition cars. demand for fuel as a result of the global spreading of the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (commonly referred to as coronavirus disease 2019 (COVID-19)), were the major contributors. More than 80% of crude oil is processed for the production of gasoline, diesel, and jet fuel. Travel restrictions for preventing the virus outbreaks (Wilson and Chen, 2020) make grounded airlines and fewer cars on roads and consequently lower demands for fuels. During April 2020, oil took a historic nosedive when its price turned to a negative value for the first time on record. Consequently, many oil tankers floated on the sea without knowing where they are going to unload the fuel. With an average cost of holding oil at $0.2 per barrel per day, oil tankers have to spend $6.0 per barrel per month waiting for oil unloading. In May 2020, oil prices began to rise. On May 19, the WTI was trading at $32.36 while Brent was trading at $34.51 (oilprice.com). Gradual reopening of the economies and returned people to commute to work, preferring their own vehicles to public transportation, has increased the demand. Low oil price was another reason for increasing the demand, e.g., the China National Petroleum Corporation (CNPC) set an increase in crude oil imports by 2% in 2020. On the other hand, the oil supply has decreased and this helps oil prices come to life. An example of this decrease is falling oil production in the US from 13.1 million bpd on March 2020 to 11.6 million bpd in May, according to the Energy Information Administration. On the other hand, Iraq, the OPEC’s second-largest oil producer, did not comply with production cuts and according to the OPEC+ deal in early May (reported by Monthly Oil Market Report (MOMR)), Iraq needed to cut ca. 1 million bpd of its production. Fig. 2 shows the global ethanol production from 2007 to 2019. Global ethanol production grew from 13 billion gallons in 2007 to up to 29 billion gallons in 2019. The United States and Brazil are the major ethanol producers in the world, dominating up to 83% of total production in 2019. For a long time, Brazil has been the top producer of ethanol; Chapter 14 • Production of alcohols by filamentous fungi 437 35 USA Billions of Gallons 30 Brazil European Union China Canada Rest of World 25 20 15 10 5 0 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Last updated: May 2020 FIG. 2 Global ethanol production by country or region. Renewable fuel association (https://ethanolrfa.org/statistics/ annual-ethanol-production/). but recently, surpassed by the United States. However, sugar and ethanol industries in Brazil are responsible for providing 2.3 of the country’s domestic gross product and this country is the main exporter of ethanol in the world (Basso et al., 2011). Among countries, China is the third-largest ethanol production; however, it only accounts for the production of ca. 4% of the total production (Wu et al., 2021). Nowadays, ethanol is mainly produced via fermentation routes using plant-derived substrates. Therefore, the term “bioethanol” which is used to apply, is not very common in the current literature. 2. Feedstocks for fermentative production of alcohols The carbon source for fermentative alcohols production is generally obtained from plants. Feedstock cost is reported as a major contributor to final alcohols price. A European Commission estimated that 48%–60% of the cost of ethanol is due to feedstocks used (Moscoviz et al., 2021). Depending on its origin and structure, different generations of feedstocks have been developed. Sugar- and starch-based substrates are considered as the firstgeneration feedstocks and have the most direct bioconversion process to alcohols. Simple sugars, e.g., glucose, can be easily fermented to alcohols; however, in order to have an economically viable process, cheaper carbohydrates are envisaged (Rosales-Calderon and Arantes, 2019). A major by-product of sugar industries that is widely used for alcohols production is molasses. Molasses is a dark brown and viscous liquid obtained after crystallization of sucrose derived from sugar cane and sugar beet. In sugar industries, an average of 0.38-ton molasses is generated by the production of 1 ton of sugar, and the annual production of molasses reaches up to 55 million tons. It is characterized by having less than 20% water, 45%–60% sucrose, 2%–20% glucose and fructose, and small amounts of phosphorus, potassium, magnesium, sulfur, copper, and zinc. Even a traditional use of molasses has been cattle feeding, nowadays, most ethanol-producing distilleries in Brazil rely on molasses as the raw material, and almost 90% of molasses is used for 438 Current Developments in Biotechnology and Bioengineering manufacturing alcohol (Basso et al., 2011; Jamir et al., 2021; Zhang et al., 2021). After simple sugars, starch is the most readily digestible renewable carbon source for bioconversion purposes. However, an extra process of enzymatic- or acid-based hydrolysis is required in order to obtain fermentable sugars from starch (Torabi et al., 2020). The corn- and wheatderived starch are used for the production of ethanol in the United States and Europe, respectively. Notably, first-generation ethanol currently dominates the market, as more than 97% of ethanol is produced using these feedstocks (Moscoviz et al., 2021). Considering lignocellulosic substrates as the second-generation feedstocks for alcohol production has several advantages over the first-generation feedstocks, e.g., availability, low cost, high abundance, and no competition with human food. Lignocelluloses contain a matrix of cellulose (30%–50%), hemicellulose (10%–30%), and lignin (15%–30%) as the major carbohydrates and small amounts of inorganic matter, oils, and proteins. Potential sources of lignocelluloses include agricultural residues (corn stover, wheat straw, rice straw, and rye straw), energy crops (sorghum, switch grass), forestry residues, and a fraction of municipal solid waste. However, releasing fermentable sugars dumped in their complex structure is not as straightforward as starch-based substrates. A “pretreatment” step is required in order to open up the complex structure of lignocelluloses and make them amenable for hydrolysis. Pretreatment methods are categorized as “physical,” “chemical,” “physio-chemical,” and “biological” processes (Schubert, 2020). The changes that occurred in lignocellulosic structure during pretreatment are dependent on the pretreatment and lignocellulose types. In general, lowering cellulose crystallinity, partial hydrolysis of cellulose and hemicellulose, and removal of lignin, are the main effects of pretreatment on lignocellulosic structure (Satari and Jaiswal, 2021). This extra step along with the downstream processes for detoxification and high price of hydrolytic enzymes, make the process of second-generation alcohols production infeasible and research studies are on the way for its commercialization (Satari et al., 2019a). Algae are third-generation feedstocks for ethanol production, and depending on their size, they are categorized as micro- and macroalgae. The high carbohydrate content of some algal species make them suitable feedstocks for alcohols production; however, the production of simple sugars from algal biomass requires cell cultivation, harvest, fractionation, and finally hydrolysis of carbohydrate polymers. The extra processes make alcohols production from algae more challenging than that of lignocelluloses (da Maia et al., 2020; Satari and Jaiswal, 2021). 3. Production of ethanol by filamentous fungi Fermentative ethanol is obtained by the following simple reactions. C6 H12 O6 5C5 H10 O5 microorganisms ) microorganisms ) 2C2 H5 OH + 2CO2 5C2 H5 OH + 5CO2 Chapter 14 • Production of alcohols by filamentous fungi 439 C6H12O6 represents a hexose, e.g., glucose, and C5H10O5 is a pentose, e.g., xylose. Based on the stoichiometry of these reactions, a maximum 0.51 g ethanol is obtainable from each gram of sugars. In the production of ethanol from lignocellulose-derived glucose, the yield is usually calculated according to the following formula. Ethanol yield ð%Þ ¼ ½EtOHŠ  100 ½BiomassŠ  f  1:111  0:51 In this equation, [EtOH] is ethanol concentration at the end of fermentation period (g/L), [Biomass] is the biomass concentration (g/L) (DWB), 1.111 is the hydration factor of glucan to glucose, and f is the glucan fraction in substrate (Satari et al., 2018). A general process for the production of different generations of ethanol is shown in Fig. 3. Enzymatic hydrolysis and fermentation of starch and the next-generation feedstocks can be performed separately or via an integrated process called simultaneous saccharification and fermentation (SSF). In the SSF process, the microorganisms and hydrolytic enzymes are working simultaneously and fermentation of sugars occurs as the sugars are produced. Generally, integrated processes have been developed in order to increase the overall yield by eliminating the negative effect of initial high sugar concentration. In developing second-generation ethanol (and butanol) production, pretreatment, enzymatic hydrolysis, and fermentation, can be consolidated in a single process, i.e., consolidated bioprocessing (CBP) (Salehi Jouzani and Taherzadeh, 2015). First-generation (sugars and starch) Hydrolysis (amylase) Second-generation (lignocelluloses) Third-generation (microalgae) Pretreatment Cell fractionation (milling, enzymatic hydrolysis, ...) (physical, chemical, physico-chemical) Hydrolysis Hydrolysis (dilute-acid, cellulase) (amylase, cellulase) (distillation, pervaporation, dehydration) FIG. 3 Ethanol production process from different generations of feedstock. 440 Current Developments in Biotechnology and Bioengineering In developing a successful ethanol distillery, finding a suitable fermentative microorganism has great importance. In second-generation ethanol production, the microorganism must be able to ferment a variety of hexoses and pentoses in the lignocellulose-derived hydrolysates. Besides, as inhibitory by-products are usually generated during pretreatment of lignocelluloses, the microorganism should have tolerance to a reasonable concentration of them. Moreover, tolerance to a high ethanol titer and metabolic intermediates, high yield and productivity, and easy production and handling are other merits of a suitable microorganism. Saccharomyces cerevisiae, Escherichia coli, and Zymomonas mobilis possess some of these characteristics, which were extensively used in ethanolic fermentation (Adegboye et al., 2021; He et al., 2014). 3.1 Production of ethanol by zygomycetes Within the Zygomycetes fungi, which is a group of Zygomycota, some species in the order Mucorales are known to ferment sugars to ethanol with a high yield and productivity. Ethanol producing Mucorales are different in terms of growth requirements, e.g., carbon type assimilation, oxygen requirement, and optimal growth temperature. Among the Mucorales, Mucor indicus, Mucor circinelloides, Rhizopus oryzae, some species of Absidia and Rhizomucor have received great attention because of their ability to produce ethanol from a variety of substrates (Ferreira et al., 2013; Karimi and Zamani, 2013; Rodrigues Reis et al., 2019; Satari and Karimi, 2018). Table 1 summarized some results of ethanol production from different carbon sources by Mucorales. Different ethanol-producing Mucorales have high potential in ethanol production from first- and second-generation feedstocks, as shown in this table. The process involves the cultivation of fungal cells in agar plates, containing agar, peptone, and glucose, in order to propagate fungal hyphae and spores. Afterward, the generated cells were aseptically transferred to fermentation media containing carbon sources as well as nutrients, e.g., nitrogen, phosphorous, magnesium, and calcium sources, and some other elements. The pH of media is adjusted to ca. 5.5, and the temperature is kept at around 32°C (Satari and Karimi, 2020). During the fermentation, the fungal shape, mass, or size, increase, and the carbon sources are converted to fungal biomass and ethanol. Ethanol-producing Mucorales are dimorphic, and depending on fermentation conditions, e.g., aeration, and spore number inoculation, their morphology can switch between yeast-like and filamentous form (Lennartsson et al., 2009; Satari et al., 2016). Fungal filaments can grow as mycelial clumps or form spherical pellets (Fig. 4). Pellets restrict the mass transfer from fermentation media to the cells, and the formation of mycelia is associated with problems such as clogging the probes and wrapping around the impellers. However, collecting and reusing the filaments is more facilitated in comparison with the yeasts in the fermentation broth. The fungal biomass is a valuable by-product of fermentation. This valuable by-product can be directly consumed as fodder or used to extract valuable chemicals, e.g., chitosan, fatty acids, and mycoproteins (Edebo, 2008; Satari et al., 2016). Besides, the potential of Chapter 14 • Production of alcohols by filamentous fungi 441 Table 1 Ethanol production by ethanol-producing Mucorales from different substrates. Zygomycetes fungi Substrate Ethanol yield or Fermentation condition concentration Mucor circinelloides Glucose R. oryzae M. indicus Glucose Xylose Glucose Absidia spp. Glucose Rhizomucor pusillus Rhizopus sp. Xylose Submerged batch cultivations, aerobic growth Batch cultivation, aerobic condition Batch cultivation, aerobic condition Submerge fermentation, aerobic Aerobic M. indicus M. indicus M. indicus a Hydrolysates of wheat straw Mixed sugars derived from corn stover Glucose Hydrolysates of rice straw Anaerobic References Up to 0.34 g/g sugar €bbehu €sen et al. (2004) Lu 0.41 g/g sugar 0.07 g/g sugar 0.40 g/g sugar Millati et al. (2005) 383 mg/g Wikandari et al. (2012) 0.18 g/g Komeda et al. (2015) 0.40 g/g sugar FazeliNejad et al. (2016) Aerobic cultivation, batch 0.38 g/g sugar mode Aerobic Up to 0.45 g/g sugar Anaerobic Up to 0.46 g/g sugar SSSFa 99.4 g/L and yield of 89.5% Sues et al. (2005) Shafiei Alavijeh et al. (2020) Abasian et al. (2020) Molaverdi et al. (2019) Solid-state simultaneous saccharification and fermentation (solid-state fermentation: fermentation at low moisture content, i.e., <15%). the fungal biomass extract as a replacement with expensive yeast extract in industrial fermentation processes was recently reported (Asachi and Karimi, 2013). Besides ethanol yield, ethanol concentration is also important as a high concentration of obtained ethanol can facilitate the downstream separation. As mentioned earlier up to 0.51 g/L ethanol from 1 g/L hexose is achievable in theory, even it is not possible to obtain this in practice. Final ethanol concentration is dependent on initial substrate loading, even though a high initial sugar concentration inhibits the performance of the fungi. Production of by-products, e.g., glycerol, fungal biomass, and organic acid, decreased final ethanol yield and concentration. Up to 19.5 g/L ethanol was achieved using M. indicus in a synthetic media with 50 g/L glucose (Safaei et al., 2015). Notably, some ethanol-producing Mucorales secrete extracellular cellulytic enzymes, for example, during solid-state fermentation. These fungi are suitable microorganisms for ethanol production via CBP process. However, this ability has not fully been examined yet (Behnam et al., 2019; Satari and Karimi, 2018). 442 Current Developments in Biotechnology and Bioengineering FIG. 4 Formation of 3–4 mm pellets in cultivation of R. oryzae on citrus waste free sugars for ethanol and fungal biomass production (Satari, 2016). 3.2 Production of ethanol by ascomycetes Like Zygomycetes, filamentous Ascomycetes fungi, e.g., Fusarium, Aspergillus, and Neurospora species, have ethanol production ability (Ferreira et al., 2016). Similar to Zygomycetes, some Ascomycetes can produce ethanol in a CBP process and can assimilate lignocellulosic-derived xylose. For example, Fusarium oxysporum was reported as a fungus with a high potential for ethanol production from lignocelluloses through CBP (Ali et al., 2016). Fusarium oxysporum was reported to be able to secret a variety of hydrolytic enzymes including peptidase, xylanase, pectinase, β-glucosidase, amylase, and invertase (da Rosa-Garzon et al., 2019). Table 2 summarized the results of recent research on ethanol production by Ascomycetes fungi from different carbon sources. The processes for ethanol production are similar to those by Zygomycetes. In terms of ethanol titer, Neurospora intermedia was reported to be able to produce up to 18.5 g/L ethanol (Nair et al., 2017a). While the production of fungal biomass is inevitable in ethanolic fermentation using Ascomycetes, the focus of some studies has been exclusively on this. Some Ascomycetes, e.g., Neurospora intermedia and Aspergillus oryzae, are edible and can accumulate substantial amounts of mycoproteins in their bodies (Nair and Taherzadeh, 2016). Besides, the fungal biomass Chapter 14 • Production of alcohols by filamentous fungi Table 2 443 Ethanol production using Ascomycetes from different carbon sources. Fermentation condition Ethanol yield or concentration References Batch cultivation Continuous cultivation SHF, batch mode 3.5 g/L 5.0 g/L Ferreira et al. (2015) Ethanol yield of 94% CBP Up to 37 g/L ethanol (ca. 4% ABV) Nair et al. (2017b) Wilkinson et al. (2017) Miscanthus CBP wheat bran stillage-fiber SSF Up to ca. 40 mM ethanol 81% yield 91% yield Waters et al. (2017) Nair et al. (2017a) Ascomycetes fungi Substrate Neurospora intermedia Thin stillage Neurospora intermedia Hydrolysates of wheat straw Brewers spent grains Aspergillus oryzae + Saccaromycer cerevisiae Neurospora crassa Neurospora intermedia can be used for the extraction of natural pigments with a wide application in the food industry (Gmoser et al., 2017). 4. Metabolic engineering of filamentous fungi for production of ethanol In developing a successful second-generation alcohol production, the key steps are pretreatment, enzymatic hydrolysis, and conversion of the obtained sugars to the desired product. A major problem associated with some pretreatment methods, e.g., dilute-acid pretreatment, is the generation of some inhibitory by-products that can severely restrict € nsson the performance of hydrolytic enzymes as well as fermentative microorganisms ( Jo et al., 2013). Even though several techniques have been reported in the related literature to detoxify these inhibitors, the burden of additional costs of detoxification may hinder the process economy, especially in the production of ethanol with a narrow marginal profit € nsson and Martı́n, 2016). In the second step, cellulases and hemicellulases produced ( Jo from different bacteria, fungi, and yeasts are responsible for the hydrolysis of lignocelluloses and the liberation of monomeric sugars. Obtaining a high-yield sugar in this step is dependent on using robust hydrolytic enzymes with high activity. In the third step, directing carbon flow in metabolic pathways for the production of the maximum amount of desirable alcohol and minimum amount of unwanted by-products is sought. Production of specific hydrolytic enzymes for lignocellulosic breakdown and production of sugars is performed by engineered microorganisms (de Paula et al., 2019). Modification of promoters and transcription factor for promotion of a gene expression related to the production of hydrolytic enzymes, and protein engineering strategies have been considered in the previous chapters. Developing robust microorganisms with high tolerance to 444 Current Developments in Biotechnology and Bioengineering pretreatment-derived inhibitors as well as production of a specific product with a high carbon yield are other targets of engineering microorganisms. The basis of metabolic engineering strategies is on alteration of the flow in metabolic pathways in order to produce a desired product. Elimination, prevention, or removal of pathways led to the production of an undesirable by-product, and expression of specific enzymes responsible for the production of a product are the focus of metabolic engineering strategies. Here, we summarized the recent achievements on metabolic engineering of filamentous fungi for the identification of genes and mechanisms of controlling the expression of genes for the production of a target product as well as promoter engineering. Zymomonas mobilis is the most studied bacterium for metabolic engineering purposes because of its capability to produce ethanol from various sugars, e.g., glucose, fructose, and sucrose (He et al., 2014). Insertion of ethanol production pathway genes from Z. mobilis gave the ethanol production ability to E. coli. Later, several engineering techniques were performed on Z. mobilis in order to produce ethanol from pentoses (Mohagheghi et al., 2014; Yanase et al., 2012). Alleviation glucose catabolic repression, and therefore cofermentation of mixed sugars is another aspect of metabolic engineering as most ethanol-producing microorganisms, including industrial the ethanol producer, Saccharomyces cerevisiae, tend to first consume glucose in the fermentation broth. A recent technique of genome editing by clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system can be used for genome editing of eukaryotic cells (Ran et al., 2013; Zhang and Showalter, 2020). CRISPR/Cas9 genome editing is a tool for precise gene insertion, gene deletion, and gene replacement. There are available some studies on using this technology to engineer filamentous fungi, e.g., engineering A. niger for the production of specific hydrolytic enzymes for using in CBP (de Paula et al., 2019). In an attempt to simultaneously ferment glucose and cellubiose to ethanol, Li et al. (2020) up to four times improved ethanol production from cellubiose using Myceliophthora thermophila after a couple of genetic engineering. Feldman et al. (2019) identified and manipulated genes associated with tolerance or degradation of furfural in Neurospora crassa. They obtained 30% more tolerance to furfural in using carboxymethyl cellulose as the sole carbon source. Some ethanol-producing mucoralean fungi, e.g., M. indicus and R. oryzae, cannot ferment sucrose in fermentation media. Engineering these fungi to obtain this ability would be a hot topic in future works. 5. Ethanol recovery and concentration In fermentation broth, ethanol is typically produced with concentrations of 8%–16%, because a higher amount of ethanol is suppressive to microorganisms. Distillation is the most commonly used methanol for the concentration of ethanol up to 90%–95%. The fermentation broth is first centrifuged to remove microorganisms and other suspended solids. The obtained clarified broth, known as beer or wine, is fed to a two-column distillation unit. Wine contains ethanol and small amounts of volatile compounds as well Chapter 14 • Production of alcohols by filamentous fungi 445 Rectifying column Microorganisms and suspended solids Beer Ethanol (50-55%) Centrifuge Fermentation broth (8-16% ethanol) Stripper column Ethanol (95%) Stillage Water FIG. 5 Ethanol concentration process by distillation. as non-volatile higher alcohols, e.g., amyl alcohol, 1-propanol, and isobutanol. Ethanol is separated in the first column, known as stripper column, based on the difference of its volatility to water and is concentrated up to 50%–55%. Concentrated ethanol is then passed to the second column, known as rectifying column, in order to be concentrated to 90%–95% (Fig. 5). A higher concentration of ethanol is not possible by distillation as ethanol-water mixture forms azeotrope at 95% ethanol concentration (Karimi and Chisti, 2017; Satari et al., 2019b). Up to 99.9% ethanol is required for petroleum blend in order to prevent the formation of biphases. Ethanol at its azeotropic concentration is passed through molecular sieve in order to obtain the final concentration. The technology for distillation of ethanol is pretty mature; however, this process suffers from high energy requirement for evaporation of a high amount of water. Pervaporation (PV) is a membrane-based separation technology that can be used for ethanol recovery with advantages of lower energy consumption and simple equipment. In PV, a desired product permeates with a higher rate in a membrane and evaporates into vapor on another side of membrane, resulting in enrichment of the product in the permeate side (Fig. 6) (Peng et al., 2021). The performance of different membrane materials include polymeric, inorganic, and mixed matrix membrane is comprehensively reviewed by Peng et al. (2021). 6. Fermentative production of butanol Even though the biological production of ethanol is an important bridge between carbohydrate resources and a liquid biofuel, fuel grade ethanol has some weak points like its relatively low energy density, high solubility in water, and limitation in blending with gasoline. In this regard, the old ABE fermentation process, which had been abandoned 446 Current Developments in Biotechnology and Bioengineering Water Ethanol FIG. 6 The mechanism of ethanol separation by pervaporation. after the world war II due to poor economic compatibility, was suggested as a natural pathway to obtain 1-butanol as an advanced biofuel (Amiri, 2020). The gram negative and strictly anaerobic bacteria from Clostridia family such as Clostridium acetobutylicum and Clostridium beijerinckii are traditionally used for ABE fermentation. However, the reviving of this process is significantly tied with the success in strain development to addressing the important drawbacks of the bacteria (Amiri et al., 2016). Even though limited studies were devoted to engineering filamentous fungi for higher alcohol production until now, the advantageous filamentous fungi are potential hosts for expressing Clostridia genes expressing the metabolic pathway for 1-butanol production (Adegboye et al., 2021). In addition, the advances in producing non-natural higher alcohols in Escherichia coli and S. cerevisiae might be implemented for developing alcoholproducing filamentous fungi. This potential of filamentous fungi is presented in this section. ABE fermentation pathway through which acetone, butanol, and ethanol, are produced in a solventogenic phase after triggering the pathway by pH reduction in the previous acid€ tke-Eversloh and Bahl, 2011). The sensitivity of the solvent producogenesis metabolism (Lu ing Clostridia to a number of culture conditions make the fermentation process complicated (Amiri and Karimi, 2018). For instance, in the presence of a trace of oxygen in the medium, butanol production is completely stopped ( Jones et al., 1982). In addition, these microbes are highly sensitive to phenolic compounds typically present in lignocellulosic hydrolysates (Amiri and Karimi, 2018). The inhibition by medium ingredients might be resolved by the detoxification process, whereas the product inhibition significantly delimits the final titer of the product. It has been found that alcohols at a threshold concentration interfere with the permeability of the cell membrane decreasing the ionic potential of the cell (Lam et al., 2014). The wild solvent producing Clostridia species typically produce less than 15 g/L butanol, which significantly increases the energy consumption in the downstream process for obtaining fuel-grade 1-butanol (Amiri, 2020). Chapter 14 • Production of alcohols by filamentous fungi 447 Considering the inherent drawbacks of the Clostridia, the idea of overexpression of clostridial genes in more robust microorganisms has been evaluated by different researchers. E. coli and S. cerevisiae are the most studied hosts for higher alcohol production. S. cerevisiae with repressed differentiation showed filamentous growth in presence of isoamyl alcohol and 1-butanol (Lorenz et al., 2000). Furthermore, a pseudohyphal morphology was seen in S. cerevisiae affected by 1-butanol (Lorenz et al., 2000). Owing to their advantageous characteristics to secret large amounts of hydrolytic enzymes, filamentous fungi have been attracted interest as hosts for gene expression (Nevalainen et al., 2005). El-Kady et al. (1995) showed the accumulation of alditols and sugar alcohols from five species of Aspergillus. However, the titer of the alcohols was less than 350 mmol/L. Wang et al. (2018) developed the first filamentous fungal fatty alcohol-producing cell factory using the GFP-fusion coupling fluorescence-activated cell sorting platform, where 2 mg hexadecanol per gram biomass was produced by filamentous fungus Trichoderma reesei. CBP, symbiotic cocultures, and two-step fermentation are important processes through which filamentous fungi can be utilized along with solvent producing Clostidia for improved butanol production. Ozturk et al. (2020) developed a two-step fermentation by amylase producing Aspergillus oryzae and C. acetobutylicum for butanol production from cooked rice leading to the production of 11 g/L butanol and 18g/L total ABE. In this study, it was shown that the weak point of the solvent producing bacterium in exerting relatively poor amylolytic activity could be covered by utilizing amylase-producing filamentous fungi. Recently, development of synthetic symbiosis of microorganisms in the form of cocultures has attracted interests as a powerful tool for designing biological systems for the enhancement of alcohols production. Considering their unique ability to exerting a wide range of enzymes, filamentous fungi are advantageous partners for solvent producing Clostridia to form powerful cocultures. Nesterenkonia sp. strain F. was recently evaluated as an amylase producing partner for a powerful coculture with C. acetobutylicum leading to improved starch-based butanol production under aerobic conditions (Ebrahimi et al., 2020). The idea of direct utilization of a lignocellulosic waste based on co-cultivation of cellulase producing strains has been implemented for direct ethanol production from rice straw, where coculture of two fungi belonging to Mucor circinelloides led to 1.28 g/L ethanol production (Takano and Hoshino, 2012). CBP was also suggested to simultaneously utilize enzyme producing filamentous fungi with alcohol producing microorganisms (Amore and Faraco, 2012). Cellulase producing filamentous fungi especially Talaromyces emersoni (Grassick et al., 2004), Chaetomium thermophilum (Millner, 1977), Hermoascus aurantiacus (Gomes et al., 2000), Mycelioph thorathermophila (Roy et al., 1990), Thielavia terrestris, and Corynascus thermophilus (Maheshwari et al., 2000) have high potential for utilization in a CBP for ethanol production from lignocellulose. Besides the direct utilization of fungi in the fermentation process, they may indirectly improve the process of alcohol production through delignification of lignocellulose by “white-rot” (Akin et al., 1993), detoxification of hemicellulosic hydrolysates by filamentous soft-rot fungus degrade inhibitors (Parawira and Tekere, 2011), or enrichment of the fermentation wastes (Vuong et al., 2020) (Fig. 7). 448 Current Developments in Biotechnology and Bioengineering Alcohol Waste By products Impurities Pretreated biomass By products Water Pretreatment: Delignification by "white-rot" can significantly improve the process (Moreno et al., 2015). Detoxification: The filamentous soft-rot fungus degrade inhibitors of hemicellulosic hydrolysate (Parawira and Tekere, 2011). Agent recovery Enzymatic hydrolysis: Exerting amylase (Ozturk et al., 2020) or cellulase (Takano and Hoshino, 2012) enzymes for degradation of wastes. Fermentation: Engineered fungi, Co-culture (Takano and Hoshino, 2012) or consolidated bioprocessing (Amore and Faraco, 2012) by filamentous fungi. Pre-fractionator Alcohol recovery Evaporators/dryers: Enrichment of fermentation waste by fungi like Trichoderma Harzianum (Vuong et al., 2020). FIG. 7 Potential use of filamentous fungi in the alcohol production process. 7. Conclusions and perspectives Filamentous fungi have a high potential in the production of alcohols. For ethanol production, Zygomycetes and Ascomycetes can produce high-yield ethanol. They show a switchable micro- and macro-morphology that can be adjusted for industrial applications. Moreover, a variety of sugars derived from lignocelluloses can be fermented by them. Besides, a reasonable tolerance to the inhibitors generated during pretreatment of lignocelluloses is another trait of these fungi. Their application for use in CBP is a relatively new aspect that worth examining in future research studies. Applying new genetic engineering tools can give them new features and help to develop more robust fungi. Furthermore, filamentous fungi could play important roles in the industrial production of higher alcohols like n-butanol as an advanced biofuel. In addition to midstream utilization of fungi for n-butanol production through CBP, symbiotic cocultures, or two-step fermentation, the fungi may play important roles in upstream or downstream processes like delignification of lignocellulose, detoxification of hydrolysates, or enrichment of the fermentation wastes. References Abasian, L., Shafiei Alavijeh, R., Satari, B., Karimi, K., 2020. Sustainable and effective chitosan production by dimorphic fungus Mucor rouxii via replacing yeast extract with fungal extract. Appl. Biochem. Biotechnol. 191 (2), 666–678. Chapter 14 • Production of alcohols by filamentous fungi 449 Adegboye, M.F., Ojuederie, O.B., Talia, P.M., Babalola, O.O., 2021. 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Food Chem. 346, 128860. This page intentionally left blank 15 Biological production of organic acids by filamentous fungi Vivek Narisettya, G. Renukab, K. Amulyac, Kamalpreet Kaur Brard,e, Sara Magdoulid,e, Parameswaran Binodf, Vinod Kumara, S. Venkata Mohanc, Ashok Pandeyg,h, and Raveendran Sindhuf a S C H OOL OF W AT E R , E NE R GY , AND E NV I RONMENT, CRANFIELD UNIVERSITY, CRANFIELD, UNITED KINGDOM b DEPARTMENT OF MICROBIOLOGY, PINGLE GOV ERNMENT DEGREE C OL L E G E F O R W OME N, WA R A N G A L , I N DI A c BIOE NGINEERING AND ENVIRONMENTAL SCIENCES, DEPARTMENT O F E NERGY AND ENVIRONMENTAL ENGINEERING, CSIR-INDIAN I N STI TU TE O F CH EM I CAL T ECHNOLOGY, HYDERABAD, I NDIA d DEPARTMENT OF CIVIL ENGINEERING, LASSONDE SCHOOL O F E NGINEE RING, YORK UNI VE RSIT Y, TORONTO, ON, CANADA e INDUSTRIAL WASTE TECHNOLOGY CENTER, ABITIBI TEMISCAMINGUE, QC , CANADA f MICROBIAL PROCESSES AND TECHNOLOGY DIVI SI ON, CSIR -NATIONAL I NSTI TUTE FOR INTERDISCIPLINARY S CIENCE AND TECHNOLO GY (CSIR-NIIST), THIRUVANANTHAP URAM, KE RALA, I NDI A g CENTRE FOR I NNOVATION AND TRANSLATIONAL RESEARCH, CSIR-INDIAN INST IT UT E O F TOXI COLOGY RESE ARCH, LUCK NOW , I NDI A h SUSTAI NABILI TY CLUSTER, SCHOOL OF ENGINEERING, UNIVERSITY O F PE TR OLE UM AND E NE RGY STUDIES, DEHRADUN, INDIA 1. Introduction Filamentous fungi are microscopic eukaryotic organisms displaying fine filamentous growth on various solid substrates (Yang et al., 2016). These microorganisms are employed in the production of various industrially important metabolites like enzymes, vitamins, antibiotics, hormones, steroids, and organic acids. The commercial status attained for the production of enzymes and organic acids are excellent examples of fungal biotechnology (Porro and Branduardi, 2017; Vassilev, 1991; Yang et al., 2017). However, the production of penicillin is well-known compared to organic acids production, as these metabolites do play a less role in human well-being. Among the commodity chemicals low molecular weight organic acids produced by these filamentous fungi have significant importance (Liaud et al., 2014). Currently, several organic acids like lactic acid, citric acid (CA), and itaconic acid (IA) are commercially produced by Rhizopus oryzae, Aspergillus niger, and Aspergillus terreus, respectively. These organic acids have applications as food additives, chelators, acidulants, as chemical building blocks in polymer production, textile paint, and pharmaceutical industries (Porro and Branduardi, 2017). It was observed that filamentous fungi produce these organic acids in hundreds of grams per liter with >80% efficiency in submerged cultivations. So, we might get a though, how the fungus grown in the natural ecosystem has such a high efficiency of organic acid production? May be the organic acid Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00005-3 Copyright © 2023 Elsevier Inc. All rights reserved. 455 456 Current Developments in Biotechnology and Bioengineering biosynthesis of fungus is not regulated in an organized manner, hence placed in the fermentation media with high substrate concentrations, the flux of the carbon will be toward acid synthesis. But there are advantages for the fungus in producing and adapting to change in the external environment because of these acids, first, these organic acids have chelating properties, which would help in chelating of metal compounds in the environment and increase solubility. Second, with acidic pH environment the bacterial contamination can be avoided (Alonso et al., 2015; Karaffa and Kubicek, 2019). Considering the global population, industrialization, environmental protection, food security, and meeting the needs, the bio-based production of these organic acids through the concept of biorefinery wherein renewable and sustainable lignocellulosic residues will be used as feedstock is inevitable. As filamentous fungi are known to grow on multiple feedstocks, they are considered as suitable candidates for developing bioprocesses for organic acid biosynthesis in commercial scales. This chapter discusses the aspects of fungal organic acid production, the biochemistry, genetics, metabolic, and process engineering approaches carried out to improve the end product titers to attain the commercial potential. The organic acids considered are itaconic, gluconic, citric, and oxalic acid (OA). 2. Itaconic acid IA also known as 2-methylidenebutanedioic acid, is an unsaturated dicarbonic acid with two pKa values, pKa1  3.84, and pKa2  5.55 at 25°C (Krull et al., 2017). IA has significant commercial interest as monomer or additive in the manufacturing of fiber, resins, lattices, plastic, detergents, rubber, paint, surfactants, lubricants, and bioactive compounds. Due to the presence of an unsaturated and highly reactive dicarboxylic acid functional group, the acid can be involved in various complex organic reactions like esterification, polymerization and anhydride formation (Saha et al., 2017). IA occurs in non-dissociated form in solution with pH <2.0 and is doubly dissociated in pH > 7.0 and in between pH 2.0–7.0, a mixture of dissociated and undissociated forms exists. The global IA production was over 41,400 tonnes worth 74.5 million USD in 2011 and is expected to improve 5.5% and worth 204.6 million USD by 2023 (Cunha da Cruz et al., 2018). In traditional chemical process, IA can be synthesized by pyrolysis of CA and subsequent hydrolysis of the resulting anhydride. Other approaches include oxidation of mesityl oxide and isomerization of the intermediate CA to IA. However, in the chemical process, the feedstocks catalysts and the process conditions used for IA production were not found to be economical and environmental friendly compared to the biological processes (Bafana and Pandey, 2018). 2.1 Microorganisms, metabolism, and physiology of itaconic acid biosynthesis Various fungal strains like Aspergillus terreus, Ustilago maydis, Candida sp., and Pseudozyma antarctica are reported IA producers. A. terreus and U. maydis, are potent strains and Chapter 15 • Organic acids by filamentous fungi 457 Table 1 Summary of organic acids production from various carbon sources by various fungal strains. Feedstock/ substrate Cultivation conditions (pH, Temp °C) Titers (g/L) Yield (g/g) Productivity (g/L h) References Glucose Food waste Glucose Glucose 3.4, 35 2.3, 45 2.3, 45 6.5, 30 162 41.1 44.7 75.7 0.46 0.27 0.3 0.54 0.99 0.19 0.20 0,53 Krull et al. (2017) Narisetty et al. (2021) Narisetty et al. (2021 Becker et al. (2021) Glucose Glucose 6, 28 6, 28 56.25 39.69 0.46 0.33 0.39 0.27 Ahmed et al. (2015) Ahmed et al. (2015) Pomegranate peel wastes Sugar beet molasses 8, 25 351 NG 1.79 6, 30 68.8 NG 0.4 Roukas and Kotzekidou (2020) Ozdal and Kurbanoglu (2019) A. niger Lactose 6.5, 30 26.62 0.26 0.18 A. niger Cashew apple juice 6, 30 106.75 0.53 Microorganism Itaconic acid A. terreus DSM 23081 A. terreus BD A. terreus BD Ustilago maydis MB215 Gluconic acid Penicillium puberulum Penicillium frequentans Citric acid A. niger B60 A. niger Oxalic acid NG Mandal and Banerjee (2005) Betiku et al. (2016) the model organisms evaluated for investigating the biosynthetic pathways and process conditions (Table 1; Bafana et al., 2017; Bafana and Pandey, 2018). IA is synthesized from cis-aconitic acid, the intermediate of tricarboxylic acid cycle (TCA) cycle and the pathway was proposed by Bentley and Thiessen in 1957. In the central carbon metabolism, glucose or other carbon source is assimilated through glycolysis, and pyruvate is generated, followed by dehydrogenation to produce acetyl-CoA. Later in mitochondria, acetyl-CoA and oxaloacetic acid form citrate via citrate synthase. In the subsequent reaction, citrate is dehydrated to cis-aconitic acid, which is transported to cytosol through putative mitochondrial tricarboxylic acid transporter (MTTA). In the cytosol, cis-aconitic acid in the present of cis-aconitic acid decarboxylase (CAD) [EC 4.1.16] is converted to IA. From the cytosol, IA is transported through major facilitator superfamily (MFSA) transporter into the extracellular environment. In addition to the cadA gene, few other regulatory genes were observed as cluster, which transcribe to mitochondrial TCA transporter (mtt1), a membrane permease and a transcription factor, that could regulate the pathway expression (Kuenz and Krull, 2018; Zhao et al., 2018). In U. maydis, trans-form of aconitic acid was observed to be the precursor to produce IA. cis-Aconitic acid is transported into the cytosol and converted into transform before decarboxylation into IA. Hence in the biosynthesis of IA from glucose or 458 Current Developments in Biotechnology and Bioengineering other carbon sources the key enzyme is cis-aconitate decarboxylase (CAD) encoded by cadA gene (Wierckx et al., 2020). 