4
Yeasts Biodiversity and Its Significance: Case
Studies in Natural and Human-Related
Environments, Ex Situ Preservation,
Applications and Challenges
Enrique Javier Carvajal Barriga et al.*
Centro Neotropical para la Investigación de la Biomasa, Colección de Levaduras Quito
Católica, Pontificia Universidad Católica del Ecuador, Quito
Ecuador
1. Introduction
Yeasts are a group of microorganisms that belongs to the Fungal Kingdom. These unicellular
fungi are distributed between the Basidiomycota and Ascomycota Phyla, being a paraphyletic
group. Since 1865, its study has experienced a very important advance in terms of its
understanding, characterization and taxonomic accommodation. Nevertheless, it is estimated
that about 99% of the potential biodiversity of this group of eukaryotic microorganisms is still
unknown. That is why there is a need for increasing efforts to study yeast biodiversity,
especially in mega diverse countries from the tropical regions of the planet.
To date, the majority of yeast species catalogued have been discovered in countries from the
Northern hemisphere. Relatively few studies dedicated to yeast biodiversity have been done
in tropical zones of the planet and in Southern hemisphere countries that embrace abundant
and diverse ecosystems. A number of case studies of these approaches to yeast biodiversity
are presented in this chapter, including the discovery and subsequent description of novel
yeast species recently isolated in Ecuador, Brazil and Argentina. The chapter will also deal
with the biodiversity of yeasts found in industry-influenced environments in Spain.
Moreover, ex situ preservation of yeast isolates for further characterization by physiological,
morphological and molecular techniques is a fundamental issue in terms of the
* Diego Libkind2, Ana Isabel Briones3, Juan Úbeda Iranzo3, Patricia Portero1, Ian Roberts4, Steve James4,
Paula B. Morais5 and Carlos A. Rosa6
1Centro Neotropical para la Investigación de la Biomasa, Colección de Levaduras Quito Católica, Pontificia
Universidad Católica del Ecuador, Quito, Ecuador
2Laboratorio de Microbiología Aplicada y Biotecnología, Instituto de Investigaciones en Biodiversidad y
Medioambiente (INIBIOMA), CONICET-UNComahue, Bariloche, Argentina
3Laboratorio de Biotecnología en Levaduras, Universidad de Castilla La Mancha, Ciudad Real, España
4Institute of Food Research, National Collection of Yeast Cultures, Norwich, United Kingdom
5Laboratório de Microbiologia Ambiental e Biotecnologia, Campus Universitário de Palmas, Universidade Federal
do Tocantins, Palmas, TO, Brazil
6Departamento de Microbiologia, ICB, Universidade Federal de Minas Gerais,
Belo Horizonte, MG, Brazil
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Changing Diversity in Changing Environment
understanding and preservation of the biodiversity. Yeast culture collections play a
fundamental role not only as the repositories for invaluable yeasts strains (germplasm), but
also as platforms for biotechnology exploitation. Experiences and challenges that several
yeasts collections are facing will also be discussed in this chapter.
2. Biodiversity of Yeast, ¿What does it mean?
The term biodiversity is an abstract expression of all aspects of the variety of life (Gaston,
1996); from bio-molecules to the variety of different species populations and communities of
species. Variation is the essence of biology. Thus, biodiversity is an intrinsic feature of life.
Under this focus, biodiversity loss is one of the main global concerns. This, loss can be
produced by a number of different factors related to human activities and to natural events;
where competition at the intra- or inter-specific levels and even at the molecular scale,
reminds us the real drama of life, where Darwin’s “The Origin of Species” reaches the nerve
of this fundamental issue of living organisms.
Nonetheless, an undefined number of yeast species losses can be caused due to the
perturbation of habitats by humankind. As Dr. Steve James from the National Collection of
Yeast Cultures in the United Kingdom points out, talking about the importance of yeast
biodiversity surveys: “It’s a race against time. We know that massive loss of species
diversity is occurring worldwide. Our efforts are thus focused on characterizing and
subsequently preserving what remains.”
Global-scale conversion of tropical rainforests and agricultural intensification are major
causes of biodiversity loss (Chapin et al, 2000; Hoekstra et al, 2005). Extinction is the final
result of a process that starts with the vigor’s weakening of certain populations. The most
undesirable and irreparable effect is the complete loss of all (component) populations of a
single species. This effect is uniquely evident when the fragmentation and perturbation
degree of natural micro- and macro-landscapes overwhelms the “decisive threshold” (Pimm
and Raven, 2000).
Some microbes do seem to be restricted to very particular environments and are endangered
in as much as these environments are threatened; microbes intimately associated with other
organisms share (partially) the biogeographies of their hosts. As far as they are speciesspecific, they could potentially become extinct along with their hosts (Weinbauer and
Rassoulzadegan, 2007).
We still do not have any definitive evidence of the extinction of any yeast species: it is very
hard to determine that. Nonetheless, we can presume that a number of co-evolving yeasts
species have probably become extinct along with their plant or animal hosts. Studies made
on bumble bees demonstrate that insects play a crucial role, not only in yeast dispersion, but
also acting as a type of “wet nurse” during winter, when environmental conditions are very
harsh and no flowers are present in the fields (Byrsch, 2004). In this way, extinction or
weakening of insects populations can ultimately lead to the extinction of certain insectdependent yeasts species, at least locally.
As biodiversity is not only referent to the living organism by itself, but also to the diversity
of its strains and varieties. Likewise, the molecular variety of yeasts is both huge and
extremely dynamic. The occurrence of a great variety of yeast strains is the result of the high
mutation rate that provides these microorganisms with the ability to adapt to different
environments. Mutation rate is an important parameter in evolution. It dictates the speed of
adaptation in populations with beneficial mutations; in the absence of such mutations it sets
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57
the equilibrium fitness of the population (Gregory et al, 2007). The loss of varieties in yeast
strains is also a concern, but, at the same time is an issue that we cannot solve and probably
we don’t need to try solving.
The search for yeasts species/strains with economic potential is a way to preserve those
genetic varieties that are worth being kept and used in a wide range of applications.
Gregory et al in 2007 found that in S. cerevisiae the mutation rate per gene is in the order of
10-10/base pair/generation. It is important to note that different DNA regions in living
organisms have different variability. These mathematical approaches can be used to
estimate the evolutionary distance in terms of time between different strains. As for S.
cerevisiae, it has been possible to determine, based upon the nucleotide variations of several
genes belonging to different strains, that this yeast species was most likely first
domesticated about 11,900 years ago (Fay and Benavides, 2005). The study of ancient
dormant yeast strains/species, although still in its infancy, is nevertheless a field that offers
the opportunity to help better understanding of microbial biodiversity over time (Gomes et
al., 2010).
Dormancy in yeasts and other microorganisms plays a key role to help keep a seed bank for
the future (Jones and Lennon, 2009). The biodiversity of microbial communities, of which
yeasts are an integral part of has important implications for the stability and functioning of
managed and natural ecosystems. Dormancy is one trait that allows species to contend with
temporal variability of environmental conditions. This “bet-hedging” strategy allows
dormant individuals to become members of a “seed bank”, which can contribute to the
diversity and dynamics of communities in future generations (Turner et al. 1998; Caceres
and Tessier, 2003). The recovery of dormant yeast species from archaeological pieces as well
as paleontological rests provides a means of reviving species or strains that were probably
destined to become extinct (Gomes et al., 2009). The techniques and the approaches already
done in this field will be outlined later in this chapter.
Yeasts are also adapted to dispersion and then survival. One example of this is the crossshaped yeast Metchnikowia gruessii that is dispersed by bees visiting flowers during its
feeding periods in the day. This cross-formed yeast species is adapted to the glossa or
tongue of the bees and so use the insect as a means of dispersal from one flower to the next.
As for the studies carried out by Byrsch in 2004, this species is highly successful, very
common and forms predominant populations in nectar of certain central European flowers.
The best way to get into the study of yeasts in natural environment is using ecological
criteria: yeasts occupy a diverse variety of micro-ecosystems and are well adapted to a wide
range of weathers, altitudes, substrates and geographical locations. It is possible to find
yeasts in glaciers, high salinity lakes, water, soil, air, intestines of a variety of vertebrates and
invertebrates, and even in acid waters (Russo et al, 2010) and marine deep-sea environments
(Nagahama Takahiko, Biodiversity and Ecophysiology of yeast). The proper way to study
yeast diversity and its function in communities is by gaining an understanding of their role
in communities, so we can predict the occurrence of certain species based on the features of
the micro- and macro-landscapes.
Yeast species such as S. cerevisiae have been used by humankind throughout history for the
production of fermented foods and beverages around the world. That is why yeasts are
intimately linked to our day-to-day activities related with culture, economy and nutrition.
Moreover, certain yeast species are linked to human diseases, while others form part of the
intestine’s micro-flora in both vertebrates and invertebrates.
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Changing Diversity in Changing Environment
Nevertheless, a relatively small number of yeast species are currently being used in
industry, while, a large number of species collected from natural environments and humanrelated micro-ecosystems are still being studied and classified. Yeast taxonomy deals with
the classification and accommodation of species that are being discovered in an ever
increasing number year after year.
2.1 Yeast diversity in numbers
It is believed that only 1% of all extant yeast species is currently known. From 1820 up to
2011 the number of described yeasts has increased dramatically. By 2005, more than 2500
yeast species were published. This number of species already named includes synonyms
which are being taxonomically re-accommodated. Currently there are approx. 1500
recognised yeast species, which means the expected number of yeast species on Earth
would be around 150,000. Large territories of Africa, Antarctica, Asia, Australia and Latin
America are mainly virgin (Hawksworth, D.L, 2004). These new and hardly explored
habitats represent rich sources of fungal biodiversity still awaiting discovery. To date,
relatively little work has been carried out in this field in South American countries like
Argentina, Brazil and Ecuador. The yeast diversity in such countries is potentially huge.
For example, over 200 new species of yeasts have been found amongst 650 isolates from the
guts of beetles (Suh et al. 2004, Suh & Blackwell 2005). Coleoptera species are floricolous
insects and tree flux communities whose species number about 350,000. Nevertheless, not
all beetle species harbour yeasts, and so its number must to be first established to predict a
possible overall estimate of yeast related with them (Lachance, 2005 Yeast biodiversity and
Ecophysiology).
Molecular techniques used since 2000 have greatly boosted the number of new species
identified. Molecular analyses of the variable D1/D2 regions of the 26S rDNA, 18S, 5.8S and
mitochondrial small subunit rDNAs gene, as well as ITS sequencing and RFLP-ITS are very
useful ways to identify yeast species and invaluable tools for phylogenetic studies
(Kurtzman and Fell, 2005 biodiversity and Ecophysiology). These molecular techniques,
combined with microbiological and physiological tests, are being used to characterize yeast
isolates and species. Most of the analyses have used rDNA sequences, however, we now
know that there are no universal criteria to distinguish between genera.
Communities of yeasts are affected by natural selection which eliminates deleterious
mutations and rapid fixation of adaptive alleles, just as the environment determines whether
or not a species can become established within a community (Lachance, 2006). In these
terms, we can understand that events of speciation and/or extinction are occurring in yeasts
around us all the time at a relatively high rate due to its remarkable rate of reproduction,
mutation and adaptation to changing situations.
Diversity between strains of the same species, such as Saccharomyces cerevisiae, has also been
studied by molecular methods. It is well known that there are variations in strains and some
metabolic abilities/disabilities are not necessarily linked to the species but rather to the
strains (i.e. strain variable). With few exceptions, only one strain or an individual of a
particular species is sequenced while hundreds of other variants, which may be important to
public health, scientific research, or commercial applications, remain un-deciphered
(Winzeler et al, 2002). The use of microarrays of whole genomes divided into 25mers helps
to find variations between different strains of a single species, in as much as a single base
substitution in these 25mers (especially those found in the center of the sequence) disrupts
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hybridization (Chee et al. 1996; Gingeras et al. 1998; Troesch et al. 1999; Lockhart and
Winzeler 2000). Single Feature Polymorphism SFP assessment carried out on 14 different
strains of S. cerevisiae yielded 11,115 variations, which demonstrates the huge genotypic
variation between strains of a single species and the opportunities these variations offer for
the research (Winzeler et al, 2002).