2.2 Production and process conditions Although diverse microorganisms were identified and genetically constructed for IA production, A. terreus has been the organism of choice for industrial-scale production, mainly due to its tolerance to low pH, and the ability to accumulate higher IA titers and yield. In 1931, Kinoshita first reported the accumulation of IA in sucrose fermentation using Aspergillus sp., and later named the strain as A. itaconicus. Later in 1960 Lockwood and Reeves at the National Center for Agricultural Utilization Research (NRRL) isolated, identified and named A. terreus NRRL 1960, which is still considered as one of the most potent strains available for IA production from glucose. The A. terreus strain was observed to accumulate >120 g/L IA based on the feed concentration. During IA biosynthesis process parameters like physiological pH, temperature, aeration or oxygen concentration, and nutrient components play a significant role. For A. terreus, the optimal pH for IA production is between 1.8 and 2.2, but in an interesting approach, Hevekerl and associates, adjusted the pH to 3.0, after 2.1 days of cultivation, which resulted in 146 g/L IA, with 0.48 and 0.81 mol/mol productivity and yield, respectively (Hevekerl et al., 2014). A shift in the pH from 1.8 to 3.0 has posed a positive impact on the product titers. The pH < pKa2 resulted in cessation of growth and metabolite production, due to permeation of undissociated IA into the microbial cell changing the intracellular pH by acidifying the cytosol (Wierckx et al., 2020). Even the undissociated form of IA could enter the microbial cell by passive diffusion and was observed to inhibit isocitrate lyase, disrupting the anaplerotic glyoxylate cycle. Dissolved oxygen (DO) plays an important role in mycelial growth and IA accumulation. IA biosynthesis is a strict aerobic process, which requires 1.5 mol of O2 per mole of IA produced from glucose. It was also observed that interrupting the aeration for 10 min resulted in cessation of IA production and later the production re-commenced after 24 h of incubation. During reduced or low concentrations of oxygen available for the microbial cells, the NADH cannot be oxidized and accumulated intracellularly, which also results in the depletion of ATP levels, inhibiting fungal growth and metabolism. More contrary situation was the inhibition of citrate synthase and phosphofructokinase enzymes by accumulated NADH. Hence uninterrupted aeration and agitation is required for adequate IA biosynthesis and accumulation (Bafana et al., 2019; Bafana and Pandey, 2018; Hosseinpour Tehrani et al., 2019; Saha et al., 2019). Apart from process parameters, various media components like carbon source, nitrogen and phosphorous concentrations, and micronutrients were observed to affect IA accumulation. Kuenz and associates developed a simple media (g/L) with glucose (180), KH2PO4 (0.1), NH4NO3 (3.0), and CaCl2 (5.0) that provided high IA titers, yield and productivity (Kuenz et al., 2012). It was reported that a nitrogen and phosphate limitation triggered IA accumulation. During phosphate limitation, the levels of ATP drop, Chapter 15 • Organic acids by filamentous fungi 459 impairing oxidative phosphorylation and increasing the carbon flux toward glycolysis and TCA cycle. This enhances substrate level phosphorylation, and NAD(P)H regeneration, as the carbon flux is more toward TCA than respiration, promoting IA accumulation. Whereas in nitrogen rich conditions, ammonium ions was observed to inhibit phosphofructokinase (fructose-6-phosphate + ATP ! fructose 1,6- bisphosphate + ADP), which may alter the carbon flux through Embden-Meyerhof-Parnas (EMP) pathway. A similar phenomenon was observed in all the IA producers like U. maydis, P. antarctica, and € chs, 2013; Saha et al., 2019). Furthermore, IA production is A. terreus (Klement and Bu influenced by other micronutrients like calcium, iron, manganese, and copper production in the absence of manganese ions growth was in the form of pellets, whereas in the presence of manganese the culture grew in the form of long hyphae with branching filaments (Kuenz and Krull, 2018; Saha, 2017). 2.3 Strain engineering and process modifications Several eukaryotic microorganisms, such as Yarrowia lipolytica, Saccharomyces cerevisiae, and Aspergillus niger, were developed for IA production. The titers produced by these recombinant strains were not in comparison with the commercially viable A. terreus strains. But the metabolic engineering has unlocked the innovative pathway of integrating the saccharification and fermentation in fungal strains, which could reduce the fermentation time, and prevent the inhibitors generated during high temperature and pressure pretreatment procedures. In U. maydis, by the overexpression of transcriptional regulator (ria 1), deletion of itaconate oxidase (cyp 3) and by optimizing the process conditions, the strain was able to accumulate 80 g/L IA in 16 L bioreactor in a fed-batch mode of fermentation (Demir et al., 2021). Currently, the utilization of second-generation lignocellulosic feedstocks as the carbon sources is of primary interest. The fungal strains have an ability to digest the polymeric cellulose to fermentable sugars, but these strains are sensitive to inhibitors like furfurals, phenols and fufuryl alcohols produced during the initial chemical pretreatment and enzymatic hydrolysis (Saha et al., 2019). Hence, strain development toward utilization of these renewable cost-effective feedstocks would be beneficial in the industrial perspective. A strain of A. terreus AFYSZ-38 was developed by protoplast fusion between the IA hyper producing strain and the strain resistant to fermentation inhibitors from the hydrolysates. The mutant strain was able to produce 41.5 g/L IA in an fed-batch mode and 22.43 g/L in simultaneous saccharification and fermentation strategy utilizing bamboo shoot feedstock (Yang et al., 2020). Starch-based feedstocks are also significant industrially based on the geographic locations and source availability. A new strain A. niveus MG183809, was able to utilize corn starch and produce 15.65 g/L IA, but further strain engineering and process modifications can be carried out to improve the IA titers (Gnanasekaran et al., 2018). Despite the high titers of IA by A. terreus, the strain still has limitations due to product mediated growth inhibition, lower carbon flux toward IA and improved excretion of IA into 460 Current Developments in Biotechnology and Bioengineering the extracellular space. With the developed omics tools and techniques, the underlying mechanisms can be further investigated to address these limitations and construct an industrially economical strain. 3. Gluconic acid Gluconic acid (GA) or pentahydroxy hexanoic acid (C6H12O7) is a non-volatile, noncorrosive, and non-toxic mild organic acid with pKa of 3.86. It imparts sour taste in foods like wine and fruit juices (Zhang et al., 2016; Zhou et al., 2019). GA and its salts find wide application in food industry, pharmaceuticals, textile, and leather industry due to its unique properties like low toxicity, and corrosiveness, its metal ions sequestering capability, and biodegradability (Pal et al., 2016). GA is used in meat and dairy products as a leavening agent. It is also used as a flavoring agent and in formulation of various food products. Hence, in a due course of 20 years, the increasing demand for this organic acid summed to production capacity of more than 60,000 tonnes per year with a global market value of $ 1 billion USD in 2020 and expected to increase with CAGR of 5% to $ 1.9 billion USD by 2028. Commercially GA can be produced by three different approaches, namely, (a) chemical oxidation of glucose with a hypochlorite solution (b) electrolytic oxidation of glucose solution in the presence of gold catalysts (c) fermentation process with specific microorganisms. However, the microbial fermentation process is a more advantageous technique for GA production since the inevitable side reactions stemming during the chemical production processes may be avoided. 3.1 Microorganisms, metabolism, and physiology of gluconic acid biosynthesis A wide group of filamentous fungi have the ability to produce GA on a large-scale. The industrial production process is carried out in batch cultivation using several fungal species belonging to different fungal genera like Aspergillus, Penicillium, Fusarium, Mucor, and Geliocladium. Conventional screening protocols, like plate assay for acidification, were employed to isolate potential indigenous fungal strain for commercial production of GA. The biological process involves dehydrogenation of glucose catalyzed by glucose oxidase [EC 1.1.3.4] to produce GA. The metabolism involves oxidation of aldehyde group on the C1 of glucose to a carboxyl group resulting in the production of glucono-delta-lactone (GDL) (C6H10O6) and hydrogen peroxide (H2O2). The glucose oxidase is a FAD-dependent flavoprotein and a high concentration of glucose and oxygen at pH 5.5 favor this reaction. Further, spontaneous hydrolysis results in GA, mediated by lactone hydrolysing enzyme glucose dehydrogenase, and peroxidase (Pal et al., 2016). Chapter 15 • Organic acids by filamentous fungi 461 3.2 Production and process conditions Production of GA through biological process is impacted by physiological conditions and media parameters like aeration (KLa), agitation, pH, substrate (glucose), and nitrogen concentrations. The optimal conditions for GA production were observed as follows: glucose (110–250 g/L); nitrogen and phosphorous (20 mM); pH (4.5–7.0); and very high aeration rate at elevated pressure (4 bar), that could increase the DO concentration in the aqueous phase. Along with the glucose, oxygen is also considered as the key substrate in GA biosynthesis, as glucose oxidase uses molecular oxygen for the conversion of glucose. Hence the availability of oxygen in the external medium, its concentration and the oxygen transfer coefficient are of high importance. As the reaction progresses 0.5 mol of O2 is required per mole of glucose, for producing 1 mole of GA. This suggests that the process is highly oxygen consuming that can be supplemented by atmospheric compressed air or pure oxygen (Pal et al., 2016). In A. niger, GA accumulation depends on the pH and the optimal pH is between 4.5 and 7.0. If the pH drops below 3.5, CA production is triggered, and the carbon flux is diverted toward the TCA cycle. It was observed that at pH 5.6, glucose oxidase has 100% activity, whereas at pH 2.0 and 3.0 its activity dropped to 5 and 35% (Zhang et al., 2016; Zhou et al., 2019). A wide range of carbon sources like sugarcane molasses, grape, banana must, whey permeates, breadfruit, agro-residual biomass, which are rich in glucose were used for the production of GA through biochemical process. In a study, a perishable fruit from Nigeria, breadfruit hydrolysate with 120 g/L glucose was used for production of 109.9 g/L GA with 0.96 g/g yield using A. niger (Ajala et al., 2017). Similarly using corn stover hydrolysate 76.67 g/L of GA was produced with 0.94 g/g yield (Zhang et al., 2016). 3.3 Strain engineering and process modifications A wild type of strain can perform according to its metabolic efficiency, and all the strains cannot provide the theoretical maximum yields. However, the strain or the process can be improved by metabolic engineering techniques and process optimizations. Glucose oxidase is the rate limiting enzyme for the production of GA. Considering this an industrially feasible host Pichia pastoris, was used for heterologous expression of glucose oxidase, with increased thermostability. The resulted enzyme was able to convert 324 g/L glucose to GA (Mu et al., 2019). A cell-free enzymatic process with glucose oxidase and catalase derived from the filamentous fungus can perform better in comparison to cell mediated bioconversions. Nearly 100% glucose is converted to GA under the appropriate conditions by employing the enzymatic process. This method is also approved by the FDA as no product purification steps are required for the recovery of GA (Mu et al., 2019). As the fungal enzymes or cells produced GA in accordance with the maximum theoretical yields, most of the research was concentrated on process improvements and the use of sustainable feedstocks. Immobilized cells of A. niger in polyurethane foam produced 92 g/L of GA from 1 L of whey permeate consisting of 0.5% glucose, and 9.5% lactose (Mukhopadhyay et al., 2005). Hence with the available genetic tools for fungal engineering, 462 Current Developments in Biotechnology and Bioengineering and the whole genome sequences, either the homologous overexpression of rate limiting enzymes, and deletion of by-product pathways could improve the GA titers and yield. 4. Citric acid CA or 2-hydroxy-propane-1,2,3-tricarboxylic acid (C6H8O7) is a natural, nontoxic, tricarboxylic organic acid formed in the metabolism of aerobic organisms (Amato et al., 2020). Naturally, it is found in citrus fruits like oranges, lime, lemon, berries, grapes, etc. (Papagianni, 2007). Due to its remarkable physico-chemical properties and environmentally benign nature, it is a widely used industrial acid with numerous applications in the food and beverage, pharmaceutical, personal care, detergent industry, and others (Ciriminna et al., 2017). In 2025 its global market is estimated to be US$ 3.6 billion, with a CAGR of 5.24% (Mores et al., 2021). In the food industry, it is used as an acidulant to prevent oxidative deterioration of food products such as sweets and soft drinks (Berovic and Legisa, 2007). In the pharmaceutical industry, it is used as a flavoring agent to enhance palatability and is also used as a cross-linking agent in films for controlled release of drugs (Dhillon et al., 2011). CA in the form of sodium citrate is also used as an anticoagulant in blood transfusions. Due to its low pH, in the cosmetic industry, CA is also used in astringent lotions. In addition, in the chemical industry, it is used to remove metal oxides from surfaces, tanning of leather, electroplating, etc. (Soccol et al., 2006). CA has also been widely employed in other applications like biodegradable packaging materials, as a disinfectant, extracting agent and in environmental remediation. 4.1 Microorganisms, metabolism, and physiology of citric acid biosynthesis Numerous fungi have been utilized for the production of CA using a variety of substrates. Various species of Aspergillus such as A. niger, A. awamori, A. foetidus, A. wentii, A. fenicis, A. fumaricus, A. fonsecalus, A. luchensis, A. usumii, A. aculeatus, A. phoenicis, A. saitoi, A. carbonaries and Trichoderma viride and Mucor pyriformis have been reported to produce significant amounts of CA (Show et al., 2015). Among these strains, A. niger is the preferred choice of microorganism for CA production at the industrial scale (Table 1). This is mainly because of the versatility of the organism to use multiple substrates, ease of handling and high product yields (Behera, 2020). The phenomenon of CA production by A. niger has been proposed via many theories, however no concrete explanation is available. It is assumed that accumulation of CA occurs due to an induced abnormality in (CA cycle of A. niger) (Angumeenal and Venkappayya, 2013). TCA cycle is a multienzyme catalyzed cyclic series of reactions in which the acetyl group of acetyl-Co enzyme is utilized to yield CO2 and protons. Carbohydrates are converted to pyruvate via the glycolytic pathway, which is then converted to acetyl-CoA and CO2 (Behera et al., 2021). Then, acetyl-CoA is condensed with oxaloacetate via citrate synthase enzyme to form citrate and CoA is Chapter 15 • Organic acids by filamentous fungi 463 released. In this pathway O2 is consumed to regenerate NADH formed during glycolysis to NAD+, thus generating ATP. Under specific environmental conditions, CA is overproduced mainly due to the enzymatic reactions in the TCA cycle and its production is also dependent on the co-factors associated with the enzymes (Show et al., 2015). In fungi, citrate biosynthesis occurs in the cytosol as well as in the mitochondria (Karaffa and Kubicek, 2003). After two molecules of pyruvate are formed via the conversion of D-glucose in the cytosolic glycolytic pathway, one molecule is transported into the mitochondria while the other enters the reverse TCA (rTCA) pathway. In the mitochondria, it is converted to acetyl-CoA and the malate in rTCA again enters the mitochondria via a malate-citrate antiporter (CTP) and is further converted into citrate through the TCA cycle. Citrate is then transported out of the mitochondria via counter transport of malate, thus leading to the accumulation of citrate (Karaffa and Kubicek, 2003). Through this pathway, maximum theoretical yield of citrate is 1 mol per mol glucose, if pyruvate originates from glycolysis. 4.2 Production and processing conditions The type of fermentation technique used can also have a significant influence on the yield of CA. Production of CA using fungi can be carried out through three different fermentation conditions, which include surface, submerged, and solid-state fermentation (Soccol et al., 2006). Surface fermentation was initially used to produce CA at the industrial level. However, over the past few years submerged fermentation has gained more popularity (Show et al., 2015). Surface fermentation is usually carried in two phases, wherein in the first phase, the fungus is grown as a mycelia mat on the surface of the media followed by the formation of CA via utilization of carbohydrates present in the media (Show et al., 2015). The CA formed is then extracted by washing the mycelia mats. Surface fermentation requires lower installation and energy costs but is labor intensive, sensitive to alterations in the media composition and often prone to contaminations (Soccol et al., 2006). Solidstate fermentation offers advantages of using lower water and energy, less wastewater generation and enables the use of diverse renewable feedstocks for the production of CA. However, the use of intense labor during loading, unloading, and cleaning stages might incur high operational costs and longer fermentation times (Mores et al., 2021). Submerged fermentation on the other hand mandates the use of complex equipment, which can increase the initial installation costs and in addition foaming occurs during fermentation (Mores et al., 2021). Nevertheless, it offers higher productivity and yields; it is less sensitive to changes in the medium composition, providing scope for utilizing wider range of substrates. Automation of the process can help in lowering the costs, standardization of the procedure, reduce labor and prevent contamination. Considering these advantages, submerged fermentation is extensively being used at the industrial scale for CA production. More recent studies indicate that innovations in CA fermentation can also be achieved by fungal immobilization methods, multi-step processes and the utilization of renewable feedstocks such as waste in place of conventional sugars. 464 Current Developments in Biotechnology and Bioengineering CA production was discovered more than 100 years ago and since then many studies have been carried out to enhance its fermentation process (Papagianni, 2007). Fermentation conditions for CA were established during the 1930s and 1940s, wherein various critical factors were evaluated. The accumulation of CA is predominantly influenced by the medium composition, particularly in submerged type of fermentation (Mores et al., 2021). In addition, the type of carbon source and its concentration, nitrogen and phosphate limitation, pH, aeration, trace metals concentrations and morphology of the microorganisms also play a significant role in CA fermentation (Hu et al., 2019). It has been reported that certain nutrients such as carbon source, oxygen or protons should be in excess, while nitrates and phosphates should be in limiting levels and trace elements such as manganese should be well below threshold levels. Several studies have been carried out using different carbon sources, and it was shown that CA yield is directly affected by carbon source (Dhillon et al., 2011). Monosaccharides and disaccharides are usually preferred since they can be rapidly utilized by the fungus in comparison to polysaccharides, which take more time to decompose. Sugars such as glucose, sucrose, fructose, maltose, and mannose were very effectively utilized by A. niger for CA production (Amato et al., 2020; Karaffa and Kubicek, 2019). This strain was able to utilize these sugars at a concentration range of 120–180 g/L. The yield of CA is higher when sucrose is utilized as a substrate in comparison to glucose, fructose, and lactose (Angumeenal and Venkappayya, 2013). The higher yield obtained with sucrose can be attributed to the strong extracellular mycelium-bound invertase of A. niger that effectively hydrolyzes sucrose at low pH (Karaffa and Kubicek, 2003). High sugar concentrations favor CA production since it inhibits the activity of α-ketoglutarate dehydrogenase enzyme, while low concentrations of carbon reduce the size of the mycelium affecting its shape. In industrial fermentations, glucose from starch hydrolysis, sugar cane by-products, sugar beet molasses, agro industrial wastes such as fruits, vegetables, lignocellulosic biomass, etc. are the most widely used carbon sources. Nevertheless, certain bottlenecks exist due to the need for pretreatment or difficulty in scale-up in the case of solid-state fermentation. The limitation of nitrogen and phosphorous is another crucial factor, since concentration of nitrogen greater than 0.25%, favors OA accumulation and decreases CA production. The type of nitrogen source also plays a role in CA synthesis and growth of the fungus. While ammonium nitrate reduces the duration of vegetative growth, ammonium sulfate promotes a longer period of vegetative growth (Papagianni, 2007). Supplementing salts of ammonia such as ammonium sulfate/nitrate, urea, etc. to the medium help in lowering the pH of the system, thus favoring CA production (Mores et al., 2021). Heavy metals such as Zn, Mn, Fe, and Cu are also added to liquid cultures during CA production. Metal ions can act as co-factors for the enzymes and hence controlling trace element concentration can regulate the enzyme activities for CA production (Angumeenal and Venkappayya, 2013). However, the concentration of these ions should be maintained below 1 mg/L as they tend to inhibit the activity of certain enzymes and might affect the cell morphology. Due to the accumulation of organic acids, the pH of the medium changes continuously. For inhibiting the formation of by-products such as oxalic and GAs, the pH of the medium should be Chapter 15 • Organic acids by filamentous fungi 465 maintained at 3.0 (Behera, 2020). At such low pH conditions, the chances of contamination are also much lower, and recovery of CA becomes easier. At the beginning of fermentation, the pH of the system should be above 5.0 to favor the formation of mycelium. As the biomass growth occurs during the first 48 h the pH of the system drops to 3.0 and CA production begins. Furthermore, DO also plays an important role during CA production. High DO can be regulated through agitation, aeration and culture time during fermentation. Addition of alcohols such as methanol or ethanol also has a positive effect on CA production due to its inhibitory effect. The concentration of the alcohol used is dependent on the type of the organism used and the media composition. Addition of alcohols causes a change in the lipid composition of the cell membrane of the fungi, thus affecting growth and sporulation. The concentration of the alcohols in the range of 1–5% neutralize the negative effect of the metals ions during CA production (Amato et al., 2020). 4.3 Genetic and process engineering strategies Apart from synthesis of CA using naturally producing strains, inducing mutations in these natural producers using physical and chemical agents has been extensively studied (Chroumpi et al., 2020). The commonly used method is to induce mutations in the parental strains, using mutagens such as gamma radiation, ultraviolet (UV), and chemical mutagens. Chemicals such as diethyl sulfonate (DES), N-methyl-N-nitrosoguanidine, ethidium bromide, etc., are well-known chemical mutagens. For the identification of mutated/improved strains enzyme diffusion zone analysis is usually performed and for the selection of improved strains single spore technique and passaging are the two widely employed techniques. Often for hyperproduction, hybrid methods involving UV and chemical mutagens are used. In addition, other techniques such as genome editing, metabolic engineering to reduce the formation of by-products and generating mutants with an ability to overproduce CA has been explored (Chroumpi et al., 2020). Systems metabolic engineering is another tool for developing a new synthetic pathway and introduce it into A. niger to enhance CA synthesis (Tong et al., 2019). It was reported that deletion of genes that are responsible for ATP-citrate lyase synthesis enhanced CA production while deletion of two cytosolic ATP citrate lyase subunits reduced CA production, growth pigmentation and conidial germination in A. niger (Chen and Nielsen, 2016; Meijnen et al., 2009). Conversion of fructose 6-phosphate into fructose 1,6-bisphosphate is considered a crucial controlling step for glycolysis metabolic flux via the allosteric inhibition or activation. This is carried out by the essential enzyme PFK which uses magnesium as a co-factor. It has been observed that single site mutations in this enzyme depicted 70% more CA production than the control strain. It was also observed that deleting glucose oxidase (goxC) and oxaloacetate acetyl hydrolase (OAH) (prtF) genes could lower the production of OA in the medium at pH 5.0 and in the absence of Mn2+ (Behera, 2020). Despite these strategies, metabolic engineering still presents several challenges due to the complexity of the regulation metabolism for CA accumulation and the inability to use a common metabolism engineering operating tool. 466 Current Developments in Biotechnology and Bioengineering 5. Oxalic acid OA or ethanedioic acid (C2H2O4) is a strong organic acid with two pKa values, pKa1: 1.27; pKa2: 4.27. OA has major applications in pharmaceutical, textile and leather, metal processing, agriculture and commodity chemical industries. OA has been reported in mineral weathering, nutrients acquisition, wood degradation, and metal tolerance (Schmalenberger et al., 2015; Xing et al., 2020). Due to the increased demand of OA in hydrometallurgy and as a commodity chemical, diverse fungal strains have been investigated for improved production and commercialization. Due to its low molecular weight, OA has significant role in metals speciation and mobility (Etteieb et al., 2021). Besides, it has a low solubility and forms metal complexes. It stimulates metal precipitation by lowering pH value of the medium (Gadd et al., 2014). It profoundly regulates the biogeochemical cycles and nutrient cycling for microorganisms and plants. In 2017, the global OA market size reached $616.3 million USD and was projected to increase with CAGR of 3.4% by 2025. In comparison to the other metabolites, the production of OA has always been considered ineffective during CA fermentation. Like CA and its potential use in lipid production (Magdouli et al., 2018, 2020), recent attention has been paid to the fungal production of OA and process optimization. OA plays a catalytic role during pretreatment of lignocellulosics and can efficiently hydrolyze hemicellulose (Saini et al., 2020). Traditional chemical synthesis of OA involves heating of sodium formate followed by acidification through H2SO4. 5.1 Microorganisms, metabolism, and physiology of oxalic acid biosynthesis Several species such as Aspergillus niger, Fomes annosus, Amyloporia xantha, Acremoniun sp., Tyromyces palustris, Phanerochaete chrysosporium, Coriolus versicolor, Sclerotium rolfsii, Fusarium sp., Puxillus involutus, Coniophora puteana, Coniophora marmorata, and Poria vaporaria have been employed for OA production. Most of these fungal strains are not able to produce commercially acceptable volumes of OA. A. niger has been considered as a potent strain for commercial production because it produces higher levels of OA as compared to other strains (Amato et al., 2020; Han et al., 2007). The fungal strains are reported to use lactose, sucrose and glucose for OA production (Table 1; Kobayashi et al., 2014). OA can be synthesized via three metabolic pathways in fungi (a) the cytoplasmic pathway; (b) the TCA pathway; and (c) the glyoxylate pathway. In the case of the cytoplasmic pathway, oxaloacetate produced as an end product of the EMP pathway, undergoes hydrolysis into oxalate and acetate catalyzed by cytosolic oxaloacetase [EC 3.7.1.1] in the glyoxylate pathway (Han et al., 2007). Prior to entering into the TCA cycle, pyruvate is first oxidized into acetyl-CoA by the pyruvate dehydrogenase multienzyme complex (PDC) €kela € et al., 2010), followed by cleavage of oxaloacetate and then enters to mitochondria (Ma Chapter 15 • Organic acids by filamentous fungi 467 by the oxaloacetate hydrolase (OAH) [EC 3.1.1.44] into OA. In the case of the glyoxylate pathway, OA is synthesized through the hydrolysis of citrate mediated by glyoxylate dehy€kela € et al., 2010). drogenase [EC 1.2.1.17]. This process occurs in the glyoxysomes (Ma Once produced, OA is excreted into extracellular medium via oxalate transporters. In F. palustris, a well-known producer of higher titers of OA, specific ATP-dependent FpOAR transporter, characterized as membrane protein with six transmembrane domains is present (Watanabe et al., 2010). 5.2 Production and process conditions OA production is affected by numerous factors including temperature, pH, inoculum size, minerals and medium type containing carbon, nitrogen, and phosphorous components. Various research studies have been carried out to optimize the abovementioned process parameters for improving OA yields (Table 1). Although, temperature has no direct effect on OA production, unsuitable incubation temperatures delay the process performance. For instance, if the temperature is <30°C, the process duration should be extended to achieve a maximum yield of OA (Brown et al., 2018). Therefore, OA production should be carried out at 30°C or above. pH of the medium is one of the most crucial factors influencing the OA fermentation process (Papagianni, 2007). The optimum pH value for highest OA production by A. niger (183 mM and 64.3 g dm 3) is 6.0, due to elevated glycolytic flux at high pH (Walaszczyk et al., 2018). However, Penicillium ochrochloron CBS 123824 and Clarireedia jacksonii were reported to produce maximum levels of OA at pH 7.0 (Townsend et al., 2020; Vrabl et al., 2012). OA production is inhibited at a pH 2+ lower than 3.0. As of micronutrients,
Manganese (Mn ) acts as a co-factor for OAH 2 and bicarbonate ðHCO3 Þ ions also favor OA proenzyme. Similarly, carbonate CO3 duction (Granados-Arvizu et al., 2019) and the addition of calcium (Ca2+) and copper (Cu2+) ions stimulate OA biosynthesis., and the crystals of calcium oxalate was observed in the fungal hyphae (Fomina et al., 2005; Guggiari et al., 2011). Size of the inoculums is another factor that governs OA synthesis. Inoculum size of 5  106 spores/mL of A. niger yielded a maximum of 183 mM OA (Mandal and Banerjee, 2005), while inoculum size ranges from 1.82  105 to 1.51  106 produced only 1–7 mM OA (Brown et al., 2018). Furthermore, the composition of the media is also a crucial factor for enhancing OA biosynthesis. Utilization of sucrose or glucose by the fungus for biosynthesis of GA lowers the yield of OA, whereas lactose as a substrate yields high volumes of OA. Penicillium ochrochloron CBS 123824 depicted increased OA production under excess carbon source and limited concentrations of phosphate/ammonium ðNH 4 + Þ at pH value 7.0 (Vrabl et al., 2012). When NH 4 + is present in the medium, the charge balance for the proton efflux through the plasma membrane will be maintained, and after NH+4 depletion the proton flux is balanced by, the excretion of organic acids. Moreover, nitrate was also found to improve OA production in many fungal species, for example, Paxillus involutus (Schmalenberger et al., 2015). 468 Current Developments in Biotechnology and Bioengineering 5.3 Strain engineering and process modifications Efforts also have been made to increase the OA production through metabolic engineering of the potential fungal strains (Kobayashi et al., 2014). A fungal strain, A. niger transformant with overexpression of the oahA gene produced around 318 mM of OA in a medium containing 30 g/L of glucose. Generally, oahA gene regulates the production of OAH, which catalyzes the breakdown of oxaloacetate into OA. Kobayashi et al. (2014) also obtained a high yield (321 mM) of OA by growing A. niger transformant in a glucose media. Yoshioka et al. (2020) have generated potential A. niger OA producers by genetic engineering of alternative oxidase [EC 1.10.3.11] (aoxA) gene. In A. niger, cyanide (CN) insensitive respiration pathway plays a major role in CA production, through respiratory pathway, and the CN pathway comprises only AOX enzyme. The AOX bypasses the complex III and IV during the electron transport chain and transfers electrons from ubiquinol to oxygen molecules without proton pumping. Hence, CN pathway can be considered as NADH reoxidizing pathway depicting higher AOX activity and greater e substrate assimilation through glycolysis. An OA producing strain EOAH-1, generated by overexpression of the oahA and aoxA gene from A. niger WU-2223L resulted in 28 g/L of OA from 30 g/L glucose on 7th day of cultivation (Yoshioka et al., 2020). This suggests that AOX gene also plays a pivotal role in metabolic engineering for achieving efficient OA production. With well-established molecular and omics tools, expanding the studies on different variants of homologous and heterologous enzymes with high protein expression and activity could consequently result in construction of an efficient OA producing strain. Further media and process engineering with scale-up conceptualization could lead to commercialization of the processes (Figs. 1–4). 6. Conclusions and perspectives Organic acids were understood to play an important role in day-to-day life, beginning from food, pharmaceutical, textile, packaging, plastics and even in mining and metallurgy sectors. Although various organic acids were observed to be produced by bacteria, yeast FIG. 1 Chemical structures of (A) itaconic acid; (B) gluconic acid; (C) citric acid; and (D) oxalic acid. Chapter 15 • Organic acids by filamentous fungi 469 FIG. 2 Biochemical pathway for itaconic acid production. Abbreviations: ACO, aconitase; CAD, cis-aconitic acid decarboxylase; MTT, mitochondrial tricarboxylic acid transporter; MFS, major facilitator superfamily transporters. FIG. 3 Biochemical pathway for gluconic acid production. and fungi, the fungal-based CA, and IA are mainly produced in an industrial scale. This suggests that the role of filamentous fungi for the production of organic acids cannot be disputed and they dominate the current organic market. Current research trends are devoted to understand the increasing diversity among fungi and explore this kingdom for the production of organic acids. Potential novel species for organic acids production can be assessed by the available high throughput culturing and screening tools. Furthermore, since the biochemical pathways and the genetics behind the production of these organic acids are well established in these organisms the tools to modify the portfolio of organic acids can be further improved. The available genome information will aid in 470 Current Developments in Biotechnology and Bioengineering FIG. 4 Biochemical pathway for citric and oxalic acid production. Abbreviations: ICL, isocitrate lyase; MS, malate synthase; MDH, malate dehydrogenase; OAH, oxaloacetate hydrolase. identification of metabolic pathways, the catabolic enzymes available in the organism, its promoters and regulatory genes. It can also be used to assess the expression and translation of functional gene products under different fermentation conditions. Product formation can be improved by minimizing non-producing cells and engineering fungal heterogeneities during submerged cultivation. Research efforts can also be directed toward the development of filamentous fungal genomes devoid of unwanted genes and gene clusters with the long-term perspectives to generate minimal genomes, the identification of secondary metabolites hidden in the fugal genome by integrated bioinformatics and development of controlled co-cultivation devices. Another key field of future research would be to increase the strain robustness since reducing the cost of process is an important aspect for industrially viable processes. Furthermore, since A. niger has been shown to be an efficient producer of different organic acids it can be used as a “one stop solution” and the concept of multipurpose cell factory can be established. Moreover, since it exhibits good growth on different substrates including biomass based on starch, cellulose, or proteins, it could provide a platform strain for diverting carbon from many wastes toward organic acid production. For effective utilization of these organic materials mixed culture fermentations can be employed, as the natural metabolic synergy that exists among these organisms may be ideal for production of a myriad of lignocellulolytic enzymes and value-added molecules within a biorefinery. However, while using mixed cultures challenges such as controlling the consortium and providing identical fermentation conditions for multiple species needs to be further explored. Additionally, the goal must be to obtain as much value as possible in biorefinery approach by exploring different target chemicals along with the desired product and develop novel, inexpensive, and environmentally benign ways of product extraction. 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Overexpression of the gene encoding alternative oxidase for enhanced glucose consumption in oxalic acid producing Aspergillus niger expressing oxaloacetate hydrolase gene. J. Biosci. Bioeng. 129, 172–176. https://doi.org/10.1016/j.jbiosc.2019.08.014. Zhang, H., Zhang, J., Bao, J., 2016. High titer gluconic acid fermentation by Aspergillus niger from dry dilute acid pretreated corn stover without detoxification. Bioresour. Technol. 203, 211–219. https://doi.org/ 10.1016/j.biortech.2015.12.042. Zhao, M., Lu, X., Zong, H., Li, J., Zhuge, B., 2018. Itaconic acid production in microorganisms. Biotechnol. Lett. 40, 455–464. https://doi.org/10.1007/s10529-017-2500-5. Zhou, P., Yao, R., Zhang, H., Bao, J., 2019. Unique glucose oxidation catalysis of Gluconobacter oxydans constitutes an efficient cellulosic gluconic acid fermentation free of inhibitory compounds disturbance. Biotechnol. Bioeng. 