Furthermore, Single Nucleotide Polymorphism (SNP) analysis is revealing relationships
within strains of a single species. Moreover, the analysis of variation in gene content,
nucleotide insertions and deletions, copy numbers and transposable elements are all
contributing to reveal the intricate relationships between yeast species and strains (Liti, 2009).
In other words, biodiversity of yeasts at intra- and inter-specific levels is a big endeavor that is
still very much in its infancy taking into account the huge diversity of yeasts.
In order to ascertain and classify the yeast biodiversity in nature in an affordable way it is
necessary to investigate the multiple and varied micro-ecosystems represented by substrates
that may be used as a source of nutrients by yeast as well as platforms for their dispersion.
From beetle guts, to flower nectar or rotten woods, there still remains a huge field to be
examined in order to identify and characterise novel and known yeast species: their
distribution, ecologic relationships and the understanding of the aspects involving the
yeasts natural history, and feasible uses as biotechnological work horses. South America is a
region that offers great potential in terms of biodiversity (macro and micro), where yeasts
are being isolated from habitats that never were sampled before. Initial results from a survey
run by an international consortium from Brazil, Spain and Ecuador in the Galapagos Islands
at the end of 2009 (data not published) is beginning to reveal the diversity of yeasts present
in various substrates such as flowers, cacti, rotten wood, turtle’s faeces, marine iguana
faeces, and other substrates located in four different islands. This kind of expedition has also
been done in other South American countries such as Argentina and Brazil. Several novel
species have been identified in such surveys.
At this point, the identification and characterization of yeast isolates and its preservation is a
task that may be accomplished by researchers. Yeast culture collections play the leading role
in keeping the rich diversity of yeasts for current and future applications as well as genetic
reserve. Some aspects of the biodiversity studies, yeasts preservation, novel yeast species
description and its taxonomic accommodation and biotechnology applications will be
developed in this chapter.
3. Ecology and biodiversity of yeasts
3.1 Yeast-insect interactions as example of biodiversity studies
In general, yeasts are suspected to engage in intimate symbiotic relationships with insects,
although the nature of the interaction remains elusive in most cases (Lachance, 2006).
Several examples of yeasts associated with insects have been reported in recent years (Rosa
et al., 2003; Lachance et al. 2005; Starmer & Lachance, 2011). In most cases, the insects vector
the yeasts and use these microorganisms as a food source. The fruit flies of the genus
Drosophila eat yeast, digesting vegetative cells but passing spores through the gut intact and
viable (Colluccio et al., 2008). Yeasts have been also described as endosymbionts in
mosquito populations, lacewings, beetles and homoptera (Ganter 2006; Ricci et al. 2011). The
insects rely on yeasts for various metabolic functions, including synthesis of amino acids,
vitamins, lipids, sterols and pheromones, degradation of nutritional substrates, and
detoxification of compounds (Suh et al. 2003; Starmer & Lachance, 2011).
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Changing Diversity in Changing Environment
Geographic gradients were identified in Candida ipomoeae and Metschnikowia borealis that are
found in association with nitidulid beetles that visit short-lived flowers of morning glories
and a few other plant families, indicating that historical and climatic factors play a role in
shaping the populations (Lachance et al. 2001). Highly specific associations between
floricolous nitidulid beetles and various yeasts, including those in the Metschnikowia clade,
have been documented worldwide (Lachance et al. 2001b, 2005). Metschnikowia and related
species associated with nitidulid beetles are presumed to have co-speciated with the insects
(Lachance et al. 2005; Lachance, 2006). Lachance et al. (2001) suggest that Conotelus spp.
adults feed on the nectar of ephemeral flowers and in doing so, deposit yeasts together with
their fecal material in the corolla. The yeasts grow at the expense of nutrients present at the
surface of the corolla. The transmission of yeasts is probably horizontal, through crosscontamination at feeding sites or possibly during copulation. One possible role of the yeasts
is to assimilate low complexity carbon and nitrogen sources present in the flower and thus
provide the beetle larvae with a diet that contains essential nutrients such as lipids (Nasir
and Noda 2003).
More recently, a highly diverse yeast assemblage was found in the gut of various beetles
families (Suh & Blackwell, 2004;) especially phytophagous Coleoptera, Homoptera,
Hemiptera, Isoptera and Lepidoptera (Suh et al., 2005; Ganter, 2006; Lachance, 2006; Starmer
& Lachance, 2011). In particular, it is well known that bark beetles of the weevil subfamily
Scolytinae increase their host-colonizing potential by means of symbiotic relationships with
fungi, which are carried within specialized structures called the mycangia, or on the body
surface (Ganter, 2006). About 200 apparently undescribed species have been discovered so
far from the gut of basidioma-feeding beetles, and many of those yeasts form independent
clades in Saccharomycotina that have not been recognized previously (Suh et al. 2005). For
example, more than 40 new beetle-associated yeast species were reported recently to form
several major clades near C. tanzawaensis, Meyerozyma guilliermondii, C. mesenterica, and C.
membranifaciens, and each of these clades was composed almost exclusively of insect
associates (Suh & Blackwell 2004, 2005, Suh et al. 2004b, 2005).
The relationships of yeasts and insects are being discovered as studies expand: the brown
planthopper (BPH), Nilaparvata lugens, harbors yeast-like symbiotes (YLS), especially in
mycetocytes formed by fat body cells found in the abdomen. Pichia-like and Cryptococcuslike symbiotes may present a potential for biological control of this insect pest (Ganter,
2006). Also, the mutual relations of fungus-growing ants, their fungal cultivars, and
antibiotic-producing bacteria suffers the interference of a black yeast counterpart that
acquires nutrients from the ants’ bacterial mutualist, and suppresses bacterial growth.
Several yeast species were isolated from fungus garden and waste deposit of these ants, and
could play an important ecological role in these substrates (Pagnocca et al. 2010).