116, 2191–2199. https://doi.org/10.1002/bit.27020. This page intentionally left blank 16 Production of antibiotics by filamentous fungi Parameswaran Binoda, Raveendran Sindhua, and Ashok Pandeyb,c a MICROBIAL PROCESSES AND TECHNOLOGY DIVI SI ON, CSIR -NATIONAL I NSTI TUTE FOR INTERDISCIPLINARY S CIENCE AND TECHNOLO GY (CSIR-NIIST), THIRUVANANTHAP URAM, KE RALA, I NDI A b CENTRE FOR I NNOVATION AND TRANSLATIONAL RESEARCH, CSIR-INDIAN INST IT UT E O F T OX ICOLOGY RE SEARC H, LUCK NOW, I NDI A c SUSTAI NABI LI TY CLUSTER, SCHOOL OF ENGINEERING, UNIVERSITY O F PE TR OLE UM AND E NE RGY STUDIES, DEHRADUN, INDIA 1. Introduction The production of antibiotics using microorganisms is one of the attractive and economically feasible approaches in drug and pharmaceuticals sectors. Till now, more than 10,000 antibiotics have been reported to be produced by microbes. Among the microbes, filamentous fungi are the well-known and the first recognized microorganisms for antibiotic production. The primary use of these antibiotics is to treat infectious diseases in humans and some of them are used in veterinary and agricultural applications. More than half of all antibiotics identified so far are produced by actinomycetes, 10%–15% are produced by nonfilamentous bacteria and about 20% are produced by filamentous fungi (Demain, 2014). Compared to bacteria, fungi are less explored for the production of secondary metabolites like antibiotics. A study on the genome analysis of 24 different fungi showed several genes responsible for the production of antibiotics which shows the immense potential of fungi to produce a large variety of natural antibiotics (Nielsen et al., 2017). Filamentous fungi are known to produce a wide range of antibiotics. Major contributions of antibiotic market are β-lactam antibiotics which constitute penicillins, cephalosporins, clavulanic acid, and carbapenems. Among these, filamentous fungi plays a pivotal role on the production of penicillins and cephalosporins (Demain and Martens, 2017). Antibiotics production can be fine-tuned by manipulating the nutrient media used for fermentation. Since a significant cost for production of antibiotics is contributed by the carbon source, several research and developmental activities are going on to reduce the overall process economics. Several studies revealed that glucose as well as other carbohydrates interferes with antibiotics production. Glucose is an excellent carbon source which promotes growth but it interferes antibiotics production. These occur due to catabolite repression and vary depending on the microbial strain used for the production of antibiotics. Studies revealed that some proteins and protein complexes are required for the effect of carbon source on antibiotic synthesis (Sanchez et al., 2010). Alberti et al. (2017) Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00019-3 Copyright © 2023 Elsevier Inc. All rights reserved. 477 478 Current Developments in Biotechnology and Bioengineering reported natural products from filamentous fungi and its production by heterologous expression. There are several advantages for heterologous expression for enhanced production of secondary metabolites like antibiotics by filamentous fungi. In future, filamentous fungi play a significant role as sources of new antibiotics through synthetic biology. This chapter provides an insight to importance of filamentous fungi in the production of different antibiotics, the mode of action, strategies adopted for production, metabolic engineering strategies adopted for improved production were discussed. 2. History The first known use of antibiotics dates back around 2500 years ago where the Chinese used moldy curd of soybeans to threat infections. Also, moldy cheese was used by Chinese and Ukrainian peasants to treat infected wounds. This treatment was considered as a standard treatment for human infections at that time and it was very effective. As early as 350 AD, the Sudanese-Nubian civilization used a type of tetracycline antibiotics. Crude plant extract and cheese curds were used by Europeans to treat infection during the Middle Ages. Until the 20th century, the action of antibiotics was not known. The modern era of antibiotics started in 1877, when Louis Pasteur and his coworkers discovered that the material derived from microorganisms could prevent the growth of other diseasecausing microbes. The first report of filamentous fungi producing antibiotics was published by Alexander Fleming in 1929. He had isolated a chemical, which he named “penicillin,” from a mold, Penicillium notatum. “The penicillin had prevented the growth of a neighboring colony of germs in the same Petri dish” he wrote in his research findings, though he was unable to purify the penicillin, he is the first person to publish germ killing power (Lax, 2004). Later in 1938, Florey, Ernst Chain and Norman Heatley of Oxford University, developed methods for extraction and purification of penicillin and proved its power as a drug to prevent bacterial growth by conducting controlled experiments on variously infected mice. At about the same time, Fleming described in vitro tests comparing various sulfonamides with penicillin. Penicillin proved to be more potent than the sulfonamides, uninhibited by pus and products of infection, and was apparently nontoxic. In 1942, Harold Raistrick and coworkers isolated an antimicrobial compound, stipitatic acid, from Penicillium stipitatum (Birkinshaw et al., 1942) and the structure of this compound was identified in Dewar (1945). In 1943, a laboratory worker Mary Hunt found another species of Penicillium, Penicillium chrysogenum which could produce high titers of penicillin which is the backbone for its commercial production. Later several high yielding strains were reported which were produced by mutation induced by UV and X-rays. This made the emergence of antibacterial drug industries and by 1970s, more than 270 antibiotics had been produced (Goozner, 2004). In the following years, the large use of several antibiotics and nonrational use of broad-spectrum antibiotics resulted in multidrug-resistant strains which created market opportunities to develop new antibiotics (Drugwatch, 2002; Livermore, 2004; Spellberg et al., 2004). Chapter 16 • Production of antibiotics by filamentous fungi 479 3. Filamentous fungi producing antibiotics Since the discovery of penicillin, the Kingdom fungi are well attracted by the researchers for exploring valuable antimicrobial and other bioactive substances. Several filamentous fungi are reported for the production of different kinds of antibacterial substances. The uncontrolled use of antibiotics leads to development of antibiotic resistance. This made the researchers to look for novel antimicrobial substances from natural sources. Among them, the filamentous fungi attracted as an important source for antibiotic production as this group of organisms is known to produce wide varieties of bioactive molecules. Most of these antimicrobials have been identified from Ascomycetes fungi, such as Aspergillus and Penicillium and a few from Basidiomycetous fungi (Bala et al., 2011). More than 30% of isolated metabolites belong to Aspergillus and Penicillium
rdy, 2005). Aspergillus fumigatus (Fumigatin and Fumigacin), Aspergillus clavatus (Be (Clavacin), Penicillium citrinum (Citrinin), Penicillium sp. (Puberulic acid, puberulonic acid), P. spinulosume (Spinulosin), Phacelotrema claviforme (Claviformin), Gliocladium fimbriatum (Gliotoxin) are some of the examples of reported antimicrobial secondary metabolites from filamentous fungi. In addition to antibacterial agents, there are several filamentous fungal metabolites reported as antiplasmodial agents. Stipitatic acid and other tropolones such as puberulic acid are produced by Penicillium puberulum and Penicillium aurantio-virens which are proved to be a potent antiplasmodial agent (Birkinshaw and Raistrick, 1932). Marine ecosystem also provides a unique niche of filamentous fungi producing several antibiotic secondary metabolites which are not found in terrestrial counterparts. β-Lactam type natural antibiotic, cephalosporin C, was isolated from a Cephalosporium species (Abraham et al., 1953; Newton and Abraham, 1955). This fungus was isolated from the Sardinian coast and represents the first antibiotic from a marine source. Another antibiotic isolated from an Aspergillus sp. from a marine sample produced gliotoxin which is chemically a diketopiperazine. This was the first diketopiperazine type antimicrobial compound isolated from a fungal species of marine habitat (Okutani, 1977). Later, many marine and terrestrial fungi are reported to produce gliotoxin and related diketopiperazines (Okutani, 1977). This includes Auranomides A and B from Penicillium aurantiogriseum, and aspergilols A and B from a deep-sea Aspergillus versicolor (Song et al., 2012; Wu et al., 2016). Fusidanes are a group of antibiotics isolated from fungal species belonging to triterpenes. Around 18 fusidane-type antibiotics have been reported which are produced naturally by many fungal species (Zhao et al., 2013). Majority of antibiotics producing fungi belongs to the sub phylum Pezizomycotina. Cephalosporin P1 was first identified in 1951 from the culture filtrate of Acremonium chrysogenum (Burton and Abraham, 1951). Fusidic acid is produced from Fusidium coccineum which is widely used against bacterial infections by Staphylococcus species (Godtfredsen et al., 1962; Daehne et al., 1979; Fernandes, 2016). 480 Current Developments in Biotechnology and Bioengineering 4. Production of antibiotics The major production processes of antibiotics from filamentous fungi are through fermentation. The first step in the production process is isolation and identification of a potent fungal strain which produces the antibiotics. The antibiotic properties need to be evaluated and confirmed before the actual production process. Various fermentation strategies such as solid-state and submerged fermentation (SmF) can be adopted for antibiotic production. The various process involved in the antibiotic production from filamentous fungi are schematically represented in Fig. 1. 4.1 Isolation and screening of microbes The first step in the production of antibiotics from filamentous fungi is the identification of fungi which can produce fairly good amount of antibiotics. Screening like primary and secondary screening will be employed to determine the performance of antibiotics as well as to find out the mode of action. Therapeutic index will also be evaluated and those with acceptable therapeutic index will be used for clinical trials. The conventional method of screening of filamentous fungi for novel antibiotics is tedious and time consuming. Chen et al. (2012) reported a screening strategy employing a mixed agar plate culture (MAPC) which serves as a potential tool for search of novel antibiotics from filamentous fungi. Potato sucrose agar was employed for screening fungal strains. One of the major limitations for high-throughput screening (HTS) where selection is based on the targets in vitro rather than the whole cell was found ineffective. Hence, there is an urgent need to develop novel strategies for screening next generation antibiotics from filamentous fungi which are a potential source for several antibiotics. Mixed agar plate cultures (MAPCs) have several advantages over traditional whole cell screening. The major advantage is simple and convenient and increases the probability of discovering novel antibiotics from filamentous fungi under laboratory conditions. This strategy can select potential candidates by eliminating less potent samples in the first round of screening itself. In conventional strategy, sample preparation itself is time consuming where the samples have to suspend in sterile water, vortexed, and dilutions to be prepared for cultivation. 4.2 Antimicrobial assay To estimate the antimicrobial activity of fungal extract, several assay methods are used. These are explained below. • Agar disc diffusion method—This is a simple method to check the antimicrobial activity. Here the culture extract is dropped in to discs at various concentrations and it is incubated with the test bacteria in agar plates. Inhibition zone diameter (IZD) will be observed after a specific incubation time. Chapter 16 • Production of antibiotics by filamentous fungi FIG. 1 Schematic representation of antibiotic production. 481 482 • • • • • • • • • Current Developments in Biotechnology and Bioengineering Agar well diffusion method—This method is very similar to agar disc diffusion method. Here wells are made in the agar plates and the culture extract is poured in the wells at a fixed concentration. The antibacterial activity is measured by the clear zone developed as mentioned in agar disc diffusion method (Valgas et al., 2007). Microdilution method—In this method, microtiter plates are used for testing the antimicrobial activity. Microtiter wells are first filled with broth containing different concentrations of the culture extract and it is then inoculated with test bacteria. After the specified time of incubation, the minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC), or a concentration that inhibits 50% of the bacterial growth (IC50) is calculated (Soothill et al., 1992). Another method is the incorporation of the extract in the culture medium and determination of bacterial colonies. Evaluation of colony forming units (CFUs) will provide the antibacterial properties. Agar plug diffusion method—This method is used to check the antagonism between microorganisms. Here, the fungus is first cultured in an agar plate and while growing it secretes the bioactive molecule which diffuses into the agar medium. This agar is cut aseptically and plated in another agar plate which contained previously cultured test microorganism or bacteria. The antimicrobial activity can be checked by measuring the clear zone. Cross-streak method—Antagonism of microbes will be evaluated by this strategy. The microbial strain of interest is incubated for specified time in agar plate at the center and the plate is now inoculated with the test organisms by single streak perpendicular to the central streak and incubates it for further periods. The antimicrobial activity is analyzed by observing the formed clear zone. Poisoned food method—This method is used for the evaluation of antifungal effect against molds. Extract at a desired concentration is mixed with agar before pouring into Petri plates. After specified time of preincubation, the mycelia disc is placed in the center of the plate and incubated. Diameter of fungal growth will be checked in both control and test samples. Direct bioautography—In direct bioautography, the antimicrobial agents are developed in thin-layer chromatography (TLC) plate and the plate is sprayed with the extract and it is incubated at 25°C for 48 h under humid condition. The developed plate is visualized by using various visualizing agents like p-Iodonitrotetrazolium violet. Agar overlay bioassay—where developed TLC plate (as done in direct bioautography) is covered with a molten seeded agar medium. After incubation plate is stained with tetrazolium dye. Time-kill test—This method provides antimicrobial effect either based on time or concentration. The test is done using three test tubes containing test organism in culture broth and in first and second test tubes, the fungal extract is added and third one is kept as control. After incubation at required time, the dead cells are calculated by plating. Chapter 16 • Production of antibiotics by filamentous fungi • • 483 Flow cytofluorometric assay—This method involves the detection of damaged cells using flow cytometer. The advantage of this method is that all cell types dead, viable, and damaged could be identified. The antimicrobial effect and antimicrobial resistance can be estimated by this method. In addition to this, the method gives reproducible results within a short time in 2–6 h. Bioluminescent methods—This method is based on the capacity to measure adenosine triphosphate (ATP) produced by bacteria or fungi. The amount of ATP in all living cells is more or less constant and the quantification of ATP provides the state of the cells. In presence of ATP and luciferase, D-luciferin is converted to oxyluciferin which emits light. This is the major principle of this method. The quantity of emitted light is directly proportional to the viability of the cells. 4.3 Fermentation process Fermentation techniques are followed for the large-scale production of antibiotics. The production media is an important element in all fermentation processes for the antibiotic production using filamentous fungi. Both solid-state and submerged fermentation are used for the production. Carbon and nitrogen are the main constituent and in addition to other inorganic macro and microelements are used depends on the organism and antibiotics of interest. 4.3.1 Submerged fermentation (SmF) for the production of antibiotics SmF is carried out in liquid medium. The major limitations or drawback of this strategy is the generation of large amount of effluent and the product is obtained in dilute form. This makes SmF economically nonviable. Several studies revealed that more than 50%–60% of the overall cost can be reduced by employing SSF. Large volume of fermented broth is to be concentrated by ultrafiltration, centrifugation to be carried out for harvesting the cells. Dater (1986) reported that these unit operations contributes to 48%–76% of the overall process economics. Examples of antibiotics produced by SmF include bacitracin and penicillin. Biosynthesis of bacitracin by Bacillus licheniformis BCL 21 in SmF was reported by Tahir et al. (2012). Highest antibiotic production was observed with M1 medium (245.5 IU/mL). The optimum conditions were 6% inoculums concentration, pH 8.0 and incubation temperature of 37°C. 4.3.2 Solid-state fermentation (SSF) for the production of antibiotics Solid-state fermentation (SSF) involves growing of microbes on substrates without the presence of free flowing liquid. It is usually performed with cheap and easily available substrates like wheat bran and rice bran. There are several advantages of SSF, this includes low capital investment, low wastewater generation, low energy requirement and product is obtained in concentrated form. The major limitation of this technique is the contamination issues and heat buildup during the fermentation process. Majority of SSF uses 484 Current Developments in Biotechnology and Bioengineering agroindustrial residues as substrates for fermentation. Examples of antibiotics produced by SSF include penicillin, paomomycin, rifamycin, and cephalosporin. El-Housseiny et al. (2021) reported paramomycin production by Streptomyces rimosus NRRL 2455 by adopting SSF. Paromomycin is a broad spectrum antibiotic against Gram-positive, Gramnegative bacteria and protozoa. Media engineering was carried out for improving the production. A comparative study on SSF and SmF were also done. SSF gave better production of paromomycin in comparison to SmF. This is the first report on paromomycin production by SSF. Antibiotics production using low cost substrates was reported by Al-Farraj et al., (2020). Streptomyces sp. isolated from mangrove is used in this study. Different waste biomass such as apple pomace, pineapple peel, orange peel, rice bran, wheat bran, green gram husk, banana peel, pomegranate peel, and black gram husk were evaluated for antibiotic production by Streptomyces sp. The study revealed that wheat bran served as the best substrate for antibiotics production (209 U/g) by Streptomyces sp. and lowest production was observed with pomegranate peel (43 U/g). SSF and production of rifamycin SV using Amycolaptopsis mediterranei was observed by Nagavalli et al. (2014). The study revealed that under optimized conditions the strain could produce 20 g rifampicin SV per kg of dry substrate under SSF. There was a fivefold increase in production of rifampicin SV. Optimum conditions of SSF were deoiled cotton cake with a solid loading of 10% (w/w), pH 7.0, incubation temperature of 30°C, and inoculums concentration of 30% (v/w). Tabaraie et al., (2012) evaluated cephalosporin production by SSF and SmF. Acremonium crysogenum was used in this study. The results indicate that better production was observed in SSF in comparison with SmF. It is easy to maintain optimum fermentation conditions in SSF. Cephalosporin production is 376.6 μg/mL in SSF; while in SmF, it is 315.7 μg/mL. SSF for the production of meroparamnycin from Streptomyces sp. was evaluated by Moustafa et al. (2009). Five different substrates like wheat bran, rice, quaker, bread, and ground corn were evaluated for antibiotic production. The study revealed that wheat bran serves as the potential substrate for antibiotic production. Majority of the antibiotics are commercially produced by employing SSF strategy due to their inherent advantages of high product yield, less energy requirement, and minimal wastewater generation. 4.4 Extraction and purification Based on the fermentation condition and antibiotics of interest, various extraction, and purification methods are followed. For water-soluble antibiotics, ion-exchange resins are used and for oil soluble antibiotics, solvent extraction methods are preferred. In solvent extraction, the fermentation broth is treated with organic solvents that dissolve antibiotics (Elander, 2003). 4.5 Refining The final form of antibiotics to be sold in market varies based on its application. It is made as solution form for the purpose of intravenous injection or as gel form for incorporating Chapter 16 • Production of antibiotics by filamentous fungi 485 in to topical ointments. It is also made as solid powder for making capsules. Depends on the final form, various refining steps are followed. 4.6 Quality control Quality control serves a pivotal role in the production of antibiotics. Since it involves fermentation process, care should be taken to make it absolutely free from any microbial or other contaminants. To ensure this, the production media and the entire fermenter should be sterilized properly before its production. The entire unit operation should be monitored properly and quality of all components should be checked on regular basis. Antibiotic production is highly regulated by the government in most countries. In the United States, it is regulated by Food and Drug Administration (FDA). In India, the antibiotics are under the purview of the Central Drugs Standard and Control Organization (CDSCO) under the Ministry of Health and Family Welfare (MoHFW). Antibiotics comes under Schedule H and it is to be sold only under doctor’s prescription. 5. Examples of fungal antibiotics Due to the difference in structure and degree of affinity to target sites within bacterial cells, different antibiotics have different mode of action. Based on the mode of action, antibiotics can be divided in to following groups. • Inhibitors of cell wall synthesis This group of antibiotics inhibits the synthesis of cell wall of bacteria and hence controls its multiplication. Humans and animals lack cell walls, but cell wall is very critical for the survival of bacteria. The bacterial cell wall is made of peptidoglycan which is a long sugar polymer. It forms cross-linking by the action of an enzyme, transglycosidases. This group of antibiotics competitively inhibits the action of transpeptidase and hence it affects the cell wall synthesis. Examples include penicillins, cephalosporins, bacitracin, vancomycin, etc. β-Lactam antibiotics and glycopeptides antibiotics play an importance role in inhibition of cell wall synthesis. Antibiotics such as D-cycloserine and fosfomycin inhibit cell wall biosynthesis—stage 1: the cytoplasmic stage. Antibiotics like tunicamycin and uridyl peptide inhibits cell wall biosynthesis—stage 2: the membrane associated stage. Antibiotics like glycopeptides, bacitracin, β-lactams, and glycopeptides inhibits cell wall biosynthesis— stage 3: the extra cytoplasmic stage. The major difference between Gram-positive and Gram-negative bacteria is due to their cell wall difference in the components. Grampositive bacteria have a thick peptidoglycan layer and a lipid outer membrane is absent. While Gram-negative bacteria have a thin peptidoglycan layer and have a lipid outer membrane. Since the cell wall of Gram-positive bacteria is simple they are permeable to a wide range of antibiotics. Mycobacterial cell walls have a thick lipid layer which acts as a barrier for antibiotics. 486 • Current Developments in Biotechnology and Bioengineering Inhibitors of cell membrane function Bacterial cell membrane acts as permeability barrier for most molecules and disruption of this membrane results in the leakage of essential molecules endangering the survival of bacterial cells; the antibiotics of this class disrupt the cell membrane. This class of antibiotics is not so selective because both prokaryotes and eukaryotes possess cell membrane and hence this class of antibiotics acts against these cells. Most of the antibiotics of this class are toxic to mammalian cells and are mostly used in topical applications. Examples include polymixin B, colistin, etc. • Inhibitors of protein synthesis Protein synthesis is an essential step for the survival and multiplication of all living cells including bacteria. This class of antibiotics binds on 30s or 50s subunit of ribosomes and inhibits the protein synthesis. This leads to metabolic disruption and the death of the bacterial cells. Examples include aminoglycosides, macrolides, lincosamides, etc. • Inhibitors of nucleic acid synthesis Some antibiotics bind to various processes involved in DNA and RNA replication. This affects the normal synthesis of genetic elements and hence affects the bacterial multiplication and survival. These include quinolones, metronidazole, rifampin, etc. • Inhibitors of other metabolic processes Several antibiotics act on the cellular metabolic processes and thus control the bacterial growth. For example, antibiotics like sulfonamides disrupt the folic acid pathway. It inhibits enzymes which are essential for the production of folic acid, which is necessary the production of precursors important for DNA synthesis in bacterial cells. Since this pathway is absent in humans, these antibiotics will not affect metabolism in humans. 5.1 Penicillin Penicillin is a family of antibiotics (Fig. 2). The biosynthetic penicillin is natural penicillin that is harvested from the mold through fermentation (penicillin G). Semisynthetic derivatives of penicillin consist of basic penicillin structure with chemical modification by removing the acyl group to leave 6-aminopenicillanic acid and then adding acyl groups. In ampicillin, an amino group is added to the penicillin structure. The family includes penicillin F, penicillin G, and penicillin X, as well as ampicillin, amoxicillin, nafcillin, and ticarcillin. Even though this is the first natural antibiotics, it is also produced synthetically. It majorly prevents the growth of Gram-positive bacteria, but a few are active against Gram-negative bacteria. It prevents the formation of peptidoglycan in Gram-positive bacteria and causes the bacterium to swell and burst. The organisms’ sensitive to penicillin includes Clostridium welchii, Clostridium septicum, Clostridium oedematiens, Corynbacterium diphtheriae, Streptococcus pyogenes, Streptococcus viridans, Streptococcus pneumoniae, and three types of staphylococci. Chapter 16 • Production of antibiotics by filamentous fungi 487 FIG. 2 Structure of biosynthetic penicillin (penicillin G) and other semisynthetic derivatives. The organisms which are susceptible in vitro to penicillin include the Pneumococcus, Staphylococcus, Meningococcus, Gonococcus, and Lactobacillus, Staphylococcus haemolyticus, Staphylococcus viridans, Bacillus subtilis, Clostridium welchii, Vibrio septicus, Clostridium histolyticum, Lactobacillus sporogenes, Clostridium oedematiens, Clostridium sordellii, and Cryptococcus hominis. Resistant organisms in vitro included Haemophilus influenzae, Balantidium coli, Monilia albicans, and Candida krusei. 5.1.1 Mode of action The mode of action of penicillin on bacteria is by preventing the formation of new cell walls and inhibits the bacterial growth. Structurally, penicillin contains four-member, nitrogen-containing beta-lactam ring. This will target penicillin-binding proteins which are present on cell membrane and plays a pivotal role in cross linking of bacterial cell wall. This will bind to penicillin binding proteins and prevent peptidoglycan cross-linking in cell walls. This will damage cell wall and water enters the cell and cause autolysis finally leading to cell death (Fig. 3). 5.2 Cephalosporin These are first reported from Cephalosporium which acts against a broad spectrum of bacteria and hence used for the treatment of infections of various organs (Fig. 4). Several research and developments activities were going for improving its structure to make it more effective against a broad spectrum of microbes. Till date, five generations of Cephalosporin are available. The first generation is active against Gram-positive bacteria. Second generation is active against Gram-negative bacteria. Third generation is effective against treatment of infections associated with skin and soft tissues. Fourth generation has more potential against Gram-negative bacteria in comparison with the second generation. Fifth generation is active against methicillin-resistant Staphylococcus aureus (MRSA) and Gram-positive bacteria. 488 Current Developments in Biotechnology and Bioengineering FIG. 3 Action of penicillin on Gram-positive bacterial cell wall. FIG. 4 Structure of Cephalosporin C. 5.2.1 Mode of action These are bactericidal in nature and the mode of action is similar to penicillin. It inhibits enzymes involved in peptidoglycan formation. 5.3 Fusidane Fusidane-type antibiotics (Fig. 5) act against Gram-positive bacteria. This group is represented by fusidic acid, helvolic acid, and cephalosporin P1. It is a steroid and it inhibits the protein synthesis by inhibiting the enzyme pantothenate kinase (Tanaka et al., 1969; Cundliffe, 1972; Mazumder, 1975). It acts as a bacteriostatic and bacteriocidal depending on the concentration (Verbist, 1990; Lv et al., 2017; Cao et al., 2019, 2020). The first report on the production of helvolic acid was in 1942 where Wilkins and Harris reported that culture filtrates of Aspergillus fumigatus, mut. Helvola Yuill, showed antibacterial activity. Later Chain and coworkers isolated and crystallized this compound and chemical structure and biological properties have been established and they named the compound as helvolic acid (Fig. 5). Helvolic acid is in general active against Gram-positive and almost inactive against Gram-negative organisms. The dilution at which it inhibits growth is influenced by Chapter 16 • Production of antibiotics by filamentous fungi 489 FIG. 5 Structure of Fusidane-type antibiotics. the number of organisms in the inoculum. Its action is predominantly bacteriostatic (Chain et al., 1943). Biosynthesis of fusidane-type antibiotics occurs by putative biosynthetic gene cluster consisting of nine genes of Aspergillus fumigatus Af293 (Mitsuguchi et al., 2009; Lodeiro et al., 2009). Helvolic acid was also isolate from an entomopathogenic fungus, Metarhizium anisopliae HF293, when grown in insect-derived material (Lee et al., 2008). The first report on the production of cephalosporin P1 by Cephalosporium sp. was reported by Burton and Abraham (1951). Structurally it resembles helvolic acid. It showed activity against S. aureus. 5.3.1 Mode of action Fusidic acid is effective against staphylococci, including both methicillin sensitive and resistant strains and Gram-positive anaerobes such as Clostridium difficile and Clostridium perfringens, Propionibacterium acnes and Peptostreptococcus (Collignon and Turnidge, 1999). It is not active against the Enterobacteriacae (Elkins and Nikaido, 2002; Stock, 2003; Stock and Wiedemann, 2001; Stock et al., 2001). Resistance in vitro has been demonstrated for Borrelia burgdorferi (Hunfeld et al., 2001) and Yersinia enterocolitica (Stock et al., 2002). Fusidic acid is a protein synthesis inhibitor. It inhibits conversion of GTP to GDP which subsequently affect protein synthesis. 5.4 Asperchondols Asperchondols are phenolic bisabolane sesquiterpenes showing antibacterial properties isolated from Aspergillus sp. (Liu et al., 2017). The vacuum liquid chromatographic 490 Current Developments in Biotechnology and Bioengineering fraction showed inhibitory effect against Enterococcus faecium ATCC35667 and other eight human pathogenic bacteria. 5.5 Aspochalasin This is an antibiotic isolated from Aspergillus microcysticus (Naruse et al., 1993). This compound is structurally related to cytochalasins, well known mycotoxins. Aspochalamins A-D has been isolated from Aspergillus niveus. Aspochalasin were isolated from the mycelium of this fungus which also shed good antimicrobial activity. They exhibit low antimicrobial activity against Gram positive bacteria and cytoxic activity toward different tumor cell lines (Gebhardt et al., 2004). Aspochalasin has also been isolated from Aspergillus flavipes (Ratnaweera et al., 2016). 5.6 Aspergillin This antibiotic was extracted from Ammophilus fumigatus in 1944 (Soltys, 1944). This showed inhibition against Mycobacterium tuberculosis, but inactive against Staphylococci. The study shows that Aspergillin is nontoxic for experimental animals. 5.7 Fumagillin Fumagillin was isolated from A. fumigatus (H-3) (Hanson and Eble, 1949). Eble and Hanson (1951) reported antibacterial activity against fumagillin. Killough et al. (1952) reported its role in the treatment of human intestinal amebiasis and microsporidiosis caused by Enterocytozoon bieneusi (Conteas et al., 2000). 5.8 Claviformin Claviformin is reported from A. giganteus (Wehm) by Florey et al. (1944). It is active against a wide array of Gram-positive and Gram-negative bacteria (Bennett and Klich, 2003). 5.9 Tropolone derivative Tropolone are group of compounds showing bacteriostatic and bactericidal activities. Some of the examples of Tropolone compounds include puberulic acid, viticolins, and stipitatic acid. Since this compound exhibits toxicity, this has not been used in the treatment of human infection. Viticolins are antimalarial agents isolated from Penicillium viticola. Stipitatic acid is isolated from Talaromyces stipitatus (Penicillium stipitatum) (Davison et al., 2012). Some of these tropolones are broad-spectrum antibacterial agents effective against both Gram-positive and Gram-negative bacteria (Trust, 1975). The natural tropolones are isolated from Cordyceps sp. BCC 1681 (Seephonkai et al., 2001). The bifunctional metalloenzyme, CapF, required for synthesis of capsular polysaccharides in certain pathogenic bacteria such as S. aureus. The capsular polysaccharide is a mucous layer in these bacteria which helps in infection and immune evasion. This enzyme is a target for antibiotic action. Tropolones are reported to inhibit CapF in S. aureus (Nakano et al., 2015). Chapter 16 • Production of antibiotics by filamentous fungi 491 5.10 Other antibiotics from filamentous fungi Gigantic acid was isolated from A. giganteus (Wehm) by Philpot (1943). Dihydrogeodin was isolated from the fungus A. terreus var. aureus. This compound showed antibacterial activity against B. subtilis (IFO-3513) (Inamori et al., 1983). CJ-17, 665, was isolated from the fermentation broth of A. ochraceus (CL41582) and it showed inhibitory effect against multidrug-resistant S. aureus (MDRSA), S. pyogenes, and Enterococcus faecalis (Sugie et al., 2001). Ascochlorinis is an antifungal antibiotic isolated from the culture broth of one Cylindrocarpon strain (Kawaguchi et al., 2013). 5.11 Metabolic engineering One of the major limitations of antibiotic production from fungi is the low product yield. Hence, various metabolic engineering strategies are adopted to improve the antibiotic production. Using the efficient gene editing tool like CRISPR-Cas9, different fungal model organisms have been developed (Nødvig et al., 2015; Matsu-ura et al., 2015; Pohl et al., 2016). A better understanding on the biosynthetic pathway of antibiotic production in fungi is the first and foremost step in metabolic engineering. A detailed understanding on biochemical pathway is needed and radiotracer technique is used to find unknown pathways (Anesiadis et al., 2008). The use of computational approach is the second step where in silico models are developed based on the available information about particular metabolic pathway. Several cephalosporin intermediates have been produced from the penicillin pathway of Penicillium chrysogenum (Crawford et al., 1995). Genome sequencing combined with genome mining and other computational tools have been currently used to develop better producing strains. The following approaches are employed. Penicillium chrysogenum is the strain which is widely used for the production of penicillin. The yield by the wild strain is very less. Studies revealed that most of the secondary metabolite associated genes were either silent or poorly expressed. For improved production of the antibiotics several genetic manipulation strategies were employed. Advanced genome editing tools helped for efficient manipulation of complex fungal cell factories. Pohl et al. (2016, 2018) reported genome editing tools like homologous recombination as well as CRISPR/cas9 helped in more advanced engineering of Penicillium chrysogenum. 5.11.1 Targeted approaches This is a simple approach for identifying the gene cluster involved in the synthesis of antibiotics. The target antibiotics production pathway is compared with the number of similar gene clusters between two or more species producing the compound and narrow down the number of candidate gene clusters responsible for the antibiotic synthesis. This is then compared with the retro-biosynthetic analysis to deduce the enzymes and precursors responsible (Chooi et al., 2010; Gao et al., 2011; Cacho et al., 2015). 5.11.2 Untargeted approaches In untargeted approach, the biosynthetic potential is assessed based on correlating all detected gene clusters to databases which links gene clusters and compounds. 492 Current Developments in Biotechnology and Bioengineering 5.11.3 Metabolomics approaches In this approach, the structure of unknown compound is detected by mass spectrometry and it connects to the corresponding gene cluster in a sequenced genome. One example on the metabolic engineering approach to develop fungal antibiotics is the identification of genes responsible for the synthesis of stipitatic acid using a combination of genetic and chemical methods. In this approach, the synthetic pathway is blocked at different steps and analyzed the formation of tropolone structure at the molecular level (University of Bristol, 2012). With the advent of new molecular and metabolic engineering techniques, the yield of antibiotics has been improved from various fungal sources. The main rate-limiting steps have been identified and overcome through various approaches. Overexpression of cefEF gene improved Cephalosporin C production by 15% (Skatrud et al., 1989). The industrially important fungi like P. chrysogenum and Cephalosporium acremonium are proved to be a best tool for manipulation of biosynthetic pathways. New type of penicillin, Penicillin V has been synthesized by transformation of Neurospora crassa and Aspergillus niger containing Penicillium chrysogenum penicillin biosynthetic genes (Smith et al., 1990). 6. Conclusions and perspectives Filamentous fungi are potential source of several antibiotics that are effective against several bacterial species. The unique ability of fungi to grow in controlled condition and easiness in scale-up of the fungal cultures though various fermentation approaches makes them an excellent tool for the large-scale production of antibiotics. Novel fungal species can be screened from various habitats to find novel antibiotics. This is very important in the scenario of highly developed antibiotic resistance by various bacterial species. The novel natural antibiotics from fungi could be a remedy to control bacteria which are resistance to the currently available antibiotics. The advent of molecular biology and metabolic engineering techniques made easy to develop new biosynthetic pathways and express the genes in various bacterial and fungal species to produce antibiotics. More studies are required in these directions to develop broad spectrum antibiotics from filamentous fungi. Acknowledgment One of the authors (Raveendran Sindhu) acknowledges Department of Science and Technology for sanctioning a project under DST WOS-B scheme. References Abraham, E.P., Newton, G.G., Crawford, K., Burton, H.S., Hale, C.W., 1953. Cephalosporin N: a new type of penicillin. Nature 171, 343. Alberti, F., Foster, G.D., Bailey, A.M., 2017. Natural products from filamentous fungi and production by heterologous expression. Appl. Microbiol. Biotechnol. 101, 493–500. Chapter 16 • Production of antibiotics by filamentous fungi 493 Al-Farraj, D.A., Varghese, R., Vagvolgyi, C., Elshikh, M.S., Alokda, A.M., Mahmoud, A.H., 2020. Antibiotics production in optimized culture condition using low cost substrates from Streptomyces sp. AS4 isolated from mangrove soil sediment. J. King Saud Univ. Sci. 32, 1528–1535. Anesiadis, N., Cluett, W.R., Mahadevan, R., 2008. Dynamic metabolic engineering for increasing bioprocess productivity. Metab. Eng. 10 (5), 255–266. 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dric Delattrea,b, Gustavo Cabrera-Barjasc, Aparna Banerjeed, Saddys Ce Rodriguez-Llamazarese, Guillaume Pierrea, Pascal Dubessaya, Philippe Michauda, and Akram Zamanif