Yeasts have also been reported associated with several species of bees, including social and
solitary bees (Pimentel et al. 2005; Ganter 2006; Lachance et al. 2011). The majority of bee
species, of which there are approx. 20,000 species (Michener 2000), have never been
examined for the presence of yeasts (Rosa et al. 2003). The clade Starmerella, that includes
two teleomorphic species and several asexual Candida species, has been isolated from honey,
provisional pollen, nectar and waste deposits in hives and nests of several bee species (Rosa
et al. 2003). The nature of the possible symbiosis is not known with certainty, but a role in
pollen maturation is suspected (Starmer & Lachance, 2011). These yeasts were able to
produce several extracellular enzymes that could metabolize the sugars and pollen stored in
the nests, improving their nutritional quality (Rosa et al. 2003).
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3.2 The yeasts in plant substrates: Leaves, flowers and fruits
All aerial plant surfaces, known as the phylloplane or phyllosphere are inhabited by diverse
assemblages of microorganisms, and these have profound effects upon plant health and
impact on ecosystem functions. The associations established on plant surfaces range from
relatively inconsequential or transient to substantial or permanent (Fonseca & Inacio, 2006).
The leaf surface characteristics may affect, both qualitatively and quantitatively, the
immigration of yeasts to the phylloplane (Fonseca & Inacio, 2006). Leaf surfaces are
colonized by members of several genera of saprophitic yeasts that provide a natural barrier
against plant pathogens (Fokkema et al., 1979). Leaves are exposed to rapid fluctuations of
temperature and relative humidity values, which may have an impact on the yeast
population. Large fluxes of UV radiation are also one of the most prominent features of the
leaf surface environment to which microorganisms have presumably had to adapt (Lindow
& Brandl, 2003). Many plants contain a number of compounds whose adaptive significance
may be a defense against invertebrates and microorganisms (Robinson, 1974). These
compounds also act, in some cases, as selective agents which shape the yeast community
composition (Starmer & Lachance, 2011). Some yeast species isolated from fruits have a
potential use as antagonists and can serve as a biological control against post-harvest decay
fruit diseases (Ippolito and Nigro, 2000; Seibold et al., 2004).
Flowers and other parts of plant species belonging to the Convolvulaceae, Bromeliaceae and
Heliconiaceae families are rich sources of novel yeast species. Most of the novel yeast
species isolated from these plants belong to the Metschnikowia, Wickerhamiella and Starmerella
clades (Lachance et al. 2001; Ruivo et al. 2005; Rosa et al. 2007; Barbosa et al. 2011). In
ephemeral flowers of the Convolvulaceae, the yeasts are transported by pollinating and
non-pollinating flies, beetles and bees that deposit them in the corolla (Lachance et al. 2001).
In the longer-lasting flowers of the Heliconiaceae, yeasts are probably introduced by a
different and more diverse set of animal vectors and they may grow on the sugary
compounds present in nectar (Barbosa et al. 2011).
Most yeast species isolated from flowers are supposedly nectar-inhabiting yeasts. Dense
yeast communities often occur in the floral nectar of animal-pollinated plants, where they
can behave as parasites of plant-pollinator mutualisms (Brysch-Herzberg 2004; Canto et al.
2008; Herrera et al. 2008, 2009 de Vega et al. 2009). Nectar yeasts, particularly at high
densities, induce metabolic degradation of nectar, which can be detrimental to plant
reproduction through reduced pollinator service (Herrera et al. 2008). This might originate
selective pressures on plants to defend their nectars from exploiters through, e.g. the
production of antimicrobial secondary compounds (Irwin et al. 2004). Metschnikowia
reukaufii, M. gruessii, C. bombi, K. dobzhanskii, Hanseniaspora sp., H. osmophila, Saccharomyces
bayanus, Cryptococcus saitoi and Crypt. friedrichii were the most frequent yeasts isolated from
these substrates. The osmotic stress associated with the nectar high sugar concentrations is
probably a limiting environmental factor together with the presence of secondary
compounds (Nicolson et al. 2007; González- Teuber & Heil 2009). Since, the low species
diversity prevailing in nectar yeast communities so far studied could reflect a generalized
environmental filtering. Very low nitrogen content, another characteristic feature of floral
nectars (Nicolson et al. 2007), may be yet another factor limiting the suitability of floral
nectars as habitats for yeasts other than highly specialized nectarivores. A combination of
osmotolerance, tolerance or resistance to secondary compounds and efficient nitrogen use
possibly allows these specialists to exploit floral nectar.
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Changing Diversity in Changing Environment
Another aspect of the interaction of yeasts and nectar-producing plants is related to the
fermentation of nectar sugars by yeasts. In cool environments floral warming can benefit
both the plants (e.g. by faster growth of pollen tubes) and the pollinators (by providing a
heat reward), and yeasts can become important floral warming agents for plants living in
shady forest, which are unable to use direct sunshine to warm their flowers. Floral warming
by yeasts and the attractiveness provides an example whereby yeasts in nectar could under
some circumstances benefit plants, pollinators or both. Also, the abundant alcohol
accumulating in the nectar of a tropical palm as a consequence of yeast metabolism may
ultimately enhance the attractiveness of inflorescences to alcohol-seeking mammalian
pollinators (Wiens et al. 2008).
Decaying fruits are an important microhabitat for several yeast species (Morais et al. 2006;
Starmer & Lachance, 2011). These ephemeral substrates are among the most important sites
of oviposition and sources of nutrition for larval and adult stages of insects, which vector
the yeasts to new substrates (Ganter, 2006; Morais et al., 2006). Yeast communities on fruits
of one development stage turn out to be more similar when they are located closer to each
other. The similarity of neighboring groups of fruit and on neighboring trees depends cell
migration and cross-contamination of fruits with yeast cells. So, distinctions in the yeast
community structure in different geographical regions can be explained by differences in
the conditions of their formation (Slávikova et al., 2009). Evidently, propagation through cell
transfer should play an important role in formation of microbial groups on accessible
substrata during a limited period of time, such as juicy fruits, flower nectar, animal
excrement, etc. In works on yeast ecology, it was suggested that contamination plays the
initial role in formation of the specific structure of yeast in such communities, in particular,
directed phoretic transportation of yeast cells to invertebrates (Morais et al. 2006; Starmer &
Lachance 2011).