CLERMONT AUVERGNE, CLERMONT AUVERGNE INP, CNRS, INSTITUT PASCAL, UNIVERSIT E CLERMONT -FERRAND, FRANCE b INSTITUT UNIVERSITAIRE DE FR ANCE (IUF), PARIS, FRANCE c UNIVERSIDAD DE CONCEPCIÓN, UNIDAD DE DESARROLLO TECNOL ÓG I C O ( U D T ) , C OR ON E L , C HI LE d CENTRO DE INVESTIGACIÓN EN E STUDIOS AVANZADO S DEL MAUL E ( CIEAM), VI CE RREC TORÍA DE INVESTIGACIÓ N Y POSGRADO, UNI VE RSIDAD CAT ÓLICA DEL MAULE, TALC A, CHIL E e CENTRO DE INVESTIGACIÓN DE P OLÍ M E R OS AV A NZ A DO S ( C I P A ) , E D I F I C I O LABORATORI O C IPA, CONCEP CIÓ N , C HI L E f SW EDIS H C ENTR E FOR R ESOUR CE RECOVE RY, UNIVERSITY O F BORÅ S, BOR ÅS , SW EDEN a 1. Introduction Fungi are a valuable group of organisms for a large range of applications in biotechnology (enzymes), foods and beverage (food coloring and flaving, edible mushrooms, nutraceutics, and fermented foods), medicine (antibiotics, antimycotics, anti-cancer, and antidiabetes agents), crops and forestry (biocontrol, mycorrhizae, and biofertilizers), waste disposal (mycoremediation, mycomaterials, and mycofumigation) and commodities (cosmetic, enzymes, organic acids, and dyes). As an example, the 90th anniversary of A. Fleming’s discovery of penicillin (the starting point of the antibiotic chemotherapy) was recently celebrated by the scientific community (Hyde et al., 2019). Fungi diverged from other organisms around 1.5 billion years ago (Berbee et al., 2020) Paleozoic began 541 million years ago and ended about 252 million years ago with the greatest extinction event in Earth story: the Premian extinction (Robison and Crick, 2021). During this era, the Cambrian explosion (541–485.4 million year ago) saw probably the colonization of lands by fungi which is uncontroversial during the Devonian, 400 million years ago. Fungi emerged as a “Third Kingdom,” including organisms that were outside the traditional dichotomy of vegetables vs animals. Fungi are heterotrophic eukaryotes sharing the following characteristics: a fibrous cell wall, a hyperdiverse cell organization ranging from unicellular organisms to syncytial filaments that form sometimes macroscopic structures, and the loss of phagotrophic capabilities (Naranjo-Ortiz and Gabaldón, 2019; Xiao et al., 2020a). The macromolecular constituents of the fungal cell wall are critical for the biology and ecology development of any fungal species (Gow et al., 2017). Other than pathogenesis, Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00001-6 Copyright © 2023 Elsevier Inc. All rights reserved. 497 498 Current Developments in Biotechnology and Bioengineering fungal CPSs are primarily studied due to their potential applications, in the food and chemical industry, as well as in pharmaceuticals. Fungal species are also reported to produce exopolysaccharides (EPSs) and intracellular PSs apart from CPSs (Wang et al., 2017). Important fungal EPSs include pullulan, scleroglucan, and botryosphaeran which have several applications in pharmaceuticals, medicine, and food industries. While still very little information is available about mechanism of biosynthesis of fungal EPSs, most of the studies have focused on optimization of the cultivation conditions in order to improve the EPSs production by different fungi (Mahapatra and Banerjee, 2013). Pullulan forms transparent edible films for coatings other than being naturally antimicrobial, antiallergenic, and prebiotic. Scleroglucan is largely used as a viscosifier for improved oil recovery and other applications. Several fungal glucans are reported to have antioxidant and antitumor properties (Luft et al., 2020). Many mushroom PSs possess immunomodulatory properties and other favorable health effects (Giavasis et al., 2019; Xiao et al., 2020b; Goldman et al., 2021). Fungal PSs are also a part of traditional medicine and diets notably in Asia (Xiao et al., 2020a). As other PSs from terrestrial plants, macro- and microalgae, animals, and bacteria, fungal PSs have crucial physiological roles as cell wall components or as pericellular matrix surrounding the cell membranes. Keeping all these in context and the importance of cell walls to pathogenic fungi in order to develop drug targets, research on synthetic oligosaccharides mimicking fungal CPSs are on its peak in the present time (Krylov and Nifantiev, 2020a, 2020b). For all the abovementioned reasons, biosynthesis, fermentative production, and downstream processing of these fungal PSs have added importance. Structurally speaking, fungal PSs are composed of monosaccharides (mainly pentoses and hexoses) belonging to only one type (homopolysaccharides) or to several ones (heteropolysaccharides) linked sequentially by glycosidic bonds leading to linear or ramified structures. In nature, up to 40–50 different monosaccharides have been identified. This wide range of monosaccharides species and possible glycosidic bonds between the anomeric carbon of one monosaccharide with any hydroxyl of the other lead to a large group of structures resulting in single/triple helices, spirals, and sheets (Kajiwara, 2005). Typically, the biochemical nomenclature of glucides requires to call them using the name of the main type of monosaccharide in its structure replacing the ending “-ose” of the sugar name by “-an”. For example, xylan for polymer of xyloses or galactan for polymer of galactoses. For heteropolysaccharides containing two or more kinds of monosaccharides, the main one should be cited as a glycan term and the other as glyco prefixes. For example a galactomannan is composed of a main chain of linked mannose ramified by galactose residues. However, numerous PSs have a supplementary common name such as cellulose or chitin or are designated by a glycan term associated to the name of the producing organism. As an example the scleroglucan is a glucan produced by Sclerotium glucaninicum. Even if the term of PSs could be used for polymers composed of a minimum of 10 monosaccharides, fungal PSs are high molecular weight polymers. Mannans, chitin, and β-glucans (including cellulose) which are the main fungal PSs are found as CPSs. Chitin is a primary component of fungal cell walls while is a minor component of yeast cell wall. It is a Chapter 17 • Production of fungal biopolymers and their advanced applications 499 water-insoluble long-chain polymer consisting of repeating unit of β-(1,4)-N-acetylglucosamines β-(1,4)-linked, also present in many organisms other than fungi such as insect or crustaceans but also cephalopods. Mannans are abundant in yeast cell wall in the form of glycoproteins mainly composed of mannose residues linked with α-(1,2), α-(1,3), α-(1,6), and β-(1,2) linkages (Ohno, 2007). Apart from the case of cellulose which is a β-(1,4)-glucan, fungal β-glucans are mainly β-(1,3)/(1,6)-glucans present in cell walls of yeast and filamentous fungi or as soluble forms (Laroche and Michaud, 2007; Ohno, 2007). However, α-glucans, galactans, galactomannans, xylomannans, xyloglucans, arabinogalactans, mannoxyloglucans, fucomannogalactans, polygalactosamide, and polyuronides have been also described as constitutive cell wall biopolymers of some fungi or
ska-Jaroszuk et al., 2020). excreted by them to the extracellular medium (Ohno, 2007; Osin This book chapter focuses on fungal biopolymers and more especially on fungal PSs. It presents the state of the art about their biochemical structures, their production, extraction, and purification processes, as well as their applications as bioemulsifiers and biosurfactants, in medicine, and for new materials development such as textiles, and construction materials. 2. Fungal cell wall Fungal cell wall is a dynamic structure, which plays extremely important roles in the growth and survival of these microorganisms. It provides both plasticity and strength to the fungal cell. Plasticity of cell wall is necessary for the cell growth and cell division. On the other hand, cell wall acts as a protective layer for the fungal cells, which determines the shape of the cells, and avoids damages due to the osmotic pressure (Lima et al., 2019; Garcia-Rubio et al., 2020). In addition to maintaining the cell integrity, fungal cell wall controls the cellular permeability, and cellular interaction with the environment such as the attachment of fungi to the surfaces and uptake of nutrients (Bowman and Free, 2006). Therefore, the cell wall is not only an outer layer of the fungus, but also is a dynamic structure that significantly affects the fungal ecology. Furthermore, composition of cell wall is significantly regulated as a response to the environmental conditions (Gow et al., 2017). An indication of the importance of the cell wall in the fungal life is the fact that a large fraction of the whole genome (20% in yeast cells) is responsible for biosynthesis of the cell wall. These include genes for assembly of the basic components, providing the substrate for wall materials and regulators for assembly process with respect to the environmental signals (Gow et al., 2017). Furthermore, fungal cell dedicate the major fraction of its energetic output to biosynthesis of cell wall and its maintenance (Yarden and Osherov, 2010). In filamentous fungi, the cell wall plays an important role in development and integrity of the cells and protects the fungal cells from abrasions and toxic chemicals. Furthermore, cell wall is responsible for inflation of the cytoplasm; and therefore, gives the cells possibility to be highly pressurized. The high pressure allows the fungi to penetrate to the substrates (Yarden and Osherov, 2010). Fungal cell wall represents about 40% of the total volume and 20%–30% of the dry weight of the fungal cells (Bowman and Free, 2006; Lima 500 Current Developments in Biotechnology and Bioengineering Cementing components Outer surface of cell wall Structural components Inner surface of cell wall Chitin β-glucan FIG. 1 Fungal cell wall biocomposite structure containing fibrous structural components (such as chitin and β-glucans) and cementing components (such as glucans, chitosan, polyuronids, proteins, lipids, minerals, and pigments). Adopted from Gooday, G. W., 1995. Cell walls. In: Gow, N.A.R., Gadd, G.M. (Eds.), The Growing Fungus. Springer Netherlands, Dordrecht, pp. 43–62. et al., 2019). Regardless of the type of fungi, the fungal cells contain two different groups of components. Structural components are located close to the inner surface of the wall whereas cementing or gel-like materials are located close to the outer surface of the cell wall (Fig. 1). These components together make a strong and flexible structure (Gooday, 1995; Gow et al., 2017). The innermost structural components make a skeletal layer composed of fibrous PSs, i.e., chitin and β-glucans. These components are loadbearing fractions of the cell wall and make up around 50%–60% of the cell wall dry weight (Lima et al., 2019). Unlike the inner layer, the outer layer of the fungal cell wall varies depending on the fungal species (Gow et al., 2017). This layer is composed of the materials responsible for gluing the structural components together, and therefore retaining of the cell wall structure, such as proteins, glucans (α and β), chitosan, polyuronids, lipids, minerals, and pigments (RuizHerrera, 2012). In other words, the fungal cell wall is a natural biocomposite material (Yarden and Osherov, 2010). As discussed earlier fungal cell wall plays crucial roles in the growth and survival of the fungi. Furthermore, this unique biocomposite material has inspired researchers for development of fungal biomaterials for different applications. According to a former classification, the fungal kingdom was classified into eight different groups based on the fibrous structural compounds of their cell wall (Table 1). According to the current classification the fungi are classified in nine groups including Opisthosporidia, Chytridiomycota, Neocallimastigomycota, Blastocladiomycota, Zoopagomycota, Mucoromycota, Glomeromycota, Ascomycota, and Basidiomycota (Naranjo-Ortiz and Gabaldón, 2019). Therefore, according to the Table 1, most common fibrous structural components of fungal cell wall are chitin-glucan, chitin-chitosan, chitin-mannan, and glucan mannan. 3. Fungal cell wall polysaccharides Fungal cell walls have been designed by the nature according to a generic pattern and billion years of evolution have created a multiplicity of divergences, both for decorating Chapter 17 • Production of fungal biopolymers and their advanced applications 501 Table 1 Classification of fungi based on the composition of their cell based on old and new taxonomic groups of fungi. Group Fibrous structural components of cell wall 1 2 3 4 5 Cellulose-glycogen Cellulose-glucan Cellulose-chitin Chitosan-chitin Chitin-glucan 6 Mannan-glucan 7 Mannan-chitin 8 Polygalactosamine-Galactan a b c Old taxonomic group (Bartnicki-Garcia, 1968) Acrasiales Oomycetes Hypochytridiomycetes Zygomycetes Chytridiomycetes Ascomycetes Basidomycetes Deuteromycetes Saccharomycetaceae Cryptococcaceae Sporobolomycetaceae Rhoditorulaceae Trichomycetes Place in the new taxonomic group (Naranjo-Ortiz and Gabaldón, 2019) Not belong in fungal kingdom anymorea Opisthosporidia Not belong in fungal kingdom anymoreb Zoopagomycota and Mucoromycota Chytridiomycota Ascomycota Basidiomycota Ascomycota and Basidomycotac Ascomycota Basidiomycota Basidiomycota Zoopagomycota https://en.wikipedia.org/wiki/Acytostelium. https://en.wikipedia.org/wiki/Hyphochytriomycetes. https://www.sciencedirect.com/topics/immunology-and-microbiology/deuteromycetes. molecules and inclusive polymers (especially PSs) composing their structures. Differences in structural features have been noticed among the vast kingdom of fungi but also morphotypes inside the same species. These changes are mainly observed as soon as fungal cell-walls are conceptually separated in different layers from the inner (structural core) to the most outer ones. According to literature, PSs represent more than 90% of fungal cell
, 2007) and their location and interactions into these layers lead to specific wall (Latge involvements such as the fungal cell life (Netea et al., 2006), mechanical and physicochemical resistances (Free, 2013), adherence capacity and biofilm production
, 2018), or 3D network behavior (Gow et al., 2017). β-(1,3)-D-glucan (Beauvais and Latge and chitin (plus chitosan) are the major PSs commonly reported in fungal cell wall (Garcia-Rubio et al., 2020). Nevertheless, glycoproteins and melanin are also described inside fungal cell wall (sometimes up to 20%–30% in dry weight) and details are available in recent reviews dedicated to fungal cell wall proteins (Free, 2013; Gow et al., 2017; Garcia-Rubio et al., 2020). The presence of β-(1,3)-, β-(1,4)- and/or β-(1,6)-D-glucan, galactomannan, α-(1,3)-Dglucan and mannoproteins is also reported as species-dependent (Ruiz-Herrera and Ortiz-Castellanos, 2019) whereas mannan, glycosaminoglycan (GAG) and β-(1,3)-Dglucan are reported for fungi producing biofilms (Fontaine et al., 2011). α-(1,4)-D-glucose € n et al., 2004). Fungal cell walls are residues were also reported at the reducing end (Gru today considered as constantly reshaped depending on biotic and abiotic conditions that make it complicated to understand and accurately describe their structural features 502 Current Developments in Biotechnology and Bioengineering (Bleackley et al., 2019). To these constraints must be added the insolubility, complexity, and presence of amorphous biomolecules which make difficult the non-destructive and high-resolution analysis of these supramolecular assemblages (Kang et al., 2018). In this context, the scientific challenges are to better understand the effect of fungal cultivation condition (see Section 4) on the structure of fungal PSs in order to control the fungal cell wall composition for different applications. Today, S. cerevisiae, C. albicans, A. fumigatus, S. pombe, N. crassa, and C. neoformans are considered as the species having the most described and well-characterized cell wall. Thus recent overviews (Free, 2013; Gow et al., 2017; Garcia-Rubio et al., 2020) should be studied to better apprehend these complexity and variability in fungal cell wall components as shown in Fig. 2 for diverses fungal species such as Candida (with mainly chitin/glucan/mannan), Pneumocystis (with mainly glucan/mannan), Cryptococcus (with mainly galactoxylomannan/glucuronoxylomannan/glucan/chitin/chitosan), Histoplasma (with mainly glucan/chitin), and Aspergillus (with mainly galactomannan/galactosaminoglucan/glucan/chitin). Glucan can represent up to 60% (dry weight) of fungal cell wall, mostly with β-(1,3) linkages (Free, 2013). The inner skeleton layer consists of branched β-(1,3)-D-glucan with 3 to 4 interchain and chitin. The existence of intrachain hydrogen bonds between chitin and glucan allows the formation of a fibrous microfibrils structure, creating a scaffold which can serve as an anchor to covalently link other components including proteins, other amorphous carbohydrates (typically 10% or less of β-(1,4)-D-glucan, β-(1,6)-D-glucan, mannan, and galactomannan) and/or polymerized phenolic compounds (melanin)
, 2007). Ruiz-Herrera and Ortiz-Castellanos (2019) have thus overviewed seven (Latge types of fungal β-glucan, i.e., (i) unbranched β-(1,3)-glucan, (ii) β-(1,3)-glucan branched in O-6 position with few single glucose residues, (iii) glucan containing both β-(1,3) and β-(1,4) structures (in Ammophilus fumigatus), (iv) complex glucan with β-(1,3), β-(1,4), β-(1,6) moieties, (v) phosphorylated β-(1,3)-glucan, (vi) highlight branched β-(1,3)-glucan by β-(1,6)-glucose units and (vii) glucans mainly composed by β-(1,6) residues (in S. cerevisiae, C. albicans, or C. neoformans). Besides, the presence of α-(1,3)-glucan is also reported even if their structure and function in fungal cell wall are not fully understood. Their positioning in the cell wall is still controversial with some reports describing their location above the cytoplasmic membrane, as a fluid moving and varying under many factors (species, developmental form, plant cultures, etc.) (Erwig and Gow, 2016). The molecular mass weight (kDa) of these α-(1,3)-glucan ranged from 72 kDa (Lentinus edodes) to more than 800 kDa (Aspergillus wentii) and can represent 2% (Pleurotus eryngii) to 57% (Laetiporus sulphureus) of dry fungal mass. The main backbone is mainly composed of α-(1,3) linkages and multiple variations of structures are reported along the backbone, as example the presence of short spacers of α-(1,4)-D-Glcp into the main core or substituted residues such as !2.3)Glcp-(1!, !3.4)-Glcp-(1 ! or !3.6)-Glcp-(1!. A recent emphasis on fungal species containing α-(1,3)-glucan, including some structural characteristics was published according to Złotko et al. (2019). While α-(1,3)-glucan are the major component in fungal cell walls of Chi n layer E-glucan layers Most outer layer (Capsule) Mannan + proteins layer E-glucan layers Chi n/chitosan layers Protec ve D-glucan + proteins layer Chi n layer E-glucan layers *** GXM GalXM * * * * * * Histoplasma species Candida species E-glucan layers Mannan (without outer chains) + proteins layer (here H. capsulatum) (a) GAG + GM for hyphae; (b) Melanin + Rodlet for conidial walls Specific pa erns for GXM Type A D-(1,3)(1,4) and E-(1,3)-E-(1,4) glucan layers Chi n layer Type B (a) Type C Type D (b) Aspergillus species Pneumocys s species Cryptococcus species (here (a) A. fumigatus hyphae / (b) Aspergillus conidium) FIG. 2 Structural organization of the main fungal cell walls well described in the literature. International Union of Pure and Applied Chemistry (IUPAC) nomenclature has been used for drawing the glycan structures: ( ), ( ), ( ), ( ), ( ), (*), and ( ) correspond to Glc, GlcNAc, Xyl, GlcN, GlcA, Gal, and Man, respectively. Other legends: ( ) corresponds to proteins, ( ) to rodlet, ( ) to melanin, ( ) to galactomannan (GM), ( ) to galactosaminoglucan (GAG), ( ) to mannan, and ( ) to mannan without outer chains. Glc: Glucose, GlcNAc: A-acetyl glucosamine, Xyl: Xylose, GlcN: glucosamine, GlcA: Glucuronic acid, Gal: Galactose, GalXM: galactoxylomannan, and GXM: glucuronoxylomannan. 504 Current Developments in Biotechnology and Bioengineering filamentous fungi and dimorphic yeasts whereas budding yeast such as Saccharomyces cerevisiae do not contain α-(1,3)-glucan (A. Yoshimi et al., 2017). α-(1,3)-glucan acts as aggregation factor (A. fumigatus or H. capsulatum) (Fontaine et al., 2011), carbon source (Yoshimi et al., 2017), inhibitor for enzyme adsorption (A. oryzae) (He et al., 2017), cell-wall € n et al., 2005), or virulence factor maintaining actor (Schizosaccharomyces pombe) (Gru (Aspergillus fumigatus, Magnaporthe grisea, Blastomyces dermatitidis, or Paracoccidioides brasiliensis) (Yoshimi et al., 2017). Chitin, polymer of β-(1,4)-linked GlcNAc, contribute to the rigidity of glycan supramolecular structures of cell wall owing to their involvement in interchain hydrogen bonding for assembling microfibrils with glucan (Ruiz-Herrera et al., 2006). Chitin-glucan core is conventionally described in the inner layer close to the plasma membrane. Chitosan, the deacetylated form of chitin, can also exist in the fungal cell wall. The ratio between chitin and chitosan changes depending on species and physiological forms. Higher content of chitosan allows increasing the solubility and enhances the flexible capacity of the tangled structures. The amount in chitin that is converted into chitosan is different between fungal species and the physiological state of cells (Free, 2013). Chitin is directly involved in (i) capsule architecture of fungal cell wall (Zaragoza et al., 2009), (ii) interaction between species/host or, (iii) antifungal drug resistance as reported for Candida species (Louise et al., 2013). Structural features of mannan are different between yeasts and molds. This obviously affects their role in the cell wall. Mannan are less rigid due to their stereochemical configuration and are thus less involved in cell rigidity (shape of fungal cell wall). They are key factors in the permeability and porosity capacities of the fungal cell wall, such as for mannan-rich outer membranes, which allow evading the immune system in the host by hiding over reactive beta glucan present in the inner layer (Hernández-Chávez et al., 2017). For instance, galactoxylomannan (GalXM) were identified for masking PSs cell walls (Gow et al., 2017). Mannan are also engaged in biofilm process formation (adhesin)
, 2018), promoting complement-independent phagocytosis, immu(Beauvais and Latge nomodulatory effects, or inducing inhibitory factors (Pinto et al., 2008). The reported structures of N- and O-mannan and oligomannan greatly changed depending on the species, (i) high mannan structures for S. cerevisiae, C. glabrata, and C. albicans, (ii) galactomannan for S. pombe, A. fumigatus, and N. crassa, or (iii) xylomannan for C. neoformans. As example, an extracellular glucuronoxylomannan (GXM) forming a soluble capsule (shed) and composed of a linear α-(1,3)-linked D-Manp backbone with β-DXylp, β-D-GlcpA, and 6-O-acetyl constituents (Warnock, 1999) have been described in C. neoformans. Fu et al. (2020) recently proposed some insight into the assembling mechanism of capsular GXM from Cryptococcus and highlighted four different chemical structures (serotype A to D). Boletus edulis produces an alkali-soluble PS mainly composed of α-(1,3)-D-glucan chains (about 67%) containing also α-(1,3)-D-mannans (28%) (Choma et al., 2018). Mannan are often composed of proteins (mannoprotein and/or peptidomannan) which can take the form of long alkali-soluble chains (in yeasts for example)
, 2007). Outer layer or short chains bound directly to glucan/chitin (in A. fumigatus) (Latge cells of Candida species are thus dense in mannoproteins, structured by a α-(1,6)-linked D-Manp backbone with various oligomannoside side chains depending on species Chapter 17 • Production of fungal biopolymers and their advanced applications 505 (Shibata et al., 2012). Peptidomannan composed of N-mannan chains (around 15 Manp residues) with some phosphodiester linkers between some oligosaccharides have also been reported in C. albicans (Krylov and Nifantiev, 2020a). Pneumocystis cell walls do not have chitin and outer N-mannan chain but still have N- and O-mannan modified proteins (Ma et al., 2016). Peptidogalactomannan are also found in Aspergillus sp. and Pen
, 2007). In A. fumigatus, a branched galactomannan, made up of a linear icillium (Latge α-mannan with a repeating α-(1,2)/α-(1,6)-linked poly-D-mannoside backbone and short chains of β-(1,5)-Galf residues attached to the mannose units through β-(1,2)-, β-(1,3)- or β-(1,6)-bonds, is linked to a core β-(1,3)/β-(1,6) glucan (Fontaine et al., 2000; Krylov and Nifantiev, 2020b). Galactomannan structures (9%–14% in dry weight) have been € n et al., also found in the cell wall of fission yeast Schizosaccharomyces pombe (Gru 2005). Obviously, other original mannan structures have been described over the years such as (i) phosphomannan composed of α- and β-(1,2)-oligomannosides; (ii) phospholipomannan in the cell surface of C. albicans (Roeder et al., 2004), (iii) lipo(galacto)mannan anchored to the plasma membrane in yeast and molds (Costachel et al., 2005), and (iv) 2-O-methyl-D-mannose residues (8%–30%) reported in the EPSs produced by Mucorales species, which are mainly composed of β-(1,4)-GlcA residues (De Ruiter et al., 1994). Dimorphic phycomycetes such as Mucor rouxii can indeed produce two types of acidic polymers depending on the growth conditions even if the fibrillar structure is still composed of chitin and chitosan (Bartnicki-Garcia and Nickerson, 1962), i.e., (i) mucoran, which are heteropolymers composed of D-GlcA, D-Man, D-Gal and L-Fuc residues, and (ii) mucoric acid or fungal β-(1,4)-glucuronan (Dow et al., 1983; Lecointe et al., 2019). Similar structures have been reported in the cell wall of Absidia cylindrospora, Mucor mucedo, or Rhizopus nigricans (Tsuchihashi et al., 1983). More details have been described by Elboutachfaiti et al. (2011). Finally, other heteropolysaccharides (GAG) are also reported and are most often composed of Galp, GalpN and GalN units. These GAGs have been described for multifunctional adhesive properties and virulence capacity (Beauvais and Latg
e, 2018). For instance, galactosaminogalactan exposed on the outer surface in A. fumigatus cells are involved as immunosuppressor for protecting the pathogen inside the host (Fontaine et al., 2010). Fungal PSs structures available in fungal cell wall from many species have been reported in the literature (Free, 2013; Gow et al., 2017; Ruiz-Herrera and Ortiz-Castellanos, 2019; Garcia-Rubio et al., 2020). However, research at the academic and industrial levels is ongoing in order to: (i) cultivate fungi in scalable processes (see Section 4), (ii) develop efficient methods to extract and purify the fungal PSs (see Section 5), and (iii) process the fungal PSs for different applications (see Section 6). 4. Effect of growing conditions on production of fungal polysaccharides As the cell wall composition varies along the fungal morphotype, the construction of the cell wall is continuously restructured in different environmental conditions and the 506 Current Developments in Biotechnology and Bioengineering presence of stress factors (Latge and Beauvais, 2014). The nutrient source optimization has always been significant for improved production of any microbial bioproducts apart from the traditional extraction processes. However, during the last 5 years, many changes have been observed in optimizing growth condition for enhancing the production of fungal bioactive compounds. In unicellular, microscopic yeast Komagataella pastoris, reduced salts content medium has resulted in higher CPSs content through bioreactor cultivation (Farinha et al., 2019). For another probiotic yeast Lipomyces starkeyi, response surface methodology (RSM) resulted in a six-fold yield of EPSs in varying environmental conditions (Ragavan and Das, 2019). In the case of another unicellular and microscopic fungus Candida utilis, scale-up of β(1,3)/(1,6)-glucan production (both shake-flask and batch culture) have been done in waste potato juice water media supplemented with glycerol as carbon source (Bzducha-Wrobel et al., 2018). Luo et al. (2017) have also earlier produced a similar kind of report for Candida glabrata-mediated PS (PS) production using pyruvate as a carbon source. Nutrient media supplementation with vegetative oils in the submerged fermentation process has already been proved beneficial for PS production. Cultivation of Ganoderma lucidum in liquid substrate supplemented with olive oil and controlled oxygenation showed 17% wet-weight increase for six-days old vegetative inoculum; while 9.6 g L 1 of dry biomass has been obtained from batch cultivation, 15.2 g L 1 has been obtained during fed batch process (Berovic et al., 2003). In case of Cordyceps militaris, solid-state fermentation (SSF) technology was adapted for enhanced PS production and rice waste liquid media was supplemented with fructose and glycerin (Xu et al., 2019). Adaptation of SSF strategy for enhanced fungal PS production is also schematically represented in Fig. 3. Recently, for CPS production by Fusarium solani, 80% rice, 5% bean dregs, 10% rice bran, and 5% corn bran with maintained moisture level at 55% in SSF resulted in crude PS yield of 3.7  0.4 g/L (Zeng et al., 2019). Other than quantity enhancement, changes in monosaccharide composition and enzymatic activity have also been observed depending on the varying carbon sources used in the fungal cultivation. The relationship between the monosaccharide composition of CPS and the activity of related enzymes were studied using a correlation coefficient-based data acquired from various culture and carbon source conditions. Changes in α-phosphoglucomutase, phosphoglucose isomerase, and phosphomannose isomerase activities have been seen to be positively correlated with Galactose and Mannose mole percentages in G. lucidum PSs, based on correlation coefficient values obtained under various culture temperatures and pH values (Wang et al., 2017). Variability in monosaccharide composition/combinations among mushroom PSs depend on culture methods and conditions, medium composition, and extraction method. Indeed, the entire structural parameters (monosaccharide composition, degree of branching, molecular weight, and chain conformation) are reported to be greatly influenced by fermentation conditions (Zhao et al., 2014). In Pleurotus pulmonarius submerged culture, each sample had mannose, galactose, and glucose as major monosaccharide constituents (Smiderle et al., 2012). In Paecilomyces hepiali, high percentage of mannose was observed under glucose carbon source, while a high percentage of glucose was observed under mannose as carbon source Chapter 17 • Production of fungal biopolymers and their advanced applications 507 FIG. 3 Schematic diagram on fermentative production of fungal PS depending on the growth conditions. Created with BioRender.com. (Wu et al., 2014). In another study with Cordyceps sinensis mycelial cultures, the monosaccharide composition also varied according to the carbon source. The monosaccharides glucose, galactose, and mannose were found in all samples, with glucose being the major component (50%–75% of total). The addition of galactose or mannose to the culture medium resulted in no notable increase and in some cases, a slight decrease in the PS content (Chen et al., 2016). In a study with Tuber melanosporum by Zhao et al. (2014), addition of metal ions to the culture medium affected PS biosynthesis. It has been explained by the fact that metal ions work as a cofactor to the enzymes involved in PS biosynthesis. Thus, the presence of Mg2+ may alter some key enzymatic reactions changing the molar ratios of the monosaccharides (Zhao et al., 2014). Addition of Mg2+ to the media where galactose, glucose, and mannose are the main monosaccharides, resulted in maximum mannose content (27.6%), but no significant change in galactose or glucose contents. Several studies have also been performed on Mucor indicus, a zygomycetes fungus, which is majorly based on the effect of phosphate in the substrate on the fungal growth and composition of its cell wall. Other authors investigated the effect of phosphorous concentration in a semisynthetic medium containing glucose and yeast extract on the chitin and chitosan production by the fungus Mucor indicus. The lower concentration of phosphates, increased the sum of chitin and chitosan (total glucosamine content) in the fungal cell wall (Mohammadi et al., 2013). Safaei et al. (2015) studied the effect of two types of plant growth hormones (indole-3-acetic acid and kinetin) and in both cases presence of 508 Current Developments in Biotechnology and Bioengineering 1 mg/L of the hormones significantly improved the chitin and chitosan production in the fungal cell wall. The presence of trace metals significantly increased the chitosan yield at an optimal phosphate and potassium concentration. In a recent study, chitin production in the cell wall of the fungus Mucor indicus was increased more than five times by addition of phosphorous and potassium compounds to a semi-synthetic medium. Furthermore, chitin nanofibers were extracted from the cell wall of this fungus (Salehinik et al., 2021). Fermentative production of fungal PSs using different growth conditions has been briefly delineated in Fig. 3, where submerged fermentation or SSF are used for cultivation of both macro- and microscopic fungi. Different factors that influence the optimization of PS production are also demonstrated to change the structural parameters of the PSs. Overall, a paradigm shift has been observed in the last decade for optimization of fungal PS production. Different factors aggravated the optimization process; submerged and SSF, fermentation strategies, nutrient sources, cultivation time, presence of ions, presence of carbohydrate or non-carbohydrate carbon sources, and fermentation parameters (pH, temperature). While the optimization has not only augmented the PS production quantity, but also changed the structural parameters of the PS in an effective way. The majority of the studies related to the growing conditions of fungal PSs were observed on unicellular fungi like Candida or yeast, and mushroom or filamentous fungi like Mucor. While optimized production and efficient extraction of PS has already brought a big change in the field of fungal biotechnology, the future of optimization might be directed through a system biology approach, where integrated genomics, metabolomics, and secretomics will play key role to proficiently detect/modulate the cellular production strategies. 5. Extraction and purification of fungal biopolymers 5.1 Fungal chitin-glucan and chitin Fungal chitinous materials are formed as chitin–glucan complex (CGC), chitin and chitosan. Chitin–glucan complex (CGC) is a copolymer composed of chitin (poly N-acetyl-Dglucosamine) and branched 1,3/1,6-β-D-glucan (D-glucose homopolymer), having a minor amount of α-D-glucan. Chitin is the second most abundant PS after cellulose; it is a structural component of the crustacean shells and the cell wall of fungi from taxonomical groups of Zygomycetes, Ascomycetes, Basidiomycetes, and Deuteromycetes (Kaur and Dhillon, 2014). CGC is a natural component of most of fungal and yeast cell walls, providing stiffness and stability to the cells (Araujo et al., 2020). It is used as a source to obtain pure chitin and β-glucan PSs. Despite its suitability for various applications, the full potential of both CGC and chitin has not been achieved yet by their insolubility in water and most common organic solvents. Alpha-chitin is the crystalline polymorph found in the fungal cell wall, which is the most abundant form of this polymer in nature. The antiparallel arrangement of chitin chains in the crystalline cells and the high number of inter- Chapter 17 • Production of fungal biopolymers and their advanced applications 509 intramolecular hydrogen bonds between the functional groups of semi-crystalline chitin result in a highly packed material (Hassainia et al., 2018; Joseph et al., 2021). Thus, in addition to single PSs, the CGC contains a large number of inter- and intramolecular bonds among polymer chains, which accounts for its insolubility in water. Regarding the large-scale preparation of chitin and CGC from fungal cell walls, it can be noticed that industrial microorganisms have fast-growing cycling and highly predictable and reproducible composition of the cell wall. Then, extracting PSs from fungi and yeast would allow greater control of the material’s physico-chemical characteristics than crustacean sources (Hassainia et al., 2018). Also, having fermentation processes all-year-round could be advantageous because the production levels can be planned according to market demand. Another benefit of extraction of CGC and pure chitin from fungal cell walls compared to crustacean shells relies upon their non-animal sources and the generation of a lower amount of pollutant residues during processing. Besides, the fungal cell wall does not contain the human allergens tropomyosin, myosin light chain, sarcoplasmic binding protein, and arginine kinase found in crustacean shells (Khora, 2016). However, before large-scale extraction of such polymers, the raw material price must be considered. Then it would be advantageous if the fungal raw material would be a by-product from another industrial operation, citric acid production using Aspergillus niger, industrial waste beer Saccharomise cerevisae yeast, or discards from the industrial mushroom production. Although, other authors (Araújo et al. 2020b) suggest the possibility of co-producing CGC and xylitol utilizing glucose/xylose-rich substrates with Komagataella (Pichia) pastoris strain. It could also be a way to modify the composition of CGCs and their molecular weight. The industrial production of fungal CGC and chitin is relatively new compared to the chitin production from crustacean due to the large availability of shell waste from fisheries industries that encourage pioneers studies and scaling-up process development. For the large-scale production of fungal chitinous materials, two primary sources are used, Aspergillus niger biomass and Agaricus bisporus mushrooms. The main extraction processes used to isolate chitinous materials include hot alkaline (NaOH 1 M) treatment for protein and β-glucan polymers removal. In some cases, it is followed by an acid (HCl, H2SO4) treatment for remaining metal elimination (Table 2). A bleaching agent can also be used for the final product clarification, depending on the fungal source and selected application. Several companies are devoted to produce and commercialize fungal chitinous materials. Among them can be mentioned Kytozyme, a company from Belgium (https://www.kitozyme.com/en/), which produces chitosan and chitin-glucan polymers from Aspergillus niger biomass. After 15 years on the market, they developed a spin-out company KiOmedPharma (https://www. kiomedpharma.com/), devoted to produce carboxymethyl chitosan (CM-chitosan) and other chitosan-based pharma products. Also, Chibio Biotech (https://www. fungalchitosan.com/) is an eight-year-old company from China. They obtained chitosan 510 Current Developments in Biotechnology and Bioengineering Table 2 Summary of fungal chitin and chitin-glucan complex polymer properties (last 6 years). Source Extraction method Komagataella pastoris Mycelium, NaOH 1 M, 65°C, 5 h Aspergillus niger Mycelium, Sacharomise cerevisae Agaricus bisporus Hericium erinaceus Pleurotus ostreatus Rhizopus oryzae Yield (%) 13.4 Mycelium, Alkaline and acid Fruit body 1 M NaOH S/L (1:30) 80°C, 2 h Fruit body NaOH 2 wt%, 85°C, 3 h 5.9–7.4 16.3 Stalks 41.1 Mycelium, NaOH 1 M, 120°C, 20 min S/L (1:50) H2SO4 1 vol%, 120°C, 20 min 13.2 Physicochemical properties References CGC glucan: chitin molar ratio (75:25), Protein 3 wt% Mw 4.9  105 Da, PI 1.7 DAc 69.9%, CI 50% Chitin, DA 74.2% Protein >6% Chitin 92.8%, DAc 54.7% Protein 3.8% Mw 4.7  105 Da Chitin DAc 63.4%–79.8% CI 63% Farinha et al. (2015) Chitin purity 97.9 DAc 77.7% Mw 2.01  105 Da CGC, CI: 58.4 Ash: 3% Chitin DAc 89.8% Mw 2.7  106 Da Liao and Huang (2019) Ferreira et al. (2020) Sun et al. (2018a) Hassainia et al. (2018) Boureghda et al. (2021) Gachhi and Hungund (2018) from two sources, Pleurotus ostreatus (oyster mushroom) fruiting body and Aspergillus niger cell walls. As a result, they obtained a range of products like chitooligosaccharides, water-soluble chitosan and CM-chitosan. Another company is Beijing Be-Better Technology Co. (http://be-bettertech.com/products), an 11-year-old company from China. They produce chitosan products from the same sources as the former company but also from crustacean ones. Finally, ChitosanLab has its production facilities in China, but their selling offices are in France. This company produces fungal chitosan from Agaricus bisporus and Pleurotus ostreatus mushrooms and A. niger fungal cell wall. Chitin and chitosan from other sources are also available. In all