3.3 Soil yeasts
Soil has been studied as a source of yeasts because of its importance in ecosystem processes
(Starmer & Lachance 2011). Yeasts have been isolated from different types of soils in diverse
climatatic regions (Botha 2006; Cloete et al. 2009; Vaz et al. 2011). Most studies have
characterized the occurrence of yeast species, suggesting that these microorganisms are
minor contributors to soil ecological processes such as carbon recycling and mineralization
(Botha, 2006; Starmer & Lachance, 2011). Yeasts occur mainly in the upper surface of soil
rich in organic compounds provided by the decomposition of plant materials. Typical soil
yeasts include species of Cryptococcus, Debaryomyces, Lindnera, Lipomyces, Rhodotorula and
Schizoblastosporion (Botha 2006, Cloete et al. 2009; Starmer & Lachance, 2011; Mestre et al.
2011, Vaz et al. 2011).
Some yeast species are associated with rhizospheric soils and can produce polyamines, such as
cadaverine and spermine that could impact upon root growth (Cloete et al. 2009). The yeasts
Rhodotorula mucilaginosa, Cryptococcus laurentii and Saccharomyces kunashirensis were able to
produce soluble and volatile exudates that stimulated the percentage spore germination and
hyphal growth of the arbuscular mycorrhizal fungus Glomus mosseae (Sampedro et al. 2004).
Alonso et al. (2008) reported the presence of yeasts tightly associated with spores of an isolate
of G. mosseae. These yeasts were able to solubilize low-soluble P sources (Ca and Fe
phosphates) and accumulate polyphosphates. Results from inoculation experiments showed
an effect of the spore-associated yeasts on the root growth of rice, suggesting potential
tripartite interactions with mycorrhizal fungi and plants (Alonso et al. 2008).
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Cloete et al. (2010) studied the role of rhizosphere yeasts as plant nutrient-scavenging
microsymbionts in roots of a medicinal sclerophyll, Agathosma betulina, grown under
nutrient-poor conditions, and colonized by Cryptococcus laurentii. The average
concentrations of P, Fe and Mn were significantly higher in roots of yeast-inoculated plants,
compared to control plants that received autoclaved yeast. According to the authors it was
the first report describing the role of soil yeast as a plant nutrient-scavenging
microsymbiont. These results suggest the potential of yeasts to improve the nutritional
quality of soils for plant growth, although occurring in small numbers when compared to
bacteria and filamentous fungi.
4. Case studies of biodiversity in natural ecosystems and human-related
environments
4.1 Report of two novel species found in Ecuador: Candida carvajalis and
Saturnispora quitensis
Ecuador is located between 1°N and 5°S on the west coast of South America. Although
relatively small in size, mainland Ecuador can be subdivided nevertheless into three
different and quite distinctive climatic regions: the Pacific coastal plain, the Andean
highlands and the Amazon basin. In addition, Ecuador possesses a fourth region, namely
the Galapagos Islands.
Climatically, the Pacific coastal plain is hot all year, with a rainy season between December
and May. In the Andean highlands, the climate is markedly cooler, varying according to
altitude. In contrast, the Amazon basin is hot, humid and wet all year round, while the
Galapagos Islands are dry, with an annual average temperature of 25°C (77°F).
To date, very little is known about the natural yeast diversity that exists in Ecuador. In an
attempt to begin addressing this scientific shortfall, and to gain a better insight into the
effectsof contrasting habitats and climate variation on yeast species distribution, a survey was
recently set up and initiated by the Colección de Levaduras Quito Católica (CLQCA) in Quito.
The aim of the project is to catalogue, characterise and compare the indigenous yeast species
found in the different ecological habitats of the four (climatic) regions of Ecuador.
Several novel species have been found since 2006, two of them are already described. In this
chapter we will be referring to these two contributions to science (James et al, 2009).
4.1.1 Candida carvajalis sp.nov. an ascomycetous yeast species from the Ecuadorian
Amazon jungle
This yeast species was isolated from rotten wood and fallen leaf debris collected at separate
sites in the central Amazonian region of Ecuador. Phylogenetically, this species belongs to
the Clavispora clade and is closely related to Candida asparagi, Candida fructus, Candida musae
and two as yet undescribed Candida species, with the six taxa collectively forming a distinct
species group. The phylogenetic placement of this species, coupled with the fact that it could
not be induced to sporulate in pure or mixed cultures on several media, led to the
conclusion that these yeast isolates belong to a novel species of Candida (James et al, 2009).
4.1.1.1 Description of Candida carvajalis sp. nov.
Candida carvajalis – this Latin-derived epithet refers to Enrique Carvajal, father of Enrique
Javier Carvajal Barriga (director at CLQCA). Although not a biologist himself, his passion
for nature has nevertheless led him to become an active collaborator in the search for novel
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yeast species in Ecuador. He collected these as well as other yeasts while on a number of
field trips to the central Amazonian region of Ecuador (Car.va.jal.is). Figure 1.
(Image courtesy of Kathryn Cross, IFR)
Fig. 1. Scanning electron microscopic image of vegetative cells of Candida carvajalisstrain
CLQCA 20-011T grown in YM broth for 1 day at 25°Cwith agitation. Scale bar = 1 µm.
On YM agar, after 2 days at 25°C, cells are spheroidal to ovoidal (3–7 to 4–8 µm), and occur
singly, in pairs or in groups. Budding is multilateral. No sexual state is observed from mixed
or pure cultures plated on corn-meal agar, Gorodkowa agar, potassium acetate agar, PDA
and YM agar. Pseudohyphae are formed (but only in CLQCA-20-011T), but true hyphae are
not formed. The type strain is CLQCA 20-011T, isolated from rotten wood, collected near the
town of Dayuma, in the central Amazonian region of Ecuador. Cultures of the type strain
and CLQCA 20-014 have been deposited with the CLQCA, Quito, Ecuador, and the National
Collection of Yeast Cultures (NCYC), Norwich, UK (CLQCA 20-011T as NCYC 3509T and
CLQCA 20-014 as NCYC 3508). The type strain has also been deposited with the CBS,
Utrecht, the Netherlands, as CBS 11361T.