cases, the fungal chitosan production know-how is protected by patents or industrial secrets. It is expected that industrial production of chitinous materials will increase in the future, considering growing raw material availability and people’s habit changes worldwide toward a vegetarian lifestyle and non-animal-derived products used in cosmetics, nutraceuticals, and organic farming. Some characteristics from CGC and chitin isolated from fungal species in last 6 years are summarized in Table 2. Chapter 17 • Production of fungal biopolymers and their advanced applications 511 5.2 Fungal chitosan Chitosan is a family of copolymers of β-1-4-linked D-glucosamine and N-acetyl-Dglucosamine in different ratios. They are traditionally obtained by thermochemical treatment of chitin isolated from the crustacean shell wastes. However, this process has some drawbacks such as: (1) environmental issues due to a high amount of pollutant chemicals used and high energy consumption, (2) limited availability of raw material (crustaceous shells), depending on the seasonal year, (3) heterogeneity of physicochemical characteristics in the end-chitosan products: degree of deacetylation (DD), degree of polymerization, protein content, among others, and (4) high-production cost (Singh et al., 2020). Fungi are an alternative source of chitosan and chitin; both are constituents of the fungal cell wall during their life cycle. Chitin is directly synthesized by fungal species, whereas chitosan is generated naturally by the deacetylase enzyme or alkali treatment that converts chitin to chitosan. Zygomycetes fungi are the only group in which chitosan is a natural component of the cell wall. Deacetylase enzymes have been isolated from fungi belong to the order of Mucorales and Saccharomycetales to convert the fungal chitin to chitosan in vitro. However, the activity of these enzymes is inhibited by the insolubility and crystallinity of the chitin (Ghormade et al., 2017). Recent advances in biotechnological tools offer promising possibilities for fungal chitosan production, with tailored properties, at an industrial scale. The Zygomycetes fungi have a huge potential for chitosan commercial production due to higher amounts of chitin and chitosan in their cell walls than other fungi classes. Several Zygomycetes strains are suitable for chitosan production such as Absidia coerulea, Absidia glauca, Benjaminiella poitrasii, Cunnighamella bertholletiae, Cunnighamella elegans, Gongronella butleri, Mucor rouxii, Mucor racemosus, Rhizopus arrhizus, and Rhizopus oryzae. These strains can produce 30–140 g of chitosan/kg dry biomass with a degree of deacetylation (DD) of 70%–90% (Ghormade et al., 2017). In addition to the strain selection, other relevant parameters to improve the quality and quantity of extracted fungal chitosan include: nature of the mycelia in solid or submerged fermentation; nutritional requirements for maximum growth; incubation time and conditions used; and chitosan extraction procedure (Kaur and Dhillon, 2014; Namboodiri and Pakshirajan, 2020). Usually, the chitosan extraction from fungal mycelia involves five steps: biomass extraction, alkali treatment, acid treatment, pH adjustment, and drying (Ghormade et al., 2017). The mild alkali treatment using 1 M NaOH at 120°C for 15–20 min is carried out to separate chitin/chitosan from the other carbohydrates, lipids and proteins available in fungal biomass. The insoluble material (chitin/chitosan is subjected to an acid treatment using, 2% (v/v) acetic acid at 90°C for 6–7 h for separation chitosan from chitin. Chitosan is soluble in acid solutions and can be recovered by increasing the pH to 8.0–9.0. The pretreatments of fungal biomass are also applied to improve the chitosan yield. Ma et al. (2021) investigated different pretreatment methods such as ionic liquid, steam explosion, and its combination to mycelium residues from citric acid fermentation. Steam explosion pretreatment (2.5 MPa for 1 min) of Aspergillus niger mycelium residues, combined with 512 Current Developments in Biotechnology and Bioengineering an ionic liquid extraction, increased the fungal chitosan enzymatic deacetylation using a Rhodococcus equi CGMCC14861 chitin deacetylase. The DD of chitosan was 1.2-fold higher than that produced from unpretreated mycelium residues. In another study (Yang et al., 2017), dilute acid-assisted steam explosion pretreatment of corn stover was effective for chitosan production from Rhizopus oryzae. Chitosan extraction was performed through subsequent alkali and acid treatments of fungal biomass, where cell-wall material was obtained by alkali treatment and chitosan was separated from the cell wall by dissolution in acetic acid. Pure chitosan was recovered by precipitation at alkaline pH. At the industrial scale, the utilization of fungal mycelium wastes resulting from biotechnological and food industries, and the improvement of chitosan yield within the fungal cell using the metabolic and process engineering strategies are two attractive approaches for the development of a cost-effective fermentation process of chitosan production (Ghormade et al., 2017; Singh et al., 2020). Both strategies are environmentally friendly due to reducing fungal waste harvest pollution and eliminating the expensive chemical deacetylation step. In last 5 years, the use of alternate inexpensive carbon sources such as cassava wastewater, steep corn liquor, and paper mill wastewater, for scaling-up fungal chitosan production have been of particular interest. Table 3 shows a summary of the current studies used waste material as a substrate for production of fungal chitosan. In conclusion, the use of low-cost fungal biomass is an unlimited economic source to fungal chitosan production. In addition, it will help to alleviate environmental pollution. The physicochemical properties of the fungal chitosan are consistent and can be controlled by changing the different fermentation variables. However, techno-economic studies of scaling up fungal cultivation and chitosan purification for industrial production must be performed to determine the economic feasibility and critical techno-economic variables that affect the profitability of fungal chitosan production. 6. Applications of fungal biopolymers The ability of the fungi for the growth on low cost substrates such as food waste, not only opens up opportunities for waste valorization but also set the stage for development of different products from the obtained fungal biopolymers (Svensson et al., 2021a). Here, different applications of fungal biopolymers will be discussed. 6.1 Bioemulsifiers and biosurfactants Bioemulsifiers and biosurfactants are surface active amphiphilic coumponds, produced by microorganisms, containing both hydrophilic and liphophilic functional groups (Alizadeh-Sani et al., 2018). Bioemulsifiers are polyphilic polymers that are effective in stabilizing oil–water emulsions and have wide applications in food industry. Biosurfactants efficiently reduce the surface tension between the air/water or oil/water interfaces and are used for several applications such as dispersion systems, personal hygiene, detergents, Table 3 Fungal chitosan form alternative biowaste resources. Fungal species Culture medium Extraction method Yield Physic-chemical attributes of chitosan a Mild alkali treatment using 1 M NaOH at 120 °C for 20 min 138 g/kg of dry fungal biomass DD ¼ 81% Mw ¼ 300 kDa Mild alkali treatment using 1 M NaOH (30 mL, v/v) at 121°C for 15 min 44.91 mg/g by L. hyalospora DD ¼ 80%–84% Mixture of cashew apple juice and cheese whey Mild alkali treatment using 1 M NaOH (1:40 w/v) at 121°C for 15 min 65–55 mg Chitosan /g of mycelial dry mass DD ¼ 75% Deuterated glycerol and d7-glucose Alkali treatment 2 N NaOH solution (1 g biomass: 100 mL solution) at 121°C for 30 min 1–7 wt% Free chitosan weight yield Aspergillus niger and Fusarium culmorum Mill potato suspension Mild alkali treatment using 1 M NaOH (30 mL/g solid) at 121°C for 30 min 19% Rhizopus oryzae AS 3.819 Xylose rich of corn stover prehydrolysate Steam explotion biomass treatment Milk alkali treatment 1 N NaOH (1:40 w/v) at 121°C for 15 min 0.09 g/g biomass. Penicillium citrinum IITG-KP1 Mucor subtilissimus UCP 1262 Lichtheimia hyalospora UCP 1266 Cunninghamella phaeospora UCP 1303 and Cunninghamella elegans UCP 1306 Yeast Pichia pastoris and Rhizopus oryzae Paper mill wastewater (lignin compounds and pentose sugars) supplemented with N-source, minerals and acetic acid Mixture of corn steep liquor and cassava wastewater DD ¼ 66%–83% Mw ¼ 90–27 kDa for A. niger, Mw ¼ 146–112 kDa for F. culmorum DD ¼ 91% Application Reference Treatment of paper mill wastewater and simultaneous chitosan production Namboodiri and Pakshirajan (2019) de Souza et al. (2020) Antimicrobial activity against Scytalidium lignicola and Fusarium. solani Berger et al. (2020) Chitosan film useful for enzyme immobilization and future neutron scattering experiments. Remotion of metallic contaminant and pesticide from water Yuan et al. (2021) CabreraBarjas et al. (2020) Yang et al. (2017) Continued Table 3 Fungal chitosan form alternative biowaste resources—cont’d Fungal species Culture medium Extraction method Yield Cunninghamella elegans SIS 41 Mixture of corn steep liquor and papaya peel juice 2% (w/v) NaOH (1:30 w/v at 90°C for 2 h/10% acetic acid (1:40 w/v) at 60°C for 6 h 37.2 mg/g Fresh cut mushrooms strain A15 Mushroom stipe offcuts 2 M NaOH at 100°C for 2 h/1% w/v oxalic acid at 100°C for 1 h 105 mg/g Rhizopus oryzae Whey salt medium and molasses salt medium Mild alkali treatment using 1 M NaOH (1:40 w/v) at 121°C for 15 min/0.05 N H2SO4 (1:100, w/v) at room temperature/ 2% acetic acid (v/v) for 24 h at 95°C 10.0%– 11.5% a Degree of deacetylation. Physic-chemical attributes of chitosan DD ¼ 86% Mw ¼ 40.8 kDa DD ¼ 87%–82% Mw ¼ 120–178 kDa Application Reference Antibacterial effects against different phytopathogenic Colletotrichum species Active edible coatings for inhibition of Saccharomyces cerevisiae yeast and Escherichia coli Ramos Berger et al. (2018) Poverenov et al. (2018) Chatterjee et al. (2019) Chapter 17 • Production of fungal biopolymers and their advanced applications 515 and agriculture (Sena et al., 2018; Luft et al., 2020). While biosurfactants and bioemulsifiers are mainly produced by bacteria and yeast, recently filamentous fungi have shown high capacity for production of these materials (Sena et al., 2018; Sanches et al., 2021). Different type of extracellular biosurfactants is reported to be produced by fungi, among which glycolipids (containing a carbohydrate part which is linked to fatty acids) and lipopeptides (containing a hydrophobic tail of fatty acids linked to a hydrophilic head of amino acids), and complexes of carbohydrates with proteins and lipids can be mentioned (Luft et al., 2020; Sanches et al., 2021). Furthermore, yeast and filamentous fungi have shown the capacity for production of different types of bioemulsifiers. Mannoproteins are one of the most important type of fungal bioemulsifiers that are available in the cell wall of yeast (Saccharomyces spp. and Kluyveromyces marxianus) and can be released by treatment of the yeast biomass with aqueous solutions under pressure in autoclave (Dikit et al., 2010; Alizadeh-Sani et al., 2018). By optimization of the fermentation conditions, using low costs substates, and more efficient methods for biopolymers recovery, it is expected that production of fungal bioemulsifiers and biosurfactants becomes more economically feasible in the future (Luft et al., 2020). Furthermore, fungal bioemulsifiers and biosurfactants can be extracted from by-products of other industries where yeast/filamentous fungi are used for production of other products such as wine (Dikit et al., 2010). Moreover, recently fungal biopolymers that are released from the fungal biomass in the process of edible mycoproteins production, Quorn™, have shown promising foaming and emonsidying properties for using in the food industry (Lonchamp et al., 2019). 6.2 Wound healing materials Application of fungal-based materials for wound healing goes back to ancient times where fungi was used to stop bleeding (Jones et al., 2020a). In 1994, Chung et al. (1994) tested effect of alkali insoluble material of A. oryzae, M. mucedo, and P. blakesleeanus on rate of proliferation of human fibroblasts and observed proproliferant activity over 13 days. These fungi belong to the division of ascomycota, zygomycota, and mucoromycota, respectively. This means their cell wall contains mainly chitin-glucan, chitin-chitosan, and chitin-chitosan, respectively. In that study, a correlation was reported between the proproliferant effect of fungal alkali insoluble material and their chitin content where P. blakesleeanus with highest chitin content (91%) resulted in highest enhancement in cell proliferation. Since 1997, most of the research studies conducted on wound healing properties of the fungal chitin have used fungal species that have chitin-glucan in their cell wall. In 1997, Su et al. (1997) used fruiting body of Ganoderma tsugae to make a membrane called Sacchachitin which was tested as a skin replacement. Ganoderma tsugae is a mushroom that belongs to the division of Basidiomycota, which contain chitin-glucan in their cell wall. The alkali insoluble material of this mushroom, containing 60% glucans and 40% chitin, was suspended in water and subjected to a wet laying process to form a membrane which was then freeze dried. The dried membrane, Sacchachitin, improved the wound healing process in rat skin compared to the control experiment where cotton gauge 516 Current Developments in Biotechnology and Bioengineering was used to cover the wound. Later, in 2004, effect of Sacchachitin membrane was investigated on the rate of wound heling process and the resulted indicated that Sacchachitin may increase the wound healing rate by earlier expression of proliferating cell nuclear antigen and type I collagen compared to the control experiments. Furthermore, Sacchachitin led to later expression of tissue-transglutaminase which is an indicator for apoptosis and maybe a sign for longer period of blood supply to the wound area which facilitates the wound healing (Chao et al., 2020). 6.3 Tissue engineering Biopolymers play an important role in the development of scaffolds that can induce the regenerative processes in the body. Nwe et al. (2009) extracted chitosan from cell wall of Gongronella butleri and developed a scaffold by dissolution of chitosan in acetic acid solution followed by freeze drying. The obtained scaffold exhibited an interconnected porous structure and showed high mechanical strength, and promoted proliferation of fibroblast cells which are necessary properties for material used in tissue engineering (Nwe et al., 2009). Narayanan et al. (2020) developed a 3D scaffolds containing chitin-glucan by growing of Aspergillus sp. on potato dextrose broth under static conditions to form a fungal mat, followed by dehydration of fungal mat using ethanol, and freeze drying. The surface associated proteins of obtained fungal-scaffolds were then solubilized through reaction with a reducing agent. Human keratinocytes were seeded on the fungal-scaffold and the results indicated deposition of extra cellular matrix components and formation of cell sheets in 14 day. This indicates the potential of the fungal-scaffold for tissue engineering applications. 6.4 Antimicrobial and preserving agent From the last two decades, the antimicrobial effect of chitosan and its derivatives has fascinated lot of attention in agriculture, pharmaceutics, medical, and food industry (Brasselet et al., 2019). Chitosan was demonstrated to be efficient inhibitor of the growth of many microbials species such as yeast, bacteria and fungi among them spoilage, phytopathogens, and pathogens species (Kiskó et al., 2005; Raafat and Sahl, 2009; Hosseinnejad and Jafari, 2016; Hu et al., 2019). By comparison with other biomolecules such as phenolic compounds, chitosan presents 30%–50% higher antimicrobial activity and a large antiseptic spectrum (Raafat and Sahl, 2009; Brasselet et al., 2019) not only on Gram-positive bacteria (Staphylococcus aureus or Bacillus subtilis) but also to Gramnegative bacteria (Pseudomonas aeruginosa or Escherichia coli). Therefore, chitosan and its derivatives have been mainly described for their ability to eliminate microorganism in biofilm and planktonic form and to reduce the adhesion and growth of microorganism: (i) on specific surface of antiseptic material (Costa et al., 2017; Campana et al., 2018) and (ii) in liquid food such as fruit juices or wine (Kiskó et al., 2005; Paulin et al., 2020). The last years in winemakers industries, fungal chitosan have been approved by European Union and International Organization of Vine and Wine Chapter 17 • Production of fungal biopolymers and their advanced applications 517 (International Code of Oenological Practices) as non-allergenic, non-toxic, and renewable natural antiseptic biomolecules for wine treatment to reduce Brettanomyces microorganisms by sedimentation process (Taillandier et al., 2015; Paulin et al., 2020). In this context, recently the antiseptic effect of fungal chitosan on Brettanomyces bruxellensis strains which are yeast and well-known as the main spoilage microbial agent in red wines has been confirmed (Paulin et al., 2020). In this study on a collection of 53 strains of B. bruxellensis, such as 13 diploid strains belong to the CBS2499 genetic group, 13 triploid strains belong to the genetic group AWRI1499, 14 triploid strains belong to the genetic group AWRI1608, and 13 strains distributed into the L14165, L0308, and CBS5512 genetic clusters were investigated. The authors particularly showed that two types of fungal chitosan (a high molecular weight fraction of 400 kDa and a low molecular weight fraction of 32 kDa) could specifically disturb and significantly decrease the cultivable Brettanomyces bruxellensis strains by flocculation/sedimentation step (Paulin et al., 2020). Concerning the real antimicrobial effect from chitosan and derivatives, lot of studies proposed diverse mechanistic approaches due to the specific interaction between the cationic charge of chitosan (R-NH3+) and the negatives charges of the bacterial surface (No et al., 2002; Raafat and Sahl, 2009; Younes et al., 2014b). As observed in Fig. 4, some antimicrobial mechanisms largely described in literature could be suggested. All the main described mechanisms could be summarized as: (i) coating of the cell wall (negatively charged) or directly the cell membrane leading to the establishment of an oxygen/nutrient barrier; (ii) alteration of membrane cell permeability and energy generating pathways leading to cellular morphological change; (iii) low molecular weight chitosan such as chitooligosaccharides (COSs) induce cell aggregation and insoluble fractions act as fining agents; (iv) COS may cross the walls and membranes to interact with Cells with external wall/polysaccharides iii. COS induce cell agregation and insoluble fractions act as fining agents ii. Alteration of membrane permeability and energy generating pathways Cells with external membrane iv. COS may cross the walls and membranes v. Damage to DNA, RNA, proteins; stress, autolysis vi. Wall and membrane disruption iii. vi. Collapse of internal membrane or total disruption of the two membranes; internal material leakage v. ii i. i. Coating the cell wall (negatively charged) or directly the cell membrane vii. Negatively charged nutrient sequestration +++++++++ ++++ ++ Positively charged chitosan FIG. 4 Schematic representation of the proposed antimicrobial mechanisms of chitosan. Adapted from Brasselet, C., Pierre, G., Dubessay, P., Dols-Lafargue, M., Coulon, J., Maupeu, J., Vallet-Courbin, A., de Baynast, H., Doco, T., Michaud, P., Delattre, C., 2019. Modification of chitosan for the generation of functional derivatives. Appl. Sci. 9(7), 1321. 518 Current Developments in Biotechnology and Bioengineering DNA; (v) damage to DNA transcription (supposed interaction between chitosan and 30S ribosomal subunit), RNA, proteins, stress, autolysis; (vi) wall and membrane disruption and finally, (vii) negatively charged nutrient sequestration. Mostly, studies indicated that the main ionic interactions between chitosan and microorganisms disturb the cell surface leading to contents leakage in bacterial growth inhibition (Raafat and Sahl, 2009; Brasselet et al., 2019). Nevertheless, other structural parameters of chitosan have also been widely described in the modulation and increasing of antimicrobial effect such as the molecular weight (Mw) and the degrees of deacetylation (DD) which are well known to control the water solubility of chitosan and derivatives (Qin et al., 2006; Brasselet et al., 2019). Therefore, many publications reported the physicochemical or enzymatic production of efficient antimicrobial chitosan derivatives with low molecular weight (such as chitooligosaccharides) and high degree of deacetylation to increase antimicrobial activities against fungi and bacteria (No et al., 2002; Qin et al., 2006; Raafat and Sahl, 2009; Younes et al., 2014b; Brasselet et al., 2019). Finally, it is important to mention that in chitosan aqueous solutions, the antimicrobial effect is strongly affected by the pH solution (range of pH 4–6) with increased effect at lower pH and decreased effect at pH near to pH 7 due to insolubility and deprotonation of chitosan (RNH3+ ! R-NH2) (No et al., 2002; Younes et al., 2014a). In this context, some chemical modification of chitosan such as carboxymethylation, and N-trimethylation have been investigated to increase antibacterial activity at pH higher than 7 (Meng et al., 2012; Shariatinia, 2018). The last decades, lots of antimicrobial studies have been reported using fungal PSs (chitin, chitosan, glucan, and their derivatives). For example, chitin and chitosan extracted from Pleurotus spp. using alkaline method gave antimicrobial effect (Johney et al., 2016). In fact, authors demonstrated that chitin and chitosan from P. florida and P. eous have: (1) antibacterial effect against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli and, (2) antifungal effect against Fusarium solani, Aspergillus flavus, and Aspergillus niger. In another study, Jeihanipour et al. (2007) investigated the antimicrobial properties of chitosan extracted from the cell wall of filamentous zygomycetes fungus Rhizopus oryzae. Authors observed the reduction of bacteria viability for Staphylococcus aureus, Klebsiella pneumoniae, and E. coli higher than 60% when they used a fungal chitosan concentration of 200 ppm. More, in this study (Jeihanipour et al., 2007) analyzed the minimum bactericidal concentration (MBC) of the fungal chitosan from R. oryzae and MBC was estimated at 700, 500, and 300 ppm for K. pneumoniae, E. coli, and S. aureus, respectively. Recently, an interesting approach was proposed to improve the antibacterial effect of fungal chitin-glucan complex by grafting of gallic acid using a free radical mediated method (Singh et al., 2019). Chitin-glucan complex was firstly extracted from the mushroom Agaricus bisporus and secondly gallic acid was covalently grafted into chitin-glucan complex with a degree of derivatization of 40% employing ascorbic acid/hydrogen peroxide reactive system. Interestingly, compared to chitin-glucan complex, gallic acid grafted chitin-glucan complex was more efficient on growth inhibition of Escherichia coli and Bacillus subtilis. Chapter 17 • Production of fungal biopolymers and their advanced applications 519 In the same strategy, Wan-Mohtar et al. (2016) chemically sulfated a β-D-(1,3)-Glucan extracted from the mushroom Ganoderma lucidum in order to obtain effective watersoluble antimicrobial agent against a panel of several bacteria well-known in human health and food topics. Therefore, β-D-(1,3)-Glucan was sulfated using dimethyl sulfoxide/urea/sulfuric acid media in order to produce a soluble sulfated β-D-(1,3)-Glucan (GS) with a degree of sulfation (DS) of 0.90 (i.e., 90 sulfate residues for 100 glucose units in the main glucan backbone). As observed, this GS showed antimicrobial effect with minimum inhibiting concentration (MIC) of 1, 2, and 5 mg/mL for Escherichia coli, Staphylococcus aureus and Staphylococcus epidermis, respectively. Furthermore, the MBC was estimated at 2, 5, and 10 mg/mL for E. coli, S. aureus and S. epidermis, respectively. Finally, it is worth mentioning that very interesting recent reviews described the high potential of antimicrobial activity of chitin, chitosan, and glucan extracted from several fungal organisms (Lehtovaara and Gu, 2011; Araújo et al. 2020b). 6.5 Textiles (textile fibers, nonwoven textiles, and leather like materials) and paper like materials from fungi Due to the unsustainable cultivation of cotton, the environmental concerns regarding use of synthetic textiles, and struggling issues in natural leather productions, sustainable textile alternatives are highly demanded. In recent years fungal polymers have been introduced as promising resources for the textile production and the fungal materials have been processed to form leather like materials, paper like materials, and textile fibers (Jones et al., 2021). Liquid state and solid state fermentations are used for production of fungal biomass and development of textiles and paper like materials. When using liquid-state fermentation, the filamentous fungi grow on liquid media in shake flasks or bioreactors and pure fungal microfibers are separated from the media at the end of cultivation. Different substrates have been used for fungal cultivation, including (semi)synthetic media (Appels et al., 2020; Attias et al., 2021). However, using byproducts and waste streams is preferred since it will contribute to a lower production cost and more sustainable process. Jones et al. (2019a, b) used molasses which is a byproduct obtained in the sugar production process for growth of filamentous fungi and development of paper € hnlein (Ko € hnlein, 2020), Wijayarathna like materials. Recently, Svensson et al. (2021b), Ko (2021), and Mohammadkhani (2021) used a suspension of bread waste in water for cultivation of filamentous fungi and development of fungal based textiles and paper like materials. SSF can also be used for cultivation of filamentous fungi using lignocellulosic materials as the substrate. However, in this method separation of the fungal microfibers from the substrate residues is not possible and the obtained fungal material has foam like properties (C.f. Section 6.6). The fruiting body of the mushrooms can also be processed to develop textile and paper like materials. The fungal textiles and paper like materials are either developed using the whole fungal biomass or just its cell wall material containing PSs, namely chitin-chitosan or chitinglucan. Fungal leather like materials is produced by processing the whole fungal biomass. 520 Current Developments in Biotechnology and Bioengineering There are already some commercialized leather alternatives from fungi. MycoTech which is an Indonesian biotechnology-based company has released several products such as wallet, watch strap, and card tags based on their mushroom leather. MyloTM is another example which is a leather alternative produced from fungal mycelium grown on sawdust. Muskin is another fungal-based leather produced from the caps of the mushroom, Phellinus ellipsoideus, through non-toxic tanning treatments (Bustillos et al., 2020). However, details of those processed are not available and on the other hand, the properties of the available fungal leather like materials still need to be improved to replace the natural leather. Therefore, research is still ongoing in order to improve the process using more sustainable materials, to enhance the properties of the fungal leather alternatives, a replacement for the fossil-based polyester textile support which is nowadays used in the in formulation of leather alternatives (Meyer et al., 2021). Appels et al. (2020)) applied a post treatment, with different concentration of glycerol, to the films of the whole mycelium of the fungus Schizophyllum and developed films with different properties from polymer to elastomer like materials. (Wijayarathna, 2021) developed leather like materials from the fungus Rhizopus delemar grown on bread waste, through tanning of the fungal microfibers, wet laying of the microfibers, and post-treatment of the fungal films using glycerol and a bio-binder. Paper-like materials can be produced from the cell wall fraction of the filamentous fungi. Alkali treatment (for example, by sodium hydroxide solution) is usually used to remove the alkali soluble materials from the fungal biomass, leaving the cell wall fraction (i.e., alkali insoluble material). The fungal cell wall material have a fibrous structure containing micro (when mycelium is used) or nano-sized (when fruiting bodies are used) fibers of chitin together with glucans or chitosan. Often the paper like materials are produced from edible fruiting body of fungi, i.e., white champignon mushroom Agaricus biporus (Nawawi, 2016). Among different fungi, alkali insoluble materials of microfibers of A. arbuscula, M. genevensis (Jones et al. (2019a, b)), T. versicolor (Jones et al. (2019a, € hnlein, 2020) have been used for production of wet laid paper-like b)), and R. delemar (Ko materials. Moreover, chitin-glucan nanofibers extracted from fruiting bodies of A. bisporus and D. confragosa have been use for development of nanopapers with high tensile strength (more than 200 MPa) which are more hydrophobic than nanopapers made using crustacean chitin (Nawawi et al., 2019, 2020b). Generally, the papers made using nanofibers exhibit higher tensile strength compared to the one made using microfibers. During the alkali treatment a significant fraction (More than 70%) of the fungal biomass is dis€ hnlein (2020) developed an enzymatic treatsolved in alkali solution and discarded. Ko ment process using protease to extract the fungal cell wall and developed wet laid materials from the enzyme treated microfibers. The enzymatic approach set the stage for recovery of the soluble fraction of fungal biomass (namely proteins) for other applications. The fungal wet laid materials usually exhibit a yellow to brownish color and heat treatments during the drying process results in darkening of the color. Bleaching of the fungal micro and nanofibers results in formation of transparent films after wet laying and drying steps. The highest tensile strength of the fungal wet laid microfibers was Chapter 17 • Production of fungal biopolymers and their advanced applications 521 € hnlein (2020) which was 71.5 MPa. For the nanopapers derived from mushreported by Ko room higher tensile strength up to 204 MPa (Nawawi et al., 2020a) has been reported. Biocomposite nonwoven textiles have also been developed using fungal microfibers and cellulosic fibers such as flax (Nawawi, 2016), hemp (Irbe et al., 2021), and viscose € hnlein, 2020) fibers. In the biocomposites, fungal microfibers act as a binder for the (Ko cellulosic fibers. Fungal leather and paper-like materials are made by random distribution of fungal micro and nanofibers. Recently, Svensson et al. (2021b) reported alignment of the fungal microfibers of the zygomycetes fungus R. delemar (grown on bread waste) along one axis and developed fungal monofilament yarns. This was done through wet spinning of a hydrogel containing fungal cell wall into a coagulation bath containing ethanol. This opens up opportunities for development of the woven fungal textiles for different applications. Mohammadkhani (Mohammadkhani, 2021) reported that the fungal wet spun filaments exhibit antibacterial properties and therefore can be a good candidate for medical textile applications. 6.6 Biocomposites for construction and packaging applications Another innovative feature of usage of fungal biopolymers is their introduction for packaging and construction applications as alternatives for traditional materials (Jones et al., 2020b). Fungal biocomposite materials are produced through SSF of the filamentous fungi on lignocellulosic materials, namely agricultural and forestry byproducts (Jones et al., 2017). Therefore, usually fungal species with the ability for synthesizing the enzymes needed for degradation of the lignocellulosic materials such as white rot fungi are used for development of fungal biocomposites (Jones et al., 2017). During the growth, fungal microfibers are formed as a binder which holds the lignocellulosic material together resulting in a biocomposite structure. Therefore, the whole fungal biomass is used in the structure of fungal biocomposite materials. The obtained biocomposite material is dried to stop the fungal growth and usually subjected to a hot/cold pressing to reduce the porosity, increase the density, and therefore enhance the mechanical properties of the biocomposite material (Jones et al., 2017; Appels et al., 2019). The fungal mycelium usually makes around 5% of the obtained biocomposites while the rest is the residues of the lignocellulosic substrates used for cultivation of the fungi (Jones et al. (2019a, b)). The properties of the obtained biocomposites depend on the type of the substrate (Elsacker et al., 2019). Using low-cost agricultural residues and wastes, such as straw, results in formation of biocomposite materials with foam-like properties. These foam like biocomposite materials are suitable for packaging applications (Jones et al., 2020b). For example IKEA (the world’s largest furniture retailer since 2008)a uses the fungal biocomposite materials as a replacement for polystyrene in their packaging.b Furthermore, getting benefit of the low density and low thermal conductivity of the lignocellulosic substrates, fungal a https://en.wikipedia.org/wiki/IKEA#cite_note-6. b https://www.intelligentliving.co/ikea-mushroom-based-packaging/. 522 Current Developments in Biotechnology and Bioengineering biocomposites are good candidates for insulation applications with properties comparable to commercial thermal insulation materials such as glass wool and polystyrene (Xing et al., 2018). Furthermore, due to presence of lignin and silica in the lignocellulosic substrates, the fungal biocomposite materials exhibit fire-retardant properties (Jones et al., 2018). Highwater absorption properties of the fungal-based biocomposites limit their use for indoor applications. There are some prototypes and few commercial products of fungal biocomposites already available. For example, furnishing items such as chairs have been prepared using the fungal biocomposites and a prototype house with 10 m height using fungal bricks has been made (Jones et al., 2020b). There are also some companies in the USA (Ecovative design), Italy (Mogu), Indonesia (Mycotech), and the Netherlands (Officina Corpuscoli), which have released fungal-based biocomposites to the market for packaging and construction applications. However, the mechanical properties of the fungal-based biocomposites still needs to be improved before the materials can be used for commercial construction applications. 6.7 Other applications of fungal biopolymers The application of the fungal biopolymers is not only limited to the area mentioned in Sections 6.1–6.6 and other applications have also been suggested. European Food Safety Authority (ESFA) has confirmed the safety of chitin-glucan to be used as a food ingredient with the purpose to increase the daily intake of fibers (EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2010). Chitin-glucan has also been recognized as safe (GRAS) by the Food and Drug Administration (FDA, USA) as a novel food ingredient (EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2010). This material is available in weight losing tablets provided by KitoZyme (Belgium). Chitin-glucan can be consumed as a dietary supplement with an intended 2–5 g/day intake. Recently, an in vivo study in rats was conducted (Alessandri et al., 2019) and concluded that chitin-glucan is a valuable novel prebiotic molecule, which could be introduced to the human diet to re-establish/ reinforce bifidobacteria colonization in the mammalian gut. On the other side, fungal chitin, chitosan, chitin-glucan, or its hydrolysate are a valuable product for wine limpidity prevention (Bornet and Teissedre, 2008). Their use improved wine safety by reducing heavy metals (Fe, Pb, Cd), and mycotoxins (ochratoxin A) levels. Fungal cell wall biopolymers, in pure form as well as their complex in the cell wall, have shown high potential for removal of heavy metals from aqueous solutions (Rouhollahi et al., 2014; Behnam et al., 2015). Several authors reported the potential applications of fungal chitin and chitin-glucan in agriculture as pathogen control agents. For example, Sun et al. (2018a) studied the influence of the postharvest treatment with fungal chitin from Saccharomise cerevisae on disease resistance against Botrytis cinerea infection in tomato fruit. The results showed that fungal chitin dipping treatment effectively produces strong resistance to B. cinerea in tomato fruit. The fruit protection occurred by eliciting several plant defense responses such as proteins (β-1-3-glucanase, chitinase, PAL, SOD, CAT), and callose accumulation in tomato peels. This finding agrees with the previous one using chitin-glucan containing Chapter 17 • Production of fungal biopolymers and their advanced applications 523 cell wall preparation from Rhodosporidium paludigenum yeast to protect pear fruits (Sun et al., 2018b). It created disease resistance against blue mold rot caused by Penicillium expansum in pear fruit and reduced the fungal germination fruit wounds. Fungal chitin and chitosan can be used in nearly all applications where the shellfish chitin and chitosan are used. For example superabsorbent materials have been developed from cell wall of zygomycetes fungi and purified fungal chitosan (Zamani, 2010; Zamani and Taherzadeh, 2012). Furthermore, hydrogels have been produced from the chitosan extracted from the cell wall of the fungus Aspergillus niger for application in controlled drug release (Muñoz et al., 2015). Recently, fungal chitosan has been successfully tested to be used as blood-clotting agent (Radwan-Pragłowska et al., 2021). Biocompatible hydrogels have also been produced from chitin-glucan extracted from the cell wall of yeast Komagataella pastoris (DSM 70877) for potential medical applications such as drug delivery (Araújo et al., 2020). 7. Conclusions and perspectives Fungal cell wall displays a unique biocomposite structure which is composed of different PSs. Chitin, glucans, and chitosan are major components of the fungal cell wall. Composition of the fungal cell wall can be controlled by selection of the substrate and controlling the cultivation conditions. Liquid-state submerged cultivation give the possibility for production of pure fungal microfibers while the solid state cultivation results in formation of a composite structure where fungal microfibers act as a binder for the residues of the substrate. Fungal biopolymers exhibit antimicrobial properties and are biocompatible. Those properties make them good candidates for biomedical applications. The whole fungal biomass, its cell wall, or its purified biopolymers have been used for development of novel sustainable textile fibers, leather alternatives, paper-like materials, and construction materials. Fungal biopolymers are also a promising source for bioemulsifiers and biosurfactants, for application in food and agriculture industries. With the inspiration of the recycling of the materials in the nature, fungi can be key elements for bioconversion of different waste materials and side streams to functional biopolymers. 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, J.L., Weiss, K.L., Pingali, S.V., Hong, K., Urban, V.Yuan, Y., Li, H., Leite, W., Zhang, Q., Bonnesen, P.V., Labbe S., Salmon, S., O’Neill, H., 2021. Biosynthesis and characterization of deuterated chitosan in filamentous fungus and yeast. Carbohydr. Polym. 257, 117637. Zamani, A., 2010. Superabsorbent Polymers from the Cell Wall of Zygomycetes Fungi (23 Doctoral thesis, monograph). Chalmers University of Technology. Zamani, A., Taherzadeh, M.J., 2012. Production of superabsorbents from fungal chitosan. Iran. Polym. J. 21 (12), 845–853. Zaragoza, O., Rodrigues, M.L., De Jesus, M., Frases, S., Dadachova, E., Casadevall, A., 2009. The capsule of the fungal pathogen Cryptococcus neoformans. Adv. Appl. Microbiol. 68, 133–216. Zeng, Y.J., Yang, H.R., Wu, X.L., Peng, F., Huang, Z., Pu, L., Zong, M.H., Yang, J.G., Lou, W.Y., 2019. Structure and immunomodulatory activity of polysaccharides from fusarium solani DO7 by solid-state fermentation. Int. J. Biol. Macromol. 137, 568–575. Zhao, W., Chai, D.D., Li, H.M., Chen, T., Tang, Y.J., 2014. Significance of metal ion supplementation in the fermentation medium on the structure and anti-tumor activity of tuber polysaccharides produced by submerged culture of tuber melanosporum. Process Biochem. 49 (12), 2030–2038.