4.1.2 Saturnispora quitensis sp. nov., a yeast species isolated from the Maquipucuna
cloud forest reserve in Ecuador
During a pilot study to survey the yeast diversity found in the Maquipucuna cloud forest
nature reserve, located 50 miles northwest of Quito, in Ecuador, CLQCA-10-042T was
isolated together with more than 70 other yeast strains. Sequence analysis of the D1/D2
domain of the LSU rRNA gene identified the isolates as belonging to 26 different species of
the genera Barnettozyma (1), Candida (6), Hanseniapora (2), Lachancea (1), Lodderomyces (1),
Metschnikowia (2), Pichia (3), Rhodotorula (1), Saccharomyces (1), Saturnispora (1), Trichosporon
(2), Wickerhamomyces (3) and Yarrowia (1). Strain CLQCA-10-042T was isolated from the fruit
of an unidentified species of bramble (Rubus sp.), and based on its physiology and ability to
produce saturn-shaped ascospores was identified as representing a Saturnispora species
(Kurtzman, 1998). Subsequent sequence analyses of the LSU D1/D2 domain and ribosomal
ITS region established that this strain belongs to a genetically distinct and hitherto undescribed species closely related to S. hagleri. The novel species is named as Saturnispora
quitensis sp. nov., in recognition of the location in Ecuador from where it was first found.
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The yeast genus Saturnispora is characterised by teleomorphic species that typically produce
one to four spheroidal ascospores ornamented with an equatorial ledge (i.e. saturn-shaped)
and have a fairly restricted physiological profile (Kurtzman, 1998). The genus is wellsupported by phylogenetic analyses based on multigene sequence analysis of the smallsubunit (SSU) and large-subunit (LSU) rRNA genes, and translation elongation factor-1
(EF-1 ) gene (Kurtzman et al., 2008). At present, the genus comprises of seven teleomorphic
species, Saturnispora ahearnii, Saturnispora besseyi, Saturnispora dispora, Saturnispora hagleri,
Saturnispora mendoncae, Saturnispora saitoi, Saturnispora zaruensis, S. serradocipensis and S.
gonsigensis (Morais et al., 2005; Kurtzman et al., 2008). Six anamorphic species, Candida
diversa, Candida sanitii, Candida sekii, Candida siamensis, Candida silvae and Candida suwanaritii,
are also accommodated within the genus (Kurtzman et al., 2008; Boonmak et al., 2009;
Limtong et al., 2010). Collectively, these yeasts have been isolated from a wide variety of
different sources and habitats including Drosophila flies (D. cardinae and D. fascioloides),
estuarine water from mangrove forest, flowers, forest soil, insect frass, marsh water,
rhizosphere of oyster grass, sauerkraut, tree bark and tree exudate (Quercus spp.), and wild
mushroom (Hygrophorus sp.) (Liu & Kurtzman, 1991; Kurtzman, 1998; Morais et al., 2005;
Boonmak et al., 2009; Limtong et al., 2010)
From an ecological perspective, S. quitensis is most similar to S. hagleri, with both species
being found in neotropical regions; S. hagleri isolated from two different species of
Drosophila (D. cardinae and D. fascioloides) collected in an Atlantic rainforest site in Brazil, and
S. quitensis from a bramble fruit collected in a cloud forest site in Ecuador (0°03’09’’ N;
78°41’06’’ W; 1668 m.a.s.l). In their species description, Morais et al. (2005) noted that of the
six identified S. hagleri strains, four were recovered from the crops of D. cardinae. This led
the authors to suggest that this yeast may colonize tropical fruits and substrates regularly
visited by these flies and utilised as a food source. To date, only a single strain of S. quitensis
has been isolated. However, it seems plausible to suppose that like S. hagleri, additional
strains of S. quitensis could, in future, be isolated from Drosophila flies and other insects
which visit and feed upon tropical fruits found in neotropical regions like Maquipucuna.
4.1.2.1 Description of Saturnispora quitensis sp. nov.
Saturnispora quitensis – The specific epithet quitensis refers to Quito, the capital of Ecuador,
near where this strain was isolated (Qui.ten.sis).
Cells are spheroidal to ovoidal (4-7 x 5-8 µm) and occur singly or in groups after growth in
YM broth for 2 days at 25°C. Budding is multilateral. Sediment is formed after 1 month, but
no pellicle is observed. Pseudomycelia or true mycelia are not formed. After 8 days on agar
media with a low nitrogen/carbon ratio (i.e. yeast carbon base with 0.01% ammonium
sulphate), conjugated cells give rise to asci containing one to two spheroidal ascospores
ornamented with an equatorial ledge (i.e. saturn-shaped) Figure 2. Ascospores are not
liberated. Conjugation takes place between individual cells, and more commonly between
cells and their buds.
4.2 Yeast species described in Argentinean environments
In recent years, numerous studies have demonstrated that Patagonian natural environments
harbor a broad biodiversity of yeasts with high scientific and technological value (Brizzio &
van Broock, 1998; Libkind et al., 2003, 2004a, 2004b, 2006, 2007, 2008a, 2008b, 2011a, Russo et
al., 2006; de García, 2007; Brizzio et al., 2007). These studies have also shown that a large
proportion of the yeast species recovered belong to undescribed taxa, in general 25 to 40
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(Image courtesy of Kathryn Cross, IFR)
Fig. 2. Transmission electron micrograph of a single ascus containing two ascospores, one of
which is ornamented with an equatorial ledge. Scale bar=1 µm.
percent of the species obtained from a certain substrate represent novel species. This is a
clear indication of the importance of conducting bioprospection studies in microbiologically
unexplored habitats of Patagonia. To date, ten novel yeast species have been formally
described from Patagonian natural environments and at least 20 additional undescribed taxa
have been found (Libkind et al., 2005a; 2009a; 2010a; de García et al., 2010a; 2010b; Russo et
al., 2010; Wuczkowski et al., 2010). Only a few salient cases are discussed here in order to
show the importance of microbial surveys in unexplored habitats.