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sko, A., Pleszczyn A report on fungal (1!3)-α-d-glucans: properties, functions and application. Molecules 24 (21), 3972. 18 Versatility of filamentous fungi in novel processes € lru Bulkan, Jorge A. Ferreira, and Mohsen Parchami, Taner Sar, Gu Mohammad J. Taherzadeh SWE DISH C ENTRE FOR RE SOUR CE R ECOVE RY, UNIVERS ITY O F BORÅ S, BOR ÅS , SW EDEN 1. Introduction Establishing a harmony between population growth and its impact on the environment is humankind’s one of the most demanding challenges. Human activity has an immensely negative effect on the natural resources cycle through over-usage of the resources and rapid waste generation. A vital tool for mitigating these adverse effects is sustainable development. Sustainability is attainable by maximizing resource utilization efficiency and minimizing waste generation (Clark and Deswarte, 2008). Bioeconomy, as sustainable development of products and processes from the utilization of renewable resources, has become the center of attention over the past years. For instance, it is one of the main sections in the Horizon 2020 framework (European Union research and innovation program). Currently, the bioeconomy’s annual turnover is 2.3 trillion euros, with 22 million people employed. The bioeconomy focus on the production of €s et al., food, feed, biofuels, and bioproducts from renewable biological resources (Tera 2014; Hassan et al., 2019). For instance, the EU goal for renewable energy share was 20% by 2020, and it has been set to 32% by 2030. The share of renewable energy in 2016 was 17% at the EU level. (Popp et al., 2021). Waste biorefinery corresponds to the concept of sustainable development, as various value-added products such as chemicals, fuels, and biomaterials are produced from different wastes. Several strategies have been developed for waste valorization via the biorefinery concept, among which using microorganisms is a prominent method (Ferreira et al., 2016). Filamentous fungi are cell factories that can produce a wide range of value-added products. Historically, filamentous fungi had a significant impact on our life. For centuries, they have been used for the production of food and beverage. In the early twentieth century, the production of penicillin, the first true antibiotic by Penicillium rubens, made a breakthrough for the development of biological production of antibiotics, € sten, 2019; Hu € ttner et al., 2020). Moreover, filamentous enzymes, and control agents (Wo fungi have been used on an industrial scale to produce different enzymes, organic acids, Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00009-0 Copyright © 2023 Elsevier Inc. All rights reserved. 533 534 Current Developments in Biotechnology and Bioengineering € sten, 2019). A few examples pharmaceuticals, and food products (Ferreira et al., 2016; Wo of fungal products and producing companies are listed below – – – – Food products (Marlow Foods, United Kingdom) Citric acid (Citrique Belge, Belgium) Malic acid (Novozymes, Denmark) Industrial-grade enzymes (Aumenzymes, India) Additionally, filamentous fungi are capable of utilizing a versatile types of materials. These characteristics, versatile substrates, and products have put the filamentous fungi in the spotlight for establishing the biorefineries. Over the past years, fungi have exclusively been explored as a core element for establishing biorefinery by utilizing various industrial wastes and returning these wastes to the production and consumption cycle as valueadded resources (Ferreira et al., 2016). Fig. 1 shows the potential of filamentous fungi in utilizing various wastes and producing different products. In this chapter, the generation and composition of eight different agro-industrial process waste were studied. Furthermore, valorizing these wastes with filamentous fungi and establishing fungal biorefinery based on those processes were evaluated. 2. Brewery waste The brewing industry is one of the main sections of the food industry, which holds a considerable market value. Global production of beer in 2018 was around 188 billion liters (beer from the malt with alcohol >0.5% v/v), with a market value of 504 billion Euros. The main by-product of a brewery is brewer’s spent grain (BSG), with the average generation of 15–20 kg wet BSG per 100 L of beer. Thus, the generated BSG in 2018 could be estimated at around 38 million metric tons (Qiu et al., 2019; Parchami et al., 2021a). BSG is the lignocellulosic solid fraction separated after filtration of wort and is rich in various nutrients such as cellulose, starch, arabinoxylans, phenolic compounds, vitamins, lignin, fatty acids, and proteins (Meneses et al., 2013; Terrasan and Carmona, 2015; Parchami et al., 2021a). Table 1 presents the typical composition of BSG. Since BSG has a high moisture nutrient-rich content, it can biologically deteriorate in a matter of few days, changing from a valuable source of nutrients to waste, posing different environmental problems. Traditionally, BSG is used as low-quality animal feed or ends up in landfills as the generation often exceeds the demand. Numerous efforts have been made to improve the utilization of BSG by producing value-added products from it through different valorization techniques. One of these techniques that have attracted considerable attention for years is BSG valorization by using filamentous fungi. Fungal products from BSG ranging from food and feed-grade products, medical products, hormones to enzymes, biopesticides, and bioethanol. There have been different studies on the production of food and feed-grade products by filamentous fungi from BSG, since BSG is considered a food-grade by-product. Edible biomass is one of the products that has been produced from BSG using filamentous fungi. Chapter 18 • Versatility of filamentous fungi in novel processes Waste streams •Wheat straw •Wheat bran •Corn straw •Rice hulls •Sugarcane bagasse •Tee waste •Brewer spent grain •Empty fruit bunches •Cheese whey •Cream •Crème fraiche •Ice cream •Whole stillage •Thin stillage •Oil waste •Meat waste •Fish waste •Wastewater Metabolites •Citric acid •Gluconic acid •Itaconic acid •Kojic acid •Oxalic acid •Malic acid •Pigments •Ethanol •Orange peel •Banana peel •Pineapple peel •Onion peel •Potato peel •Corn cobs •Carrot peel •Apple pomace •Empty fruit bunches •Coir pith •Thatch grass •Paper waste 535 • Biomass Filamentous fungi cultivation •Food and feed •Chitin •Amino acids •Lipids •Fatty acids •Sterols Enzymes •Amylase •Cellulase •Xylanase •Protease •Lipases •Phytases •Laccase •Catalase •Keratinase FIG. 1 Substrates and products prospect in a fungal biorefinery. Modified from Ferreira, J.A., Mahboubi, A., Lennartsson, P.R., Taherzadeh, M.J. 2016. Waste biorefineries using filamentous ascomycetes fungi: present status and future prospects. Bioresour. Technol. 215, 334–345. Filamentous fungi have traditionally been used to produce Asian cuisines for hundreds of years, and these strains have been classified as Generally Regarded as Safe (GRAS) such as Aspergillus oryzae, Rhizopus oryzae, Rhizopus oligosporus, Neurospora intermedia. GRAS is a designation by the United States Food and Drug Administration (FDA) that a substance considered safe and can be added to the food. The safety of the substance is determined through the scientific procedure. There are two key terms, “general recognition” 536 Current Developments in Biotechnology and Bioengineering Table 1 Brewer spent grain (BSG) chemical composition. Componentsa (%) BSGb BSGc BSGd BSGe Cellulose Hemicellulose Total Lignin Starch Protein Ash 17.5 25.3 16.74 20.9 22.7 n.d. f 16.5 26.3 20.4 n.d.f n.d. f 2.1 18.5 23 45.2 8.7 14.8 3.3 17.9 35.7 17.8 n.d.f 19.2 3.9 a Percentage of dry weight. From Parchami et al. (2021b). c From Michelin and Teixeira (2016). d From Gmoser et al. (2020). e From Torres-Mayanga et al. (2019). f Not determined. b and “qualified expert,” in this designation considered for safety evaluation of a substance. The process of approving new microorganisms is costly, strict, and time-consuming; thus, using the GRAS microorganisms is preferred in industrial processes (Waites et al., 2009; Sewalt et al., 2016). Therefore, these fungi have been used in various studies to produce biomass with applications as food and feed products. In a study by Serba et al. (2020), A. oryzae has been used to produce fungal biomass as a food source rich in protein and carbohydrates. In another work, Parchami et al. (2021a) produced protein and fiber-rich fungal biomass from BSG as a food and feed source. They used three different filamentous fungi, namely A. oryzae, R. delemar, and N. intermedia. They have reported that submerged cultivation with Aspergillus oryzae resulted in the highest increase in protein content (34.6% w/w) compared to the initial protein content of BSG. R. oryzae is another filamentous fungus that has been used for the production of biomass from BSG. Ibarruri et al. (2019) have obtained protein-rich biomass (32% (w/w) protein content) by solid-state fermentation (SSF). Wolters et al. (2016) have produced medicinal biomass from BSG using Hericium erinaceus. For centuries, H. erinaceus has been used in Chinese traditional medicine. Additionally, various filamentous fungi species are capable of secreting lignocellulose degrading enzymes, and several studies have focused on producing cellulolytic and xylanolytic enzymes from BSG (Table 2), primarily by Aspergillus sp., Rhizopus sp., and Penicillium sp. (Terrasan et al., 2010; Knob et al., 2013; Izidoro and Knob, 2014; Terrasan and Carmona, 2015; Leite et al., 2019). Leite et al. (2019) have evaluated the capacity of six Aspergillus sp., and Rhizopus sp. for lignocellulolytic enzyme production from agroindustrial wastes, including the BSG. They have reported that the BSG was the most suitable substrate for enzyme production by SSF, and Aspergillus ibericus strains were the best enzyme producers. Besides, as these fungi are cable of breaking down the structure of BSG, different phenolic compounds in BSG such as ferulic acid and p-coumaric acid could be extracted by fungal cultivation. These phenolic compounds have antioxidant and Chapter 18 • Versatility of filamentous fungi in novel processes Table 2 Production of various cellulolytic and xylanolytic enzymes from BSG. Enzyme Fungus strain Fermentation mode Operation temperature (°C) Enzyme activitya β-Xylosidase P. janczewskii SSF 28 0.18–0.25 P. janczewskii SmF 28 0.07–0.16 B. spectabilis SmF 25 0.47 P. janczewskii A. niger CECT2915 A. niger CECT2088 A. ibericus MUM 03.49 A. ibericus MUM 04.86 R. oryzae MUM 10.260 P. glabrum A.niger SSF SSF 28 25 169–370 290 SSF 25 246 SSF 25 313 SSF 25 300 SSF 25 106 SmF SmF 25 30 34 3 P. janczewskii SmF 28 5.14–13.6 P. brasilianum SSF 26.5 709 H. grisea var. thermoidea T. stipitatus A. niger J4 SmF 45 8.19–16.9 SmF SmF 37 28 2.33 9.8 Mucor sp. AB1 SSF 30 67 B. spectabilis SmF 25 8.88 N. crassa A.niger CECT2915 A.niger CECT2088 A. ibericus MUM 03.49 A. ibericus MUM 04.86 R. oryzae MUM 10.260 SSF SSF 30 25 200 57 SSF 25 51 SSF 25 51 SSF 25 62 SSF 25 18 Xylanase Cellulase 537 References Terrasan and Carmona (2015) Terrasan et al. (2010) Galanopoulou et al. (2021) Terrasan and Carmona (2015) Knob et al. (2013) Izidoro and Knob (2014) Terrasan et al. (2010) Panagiotou et al. (2006) Mandalari et al. (2008) Stroparo et al. (2012) Hassan et al. (2020) Galanopoulou et al. (2021) Xiros et al. (2008) Leite et al. (2019) Continued 538 Current Developments in Biotechnology and Bioengineering Table 2 Production of various cellulolytic and xylanolytic enzymes from BSG—cont’d Fungus strain Fermentation mode Operation temperature (°C) Enzyme activity M. thermophila SmF 47 0.11 A. fumigatus A. fumigatus Penicillium sp. Penicillium sp. A. fumigatus SmF SSF SmF SSF SSF 30 30 30 30 30 0.35 7.5 0.23 8.3 5.03 N. crassa A. niger CECT2915 A. niger CECT2088 A. ibericus MUM 03.49 A. ibericus MUM 04.86 R. oryzae MUM 10.260 P. janczewskii SSF SSF 30 25 40 3 SSF 25 93 SSF 25 4 SSF 25 9 SSF 25 1 SSF 28 0.22–0.60 P. janczewskii SmF 28 0.05–0.64 P. brasilianum SSF 26.5 0.004 Feruloyl esterase P. brasilianum SSF 26.5 0.002 SmF 45 0.17–0.47 Pectinase H. grisea var. thermoidea T. stipitatus Mucor sp. AB1 SmF SSF 37 30 0.14 137 Enzyme β-Glucosidase α-Larabinofuranosidase a References Matsakas et al. (2015) Casas-Godoy et al. (2020) Grigorevski-Lima et al. (2009) Xiros et al. (2008) Leite et al. (2019) Terrasan and Carmona (2015) Terrasan et al. (2010) Panagiotou et al. (2006) Panagiotou et al. (2006) Mandalari et al. (2008) Hassan et al. (2020) Reported as U/g for SSF and U/mL for SmF. antimicrobial activity and are conventionally extracted by using organic solvents. Using fungi remove the need for organic solvents and the possible final product contaminations with the solvents. Moreover, as a cleaner technology, it could eliminate environmental problems associated with using solvent and improve the process economy (Leite et al., 2019; da Costa Maia et al., 2020). Gibberellic acid, a plant hormone, is another fungus metabolite that affects plant growth and has a significant role and value in agriculture. Currently, gibberellic acid is produced by Fusarium fujikuroi submerged cultivation on a commercial scale (da Silva et al., Chapter 18 • Versatility of filamentous fungi in novel processes 539 2021). da Silva et al. (2021) reported that solid-state cultivation of Fusarium fujikuroi on BSG is a viable alternative way for Gibberellic acid production. Lastly, filamentous fungi could be used for ethanol production from BSG. For bioethanol production from lignocellulosic material, the primary industrial ethanol producers such as Escherichia coli and Saccharomyces cerevisiae cannot utilize this material, and a pretreatment step is required to improve the hydrolysis. This pretreatment step is a costly and energy-intensive process that has hindered bioethanol’s industrial production from lignocellulosic wastes. A strategy to bypass this technical difficulty is using filamentous fungi. As mentioned before, different filamentous species are capable of degrading lignocellulosic material, which can deconstruct the structure of BSG and release monomeric sugars in the system. The released sugar can be converted to ethanol by well-known ethanol producers such as E. coli and S. cerevisiae. Therefore, many research studies have been done on consolidated bioprocesses (CBPs) using lignocellulose degrading fungi such as Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, and Humicola insolens (Wilkinson et al., 2017). Wilkinson et al. (2017) have tested bioethanol production from BSG using different filamentous fungi paired with yeast strains. They have reported that the A. oryzae and S. cerevisiae CBP system was the best consortia for ethanol production with the yield of 94 kg pure ethanol per 1 t dry BSG. The other strategy for ethanol production is using filamentous fungi to produce ethanol directly. Xiros et al. (2008) have reported 74 kg ethanol production per 1 t dry BSG by fungal fermentation with Neurospora crassa on BSG. Above all, filamentous fungi can produce a wide range of value-added products from BSG ranging from food and feed to fuels, making it possible to establish a circular economy based on the valorization of BSG. Although, the production of food and feed-grade products seems to be more favorable as the by-products from the food industry end up back to the food production cycle. Fig. 2 presents an overview of the integration of filamentous fungi to a brewing process for valorization BSG. FIG. 2 Brewery biorefinery based on filamentous fungi products. 540 Current Developments in Biotechnology and Bioengineering 3. Fruit industry waste The fruit processing sector is one of the biggest sectors in the food industry and is responsible for generating enormous agro-industrial waste (Dhillon et al., 2013). In 2019, the global production of fruits was around 917 million metric tons. Apple, orange, and grapes were among the top five most-produced fruits, with 87.24, 78.7, and 77.14 million metric tons, respectively (FAO, 2021). Approximately 20%–30% of produced apple and orange was used in the juice industry, while grape mostly (ca. 57%) used for wine production (Kantifedaki et al., 2018; Molinuevo-Salces et al., 2020; Filippi et al., 2021). One of the main waste after processing the fruit is the pomace. Fruit pomace is the solids generated after extracting the juice, including the pulp, peel, seeds, etc. Pomace is accounting for up to 50% of the fruit’s weight for apple and orange juice production (Kantifedaki et al., 2018; Molinuevo-Salces et al., 2020), while for the wine, pomace weight is 20% of the grape weight (Filippi et al., 2021). Moreover, the peel and seeds from other products such as canned, dried, and frozen fruits are added to this amount resulting in the annual generation of million tons of solid waste, which causes various environmental problems. Since fruits (apple, orange, and grape) pomace is rich in various nutrients, the valorization of fruits pomace by microorganisms has caught attention as a strategy for handling this enormous amount of waste. The main compounds in fruits pomace are cellulose, hemicellulose, lignin, pectin, protein, minerals, and phenolic compounds (Kantifedaki et al., 2018; Awasthi et al., 2021; Balli et al., 2021). The proximate compositional analysis of fruit pomace is reported in Table 3. As microorganisms capable of degrading lignocellulosic material, filamentous fungi have been the center of attention for the production of products such as protein-rich biomass, organic acids, enzymes, pigments, antioxidants, Table 3 Compositional analysis of apple, orange, and grape pomace. Composition Apple pomacea Orange pomaceb Grape pomacec Moisture (%) Fat (%) Protein (%) Ash (%) Carbohydrate (%) TDF (%) Pectin (%) Fructose (%) Calcium Potassium Magnesium Zinc Iron 9.00 2.27 2.37 1.60 84.76 30.15 N/A N/A 126.50a 253.10a 12.60a 0.17a 0.84a 10.55 1.88 6.00 3.68 77.78 40.47 N/A N/A 411.20a 411.2a 57.00a 0.23a 0.98a 3.33 8.16 8.49 4.65 29.2 46.17 3.92 8.91 440d 1400d 130d 980d 1800d a Reported as mg/100 mL. From O’Shea et al. (2015). c From Sousa et al. (2014). d Reported as mg/100 mg. b Chapter 18 • Versatility of filamentous fungi in novel processes 541 and biofuels from fruit pomace (Dhillon et al., 2012; Ucuncu et al., 2013). Filamentous fungi are known for the high production of extracellular enzymes, and they have been extensively researched for enzyme production. Aspergillus sp. have been vastly researched for enzyme production among different fungi, especially pectin degrading enzymes. Dı́az et al. (2012) have evaluated the enzyme production by SSF of Aspergillus awamori on grape pomace. A higher pectinase production has been reported for fermentation with a mixture of grape pomace and orange peels than only grape pomace as substrate. Pyc et al. (2003) have studied the condition of enzyme production by Aspergillus niger fermentation on apple pomace. In another study, Martin et al. (2010) isolated 34 fungal strains from soil and evaluated pectinase production in SSF. About 50% of the isolated fungi belonged to Aspergillus, Scopulariopsis, Thermomucor, Chaetomium, Thermomyces, Neosartoria, and Monascus sp.. Pectinase with the highest enzymatic activity was obtained for the SSF with Thermomucor indica-eseudaticae N31 on mixture of orange peel and wheat bran. Pathania et al. (2018) have reported the production of pectinase among other enzymes from apple pomace by solid state fermentation of Rhizopus delemar F2. Cellulase and hemicellulase are other groups of enzyme that have been produced by filamentous fungi. Table 4 shows the production of various enzyme from apple, orange, and grape pomace. Organic acids are other highly demanded commodities that filamentous fungi can produce. In fact, industrial production of organic acids such as citric acid and gluconic acid by fungi is a well-established process. For instance, the fungal production of citric acid is the Table 4 Production of various enzymes with different filamentous fungi from apple, orange, and grape pomace. Enzyme Fungus strain Fermentation mode References Apple pomace β-Glucosidase Xylanase Amylase Endoglucanase Laccase Manganese peroxidase Chitinase Chitosanase Pectin methylesterase (PME) Pectinase Polygalacturonase M. phaseolina M. phaseolina R. delemar F2 A. niger M. phaseolina R. delemar F2 A. niger M. phaseolina P. chrysosporium P. chrysosporium A. niger A. niger A. niger A. foetidus R. delemar F2 A. niger A. niger SSF SSF SSF SSF SSF SSF SSF SSF SSF SSF SSF SSF/SmF SSF SSF SSF SSF Kaur et al. (2012) Pathania et al. (2018) Pyc et al. (2003) Kaur et al. (2012) Pathania et al. (2018) Dhillon et al. (2012) Kaur et al. (2012) Gassara et al. (2010) Dhillon et al. (2011) Joshi et al. (2006) Hours et al. (1988) Pathania et al. (2018) Kiran et al. (2010) Berovic and Ostroveršnik (1997) Continued 542 Current Developments in Biotechnology and Bioengineering Table 4 Production of various enzymes with different filamentous fungi from apple, orange, and grape pomace—cont’d Enzyme Fungus strain Fermentation mode A. versicolor A. awamori A. awamori A. awamori P. atrovenetum A. flavus A. oryzae T. hirsuta P. atrovenetum A. flavus A. oryzae A. giganteus A. sojae SSF SSF SSF SSF SSF SSF SSF SSF SSF SSF SSF SmF SmF Srivastava et al. (2017) Dı́az et al. (2012) A. awamori A. awamori A. awamori SSF SSF SSF Dı́az et al. (2012) References Orange pomace Cellulase Exo-polygalacturonase Xylanase Endoglucanase Laccase Polygalacturonase Exo-polygalacturonase Adeleke et al. (2012) €hmer et al. (2011) Bo Adeleke et al. (2012) Pedrolli et al. (2008) Buyukkileci et al. (2015) Grape pomace Exo-polygalacturonase Xylanase Endoglucanase oldest and most studied process. Aspergillus and Rhizopus are the most studied genus for different organic acid production. Most of these industrial processes use glucose or sucrose as the substrates. Using agro-industrial waste as a low-cost substrate for fungal production of organic acids improves the economics of the process. It could help the fungal process of less demanded acids such as oxalic, fumaric, and itaconic acid compete with chemical production routes (Magnuson and Lasure, 2004). In a study Papadaki and Mantzouridou (2019), citric acid production from grape pomace by A. niger is reported. Ousmanova and Parker (2007) evaluated the SSF production of organic acids by three different Aspergillus strains from agro-industrial wastes. They have reported that the fungi could produce multiple acids from each substrate; however, the fungal strain and substrate type affect the produced acids. After 8 days of SSF cultivation with A. niger NRRL 2001 on the apple pomace, about 800 mg/L oxalic acid and 400 mg/L citric acid were produced while no level of citric acid was reported for the cultivation on corn cob. Ousmanova and Parker (2007) showed that the produced acids by fungi could be used for extraction of lead from contaminated soil. Additionally, pigments can be produced by filamentous fungi. Pigments have been used in many different industries for adding color to different products, especially food products (Lopes and Ligabue-Braun, 2021). Initially, pigments were extracted from natural sources such as vegetables, fruits, and insects. Although, high production costs and color instability led to the development of synthetic pigments. However, due to the Chapter 18 • Versatility of filamentous fungi in novel processes 543 harmful effect of synthetic pigments on human health, there was a growing demand for natural pigments, resulting in the development of the natural color industry. With recent advances in biotechnology, microbial production of pigments as alternative ways for production of natural pigments has captured lots of attention (de Oliveira et al., 2020; Kalra et al., 2020). Filamentous fungi are microorganisms that produce a good level of pigment with high color variation. For instance, the fungus genus Monascus is reported as one of the best fungal pigment producers. Penicillium, Trichoderma, Talaromyces, and Fusarium are other species that have been vastly researched for pigment production. Kantifedaki et al. (2018) have evaluated the production of yellow, orange, and red pigment by M. purpureus and P. purpurogenum using orange pomace. They have reported significant production of red pigment by P. purpurogenum. Fungi can produce pigments with different colors and eliminate seasonal limitations associated with natural pigment production from vegetables and fruits. However, mycotoxins produced by fungi are the critical drawback of fungal pigment production. Lopes et al. (2013) have studied the effect of substrate on pigment and mycotoxins production. They have studied the pigment production capacity of 24 different fungi, out of these 24 only four fungi, namely P. chrysogenum IFL1 and IFL2, F. graminearum IFL3, M. purpureus NRRL 1992, were capable of pigment production on different agroindustrial wastes, including grape pomace. They have reported production of pigment on grape pomace only for cultivation with P. chrysogenum IFL1. Different fungi, different levels of mycotoxins such as diacetoxyscirpenol, citrinin, and fusarenone X, were obtained for cultivation with other waste. They have reported that avoiding the mycotoxin generation is possible by adjusting the culture media and condition. de Oliveira et al. (2020) have studied the application biofilm produced from T. amestolkiae submerged cultivation. They have reported the biofilm produced from fermentation broth containing the pigments showed good antioxidant activity and could be used for food packaging. Hormones, phenolic compounds, fatty acids, protein-rich biomass, and biofuels are other valuable products produced from fruits pomace by fungal cultivation. Table 5 shows the other products that have been produced by the fungal cultivation of fruits pomace. Table 5 strain. Valorization of fruits pomace by fungal cultivation, products types, and fungi Product Fungus strain Fermentation mode R. miehei P. chrysosporium C. fimbriata R. oryzae T. harzianum P. chrysosporium SSF SSF SSF SSF SSF SSF Reference Apple pomace Phenolic compounds Fruity aroma Volatile compounds Protein-rich biomass Zambrano et al. (2018) Ajila et al. (2012) Bramorski et al. (1998) Christen et al. (2000) Ortiz-Tovar et al. (2007) Continued 544 Current Developments in Biotechnology and Bioengineering Table 5 Valorization of fruits pomace by fungal cultivation, products types, and fungi strain—cont’d Fermentation mode Reference T. harzianum Co-culture of T. harzianum, A. sojae, and Saccharomyces cerevisiae SmF SmF Ucuncu et al. (2013) Evcan and Tari (2015) F. moniliforme G. fujikuroi Rhizopus sp. A. niger M. isabellina NRRL 1757 T. harzianum SSF/SmF De Oliveira et al. (2017) SmF SSF SmF SmF Ibarruri and Hernández (2019) Alemu (2013) Carota et al. (2018) Ucuncu et al. (2013) A. elegans U. isabellina R. miehei SSF Dulf et al. (2020) SSF Zambrano et al. (2018) Product Fungus strain Bioethanol Orange pomace Plant hormone Protein-rich biomass Microbial oil Bioethanol Grape pomace Fatty acid Phenolic compounds The previous research shows the possibility of producing multiple products from fruits pomace by various filamentous fungi, which fits the biorefinery concept. Thus, it can be hypothesized that a biorefinery can be established by integrating fungal processing into the fruit processing industry (Fig. 3). 4. Bioethanol industry wastes The bioethanol industry has a global production of 114 billion L ethanol in 2019, and it is the most produced liquid biofuel in the transportation sector (REN21, 2020). Bioethanol can be produced from different raw materials. First-generation ethanol is produced from sugar- or starch-based crops, while second-generation ethanol is produced from nonedible lignocellulosic sources such as agricultural residuals. This section focuses on valorizing first-generation ethanol wastes as the most industrially established bioethanol process. Dry-grind bioethanol plant is the most popular ethanol process in the world. In this process, the hexose sugars are converted into ethanol and CO2 in a fermenter. Then, the ethanol is separated from the fermentation residuals in the distillation column (Lennartsson et al., 2014; Ferreira, 2015). This residual stream is called whole stillage. The whole stillage is sent to a decanter, and the solid fraction, wet distillers grain (WDG), separated from the liquid fraction, thin stillage. The latter is concentrated in evaporators and mixed with WDG before being sent to a drier to produce dried distillers grains with solubles (DDGS). WDG and DDGS are commonly used as animal feed due to their nutrient content, such as carbohydrates, lipids, and proteins (Ferreira, 2015). WDG has Chapter 18 • Versatility of filamentous fungi in novel processes 545 FIG. 3 Fungal biorefinery for the valorization of apple, orange, and grape pomace. a short shelf life due to its high moisture content, limiting the application as a product and otherwise requiring treatment to avoid a negative impact on the environment. Therefore, the dried product DDGS more common, although it requires an energy- and costintensive process. On the other hand, animal feed ingredients’ production rate and demand are another concern that produces more demand resulting in waste. In order to produce 1 L of ethanol, ca 20 L stillage is produced (Ferreira, 2015; Rocha-Meneses et al., 2017). It means that for an ethanol plant producing 200 million L ethanol/year, 4 billion L stillage/year is produced. This nutrient-rich material contains fibers, proteins, carbohydrates. The content of stillage from an ethanol plant using wheat grains as raw material is shown in Table 6 (Bátori et al., 2015). Rocha-Meneses et al. (2017) stated that the bioethanol production wastes create an environmental concern with its BOD level ranging from 10 to 100 g O2 L 1. There have been numerous research studies about the valorization of bioethanol industry waste, and microbial conversion via filamentous fungi is one alternative way. Filamentous fungi can grow on various materials and produce a wide range of products (Ferreira, 2015; Nair, 2017). Unlike the common bakery yeast used in ethanol fermentation, filamentous fungi are capable of consuming xylose sugars. The reactions for glucose and xylose conversion to ethanol are shown in Eq. (1) (Smith et al., 2006) and Eq. (2) (McMillan, 1993): 546 Current Developments in Biotechnology and Bioengineering Table 6 Composition of wheat-based whole stillage (Bátori et al., 2015). Parameter Value pH Total solids (% w/w) Suspended solids (% w/w) Sieved solids (% w/v) Crude protein (% w/w)a Crude protein (% w/w)b 4.3  0.0 15.6  0.1 8.8  0.0 3.2  0.2 32.0  0.6 15.1  3.9 Dissolved monomers (g/L) Acetic acid Arabinose Ethanol Glucose Glycerol Lactic acid Xylitol Xylose 0.4  0.1 1.6  0.1 0.7  0.0 1.4  0.1 12.0  0.1 1.7  0.0 0.6  0.1 0.7  0.1 Dissolved saccharides (g/L)c Arabinose Galactose Glucose Mannose Xylose 6.3  0.1 1.7  0.0 12.0  0.3 2.4  0.1 9.7  0.1 Sugar polymers (g/L)d Arabinan Galactan Glucan Mannan Xylan 1.8  0.1 0.3  0.0 4.7  0.1 0.6  0.0 3.6  0.1 a Based on dry total solids. Based on dry sieved solids. c Dissolved monomers included. d From dry sieved solids. b C6 H12 O6 ƒƒƒƒƒ! 2CH2 H5 OH + 2CO2 (1) 3C5 H10 O5 ƒƒƒƒƒ! 5CH2 H5 OH + 5CO2 (2) Hence, they have been an interest of research in the valorization of fermentation residuals (Ferreira, 2015). Ferreira et al. (2014) stated that protein-rich biomass was produced from thin stillage using five different food-grade fungi. Among these five strains (Neurospora intermedia, Aspergillus oryzae, Fusarium venenatum, Monascus purpureus, and Rhizopus sp.), N. intermedia and A. oryzae provided the best result in terms of bioethanol (5 g/L) and biomass production (19 g/L), respectively. This indicates the importance of fungus strain toward the Chapter 18 • Versatility of filamentous fungi in novel processes 547 value-added product. Besides, filamentous fungi growth reduced the total solids in the remaining liquid, as well as lactic acid and glycerol. The total solids content of the medium decrease as the fungi convert polymeric sugars and other components of thin stillage into metabolites and fungal biomass. Besides, the fungal cultivation results in biomass which is entangled with the solids in the medium. Therefore, after biomass separation, the remaining liquid has less total solids concentration (Ferreira et al., 2014). In a similar study, it was stated that N. intermedia growth on thin stillage in airlift reactor (26 L) resulted in biomass which comprises 50% (w/w) protein (including essential amino acids) and 12% lipids (i.e., omega-3 and -6 fatty acids) (Ferreira et al., 2015). Fungal biomass produced on thin stillage can be used as animal feed or fish feed ingredient as protein content is up to 50% (Ferreira et al., 2015). In another study, the whole stillage was valorized by N. intermedia cultivation, where the ethanol production was higher than S. cerevisiae. A two-stage process was implemented using two different fungi strains, resulting in 7.6 g/L ethanol and 5.8 g/L biomass with 42% protein content (Bátori et al., 2015). The techno-economic perspective of filamentous fungi integration to 1st generation ethanol plant was investigated by Rajendran et al. (2016). The energy consumption of the process decreased by filamentous fungi integration, particularly in evaporator units. In a similar process, (Bulkan et al., 2020) emphasized that the filamentous fungi integrated process has the potential to provide protein-rich food and feed ingredients such as fish feed, resulting in an improved and robust economy toward raw material/product price fluctuations in comparison to conventional bioethanol plant. Downstream processing of the fungal biomass can be carried out in different ways. Sieving and the following drying process are assumed in previous experimental studies (Ferreira, 2015), while centrifugation replaced sieving in some studies (Rasmussen et al., 2014; Rajendran et al., 2016; Bulkan et al., 2020). Koza et al. (2017) studied different dewatering strategies for the fungal biomass separation, including “gravity and centrifugal sedimentation, gravity screening, a belt filter, a filter press, and centrifuge filtration.” Gravity screening coupled with a filter centrifuge is reported to result in biomass with maximum 30% solids, due to the internal water content of the fungi cell. A following thermal drying carries up the solid concentration to 90%. Metabolites produced by fungi can be recovered from the liquid remaining after fungal biomass separation. The liquid fraction is proposed to be re-used in the liquefaction unit of the process (Lennartsson et al., 2014). In some studies, it is considered to be used as backset water partially, while the rest is re-used following a multi-evaporation step (Ferreira et al., 2015; Rajendran et al., 2016; Bulkan et al., 2020). The re-use of the liquid as process water allows the ethanol produced by filamentous fungi to mix to fresh feed for bakery yeast and end up in the distillation column with the ethanol produced by bakery yeast (Rajendran et al., 2016; Bulkan et al., 2020). The economy of the bioethanol plant has the potential to be improved by enzyme production using thin stillage as raw material, according to Shahryari et al. (2019). Amylase and xylanase were produced by N. intermedia on wheat-based thin stillage. Apart from Ascomycetes and Zygomycetes, Basidiomycetes were also used for stillage valorization. Pena et al. (2012) utilized white-rot fungi in order to valorize DDGS and whole stillage 548 Current Developments in Biotechnology and Bioengineering while producing ligninolytic enzymes. Co-cultures of microorganisms are studied in order to reach the target product, i.e., feed with balanced nutrients or improved wastewater remediation of the bioethanol process (Rodrigues Reis et al., 2018). Pietrzak and KawaRygielska (2019) reported that utilization of co-cultures of edible filamentous fungi and fodder yeast Candida utilis on corn thin stillage valorization resulted in biomass comprised essential amino acids. In addition to biomass, a potential feed ingredient, ethanol, and amylase enzyme were produced. Additionally, ethanol production in yeast fermenters was improved when the liquid left after co-culture cultivation used as backset water in the primary ethanol process. There have been several studies about the production of polyunsaturated fatty acids by filamentous fungi grown on thin stillage reported by Reis et al. (2017). Liang et al. (2012) studied eicosapentaenoic acid (EPA) production from corn thin stillage and obtained 243 g/L day EPA yield at the 9th day of Pythium irregulare cultivation. The produced oil can be further utilized in feed/food supplements (Liang et al., 2012; Reis et al., 2017). Production of biocrude is another application for fungal biomass produced from thin stillage. Suesse et al. (2016) stated that similar yield and quality of biocrude could be obtained by using Rhizopus oligosporus as feedstock compared to biocrude from microalgae. Among the various studies, bioethanol industry wastewater can be converted into various valueadded products, while the bioconversion of nutrients results in naturally treated postcultivation wastewater, which has the potential to be recycled as process water. 5. Fish processing industry waste Fish processing products are an essential source of commercial products in countries such as China, India, Thailand, Indonesia, Canada, United States of America. Although the types of fish processed/consumed vary depending on the geographical location of the countries, fish species such as salmon, herring, tuna, and shellfish can be given as examples in the world. The fish processing process may differ depending on the fish type and product type in the facilities. Standard processes in industry are filleting, freezing, drying, fermenting, canning and smoking. In fish fillets and similar production processes, solid components (head, internal organs, etc.), liquids (e.g., blood), and processing water (salt brine, filleting water, etc.) could be released. The biochemical compositions of the fish processing wastes vary according to fish species, fishing season, and process type (additives, processing water source) (Aidos et al., 2002). These wastewaters having a high content of solids which are mainly nitrogen (0.5–8.4 g/kg), phosphorus, oil (up to 43 g/kg), and have high BOD5 (up to 6000 mg/L) and COD (up to104,000 mg/L) levels (Sathivel et al., 2003; Sar et al., 2020a, 2021). The salt content of fish waste could be high due to the salting process in the fish filleting process. However, this salt content (1.2%) in fish waste is much less than in fish fillets (2.4%) (Aidos et al., 2002). Fish processing wastes are an important source of pollution for the environment, and the wastewaters are directed to waste management. Except that, offal disposals (head and internal organs) can be considered as feed because of having high-protein content. Since total mercury and selenium can accumulate in the kidneys and liver in general, fish internal organs can be expressed as non-edible according to the heavy metal content Chapter 18 • Versatility of filamentous fungi in novel processes 549 (Julshamn et al., 1987). Therefore, elemental analysis is of great importance in the evaluation of internal organs as feed. Biodiesel or biogas can be produced using the fish industry wastes rich in nitrogen and carbon sources. Typically, the fish waste having high total solids is converted to biogas/methane production through anaerobic digestion. Herein, methanogenesis can be performed through fungi (Eurotiales, Sordariales, Saccharomycetales, Sporidiales, Capnodiales, Microascales, Wallemiales, and Tremellales), bacteria (Clostridia, € cker et al., 2020). Oil extracted from disSynergistia), and archaea (Methanomicrobia) (Bu carded parts of fish or fish waste can be evaluated for biodiesel production (Yahyaee et al., 2013; Garcı́a-Moreno et al., 2014; Madhu et al., 2014; Ching-Velasquez et al., 2020). Herein, ultrasound or microwave-assisted extraction, supercritical fluid extraction, and enzymatic hydrolysis methods can be used for oil extraction from fish waste (Ivanovs and Blumberga, 2017). In addition, natural pigments can be recovered from seafood (such as shrimp, lobster, crab, crayfish, trout, and salmon) having carotenoids (Shahidi and Brown, 1998; Simpson, 2007). As an alternative to conducted processes, value-added products by filamentous fungi have been investigated from the fish industry wastes (Fig. 4). Protein-rich edible biomass can be produced through some filamentous fungi and evaluated as high-value animal feed. For this purpose, it was reported that biomass, containing 35%–65% protein produced from fish processing wastes through Aspergillus oryzae and Rhizopus oryzae, can be an alternative to biogas production and waste treatment with fungal culture process integration (Sar et al., 2020a, 2021). Some fungal species can naturally produce pigments. Lopes et al. (2013) screened the 24 different fungal strains and determined four pigment producer fungal strains (Penicillium chrysogenum, P. vasconiae, Fusarium graminearum, Monascus purpureus) using agro-industrial waste, including fish meal. Natural pigments can be potentially used in textile, food, and pharmacy industries. White-rot fungi have the ability to degrade some environmental pollutants (Moredo et al., 2003). Among them, Phanerochaete chrysosporium can grow rapidly and produce ligninolytic enzymes such as manganese peroxidase (MnP), lignin peroxidase (LiP), and laccase (Barclay et al., 1993; Hatakka, 1994). Gassara et al. (2010) evaluated the ligninolytic enzyme production using industrial wastes by P. chrysosporium. Ligninolytic enzymes can be produced in pomace, brewery wastes, fishery, and pulp and paper industry sludge, while their activities were found to be low in fish wastes (Gassara et al., 2010). FIG. 4 Valorization of fish industry waste by filamentous fungi. 550 Current Developments in Biotechnology and Bioengineering Filamentous fungi (Trichoderma and Penicillium) can also be used in the hydrolysis of fish wastewaters rich in keratin and collagen due to their proteolytic activity (Martins et al., 2014). Similarly, significant reductions in the total solids, COD, and nitrogen amounts were achieved by Aspergillus oryzae and Rhizopus oryzae strains when cultivated for fungal biomass production (Sar et al., 2020a, 2021). Therefore, fish processing wastes for fungal biomass and pigment production can be transferred to fungal culture processes, providing biological treatment of the wastes. Fish waste from the fish processing industry, rich in lipids and proteins, has been generally evaluated for low-value animal feed products and methane production (Cirne et al., 2007; Nges et al., 2012; Cadavid-Rodrı́guez et al., 2019). As an alternative to these productions, fish processing wastes can be directed to fungal culture processes for fungal biomass and pigment production. 6. Oil processing industry waste Vegetable oils are generally obtained from seeds or beans via pressing or extraction methods. Soybean, rapeseed, sunflower, groundnut, cottonseed, coconut, palm, olive, corn have been traditionally consumed and commercially traded in many countries. Among them, olive and palm oil productions have been turned into automated systems instead of the traditional pressing process, and plenty of oily wastewater has been generated during the newly generated systems. 6.1 Olive oil processing industry Olive oil products as an essential vegetable oil play a significant role in the Mediterranean Basin market (Italy, Greece, Syria, Tunisia, Turkey, Morocco, Algeria, Portugal, and Argentina) (Lopez-Villalta, 1998). Olive oil is obtained by processing olive fruit in processes called discontinuous (pressing) or continuous (centrifugation) (Dermeche et al., 2013; Sar et al., 2020b). The oldest and most basic method for olive oil production is a discontinuous pressing process. In this process, a small amount of water is required to separate the oil from other components. Olive pomace consisting of olive fruits and seeds and less amount of olive oil mill water (OOMW; 40–60 L/100 kg of olives) are released along with the production of olive oil by the traditional method. Although a small amount of waste is generated, it has a higher COD amount. It also has some disadvantages, such as non-continuous processes and required manpower. Due to the disadvantages of the traditional olive oil production method, many facilities have been switched to two- or three-stage continuous processes. More hot water is used in continuous processes, and then three different fractions (olive oil, OOMW, and olive pomace) are released by following centrifugation processes. Because of the high amount of water used, a greater amount of wastewater (80–120 L/100 kg olives) is released. The three-phase system is the most widely used method because more olive oil can be produced in a short time, despite the high water consumption. OOMW is slightly acidic, contains sugars, nitrogenous compounds, fatty acids with residual oil, tannins, lignins, organic and phenolic compounds, and has high BOD and COD concentrations (Sayadi and Ellouz, 1995; Rinaldi et al., 2003; Hentati et al., 2016; Chapter 18 • Versatility of filamentous fungi in novel processes 551 Sar et al., 2020b). Similarly, olive pomace is also rich in sugars, polyphenols, and oil (Alburquerque et al., 2004; Seçmeler et al., 2018). The mainly phenolic compounds in OOMW are phenyl acids (vanillic acid, caffeic acid, p-cumaric acid, ferulic acid), phenyl alcohols (hydroxytyrosol and tyrosol), flavonoids (luteolin), and oleuropein which are potential inhibitory materials (Romeo et al., 2019). Phenolic compounds containing these wastes are important environmental pollutant sources regarding their management and disposal (Paulo and Santos, 2021). For waste minimization, various extraction methods for the recovery of phenolic compounds have been investigated, and their potential for use in areas such as food, pharmacology, and health have been researched (Torrecilla and Cancilla, 2021). In addition, both various metabolite productions and waste treatments by microorganisms have been conducted. OMW can be viewed as a suitable growth medium for lipase production because it contains olive oil residuals and simple and complex sugars. Fungal lipase production has been extensively investigated by the genera Geotrichum, Penicillium, and Fusarium (Salgado et al., 2020). Some lipolytic fungal/yeast species such as Aspergillus oryzae, Aspergillus niger, Candida cylindracea, Geotrichum candidum, Penicillium citrinum, Rhizopus arrhizus, Rhizopus oryzae, and Yarrowia lipolytica have been generally screened and cultivated in the olive waste by-products for lipase production (Lotti et al., 1998; D’Annibale et al., 2006; Gonçalves et al., 2009; Lopes et al., 2009; Abrunhosa et al., 2013). Among them, C. cylindracea (0.46 U/mL) and G. candidum (0.52 U/mL) had high volumetric lipase activities, while P. citrinum strains (4.58–5.42 U/L/h) also had high enzyme productivity (D’Annibale et al., 2006). Lipase production can be affected by growth conditions and media components such as pH, initial COD levels, supplementations (such as nitrogen, oil), and cultivation types (submerged, bioreactors) (reviewed in Table 7). D’Annibale et al. (2006) suggested that supplementation of NH4Cl (2.4 g/L) and olive oil (3 g/L) Table 7 Lipase production by filamentous fungi from olive oil processing wastes and comparison of lipase activity with COD. Strain COD levels Lipase production References C. cylindracea NRRL Y-17506 C. cylindracea NRRL Y-17506 43 g/L 50 g/L D’Annibale et al. (2006) Brozzoli et al. (2009) C. cylindracea CBS 7869 C. cylindracea CBS 7869 G. candidum NRRL Y-553 P. citrinum ISRIM 118 A. ibericus MUM 03.49 A. ibericus MUM 03.49 Y. lipolytica W29-N6 Magnusiomyces capitatus JT5 Magnusiomyces capitatus JT5 115 g/L 179 g/L 43 g/L 43 g/L 97 g/L 97 g/L 19 g/L 55 g/L 55 g/L 0.46 IU/mL 18.7a 20.4a 2200 U/L 877 U/L 0.52 IU/mL 0.38 IU/mL 2927  54 U/L 8319  33 U/L a 78 U/L 1.4 3.96 a a Lipase activity was obtained in bioreactors. Gonçalves et al. (2009) Gonçalves et al. (2009) D’Annibale et al. (2006) D’Annibale et al. (2006) Abrunhosa et al. (2013) Abrunhosa et al. (2013) Lopes et al. (2009) Salgado et al. (2020) Salgado et al. (2020) 552 Current Developments in Biotechnology and Bioengineering improved the enzyme activity of C. cylindracea NRRL Y-17506 strain. Further studies showed that besides the addition of nitrogen and oil, lipase activity of the same strain increased from 1.8 U/mL to 18.7 U/mL and 20.4 U/mL with uncontrolled pH or pH control (below 6.5), respectively (Brozzoli et al., 2009). Geotrichum candidum can be easily produced in an OMW-based medium and produce lignin-modifying enzymes concomitantly lipase enzyme (Assas et al., 2000; Gopinath et al., 2003; D’Annibale et al., 2006; Asses et al., 2009). Aspergillus species are also promising microorganism for lipase and proteinase production (Abrunhosa et al., 2013; Oliveira et al., 2016; Salgado et al., 2016). These enzymes are important in the laundry as they are the main enzymes of the detergent formulation for removing oily and proteinaceous food ci  c et al., 2011). Salgado et al. (2016) determined that high lipase (1253 U/L) stains (Grbav and protease (3700  124.3 U/L) activities were achieved by the combination of Aspergillus species (A. ibericus, A. uvarum, and A. niger) when cultivated in olive mills and wineries effluents (1:1). Olive oil mill water (OOMW) can be considered as a potential substrate for microbial fermentation processes because of its high COD and composition regarding sugars and oil content. For this purpose, Sar et al. (2020b) examined the efficiency of fungal biomass production from OOMW through various filamentous fungi (Aspergillus oryzae, Rhizopus delemar, and Neurospora intermedia) and determined that the high biomass production (8.4 g/L), which contains 15%–49% protein, was realized by A. oryzae. The compositional of fungal biomass produced by different fungi (A. niger, Paecilomyces variotii, Pleurotus floridae, P. eryngii, P. ostreatus, P. sajor-caju) contains 13%–14% protein, 6% fiber, vitamin A, vitamin E, nicotinic acid, calcium, potassium, iron, and unsaturated fatty acids. Although microbial biomass can be produced from OMWW, additional studies are needed before it can be used as animal feed due to its unknown beneficial health effects. The total amount of phenolic compounds, organic loads, and COD have been successfully reduced and decolorized using olive oil wastewater via microbial processes (D’Annibale et al., 2004; Abrunhosa et al., 2013). Thus, biological waste treatment/ improvement has also been achieved along with microbial culture studies. Usually, there has not been a specific regulation regarding olive oil mill wastewater discharge in Mediterranean countries. OOMW is generally treated using a slow rate land treatment system (Erses Yay et al., 2012). OOMW is a rich substrate source having metal ions (K, Ca, Na, Fe, Cu, Zn, Mn) and inorganic anions (Cl , H2PO4 , F , SO42 and NO3 ) (Arienzo and Capasso, 2000). Although macro and microelements are nutrient sources for agriculture or microorganisms, extensive metal ions with inorganic ions related to phenols and pollutants need purification. For this purpose, a filtration system needs to develop for removing and recovering metal ions. Alternatively, another way could be conceivable that olive oil industry by-products can be diverted to microbial production systems. In this way, it could be ensured that OOMW can be evaluated as a raw material for enzyme (mainly lipase) and fungal biomass production, and its negative effect on the environment can be reduced. Chapter 18 • Versatility of filamentous fungi in novel processes 553 6.2 Palm oil processing industry Palm oil is extracted from the ripened mesocarp of the fruits of the oil palm tree (Elaeis guineensis). The oil palm fruit is a drupe formed in tight spiky bunches (Ngando-Ebongue et al., 2012). The five leading producing countries are Indonesia, Malaysia, Thailand, Colombia, and Nigeria. Among them, Indonesia and Malaysia produce about 80% of the world’s palm oil and export more than 90% of their palm oil production (Tan and Lim, 2019). Similar to the olive oil process, the processing of palm oil releases excessive amounts of palm oil industry wastes which are palm oil mill effluent (POME), empty fruit bunches (EFBs), oil palm trunks (OPTs), and oil palm fronds (OPFs) (Mohammad et al., 2012). POME, containing some sugars, fat, and lignocellulosic waste, has high COD and BOD values (Oswal et al., 2002). The POME is an important source of phenolic compounds such as gallic acid, protocatechuic acid, p-hydroxybenzoic acid, caffeic acid, syringic acid, vanillic acid, p-coumaric acid, and ferulic acid (Chantho et al., 2016). Various filamentous fungi (Aspergillus, Penicillium, Rhizopus, Mucor, Phanerochaete, Trichoderma, Myrothecium, and Sporotrichum) grown in POME can produce industrial enzymes (cellulase and lipase) (Prasertsan et al., 1992; Rashid et al., 2009; Suseela et al., 2014; Moya-Salazarm et al., 2019; Rachmadona et al., 2021). Concomitantly to enzyme production, fungal biomass containing 40% protein by Aspergillus oryzae can be grown in POME (Barker and Worgan, 1981). A. oryzae cultivation also contributes to significant reductions in BOD (85%) and COD (75%–80%) values (Barker and Worgan, 1981). Biovalorization in terms of decolorization and reduction of COD values can be performed by using fungal strains (Aspergillus fumigatus and Trichoderma viride) (Abdul Karim and Ahmad Kamil, 1989; Mohammad et al., 2012; Neoh et al., 2013). POME contains high levels of zinc, manganese, and iron (Shavandi et al., 2012), as well as high BOD and COD content. As POME can be evaluated for enzyme (mainly lipase) production, reduction of heavy metal values can also be evaluated in addition to BOD and COD removals after fungal cultivations. 7. Potato processing industry waste Starchy agricultural products such as corn, wheat, rice, potatoes, cassava are preferred in the food industry for various purposes such as the primary food source or obtaining refined products. Among these starchy products, potatoes are both widely grown worldwide and processed on a large scale in the food industry for various purposes, such as chips and starch. During the processing of potatoes in the food industry, different types of potato processing by-products (potato pulp, potato wastewater, potato liquor, potato peel) are generated. During the potato chips production process, an excessive amount of potato processing waste (PPW) is generated by cleaning, cutting, slicing, washing, frying, and salting steps (Kot et al., 2020). Potato liquor from the potato starch production process is heated at 110°C to remove proteins. The remaining potato peel liquor (PPL) € gerl, 1994; Bergthis rich in protein and contains a high percentage of solid material (Schu aller et al., 1999). The potato wastes contain starch, cellulose, hemicellulose, lignin, 554 Current Developments in Biotechnology and Bioengineering fermentable sugars, proteins with a high COD, BOD, and solids (Malladi and Ingham, 1993; Gáspár et al., 2007; Ahokas et al., 2014). Mainly starch and other biocompounds such as damaged starch, protein, and amylose can be recovered from PPWs (Devereux et al., 2011). The chemical composition of PPWs provides a good carbon and nitrogen source for microbial processes (Fig. 5, Table 8). For this purpose, many researchers have focused on microbial productions such as ethanol, organic acid, and fungal biomass (Jin et al., 2005; Abanoz et al., 2012; Sumer et al., 2015; Souza Filho et al., 2017b, 2019; Palakawong Na Ayudthaya et al., 2018) using various potato wastes. Microorganisms that can FIG. 5 Evaluation of potato processing wastes in fungal production processes. Table 8 fungi. Production of biometabolites from starchy products through filamentous Metabolite Bioproduct Fungal strain Enzyme amylase Aspergillus niger Aspergillus niger Rhizopus oryzae Rhizopus arrhizus Rhizopus oryzae Aspergillus oryzae cellulase Organic acid Fungal biomass Lactic acid References Abouzied and Reddy (1986) Jin et al. (1998), Izmirlioglu and Demirci (2016a,b) Julia et al. (2016), Verma and Kumar (2019) Rosenberg and Krišofı́ková (1995), Huang et al. (2003), Huang et al. (2005), Jin et al. (2005) Jin et al. (1998), Jin et al. (1999), Jin et al. (2005), Souza Filho et al. (2017b) Chapter 18 • Versatility of filamentous fungi in novel processes 555 synthesize several enzymes, such as amylase and cellulase, should be included in microbial processes since PPW contains starch and cellulose. Starch-degrading enzymes, which are α-amylase, glucoamylase, and α-glucosidase, are regulated by a pathway-specific transcription factor, AmyR (Kato et al., 2002). Cellulases are produced as a multicomponent enzyme system which are endocellulases (endoglucanases), exocellulases (exocellobiohydrolases or exoglucanases), and cellobiase (β-glucosidase) (Cao and Tan, 2002; Lockington et al., 2002). The xlnR gene is the gene encoding enzymes of the cellulolytic enzymes pathway and regulates the endo-cellulase genes eglA and eglB (Van Peij et al., 1998) and exocellulase genes cbhA and cbhB (Gielkens et al., 1999) in Aspergillus niger. S. cerevisiae, which is widely used in ethanol and single-cell protein production, can consume simple sugars such as glucose. Since S. cerevisiae cannot be naturally produced in PPW, it should not be used in starch and cellulose-containing systems. The inclusion of microorganisms such as S. cerevisiae and E. coli in the process involving PPW requires additional costincreasing processes such as hydrolysis. Instead, filamentous fungi, which can naturally hydrolyze starch and cellulose, can be integrated into potato waste-containing systems and used to produce microbial products. Ethanol production from potato starch or potato waste can be performed by amylolytic fungus, Aspergillus niger, and its co-culture with S. cerevisiae (Abouzied and Reddy, 1986; Izmirlioglu and Demirci, 2016a,b). The filamentous fungi can have different metabolite and enzyme activities to hydrolyze the starch and produce metabolites through simultaneous saccharification and fermentation (Jin et al., 1998, 2005; Richter and Berthold, 1998; Oda et al., 2002). Therefore, edible filamentous fungi mainly were studied for the determination of their capacities for organic acid productions. Lactic acid is a valuable industrial organic acid used in the food, pharmaceutical, leather, and textile industries (Huang et al., 2005). Biological production of lactic acid has been widely carried out by some bacteria (e.g., Lactobacillus and Lactococcus) and fungi (e.g., Rhizopus) species (Huang et al., 2005; Li and Cui, 2010). Fungal cultivation is more advantageous than bacterial fermentation due to their ability to consume different types of raw/waste substrates, require no additional supplements, do not require pH adjustment, and easy harvest of fungal biomass (Soccol et al., 1994; Rosenberg and Krišofı́ková, 1995; Huang et al., 2005). Fungal biomass can also be assessable as a valuable product due to its high protein content. For this purpose, Jin et al. (1999) evaluated 30 different microfungal strains for screening their amylolytic activities and biomass production capacities and selected Aspergillus oryzae, Rhizopus oligosporus, and R. arrhizus regarding the biomass production yield (4.3–5.6 g/L). Further studies showed that R. arrhizus and R. oryzae could be successfully lactic acid (0.94–0.97 g/g of starch) and fungal biomass (17–19 g/L) producers, respectively Jin et al. (2005). Similarly, many researchers suggested Rhizopus arrhizus strains to have a high capacity for starch saccharification and lactic acid synthesis (Rosenberg and Krišofı́ková, 1995; Huang et al., 2003, 2005). Protein-rich fungal biomass can be produced from potato protein liquor (PPL), concentrated wastewater generated from the starch production process (Souza Filho et al., 2017b). It was determined that the fungal biomass produced by R. oryzae in an airlift 556 Current Developments in Biotechnology and Bioengineering bioreactor, as above 65 g/L of undiluted PPL, and its protein content was 70% under optimum conditions (Souza Filho et al., 2017b). Similarly, fungal biomass, having 35%–50% protein content, produced by Aspergillus and Rhizopus species can be evaluated as animal feed (Jin et al., 1998, 2005; Souza Filho et al., 2019). COD levels of the potato processing streams were successfully reduced via fungal cultivation concomitantly the metabolite production (Jin et al., 1998; Mishra et al., 2004; Souza Filho et al., 2017b, 2019). Therewith, filamentous fungi species (especially Aspergillus and Rhizopus) can be integrated into the potato waste processes. Souza Filho et al. (Souza Filho et al., 2017a) evaluated different scenarios to analyze the techno-economic and life-cycle assessments of potato peel liquor and suggested that fungal cultivation was economically preferable. 8. Sugar processing industry waste Sugar is one of the most important food products obtained by sugarcane (Saccharum officinarum L.) and sugar beet (Beta vulgaris L. ssp. saccharata) through various processes. During the process, 1 kg sugar production results in by-products such as 0.3–0.4 kg of molasses and a high amount of fibrous residue (Fig. 6) (Ramjeawon, 2004; Botha and von Blottnitz, 2006; Mashoko et al., 2010). Molasses contains fermentable sugars (mainly sucrose and less amount of glucose and fructose) and organic substances (betaine, amino acids, minerals, vitamins, trace elements) (Valli et al., 2012; Nakata et al., 2014). Molasses containing high organic matter is generally preferred as a raw material for various biochemical processes such as ethanol (Akbas et al., 2014; Reis et al., 2020), butanol (Zetty-Arenas et al., 2021), organic acid (Abdel-Rahman et al., 2020), single-cell (Nigam and Vogel, 1991), and miscellaneous productions (Oliveira et al., 2020; Zhang et al., 2021) in industry. Sugar Sugar beet Washing & Cutting Diffusion Evaporation Crystallisation Molasses Sugar cane FIG. 6 A schematic diagram for sugar production and molasses generation from the processing of sugar cane and sugar beet. Chapter 18 • Versatility of filamentous fungi in novel processes 557 Biofuels, hydrotreated jet fuel (from lipids), and ethanol (from sugars) can be generated by using sugar processing products (Kumar et al., 2018). Molasses has become the primary raw material for ethanol production in Brazil, China, and European countries (Cardona and Sánchez, 2007; Tang et al., 2010). Ethanol production (Eqs. 3 and 4) is mainly carried out by the yeast Saccharomyces cerevisiae (Pattanakittivorakul et al., 2019; Wu et al., 2020), while other microorganisms are also used for ethanol production (Sharifia et al., 2008; Yilmaztekin et al., 2008; Akbas et al., 2014). Sucrose ðC12 H22 O11 Þ + H2 O ! Glucose ðC6 H12 O6 Þ + Fructose ðC6 H12 O6 Þ (3) Glucose or Fructose ðC6 H12 O6 Þ ! Ethanol ð2C2 H5 OHÞ + 2CO2 (4) After ethanol production and distillation in such processes, what remains is called vinasse. An excessive amount of vinasse (15 kg) is released by distilling ethanol (1 kg) produced from molasses (Nair and Taherzadeh, 2016). This ethanol industry waste, which is acidic (pH 3.5–5.0), contains a high level of organic materials (50–150 g/L COD) and is a critical pollutant source for the environment (Christofoletti et al., 2013). Various filamentous fungi were included in the system for the valorization of vinasse, and their producibility was investigated (Nitayavardhana et al., 2013; Nair and Taherzadeh, 2016; Karimi et al., 2019). Accordingly, it was determined that Aspergillus oryzae and Neurospora intermedia strains can grow in vinasse, and protein-rich fungal biomass (40%–45% protein content) and extra bioethanol production were detected. Moreover, a significant amount of COD removal was also successful (Nair and Taherzadeh, 2016). Similar studies conducted with Rhizopus strains (R. oryzae and R. oligosporus), it has been reported that fungal biomass produced from vinasse contains high protein, and this fungal biomass can be considered as a commercial protein source for aquatic feeds (fishmeal and soybean meal) (Nitayavardhana et al., 2013; Karimi et al., 2019). According to these studies, ethanol production from molasses, as sugar industry byproduct, could be integrated by a second process to produce fungal biomass. Thus, integrated two processes can become more suitable for enhancing metabolite production yields and improving the valorization of sugar industry byproducts and wastes. Recently, high lipid-containing fungal biomass from sugar industry by-products has been investigated for biodiesel production (Fig. 7). Bento et al. (2020) suggested that fungal biomass production from sugarcane molasses by Mucor circinelloides could be used in biodiesel production depending on its lipid content (29%) and fatty acid composition. Reis et al. (2020) tried to grow Mucor circinelloides in a combination of molasses and vinasse media to assimilate sucrose, glucose, and fructose, and fungal biomass with 25% lipids. Fungal biomass yield and its lipid content (35%) could be enhanced via fungal (Aspergillus sp.) co-cultivation with microalgae (Chlorella sp.) in molasses wastewater (Yang et al., 2019). In addition to ethanol production, molasses can be used for various metabolites through filamentous fungi. The ligninolytic enzyme is a group of lignin peroxidases (LiP), manganese-dependent peroxidases (MnP), and laccase. Ligninolytic enzyme 558 Current Developments in Biotechnology and Bioengineering FIG. 7 A schematic representation for biodiesel production from lipid-containing fungal biomass. production by fungi (Phylosticta, Aspergillus, Fusarium, and Penicillium) have been studied. Pant and Adholeya (2007) investigated some isolated fungi to screen their ligninolytic enzyme production activities and determined that Fusarium verticillioides, Aspergillus niger, and Aspergillus flavus had the highest LiP, MnP, and laccase activities, respectively. Similarly, it is widely common to investigate the production of enzymes such as dextranase by Aspergillus fumigatus (Fadel et al., 2020), invertase by A. niger (Veana et al., 2014), βfructofuranosidase by A. tubingensis (Xie et al., 2020), laccase by white-rot fungi (Coriolus versicolor and Funalia trogii) (Kahraman and Gurdal, 2002), phytase by Sporotrichum thermophile (Singh and Satyanarayana, 2008) from molasses. Similar to enzyme production, Pleurotus ostreatus, Flavodon flavus, Geotrichum candidum were reported to be effective in studies on the decolorization of molasses (Kim and Shoda, 1999; Raghukumar and Rivonkar, 2001; Pant and Adholeya, 2007). Sugar industry products can be evaluated as conventional sugar production, synthetic fertilizer, biogas, and alcohol production. The life-cycle assessment analyzes clearly demonstrate the advantage of producing alternative products (ethanol, biogas, animal feed, and fertilizers), with a comparative study for resource savings (Contreras et al., 2009). Alternatively, fungal products can be evaluated considering their high-lipid fungal biomass and enzyme production abilities. For this, filamentous fungi can be integrated into processes containing vinasse generated from ethanol fermentation of molasses and evaluated in microbiological production. 9. Dairy processing industry Cheese whey is an important industrial by-product of the dairy industry during the cheese production process. Approximately 9 L of whey are generated by processing each kg of cheese produced (Kosikowski, 1979). Whey containing 5%–6% lactose, 1% protein, fat, and minerals (especially nitrogen and phosphorus) has high organic load, COD, and €nzle et al., 2008; Prazeres et al., 2012). Since whey is a by-product of milk, BOD levels (Ga it is easily degradable and is challenging to store and transport as it is formed in excess. Chapter 18 • Versatility of filamentous fungi in novel processes 559 Whey powder having a high concentration of lactose is formed of dried and concentrated whey. Many researchers used cheese whey and whey powder for ethanol production (Eqs. 5 and 6) because of their high carbohydrate content. Typical ethanol producer strain, S. cerevisiae cannot assimilate lactose to ethanol due to the lack of lactose hydrolyzing enzymes. Kluyveromyces and E. coli were reported to produce ethanol from cheese whey and whey powder (Kargi and Ozmıhcı, 2006; Sar et al., 2017a,b, 2019; Alves et al., 2019). In addition, fungi such as Trichoderma reesei, Aspergillus niger, Neurospora crassa, and Fusarium graminearum can assimilate lactose and produce metabolites. Ethanol production from whey by various fungi (Fusarium, Monilia, Neurospora, Mucor, and Paecilomyces sp.) have also been studied, similar to fermentation of yeast and E. coli (Singh et al., 1992). Lactose ðC12 H22 O11 Þ + H2 O ! Glucose ðC6 H12 O6 Þ + Galactose ðC6 H12 O6 Þ (5) Glucose or Galactose ðC6 H12 O6 Þ ! Ethanol ð2C2 H5 OHÞ + 2CO2 (6) Whereas fungal strains exhibited higher ethanol productivity and high sugar tolerance, bioconversion rate is slower than yeast cultivations. Nevertheless, fungal strains can be a potential candidate for ethanol production from lignocellulosic materials thanks to their enzyme (cellulases and xylanases) production capacities (Singh et al., 1992). Similarly, fungal strains can be used in lactose-containing media for ethanol production. Okamoto et al. (2019) investigated the ethanol production capacity of Neolentinus lepideus from cheese whey and expired milk. Enzyme complexes produced by Aspergillus niger for ethanol production can be used for hydrolysis for agroindustrial wastes (whey, sugarcane bagasse, and rice byproducts), and the hydrolysates can be evaluated by ethanol-producing organisms such as S. cereviase via separate hydrolysis and fermentation (SHF) (Rocha et al., 2013). Rocha et al. (2013) reported obtaining higher ethanol yield by enzyme cocktail of A. niger than Trichoderma reesei, and complex enzyme of A. niger was promising in the disposal of dairy industry wastes. Some yeast strains such as Kluyveromyces marxianus, K. fragilis, Candida pseudotropicalis, C. versatilis were able to produce microbial lipid (single cell oil, SCO) (Schultz et al., 2006; Vamvakaki et al., 2010). Similarly, some fungal species (Mortierella isabellina, Thamnidium elegans, Mucor sp., and Fusarium) can synthesize lipase and produce SCO (Mahadik et al., 2002; Vamvakaki et al., 2010; Akpinar-Bayizit et al., 2014; Chan et al., 2018; Roy et al., 2021). Herein, whey can be promising feedstocks for lipid accumulation in fungal biomass (Akpinar-Bayizit et al., 2014; Chan et al., 2018). Lipid and amino acid contents of fungal biomass can be reached up to 24% and 48%, respectively, at the optimum conditions (Chan et al., 2018; Ibarruri and Hernández, 2019). This oily biomass can be suggested for biodiesel production as it contains oleic and palmitic, or in nutraceutical applications, as it contains linolenic acid (Chan et al., 2018). The stages of biodiesel production from biomass of an oleaginous fungus are generally as follows; oil extraction from biomass and obtaining fatty acid methyl esters (FAMEs) by acid-catalyzed transesterification and esterification at 65°C for 8 h (Vicente et al., 2009). Vicente et al. (2009) 560 Current Developments in Biotechnology and Bioengineering suggested that biodiesel production by direct transformation from fungal biomass without lipid extraction is technically possible and that this process needs to be developed on an industrial scale. Various dairy wastes (such as cheese whey, milk, yogurt, cream) can be converted into fungal biomass, ethanol, and glycerol through A. oryzae and N. intermedia cultivations (Mahboubi et al., 2017b). Dairy industry wastes can also contain high amounts of fat in addition to being rich in lactose and protein. It is even more important to evaluate dairy industry wastes containing high-fat content by microbial processes, but it also makes microbial bioconversion difficult. In order to overcome these difficulties, two-stage continuous cultivation was created using Aspergillus oryzae as fat degrader and Neurospora intermedia as lactose ` me fraiche media, which are fat-rich dairy products (Mahboubi consumer in cream and cre et al., 2017a). A similar application was examined with the same fungal strains in expired milk, and it was reported that A. oryzae produced 11 g/L biomass by degrading fat and proteins; after that N. intermedia produced 7 g/L biomass consuming the remaining lactose (Thunuguntla et al., 2018). The crude protein of A. oryzae and N. intermedia biomass from dairy industry byproducts was around 30%–40% on a dry weight basis, and these protein-rich fungal biomasses can be used as feed (Mahboubi et al., 2017b). An excessive amount of dairy industry waste (50%) has been discharged into the environment without any treatment (Kolev Slavov, 2017; Bosco et al., 2018). Traditionally treatment options, activated sludge processing, is not economically due to its having high organic load and low alkalinity (Asunis et al., 2020). Industry-integrated microbial production processes are insufficient. Therefore, dairy industry wastes/byproducts can be included in fungal growth processes, including fungal biomass production, lipase enzyme, enzyme-complex production for degradation of lignocellulose compounds, and microbial oil production. 10. Conclusions and perspectives Agro-industrial wastes are generated every year in significant quantities. These wastes mostly end up in landfills cause various environmental problems. However, recent studies on cultivation with filamentous fungi has resulted in various value-added products from these wastes. These fungal processes can be integrated into the already established industrial processes. With the integrated process, valuable products are produced by utilizing the agro-industrial wastes as low-cost substrates, which reduce the production cost while eliminating the waste management expenses. Additionally, the environmental problems thereof will be mitigated. Besides, the filamentous fungi integration simplifies the process, e.g., removing the pretreatment step in bioethanol production from lignocellulosic material. It is evident that fungal valorization paves the way for reaching the biorefinery. Although to have the biorefinery within our grasp, scaling-up these waste-based fungal processes to the industrial level is a crucial step. To do so, further studies are required, such as feedstock selection and process optimization, product development, life-cycle assessment, techno-economic analysis, and the role of involved stakeholders. Chapter 18 • Versatility of filamentous fungi in novel processes 561 Lastly, the main future objective of the fungal biorefinery based on agro-industrial waste could be food production. In the reviewed processes in this chapter, many of the used fungi for chemical production were edible filamentous fungi. By using edible fungi, it would be possible to have edible biomass in addition to produced chemicals. Moreover, since agro-industrial wastes are mainly generated from food production sectors, it has a higher possibility to produce biomass with food-grade quality. Thus, further research is required to develop the process that produces biomass with food-grade quality while producing other chemicals. 