The first formal description of Patagonian autochthonous yeasts regarded two carotenoidaccumulating yeasts (also known as red yeasts) that had the ability to produce forcefullyejected spores (ballistoconidia). These yeasts belonged to the Sporidiobolales order of the
Pucciniomycotina sub-phyllum (Basidiomycota) and were described as Sporobolomyces
patagonicus and Sporidiobolus longiusculus (Libkind et al., 2005a). The sexual stage
(teleomorph) of S. longiusculus was detected and had a particular micromorphological
feature: teliospore germination gave rise to an elongated basidium, which was five to six
times longer (120–275um) than those of the other member species of the genus Sporidiobolus
(Libkind et al., 2005a). Even though all known strains of both species were collected from
subsurface water of Andean lakes, their suspected primary habitat is the surrounding
phylloplane, probably of Nothofagus spp. trees.
A similar case was that of Cystofilobasidium lacus-mascardii, a teleomorphic species of the
Cystofilobasidiales, class Agaricomycotina (Basidiomycota) of which a single isolate was
first obtained from subsurface waters of the Mascardi lake (Libkind et al., 2009a). Once it
was recognized as an undescribed species, attempts to obtain additional isolates using
specifically designed culture media for selective isolation were performed. Thus, new
isolates were found and they happened to mate with the original isolate providing the
opportunity to describe its sexual stage. Again, terrestrial environments are more likely to
be the habitat of this yeast species based on its low relative occurrence in freshwater and its
ability to produce a wide range of extracellular enzymes (Brizzio et al., 2007).
Another interesting case is that of Cryptococcus agrionensis, a novel anamorphic yeast
of the Filobasidiales (Agaricomycotina, Basidiomycota) associated with acidic aquatic
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environments of volcanic origin in North Patagonia. Due to the high acidity, these
waterbodies also contain high concentrations of toxic metals, and thus poly-extremophile
microorganisms prevail. More than seventy Crypt. agrionensis strains were isolated, mainly
from the most acidic section of the river Agrio with a pH ranging from 1.8 to 2.7 (Russo et
al., 2010). More interesting was the fact that Crypt. agrionensis was phylogenetically related
to three Cryptococcus species that constitute what has been described as the Acid Rock
Drainage (ARD) Ecoclade (Gadanho & Sampaio, 2009). The term ‘ecoclade’ refers to species
that are related phylogenetically and show salient physiological adaptations associated with
the physicochemical conditions present in their habitats. The ARD ecoclade (including C.
agrionensis) have a peculiar ecology and physiology: They are only known from acidic
environments and are highly resistant to heavy metals such as Cd2+, Co2+, Cu2+, Li+, Ni2+
and Zn2+. The discovery of Crypt. agrionensis in acidic water of volcanic origin provided
evidence that the ARD ecoclade was not restricted to abandoned mines of the Iberian Pyrite
region (origin of the previously known species) and demonstrated that members of this
ecoclade may be found in acidic environments in general, originated both naturally and
anthropically.
During our yeast biodiversity survey in the Argentinean Patagonia we came upon isolates
of Phaffia rhodozyma (sexual form, Xanthophyllomyces dendrorhous), a yeast that belongs to the
Cystofilobasidiales order (Class Agaricomycotina, Basidiomycota). This yeast has the ability
to produce astaxanthin, a carotenoid pigment with biotechnological importance because it is
used in aquaculture for fish and crustacean pigmentation (Rodríguez-Sáiz et al., 2010).
Known isolates of this species had been found in exudates of trees of the genera Betula,
Fagus and Cornus in the Northern Hemisphere, mainly at high altitudes and latitudes. We
isolated P. rhodozyma, from the Southern Hemisphere (Patagonia, Argentina), where it was
associated with fruiting bodies of Cyttaria hariotii, an ascomycetous parasite of Nothofagus
trees (Libkind et al., 2007). The Patagonian population besides possessing a different habitat
also showed distinct genetic features based on a detailed molecular comparison with known
strains from the Northern hemisphere. However, the level of genetic divergence of the
Patagonian population with respect to the remaining strains was within the intraspecific
level. In addition by comparing the molecular phylogenies of P. rhodozyma populations with
that of their tree host (Betulaceae, Corneaceae, Fagaceae, and Nothofagaceae), a good
concordance was found which suggested that different yeast lineages colonize different tree
species (Libkind et al., 2007). Hence, we hypothesize that the association of the Patagonian
P. rhodozyma with Cyttaria derives from a previous association of the yeast with Nothofagus.
This study provided a deeper understanding of Phaffia biogeography, ecology, and
molecular phylogeny, knowledge essential to the study of astaxanthin production within an
evolutionary and ecological framework.
The cases above, describing novel yeast species/populations, clearly illustrate the need to
increase the efforts to further survey the micobiota of relatively unexplored habitats such as
the emblematic Patagonia.
4.3 Yeast biodiversity in wineries, Distillerie plants and olive oil mills in La Mancha
region (Spain)
Yeast ecosystems are used as raw materials in the food industry as well as in processing: the
yeasts in grapes, musts and fermented musts in wineries, and in the piquettes, bagasse,
grape-skins and lees used as feedstocks in the ethanol industry provide an inexhaustible
supply of microorganisms.
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La Mancha is the world’s largest vine-growing region, with a surface area of around 600,000
hectares, i.e. roughly 50% of the country’s table wine. Annual grape production, comprising
entirely winemaking varieties, is around 3.6 million tonnes. This output generates roughly
600,000 tonnes of pomace or marc, produced by pressing the fermented red or white grapes
which thus contain a certain amount of sugar. Pomace generally contains plant tissue
residue: skin and pips from the pressing of red grapes, as well as stalks from pressed white
grapes. The ratio of pomace output to grape production varies considerably, depending on
the grape variety and on growing conditions. However, pomace is estimated to account for
17% of overall grape weight; within that figure, skin accounts for 8%, pips for 5% and stalks
for the remaining 4%. Pomace and/or residual sugars are used as a feedstock for ethanol
production.