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Microbial production of value-added bioproducts and enzymes from molasses, a by-product of sugar industry. Food Chem. 346, 128860. Index Note: Page numbers followed by f indicate figures, and t indicate tables. A Acetylation, 135 Acetyl-CoA, 456–457 Acremonium chrysogenum, 68t Activated charcoal, 134 Adenosine triphosphate (ATP) measurement, 173 Aeration, 222–223 in solid-state fermentation, 261–262 Aflatoxins, 105, 124, 376–378 economic impact, 108t exposure of, 107 health effect, 106t occurrence of, 125–127t structure, producing fungi and stability of, 109–123t worldwide regulation limit of, 129t AFTOL project, 78 Agar disc diffusion method, 480 Agaricus bisporus, 68t Agar overlay bioassay, 482 Agar plug diffusion method, 482 Agar well diffusion method, 482 AGF. See Anaerobic gut fungi (AGF) AGF CAZyome, 87–88 AGF protein domains, 84f, 86 Agitation, 152–153 power consumed for, 221–222 in solid-state fermentation, 262 Agriculture industry, fungi in, 43–44, 43f Agrocybe cylindracea, 40–41 Agro-industrial residues, 299, 300t Air-lift reactors, 39, 40f, 204, 224–225t, 229 configuration, 226f effect on fungal morphology, 241 Alcohols ethanol (see Ethanol) fermentative production butanol, 445–447 feedstocks for, 437–438 production process, filamentous fungi in, 448f Algae, 337–338, 438 Allomyces macrogynus, 88 Allopolyploidy, 78 α-zearalenol (α-ZEL), 377 Alpha linolenic acid (ALA), 410, 412–413 Alternaria toxins, 109–123t American Type Culture Collection (ATCC), 165 Amylases, 198–199 Anaerobic fungi, 18 in biogas production, 47 Anaerobic gut fungi (AGF), 86 Anamorph, 153–154 Anamorphic fungi, 1 Aneuploidy, 78 Animal and fish feed ingredients, filamentous fungi as compound feed fishmeal (see Fishmeal) plant protein meals, 402–404 terrestrial animal by-product meals, 404 economic and environmental aspects, 424–425 fermented soybean and soybean meal piglets fed, 421–422 in poultry, 421 fungal biomass amino acid profile, 406–408, 408t, 409f antioxidant agents, 418 broiler chicken, 421–422 cell wall components, 413–416 fat and fatty acid content, 408–413 fish fed, application in, 422–424 575 576 Index Animal and fish feed ingredients, filamentous fungi as (Continued) mammalian food, application in, 423 minerals, 416–417 nucleotides, 419–420 pigments, 417–418 prebiotics, 420–421 protein, 406–408, 407t vitamins, 419 waste streams, 423–424 fungi kingdom, 405–406 protein source ingredients, 399–400 ruminants, fermented feed application in, 421 single-cell proteins (SCPs), 404–405 Animal-pathogenic fungi, 6–7 Animal protein consumption of, 348, 348f greenhouse gas (GHG) emissions of, 343, 348–349, 349t health concerns, 343 protein content, quality, fat, and antinutrient compounds, 345, 346t sources, 344–345 world demand for, 343 Ankaflavin, 329, 330f Antarctic fungi, 16–17 Anthraquinone fungal, 334, 335f Natural Red™, 332–334 Antibiotics, 105 industrial applications, 205–206t solid-state fermentation produced, 276t, 277–278 Antibiotics production, filamentous fungi antimicrobial assay, 480–483 ascochlorinis, 491 asperchondols, 489–490 aspergillin, 490 aspochalasin, 490 carbon source, antibiotic synthesis, 477–478 cell membrane function, inhibitors of, 486 cell wall synthesis, inhibitors of, 485 cephalosporins, 477, 487–488 claviformin, 490 dihydrogeodin, 491 diketopiperazine, 479 fermentation process extraction and purification, 484 quality control, 485 refining, 484–485 solid-state fermentation, 483–484 submerged fermentation, 483 fumagillin, 490 fusidane, 488–489 fusidanes, 479 gigantic acid, 491 glucose, 477–478 heterologous expression, 477–478 history, 478 β-lactam antibiotics, 477 marine ecosystem, 479 metabolic engineering, 491–492 metabolic processes, inhibitors of, 486 microbes, isolation and screening of, 480 microorganisms, 477 nucleic acid synthesis, inhibitors of, 486 penicillins, 477, 479, 486–487 perspectives, 492 primary use of, 477 protein synthesis, inhibitors of, 486 schematic representation of, 481f tropolone derivative, 490 Antioxidant agents, 418 Aphelids, 79 morphological characters, 80t Aquatic fungi, 15–16 α-Arabino furanosidase, 210–211t Arachidonic acid, 366, 375, 410–412 Aroma compounds, 281 Arpink red™. See Natural Red™ Ascogonium, 74–75 Ascoma, 74–75 Ascomycetes biomass in animal feed, 55 in human food, 54–55 ethanol production by, 442–443, 443t metabolites production, 50 Index 577 citric acid, 51 ethanol, 50 gluconic acid, 51 itaconic acid, 51 for solid-state fermentation, 254 Ascomycetes dikaryons, 75 Ascomycota, 67–69 characteristic features of, 96 classification of, 95 reproduction, 74–75 Ascospores, 74–75 Asexual reproduction in Ascomycota, 74 in Entomophthoromycotina, 91 in Kickxellomycotina, 92 in Mucoromycota, 93–94 in Mucoromycotina, 94 in Zoopagomycotina, 91 in Zygomycota, 73 Asexual spore formation, 153–154 Ashbya gossypii, 68t, 242, 419 Asperchondols, 489–490 Aspergillin, 490 Aspergillus niger, 366, 422–423 agitation and, 152–153 citric acid production, 464–465 gluconic acid production, 461–462 oxalic acid production, 467–468 pelleted mass of, 152–153 Aspergillus sp., 68t A. awamori, 422 A. oryzae, 421–422 A. parasiticus, 377–378 A. terreus, 457t, 458–460 for citric acid production, 47, 51, 462–463 cultivation pH, 41–42 enzyme production, 197, 295, 297–298t for ethanol production, 50 for gluconic acid production, 47, 51 hydroxyanthraquinoid (HAQN) pigments, 332 for itaconic acid production, 51 lovastatin production, 45 in marine habitats, 336–337 morphology/factors affecting, 202t mycotoxins, 108–124 pigment production, 54 value-added products by, 182–183 xylanase and lignin peroxidase by, 208 Aspochalasin, 490 Assembling the Fungal Tree of Life (AFTOL) project, 67 Autopolyploidy, 78 Axial pumping impellers, 227 Azaphilone pigments mycotoxin-free Monascus red, 328–329 non-toxigenic fungal strains, Monascus pigments from, 329–331 B Bacillomycin D, 135 Ballistoconidiogenesis, 3 Basidiobolus sp., 91 Basidiomycetes, 53 biomass, 57 ethanol production from, 53–54 reproduction, 72 Basidiomycota, 67–69 characteristic features of, 96–97 classification of, 96 reproduction, 75 Basidiomycotina, 97 Batch culture, 222–223 Batch fermentation, 38 phases in, 39f systems, 308–309, 309f Batrachochytrium dendrobatidis, 6, 84–85 Beauveria bassiana, 278 Beauvericins, 109–123t Beta-glucan, 414 Bioactive compounds, 276 Bioactive pigment production, by filamentous fungi anthraquinones, 334, 335f Natural Red™, 332–334 azaphilone pigments mycotoxin-free Monascus red, 328–329 non-toxigenic fungal strains, Monascus pigments from, 329–331 food colorants 578 Index Bioactive pigment production, by filamentous fungi (Continued) hydroxyanthraquinoid (HAQN) pigments as, 326, 331–332 polyketide-Monascus-like pigments as, 326–328 marine fungi, 326, 334–338 microbial cell factories, 325–326, 326f Biobleaching, 208–209 Biochemical, 47–48 Biocomposites, construction and packaging applications, 521–522 Bio-debarking, 204–206 Biodegradation, 190 Biodiesel, 278–279 Bioethanol, 209 industry wastes, 544–548 solid-state fermentation, 279 Bio-fabrication, 47 Biofilms, 70 Biofuels, 47–48, 209–211, 272 Biogenic amines (BAs), 377–378 Bioluminescent methods, 483 Biomass evaluation direct methods for cell count, 169–170 fluorescence techniques, 172 gravimetric technique, 169 imaging and microscopy, 170–171 impedance and capacitance techniques, 171–172 near-infrared spectroscopy, 172–173 optical density, 170 indirect methods for adenosine triphosphate measurement, 173 calorimetry, 174 chitin measurement, 173–174 CO2 production and oxygen uptake rate, 174–175 Biomass, fungal, 54–57, 151 Biomaterial, 47–48 Biopesticides, 278, 278t Bio-pitching, 206–207 Biopolymers, 280–281 Bioprocess, with filamentous fungi, 198–200 factors affecting, 200 morphology, 201–203, 201f, 202t rheology, 203–204 strain screening and inoculum, 200 Biopulping, 207–208 Bioreactor(s), 37, 219 for solid-state fermentation, 38f for submerged fermentation, 40f Bioreactor design air-lift reactor, 229 bubble columns, 228 configurations, 224, 224–225t engineering fundamentals in, 220 medium aeration and kLa, 222–223 medium viscosity and rheology, 220–221 power consumed for agitation, 221–222 shear forces and shear stress, 223–224 filamentous fungi morphology and, 236 effect of, 239–241 life cycle, 237 macromorphology in submerged fermentations, 237–238 productivity, 238–239 tailoring, 241–242 fluidized-bed bioreactors, 233–234, 233f packed-bed bioreactors, 231–232, 232f rotating-bed bioreactors, 234–235, 234f stirred-tank reactors, 226–227 trickle-bed bioreactors, 229–231, 230f Biorefineries, fungal applications, 18 industries, 42–48, 43f investment and productivity improvement strategies, 48–50 value addition to organic wastes, 48 Bioremediation, 189–190 Bio-retting, 207 Biosafety level 3 (BSL-3), 162 Biosurfactants, 279–280 Blakeslea trispora, 68t Blastocladiella emersonii, 88 Blastocladiomycota, 88–89 characteristic features of, 88 Bleaching, 208–209 Index 579 Botrytis cinerea, 522–523 Brachyallomyces life cycle, 88 Branched-chain amino acids (BCAAs), 408 Brewer’s spent grain (BSG), 258, 534–538, 536t cellulolytic and xylanolytic enzymes, 537–538t fermentation with Rhizopus sp., 265, 266f Brewery waste, 534–539 Bubble column bioreactors, 39, 40f, 224–225t, 226f, 228, 299, 302f Budding, 3, 72 Burkholderia, 77 Burkholderia gladioli, 379 Butanol, fermentative production of, 445–447 C Calorimetry, 174 Candida albicans, 2 Candida famata, 68t Cantharellus cibarius, 68t Capacitance measurement, 171–172 Carbon, role of, 257 Carbon to nitrogen (C:N) ratio, 35, 257 β-Carotene, 325–326 Carotenoids, 325 Cell count method, 169–170 Cellobiohydrolase, 210–211t Cellulases, 210 Cell wall, fungal, 3 components of, 3t Cell wall, of filamentous fungi, 413 chitin, 414–415 chitosan, 415 glucans, 414 mannose, 415–416 Cephalosporin, 487–488 mode of action, 488 structure of, 488f Cereals, mycotoxins in, 124–128 Chaetoglobosin, 109–123t Chemical methods, for mycotoxins reduction, 135–136 Chemical mutagens, 465 Chitin, 414–415 Chitinases, 198–199 Chitin assay, 173–174 Chitin-glucan complex (CGC), 508–510, 510t Chitooligosaccharides (COSs), 517–518 Chitosan, 186–187, 415 extraction and purification of, 511–512, 513–514t in zygomycetes, 56–57 3-Chloropropane-1,2-diol (3-MCPD), 378 Chytridiomycetes, 83–85 Chytridiomycota (chytrids), 67–69, 81–85 morphological features of, 82–83 parasitic roles, 83 reproduction, 73 Citric acid (CA), 275, 366 applications, 462 from ascomycetes, 51 biochemical pathway, 470f biosynthesis, microorganisms, metabolism, and physiology of, 462–463 chemical structure of, 468f definition, 462 genetic and process engineering strategies, 465 global market, 462 production and processing conditions, 463–465 Citrinin, 108–124, 109–123t, 327–329, 330f Claviformin, 490 Clostridium botulinum, 378 Clustered regularly interspaced short palindromic repeats/CRISPRassociated protein 9 (CRISPR/Cas9) genome editing, 18, 444 Coagulative pellets, 237–238 Co-cultivation, 49 Codex Alimentarius, 131 CODEX set, 128–129 Coemansia, 92 Coenocytic hyphae, 1–2 CO2 evolution rate (CER), 174 Compound feed fishmeal (see Fishmeal) plant protein meals, 402–404 terrestrial animal by-product meals, 404 Concentration of dissolved oxygen, 222 580 Index Conidia, 73 Conidiobolus, 91 Conidiophores, 73 Consolidated bioprocessing (CBP), 49, 439, 539 Continuous culture, 38 Continuous fermentation systems, 308–309, 309f Cornmeal agar, 152 Cross-streak method, 482 Cryopreservation, of filamentous fungi, 160–166 factors influencing, 163–164 freezing temperatures, 164–165 protocols for, 165, 166t steps in, 165f Cryoprotectants, 163, 164t Cryptic species, 14 Culture media, 33–35, 152 design and preparation of, 35 growth chemical requirements, 33–35 macro-nutrients, 34t for isolation and enumeration of mycobiota from fermented products, 159t types of, 33 Cycloleucomelone, 334–336 Cystogenes life cycle, 88 Cytochalasins, 109–123t Cytoplasmic waves, 70–71 Czapek-Dox Agar, 159t Czapek yeast extract agar, 159t D Daedaleopsis flavida, 45 Dairy processing industry, 558–560 Deacetylases, 210–211t Degassed power number, 222 Denaturing gradient gel electrophoresis (DGGE), 156 Deoxynivalenol economic impact, 108t health effect, 106t occurrence of, 125–127t structure, producing fungi and stability of, 109–123t worldwide regulation limit of, 129t Dermocybe sanguinea, 332 Dew retting, 207 Dichloran 18% glycerol agar, 159t Dihydrosterimagtocystin, 109–123t Dikarya, 95–97, 149–150 Dimethyl sulfoxide (DMSO), as cryoprotectant, 163, 164t Dimorphic fungi, 2, 72 Dinomyces arenysensis, 83 Direct bioautography, 482 Dispersed filamentous growth, 238 Dispersed mycelia, 200–201 Dissolved oxygen, 303, 458, 464–465 concentration of, 222 Distillation, of ethanol, 444–445, 445f Diversity, fungal, 13–14 Docosahexaenoic acid (DHA), 375, 410 Downcomer, 229 Dried distillers grains with solubles (DDGS), 544–545 Dry-grind bioethanol plant, 544–545 E EAA. See Essential amino acid (EAA) Ecosystems, fungal, 14–18 aquatic ecosystems, 15–16 extremophile environments, 16–18 terrestrial ecosystems, 15 Ectomycorrhizal Basidiomycota, 97 Eicosapentaenoic acid (EPA), 410 ELISA-spectrometric method, 136 Endo β 1,4 glucanase, 210–211t Endo β 1,4 mannanase, 210–211t Endo β 1,4 xylanase, 210–211t Endomycorrhizae, 7 Enniatins, 109–123t Entomopathogenic fungi, 278 Entomophthoromycotina, 91 Environments, for filamentous fungi isolation, 154–156 Enzymatic hydrolysis, 210 Enzymes. See also Industrial enzymes for biofuel production, 272 for food and feed applications, 272 production by SSF, 272–274, 273–274t Index 581 Enzymes, fungal, 4, 54, 181–182 applications in biofuel industry, 210, 210–211t industrial, 204, 205–206t in food industry, 44–45 in pharma industry, 45 in pulp and paper industry, 46 Epicoccum nigrum, 327–328 Ergot alkaloids, 109–123t Essential amino acid (EAA), 345, 349, 373, 374t, 407, 408t, 409f Ethanedioic acid. See Oxalic acid Ethanol, 48 applications, 435, 436f from ascomycetes, 50 from basidomycetes, 53–54 definition, 435 global production, 436–437, 437f market for, 435–436 production, by filamentous fungi, 438–443, 439f ascomycetes, 442–443, 443t metabolic engineering strategies, 443–444 zygomycetes, 440–441, 441t recovery and concentration distillation, 444–445, 445f pervaporation (PV), 445, 446f from zygomycetes, 52 Euallomyces life cycle, 88 Eukaryotic protein expression systems, 311–313, 312t European Food Safety Authority (EFSA), 254, 522 Eurotium sp., 332 Exopolysaccharides (EPS), 280, 497–498 Expression system, 197 Extremophile fungi, 16–18 F Fat mimetics, 372 Fatty acids alpha linolenic acid, 410, 412–413 arachidonic acid, 410–412 docosahexaenoic acid, 410 eicosapentaenoic acid, 410 linoleic acid, 410, 412 lipids, 408–409 oleic acid, 413 saturated fatty acids, 410 unsaturated fatty acids, 410 Fed-batch fermentation, 38, 308–309, 309f Feed conversion ratios (FCR), 402, 403t Feed ingredients, filamentous fungi as. See Animal and fish feed ingredients, filamentous fungi as Fermentation, fungal, 31 factors influencing, 40–42 inoculum preparation, 36 types, 36–39, 38–40f Fermented food, filamentous fungi in, 343–344, 353–362t advantages, 351–352 flavor enhancer, 369–370 oncom, 364–366 protein. Protein, filamentous fungi safety issues harmful contaminants, 378 mycotoxins, 376–378 pathogenic bacteria, 378–379 soy sauce (see Soy sauce) tempe (see Tempe) Fermented products, filamentous fungi isolation, 156–158, 157–158t culture media, 159t Feruloylesterase, 210–211t Filamentous fungi, 1, 150 animal feed, application in (see Animal and fish feed ingredients, filamentous fungi as) applications, 150–151, 337–338, 338f biofilm production, 70 characteristics, 42 compounds in industrial application, 186, 186t cultivation (see Bioreactor design) diversity of, 184–185 enzyme production (see Industrial enzymes) growth monitoring of, 168–175 growth of, 69–70 identification of, 159–160 582 Index Filamentous fungi (Continued) industrial applications, 204–211 industrial bioprocess with, 198–200 isolation and purification of, 151–158 single spore isolation, 154 spore isolation, 153–154 microbiology of, 183–184 in microplastics removal, 189–190 nutrient uptake in, 4 organic acid, biological production of (see Organic acid production, filamentous fungi) pigment production (see Bioactive pigment production, by filamentous fungi) in pollution reduction, 187–189, 187f, 189t preservation of, 160–168 sampling and isolation from environments, 154–156 from fermented products, 156–158, 157–158t sampling procedures for, 158 solid-state fermentation (see Solid-state fermentation (SSF)) vegetative reproduction in, 3 Fish feed, fungal biomass in. See Animal and fish feed ingredients, filamentous fungi as Fishmeal, 424 amino acid profile of, 400–401, 408, 409f consumption, status of, 401, 401f definition, 400 diets, inclusion rate in, 400 economic and environmental aspects, 401–402, 403t protein, 400 Fish processing industry waste, 548–550 Flammulina velutipes, 53 Flavor enhancer, 369–370 Flax dew retting, 207 Flow cytofluorometric assay, 483 Flow cytometry, 169 Fluid dynamics, 203–204 Fluidized-bed bioreactors, 39, 40f, 224–225t, 233–234, 233f, 241, 269, 269f characteristics and limitations, 271t Fluorescence techniques, for biomass evaluation, 172 Fluorescent ELISA, 136 Fluorophores, 172 Food additives, 366–372 and feed industry, fungi in, 43f, 44–45 mycotoxins in, 124–130, 125–127t Food applications, filamentous fungi challenges, 385 enzymes, 370–372, 371–372t fermented food (see Fermented food, filamentous fungi in) flavor enhancer, 369–370 food additives, 366 food ingredients, 372 food products, 344, 344f fungal mycelium-based food, 372–373 health aspects/functional properties, 376 industrial production modern processes, 382–384 traditional methods, 379–382 lipid, 375 vs. mushrooms, 350–351 natural pigments, 366–369, 367t proteins (see Protein) vitamins, 375 Food colorants anthraquinone fungal, 334, 335f Natural Red™, 332–334 criteria for, 368 hydroxyanthraquinoid (HAQN) pigments as, 326, 331–332 polyketide-Monascus-like pigments as, 326–328 Food grade pigments, 325–326 Freeze-drying, 160–163, 166–167 Freshwater fungi, 16 Fruit industry waste, 540–544 Fumagillin, 490 Fumaric acid, 53 Fumonisin B1 (FB1), 377 Fumonisins economic impact, 108t Index 583 health effect, 106t occurrence of, 125–127t structure, producing fungi and stability of, 109–123t worldwide regulation limit of, 129t Functional amino acids (FAAs), 407 Fungal antibiotics, production of ascochlorinis, 491 asperchondols, 489–490 aspergillin, 490 aspochalasin, 490 cell membrane function, inhibitors of, 486 cell wall synthesis, inhibitors of, 485 cephalosporin, 487–488 mode of action, 488 claviformin, 490 dihydrogeodin, 491 fumagillin, 490 fusidane, 488–489 mode of action, 489 gigantic acid, 491 metabolic engineering, 491–492 metabolic processes, inhibitors of, 486 nucleic acid synthesis, inhibitors of, 486 penicillin, 486–487 mode of action, 487 protein synthesis, inhibitors of, 486 tropolone derivative, 490 Fungal biomass, 54–57 Fungal biopolymers applications of antimicrobial and preserving agent, 516–519 biocomposites, for construction and packaging applications, 521–522 bioemulsifiers and biosurfactants, 512–515 textiles, 519–521 tissue engineering, 516 wound healing materials, 515–516 chitin, 498–499, 504 exopolysaccharides, 497–498 extraction and purification of chitin–glucan complex (CGC) and chitin, 508–510, 510t chitosan, 511–512, 513–514t fungal cell wall, 499–500, 501t polysaccharides, 500–505 structural organization of, 503f heterotrophic eukaryotes, 497 macromolecular constituents, 497–498 mannan, 504–505 perspectives, 523 Fungal biorefineries agriculture industry, 43–44 biofuels, biochemical, and biomaterial production, 47–48 food and feed industry, 44–45 industries, 42, 43f investment and productivity improvement strategies, 48–50 pharmaceutical industry, 45 pulp and paper industry, 46 textile industry, 46–47 value addition to organic wastes, 48 Fungal biotechnology, 31, 32f biomass, 54–57 culture medium, 33–35 fermentation process, 36–39 metabolites, 50–54 Fungal culture medium, 33–35 design and preparation of, 35 growth chemical requirements, 33–35 macro-nutrients, 34t Fungal diversity, 13–14 Fungal ecosystems, 14–18 aquatic, 15–16 extremophile, 16–18 terrestrial, 15 Fungal enzymes in biofuel industry, 210, 210–211t industrial applications, 204, 205–206t Fungal metabolites, 50–54 Fungal morphology, 1–4 hyphae, 2f reproduction, 3 Fungal nutrition, 4 nutrient uptake, 4 Fungal one-step IsolatioN Device (FIND), 153–154 584 Index Fungal reproduction, 71–75 Fungi, 1 applications, in biorefineries, 18 classification of, 78–97, 149–150 growth of, 69–70 lifestyles of, 4–8 metabolic and genetic complexity in, 76–77 nutrition and transport in, 70–71 roles of, 67 taxonomy of, 8–13 Hibbett classification, 9–10, 10f Tedersoo classification, 10–11, 11f Wijayawardene classification, 11–12, 12f toxigenic, 130–131 FUNGIpath, 76–77 Fusaproliferin, 109–123t Fusarin C, 109–123t Fusarium sp. biomass, 372–373 in enzymes production, 296, 297–298t for ethanol production, 50 F. oxysporum, 6, 442 F. solani, 241 F. venenatum, 44, 55, 68t, 405–406 mycotoxins, 108–124, 377 Fusidane-type antibiotics, 488–489 mode of action, 489 structure of, 489f G α-Galactosidase, 210–211t Gamma-ray irradiation, 135 Ganoderma lucidum, 68t Generally Recognized as Safe (GRAS), 197–198, 254, 405–406, 534–536 Genetically modified organisms (GMOs), 150–151 Genome, 77–78 Genome sequencing, 9 Geomyces destructans, 6 Gibberellic acid, 538–539 Gigantic acid, 491 Glomeromycota, 92–93 reproduction, 74 Glucans, 414 Gluconic acid (GA) applications, 460 from ascomycetes, 51 biochemical pathway, 469f biosynthesis, microorganisms, metabolism, and physiology of, 460 chemical structure of, 468f definition, 460 production and process conditions, 460–461 strain engineering and process modifications, 461–462 Glucosamine, 173–174 β-Glucosidase, 210–211t α-Glucuronidase, 210–211t Glucuronoyl esterase, 210–211t Glycosylation, 135 Gold NPs, 47–48 Good agricultural practice (GAP), 132–133 Good manufacturing practices (GMPs), 131 Granger causality test, 70 Gravimetric measurement technique, 169 Greenhouse gas (GHG) emissions, of protein sources, 348–349, 349t Groundnut dextrose broth, 40–41 Growth angle, fungi, 69–70 Growth monitoring, of filamentous fungi, 168–169 H Haploid mating, 72 HAQN pigments. See Hydroxyanthraquinoid (HAQN) pigments Hazard analysis and critical control point (HACCP), 131, 138 Heat flow, 174 Heat transfer, in solid-state fermentation, 262–265 Heavy metals, removal of, 188–189 Hemicellulose debranching enzymes, 210–211t Heterologous expression, 197 Hibbett classification, of fungi, 9–10, 10f High-throughput screening (HTS), 480 Homologous expression, 197 Horizontal gene transfer (HGT), 78 Index 585 Hortaea werneckii, 78 HPLC-fluorescence, 136–137 Hybrid genome, 77–78 Hydrodynamics, 203–204 in fluidized bed bioreactors, 233 Hydrogenosomes, 76 Hydrothermal method, 135 Hydroxyanthraquinoid (HAQN) pigments, 326, 331–332 2-Hydroxy-propane-1,2,3-tricarboxylic acid. See Citric acid (CA) Hyperparasitic fungi, 4–5, 7 Hyperparasitism, 7 Hyperspora aquatica, 81 Hyphae, 1–2, 2f, 72, 201f I Imleria badia, 68t Immobilized fermentation, 307–308, 308f Immunoaffinity column (IAC) cleans-up spectrometric method, 136 Impedance measurement, 171–172 Industrial bioprocess, with filamentous fungi, 198–200 factors affecting, 200 morphology, 201–203, 201f, 202t rheology, 203–204 strain screening and inoculum, 200 Industrial enzymes, 272 advantages, 294 applications, 294 production, by filamentous fungi, 297–298t advantages, 294–295 agitation, 307 Aspergillus, 295 batch, fed-batch, and continuous fermentation systems, 308–309, 309f cultivation media, 310 economic aspect, 313 engineering, 310–311, 311t food industry, application in, 370–372, 371–372t Fusarium, 296 future research, 313–314 immobilization strategies, 307–308, 308f morphologies, 306–307, 306f Penicillium, 296 pH control strategy, 309–310 process engineering challenges, 303–305 protein expression systems, 311–313, 312t Rhizopus, 296 solid-state fermentation (SSF) platforms, 299, 300t, 301f submerged fermentation (SmF) platforms, 299, 301–303, 302f Trichoderma, 297–298 Industrial wastes, as fungal growth media, 181–182, 183f Infections, fungal, 6–7 Inhibition zone diameter (IZD), 480 Inoculum preparation, 200 Inoculum transfer, 36 Inonotus obliquus, 45 In-situ microscopy (ISM), 170–171 International Code of Botanical Nomenclature (ICBN), 67 International Nucleotide Sequence Database, 9 Iron oxide NPs, 47–48 Irradiation, 134 Isoflavone, 364 Isolation and purification, of filamentous fungi, 151–158 single spore isolation, 154 spore isolation, 153–154 Itaconic acid (IA) from ascomycetes, 51 biochemical pathway, 469f biosynthesis, microorganisms, metabolism, and physiology of, 456–458 chemical process, 456 chemical structure of, 468f definition, 456 global production, 456 production and process conditions, 458–459 strain engineering and process modifications, 459–460 J Joint FAO/WHO Expert Committee on Food Additives (JECFA), 333 586 Index K Kappa number, 208 KEGG, 76–77 Kickxellomycotina, 92 Koji fermentation, 364, 380 L Lactic acid, 275 from zygomycetes, 52–53, 52t Laricifomes officinalis, 45 LC-MS-MS method, 137 Leather like materials, 519–521 Lentinula edodes, 68t Lentinus edodes, 423 Lichen compounds, 8 Life cycle of Aphelids, 79 of Batrachochytrium dendrobatidis, 84–85 of Blastocladiomycota, 88 of filamentous fungi, 237 Lignocellulose, 18, 438 Lignocellulose hydrolysis, 181–182 Lignocellulosic materials (LCMs), 221 Lignocellulosic substrates, 258 Linderina pennispora, 92 Linoleic acid (LA), 410, 412 Lipases, 198–199 for biodiesel production, 279 Lipids, 375, 408–409 Long-chain polyunsaturated fatty acids (LC-PUFAs), 410 Long-term preservation, of filamentous fungi, 162–167 Lovastatin, 45 Lycopene, 277 Lyophilization, 160–161, 166–167 Lyoprotectants, 167 Lytic polysaccharide monooxygenase (LPMO), 210–211t M Macromorphology, fungal, 237–238 Macronutrients, 33–34 physiological functions of, 34t Macroscale phenomena, during SSF, 263–264, 263f Magneto-acoustic and photoacoustic spectroscopy (MA/PAS), 174 Malt extract agar, 159t Mangrove fungi, 16 Mannan, 504–505 Mannose, 415–416 β-Mannosidase, 210–211t Mann-Whitney test, 70 Marine fungi, 16 isolation of, 155 pigment production, 326, 334–338 Marteilia cochillia, 81 Masked mycotoxins, 105 Mass transfer, 223–224, 227–228, 234–235 in solid-state fermentation, 262–265 Melanin, 334–336 Membrane filtration, 155 Metabolic engineering, 491–492 metabolomics approaches, 492 targeted approaches, 491 untargeted approaches, 491 Metabolites, fungal, 45, 50, 366–372 of ascomycetes, 50–51 of basidiomycetes, 53–54 pigments and enzymes, 54 of zygomycetes, 51–53 MetaCyc, 76–77 Metarrhizium, 78 2-Methylidenebutanedioic acid. See Itaconic acid (IA) Microbiology, of filamentous fungi, 183–184 Microdilution method, 482 Micronutrients, 33–34 Microplastics, 189–190 removal using filamentous fungi, 189–190 Micropollutants, 190 Microscale phenomena, during SSF, 263–265, 265f Microsporidia, 13–14, 81 spores, 81, 82f Minerals, 416–417 Miso, 351–352, 376 Mitosomes, 81 Mixed agar plate culture (MAPC), 480 Mixing impellers, 227 Index 587 Modified mycotoxins, 105 Moisture content, 259–260 Molasses, 437–438 Molds, 105 Mollicutes-related endobacteria (MRE), 93 Monascorubrin, 329, 330f Monascus-like polyketide azaphilone (MPA) pigments, 327–328 Monascus pigments, 54, 277, 326–328 mycotoxin-free Monascus red, 328–329 from non-toxigenic fungal strains, 329–331 Moniliformin, 109–123t Monoblepharidomycetes, 85 Monomorphic fungi, 72 Morphology, fungal, 1–4 factors affecting, 201–203, 201f, 202t hyphae, 2f reproduction, 3 Mortierella sp. fatty acids production, 282 PUFA production, 282 Mortierellomycotina, 94 Mucorales, ethanol-producing, 440–441, 441t Mucor indicus, 254 chitin production, 506–508 chitosan in, 56–57 in ethanol production, 52 Mucoromycota, 93–95 Mucoromycotina, 94–95 Mushrooms, 347–348, 350–351 Myceliophthora thermophila, 444 Mycelium, 1–2 of Basidiomycota, 97 of Glomeromycotina, 93 of Kickxellomycotina, 92 Mycobiont, 8 Mycoloop mechanism, 83 Mycoprotein, 349–350, 405 on dry/wet basis, 373 essential PUFA content of, 373 health benefits, 373 Quorn, 373, 375 Mycoremediation, 187–188 Mycorrhizae, 7 Mycorrhiza helper bacteria, 190 Mycorrhizal symbiosis, 7–8 Mycotoxins, 376–378 chemistry and processing stability, 108–124, 109–123t classification, 105 definition of, 105 detection and determination by advanced technologies, 137 by chromatographic method, 136–137 by spectrometric method, 136 economic impact, 107, 108t exposure of, 107 factors affecting production, 130–131 extrinsic factors, 131 intrinsic factors, 130–131 health risk/clinical manifestation of, 106, 106t industrial applications, 205–206t lethal effect of, 106 naphtoquinone-type, 332 occurrence in food, 124, 125–127t, 128f cereals, 124–128 international regulation, 128–130, 129t spices, 128 prevention of good agricultural practices, 132–133 networking, 133–134 by storage condition, 133 production, 303–305, 304t reduction, 134–136 by biological methods, 135 by chemical methods, 135–136 by physical methods, 134–135 test procedures, 129–130 N Nanoparticles (NPs), 47–48 Naphtoquinones, 332 Natural culture media, 33 Natural Red™, 332–334 Near-infrared spectroscopy (NIR), 172–173 Neocalimastigomycota, 85–88 AGF protein domains and homologous genes, 87–88 characteristic features of, 86 Neocallimastigomycota, 76, 78, 86 588 Index Neocallimastix frontalis, 86 Net protein utilization (NPU), 345 Networking, in mycotoxin prevention and control, 133–134 Neurospora spp. for ethanol production, 50 N. crassa, 97–98 N. intermedia, 55, 442–443 Neurospora tetrasperma, genome, 77–78 Next-generation sequencing (NGS), 175 Nicotinamide adenine dinucleotide (phosphate) (NADPH), 172 Nitrogen-fixing bacteria, 190 Nitrogen, role of, 257 Nivalenol, 108–124, 109–123t Nixtamalization, 135 Non-coagulative pellets, 237–238 Non-essential amino acid (NEAAs), 407, 408t, 409f Nonwoven textiles, 519–521 Novozymes, 197, 210 Nucleophaga, 79 Nucleotides, 419–420 Nutrients, for fungal growth, 187f, 189t wastes, residuals, and wastewaters as, 185–187 Nutrition, fungal, 4, 70–71 O oCelloScope, 171 Ochratoxins, 376–377 economic impact, 108t health effect, 106t occurrence of, 124, 125–127t structure, producing fungi and stability of, 109–123t worldwide regulation limit of, 129t Oil cakes, as substrates, 258 Oil processing industry waste, 550–553 olive oil, 550–552 palm oil, 553 Oleaginous fungi, lipids from, 47 Oleic acid, 413 Olive oil mill water (OOMW), 552 Olive oil processing industry, 550–552 Oncom, 353–362t, 364–368 Operational taxonomic units (OTUs), 13 Opisthosporidia, 78–79 Optical density (OD), 170 Organic acid, 47, 366 production by SSF, 274, 274t citric acid, 275 lactic acid, 275 succinic acid, 275 Organic acid production, filamentous fungi, 455–456, 457t citric acid applications, 462 biochemical pathway, 470f biosynthesis, microorganisms, metabolism, and physiology of, 462–463 chemical structure of, 468f definition, 462 genetic and process engineering strategies, 465 global market, 462 production and processing conditions, 463–465 gluconic acid applications, 460 biochemical pathway, 469f biosynthesis, microorganisms, metabolism, and physiology of, 460 chemical structure of, 468f definition, 460 production and process conditions, 460–461 strain engineering and process modifications, 461–462 itaconic acid biochemical pathway, 469f biosynthesis, microorganisms, metabolism, and physiology of, 456–458 chemical process, 456 chemical structure of, 468f definition, 456 global production, 456 production and process conditions, 458–459 Index 589 strain engineering and process modifications, 459–460 oxalic acid applications, 466 biochemical pathway, 470f biosynthesis, microorganisms, metabolism, and physiology of, 466–467 chemical structure of, 468f definition, 466 production and process conditions, 467 strain engineering and process modifications, 468 Oxalic acid applications, 466 biochemical pathway, 470f biosynthesis, microorganisms, metabolism, and physiology of, 466–467 chemical structure of, 468f definition, 466 production and process conditions, 467 strain engineering and process modifications, 468 Oxidative stress, 418 Oxidoreductases, 5 Oxygen availability, 222 Oxygen transfer, fermentation, 42 Oxygen uptake rate (OUR), 174–175 Oxypilins, 76 Oyster mushroom, optimal temperature for, 41 Ozonation, 135 P Packed-bed bioreactors, 37, 38f, 40f, 267–268, 268f, 299, 301f characteristics and limitations, 271t for filamentous fungi cultivation, 224–225t, 231–232, 232f Palm oil mill effluent (POME), 553 Palm oil processing industry, 553 Paper like materials, from fungi, 519–521 Paramicrosporidium, 79 genes of, 81 Parasexual cycle, 72 Parasitic fungi, 4–6 Particle size, 262 Paspalitrems, 109–123t Pathogenic bacteria, in fermented food, 378–379 Pathogenic fungi, 6 Patulin economic impact, 108t health effect, 106t occurrence of, 124, 125–127t structure, producing fungi and stability of, 109–123t worldwide regulation limit of, 129t Pectinases, 198–199 Pellets, 199, 201f in submerged fermentation, 237–238 Penicillin, 45, 277, 486–487 mode of action, 487, 488f Penicillium sp., 68t in enzymes production, 296, 297–298t hydroxyanthraquinoid (HAQN) pigments, 331–332 in marine habitats, 336–337 mycotoxins, 108–124 P. chrysogenum, 491 P. marneffei, 329, 330f P. oxalicum var, 332–333 P. simplicissimum, 188–189 Penitrems, 109–123t Pentahydroxy hexanoic acid. See Gluconic acid (GA) Peptidases, 305 Pervaporation (PV), 445, 446f pH fermentation, 41–42 solid-state fermentation, 261 Pharma industry, fungi in, 43f, 45 Phenolic compounds, solid-state fermentation, 276t, 277 Phlebia sp., 53 Photobionts, 8 Phytase, 198–199 Phytohormones, 281 Pichia pastoris, 72 Pigment production, by filamentous fungi, 366–368, 367t animal feed, 417–418 590 Index Pigment production, by filamentous fungi (Continued) bioactive pigment production (see Bioactive pigment production, by filamentous fungi) carbon and nitrogen source, 369 challenges and limitations, 369 downstream process, 368 extracellular pigment production, 368 factors affecting, 369 intracellular pigment, 368 solid-state fermentation, 368 submerged fermentation, 368 Pigments fungal, 45, 54 solid-state fermentation, 276t, 277 PlaFractor process, 272 Plant proteins in aquaculture diets, 402–404 consumption of, 348, 348f greenhouse gas (GHG) emissions of, 349, 349t protein content, quality, fat, and antinutrient compounds, 345–347, 346t sources, 344–345 Plants, fungal infections in, 6–7 Pleurotus eryngii, 40–41 Pleurotus ostreatus, 40–41, 68t, 423 Poisoned food method, 482 Pollution reduction, filamentous fungi in, 187–189, 187f, 189t Polyhydroxyalkanoates (PHA), 280 Poly-3-hydroxybutyrate (PHB), 280 Polyketide-Monascus-like pigments, 326–328 Polyketide synthases (PKSs), 328–329 Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), 159–160 Polyphasic taxonomy, 159 Polyploidy, 78 Polyunsaturated fatty acids (PUFAs), 366, 373, 375, 400–401, 410, 413 solid-state fermentation produced, 281–282 Potato dextrose agar, 159t Potato processing industry waste, 553–556 Potato protein liquor (PPL), 56 Power draw, 221 Prebiotics, 420–421 Predictive modeling, of mycotoxins, 137 Preservation, of filamentous fungi, 160–168 long-term, 162–163 cryopreservation, 163–166, 164t, 165f, 166t freeze-drying, 166–167 quality control, 167–168 short-term, 161–162 Pretreatment methods, 438 Primary microplastics, 189–190 PR-imine, 109–123t Productivity, fungal morphology and, 238–239 Prokaryotic protein expression systems, 311–313, 312t Protease, 273–274t, 305 Protein(s) animal and fish feed ingredients, 399–400 fishmeal, 400 fungal biomass, 406–408, 407t plant protein meals, 402–404 single-cell proteins (SCPs), 404–405 terrestrial animal by-product meals, 404 animal protein sources (see Animal protein) filamentous fungi environmental concerns, 349, 349t essential amino acid (EAA) contents, 374–375, 374t protein content, quality, fat, and antinutrient compounds, 343–344, 346t, 347–348 smart proteins, 349–350 tempe, 343–344 insects greenhouse gas (GHG) emissions, 348–349, 349t protein content, quality, fat, and antinutrient compounds, 346t, 347 plant-based sources (see Plant proteins) recombinant heterologous, 205–206t Protein-degrading enzymes, 305 Protein digestibility-corrected amino acid score (PDCAAS), 345–347, 373 Protein-encoding genes, 159–160 Index 591 Protein expression systems, 311–313, 312t Proteobacteria, 190 Pseudomycelia, 3 Psychrophilic fungi, 155 Puccinia graminis, 97 Pulp and paper industry, fungi in, 43f, 46 Pulp industry biobleaching, 208–209 bio-debarking, 204–206 bio-pitching, 206–207 biopulping, 207–208 bio-retting, 207 PV. See Pervaporation (PV) Q Quorn, 44, 55, 327, 372–373, 405 fatty acid profile, 375 production of, 384, 384f protein content of, 346t, 347–348 R Radial pumping impellers, 227 Raimbault columns, 267–268 Reactive oxygen species (ROS), 76, 418 Recombinant deoxyribonucleic acid (rDNA), 197 Recombinant heterologous proteins, 205–206t Red pigment mycotoxin-free Monascus red, 328–329 Natural Red™, 332–334 Reproduction, fungal, 3, 71 Ascomycota, 74–75 Basidiomycota, 75 Chytridomycota, 73 Glomeromycota, 74 unicellular yeast form, 72 Zygomycota, 73–74 Respiratory quotient, 174 Retting, 207 Reverse TCA (rTCA) pathway, 462–463 Reynolds number, 222 Rhamnolipids, 280 Rheology, 203–204, 220–221 Rhizomorphs, 71 Rhizopus sp., 51 BSG fermentation with, 265, 266f chitosan in, 56–57 enzymes production, 296 fruit and vegetable discards fermented with, 264, 264f fumaric acid production from, 53 lactic acid production, 52–53, 52t R. microsporus, 77, 379 R. oligosporus, 68t, 254 R. oryzae, 405, 440, 442f, 444 Riboflavin, 419 Riser, 229 Roquefortines, 109–123t Rose Bengal agar, 159t Rotating-bed bioreactors, 234–235, 234f Rotating drum bioreactor (RDB), 37, 38f, 267f, 268–269 characteristics and limitations, 271t Rotating fibrous bed reactor (RFBR), 240–241 Rotating/stirred drum reactors, 236 Rozella sp., 79–81 Rozellidea, 79–81 Rubia tinctoria, 332 Rubratoxin, 109–123t S Sacchachitin, 515–516 Saccharomyces cerevisiae, 68t, 445–447 genome of, 77–78, 242 growth of, 69 Salmonella sp. S. paratyphi, 378 S. typhimurium, 378 Sambacide, 277–278 Sampling and isolation, of filamentous fungi from environments, 154–156 from fermented products, 156–158, 157–158t Sampling procedures, filamentous fungi, 158 Saprophytic fungi, 4–5 Saprotrophic fungi, 5 Satratoxins, 108–124, 109–123t Saturated fatty acids (SFAs), 410 Schizophyllum commune, 68t Schizosaccharomyces pombe, 3 Scleroglucan, 497–498 592 Index Secondary metabolites, 76, 275 Secondary microplastics, 189–190 Sekelan, 107 Semi-synthetic medium, 33 Sepiolite, 134 Sexual reproduction, 3, 71 in Ascomycota, 74–75 in chytrids, 73 in Entomophthoromycotina, 91 in Kickxellomycotina, 92 in Mucoromycota, 93–94 in Mucoromycotina, 94 in Zoopagomycotina, 91 in zygomycetes, 73–74 Shear forces, 223–224 Shear stress, 223–224 Short-term preservation, of filamentous fungi, 161–162 Sigmoidal growth curve, 70 Simultaneous saccharification and fermentation (SSF), 439 Single-cell genomics method, 83 Single-cell protein (SCP), 44, 404–405 Single spore isolation, 154 SmF. See Submerged fermentation (SmF) Solid-phase extraction (SPE) cleanup-fluorometric method, 136 Solid-state fermentation (SSF), 36–37, 198–199, 219–220, 251, 505–506 advantages, 37, 252 aroma compounds, 281 bioactive compounds/secondary metabolites, 275–278, 276t antibiotics, 277–278 phenolic compounds, 277 pigments, 277 biofuels production by, 278–279, 279t biopesticide production by, 278, 278t biopolymers, 280–281 bioreactors, 37, 38f, 265–270 characteristics and limitations, 271t fluidized-bed, 269, 269f packed-bed, 267–268, 268f rotating drum, 267f, 268–269 scaling-up, 265–266 spouted-bed, 269–270 tray, 266–267, 267f biosurfactants, 279–280 enzyme production by, 272–274, 273–274t advantages, 300 agro-industrial residues, 299, 300t limitation, 300 tray and packed-bed bioreactor, 299, 301f Zymotis design, 301 filamentous fungi for, 253–258, 256f fungal pigment production, 368 heat and mass transfer in, 262–265, 263f organic acids production by, 274, 274t citric acid, 275 lactic acid, 275 succinic acid, 275 phytohormones, 281 polyunsaturated fatty acids, 281–282 process control parameters, 258–259 aeration, 261–262 agitation, 262 biological factors, 259 moisture content and water activity, 259–260 particle size, 262 pH, 261 temperature, 260–261 products and current industrial applications, 270–282 reactor designs in, 235 rotating/stirred drum, 236 tray-like bioreactors, 235–236 substrates for, 252–258, 256f value-added products of, 255f Sophorolipids, 280 Soybean meal (SBM), 402–404, 408, 409f, 421–422, 424 Soy sauce, 353–362t, 370 definition, 364 fermentation, 364 health aspects, 376 names, 364 safety issues, 378 taste of, 364 traditional production methods, 380 Index 593 filtration and cooking, 382 koji fermentation, 380 moromi fermentation, 382 sortation and boiling, 380 types of, 378 Spices, mycotoxins in, 128 € rper, 69 Spitzenko Spo11, 72 Sporangia, 73 Sporangiophore, 73 Spore isolation, 153–154 single spore technique, 154 stages in, 153–154 Spores, 200, 201f Spouted-bed bioreactors, 269–270 characteristics and limitations, 271t SSF. See Solid-state fermentation (SSF) Staphylococcus aureus, 378 Starch, 437–438 Starch-based ethanol facility, 49 Statoliths, 71 Sterigmatocystins, 108–124 occurrence of, 125–127t structure, producing fungi and stability of, 109–123t Stirred packed bed bioreactor, 37, 38f Stirred-tank reactor (STR), 39, 40f, 224–225t, 226–227, 299, 302f configuration, 226f effect on fungal morphology, 240–241 Streptomyces sp., 484 Subculturing (serial transfer), 161–162 Submerged fermentation (SmF), 36–37, 198–199, 219–220, 480 advantages and disadvantages, 37–38 aeration and agitation in, 42 antibiotic production, 483 bioreactors for, 40f for enzymes production, 368 bubble column bioreactor, 299, 302f disadvantages, 301–303 fungal species, 299 stirred-tank bioreactor (STR), 299, 302f fungal pigment production, 368 modes in, 38 pellets formation in, 237–238 vs. solid-state fermentation, 252 Succinic acid, 275 Sugar, 437–438 Sugar processing industry waste, 556–558 Suspension, 199 Swiss-Prot, 76–77 Symbiotic Basidiomycota, 97 Symbiotic fungi, 4–5, 7 Synthetic culture media, 33 Synthetic dyes, bioremediation of, 46 T Talaromyces sp., 327–328 T. aculeatus, 327–328 T. albobiverticillius, 329–331 T. atroroseus, 329–331, 331f T. purpurogenus, 327–331 Taxonomy, of fungi, 8–13 Hibbett classification, 9–10, 10f Tedersoo classification, 10–11, 11f Wijayawardene classification, 11–12, 12f Tedersoo classification, of fungi, 10–11, 11f Tempe, 351–352, 353–362t, 375 definition, 351–352 essential amino acid (EAA) contents, 374–375, 374t fermentation process, 352–364 food by-products, 352 food safety concerns, 378–379 functional properties of, 376 industrial production modern process, 382–384, 383f traditional methods, 379–380, 381f protein source, 343–344 consumption rate, 351 greenhouse gas (GHG) emissions of, 343 protein content, quality, fat, and antinutrient compounds, 346t raw materials, 352 vitamin, 375 Temperature fermentation, factors influencing, 41 in solid-state fermentation, 260–261 Termitomyces clypeatus, 68t 594 Index Terrestrial animal by-product meals, 404 Terrestrial fungi, 15 Tetracycline antibiotics, 478 Tetrapolar breeding, 75 Textile fibers, 519–521 Textile industry, fungi in, 43f, 46–47 Thaumatin, 372 Thermal processing, 135 Thermomyces lanuginosus, cultivation pH, 41–42 Thermotelomyces thermophila, 68t Thin stillage, 544–545 1000 Fungal Genomes project, 9 Time-kill test, 482 Tissue engineering, 516 Toxigenic fungi, 130–131 Trametes versicolor, 53 Transport, fungal, 70–71 Tray bioreactors, 37, 38f, 235–236, 266–267, 267f, 299, 301f characteristics and limitations, 271t Tremorgen, 109–123t Tricarboxylic acid cycle (TCA) cycle, 456–457, 462–463, 466–467 Trichoderma sp. in agriculture, 44 in enzyme production, 54, 297–298, 297–298t Trichoderma asperellum, 188–189 Trichoderma harzianum, 7 as biopesticide, 278 lipase production, 272 Trichoderma reesei, agitation in, 152–153 Trichothecenes, 108–124, 109–123t, 377 Trickle-bed bioreactors, 224–225t, 229–231, 230f Tricoderma reesei, 68t Tropolones, 490 True fungi (Eumycota), 9 Tryptoquivalines, 109–123t T-2 tetraol, 109–123t 2G bioethanol, 209 U Ultraviolet (UV) mutagens, 465 Unfolded protein response (UPR), 197 UNITE, 9 Usar, 380, 381f Ustilago maydis, 2, 68t, 457–459, 457t V Valorization, 255–257 Value-added products, 48–49 by solid-state fermentation, 255f Vanillic acid, 281 Vegan-mycoprotein, 48 Vegetative reproduction, 3, 72 Versatility, filamentous fungi bioeconomy, 533 bioethanol industry wastes, 544–548 brewery waste, 534–539 dairy processing industry, 558–560 fish processing industry waste, 548–550 fruit industry waste, 540–544 oil processing industry waste, 550–553 olive oil, 550–552 palm oil, 553 perspectives, 560–561 potato processing industry waste, 553–556 sugar processing industry waste, 556–558 sustainable development, 533 waste biorefinery, 533–534 Versicolorins, 109–123t Versiconol acetate, 109–123t Viomellein, 109–123t Viscosity, 220–221 Vitamins, 375, 419 Viticolins, 490 Void space, 263 Volvariella volvacea, 68t W Waste Directive 2018/851, 255–257 Wastewater treatment, 187–189 Water activity, 259–260 Water distribution systems, filamentous fungi isolation, 155 Water retting, 207 Wet distillers grain (WDG), 544–545 White-rot fungi, 5 in pulp and paper industry, 46 in synthetic dye degradation, 46 Index 595 Whittaker classification, of fungi, 9 Whole stillage, 544–545 Wijayawardene classification, of fungi, 11–12, 12f Wine, 444–445 Wound healing materials, 515–516 X Xanthomegnin, 109–123t Xeromyces bisporus, 152 Xylanase, 198–199, 209 Xylitol, 45 Xyloglucanase, 210–211t β-Xylosidase, 210–211t Y Yarrowia, 2 Yeasts, 2 budding in, 3 genome, 77–78 reproduction, 72 for solid-state fermentation, 253–258 Z Zearalenone, 377 economic impact, 108t health effect, 106t occurrence of, 125–127t structure, producing fungi and stability of, 109–123t worldwide regulation limit of, 129t Zinc-finger transcription factors, 70–71 Zoopagomycota, 89–92 Zoopagomycotina, 91 Zoosporangia, 73 Zoosporic fungi, 70–71 genomes, 77 Zygomycetes biomass, 55–56 chitosan in, 56–57 food and feed applications, 56 cell wall, 186–187 ethanol production by, 440–441, 441t metabolites production, 51 ethanol, 52 fumaric acid, 53 lactic acid, 52–53, 52t phylogenic classification of, 89, 90t sexual reproduction in, 73–74 Zygomycota, 67–69 reproduction, 73–74 for solid-state fermentation, 254 Zygosporangium, 73–74 Zymomonas mobilis, 444 This page intentionally left blank