4.3.1 Wineries
Traditional wine fermentation is a complex, heterogeneous microbiological process involving
the sequential development of various yeasts and other microorganisms present in musts,
such as moulds as well as lactic and acetic acid bacteria. However, it is accepted that certain
strains of Saccharomyces cerevisiae, known as “wine yeasts”, are especially well adapted to this
process, and play a major role in the fermentation of grape musts; for that widely studied.
Nonetheless, it is important to remember that upto 15 different genera of non-Saccharomyces
yeasts may also be present at the start of the wine-making process, and these may contribute to
the special characteristics of individual types of wine (Pretorious, 2000).
Although most wineries now use commercial starter cultures, it is usually to spend over 60
million tonnes of active dry yeasts (ADYs); nevertheless, spontaneous alcoholic
fermentation, that is, fermentation carried out without the addition of commercial dry
yeasts, is still typical for certain wine cellars in this wine-making area. This type of
fermentation is of particular interest with a view to ascertaining the ecology of fermentation
processes in respect to Saccharomyces and non-Saccharomyces yeast strains. In the case of the
former yeasts, it is necessary to establish whether fermentation is carried out by one or
several predominant strains or whether, in contrast, there is a succession of different strains
over the course of wine-making.
Extensive ecological surveys using molecular methods of identification have been carried out
with the aim of studying winery biodiversity and then selecting new yeasts better adapted to
local fermentation conditions (Briones et al, 1996; Fernández-Gonzalez et al, 2001; Izquierdo et.
al, 1997; Querol et. al, 1992), thus allowing the behavior of the various strains to be charted
throughout fermentation.
Non-Saccharomyces yeasts, which display low fermentative capacity and low ethanol tolerance
could impart specific characteristics to the wines, and to enhance wine flavour by increasing
concentrations of the volatile compounds responsible for the fruity aroma, through hydrolysis
of aromatic precursors prompted by -glucosidase enzyme activity (Arevalo-Villena et al,
2006;) or even for the production of volatile esters.
In the cellars of this area, during the early stages of wine-making there is substantial growth of
non-Saccharomyces species. The main species found in different grapes varieties at the
beginning of the process are Candida stellata, Pichia membranaefaciens, Metschnikowia pulcherrima
Hanseniaspora uvarum/guillermondii/osmophila, Kluyveromyces thermotolerans. Torulaspora
delbrueckii and Debaryomyces hansenii were isolated at the middle of the process, and Lachancea
fermentati (formerly Zygosaccharomyces fermentati) were able to survive at the middle, or even
until the end of fermentation.
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The study of the enzymatic activities of non-Saccharomyces wine yeasts revealed that nearly
80% of the yeasts presented at least one enzyme of biotechnological interest.
Polygalacturonase was the enzyme most commonly found and was secreted by 45% of the
yeasts, whereas -glucosidase was only observed in 14% of the yeasts. Proteolytic activity
was also found in some species (Fernández-Gonzalez et al, 2000).
The analysis of restriction mitochondrial DNA is a suitable technique to study the biodiversity
of Saccharomyces wine strains. With regard to the population dynamics of Saccharomyces
strains, there was a greater variability of them at the start of fermentation; as fermentation
progressed some initial strains were succeeded and displaced by others better adapted to
environmental conditions; this succession of strains is common in spontaneous fermentation.
Saccharomyces biodiversity during vinification of the different red grape varieties is shown in
Figure 3. Of the 21 genetic profiles identified, 10 contained more than four isolates. In some
cases, as can be observed in the figure, the isolates are grouped in a dominant profile, i.e. 26%
of isolates displayed the same profile (C), suggesting that this was a common genetic pattern
in the winery; other patterns included 16% (A) or around 10% (E) of isolates, others 8% - 7%,
and some of them accounted for around 2% or 3%, while the remainder contained only one or
two isolates.
T: Tempranillo; S: Syrah; CS: Cabernet Sauvignon; PV: Petit Verdot. I: Beginning; M: Middle; F: End
Fig. 3. Genetic profiles of Saccharomyces wild strains in red grape varieties.
Some profiles were exclusive to certain varieties: profile C was isolated in abundance from
all varieties; profile J was also present, though to a lesser extent, in almost all tanks; profiles
A and B were characteristic of Cabernet and Petit Verdot, whilst M and T were typical of
Tempranillo, and E and H of Syrah.
Other cellars showed a 65% of variability respect to a genetic patterns, found some of them
repeated in some of the fermentation stages sampled. Substantial genetic differences were
recorded, a customary finding for spontaneous fermentations representative for the studied
region (Briones et al. 1996; Izquierdo et al. 1997).
In a study carried out in a single winery, situated around 1000 m.a.s.l. and whose wines
have high quality, the sampling was done in 11 both white and red fermentations of
different grape varieties (Chardonnay, Sauvignon Blanc, Cabernet sauvignon, Tempranillo,
Merlot, Syrah) collecting a total of 28 samples at different stages of fermentations. The
molecular analysis methods led to the determination of 23 different Saccharomyces mtDNA
restriction patterns from 358 isolates. The degree of variability was a good parameter with
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Changing Diversity in Changing Environment
which to evaluate the number of strains actively involved in fermentation. The variability
average found in this study (6.4%) was similar to those from previous studies: 8.6% (Querol
et al., 1994); 2.2 to 4.2% (Schütz and Gafner, 1994) and clearly lower than corresponding
results from 32, 42, 38% and 23, 23, 22% in three different cellars and two consecutive years
(Izquierdo et al. 1997), 22% (Torija et al. 2001) and 20.7% (Nadal et al. 1996).
The majority pattern found cluster the 56% of the isolated ones, followed of others one with
a 15% and a 9%. Four restriction patterns were about 3% and the rest of the patterns whose
presence was limited to one or two isolates. The main pattern was isolated in all sampled
vats and in all grape varieties, both white and red; that situation is not frequently in this
viticulture area, where the Saccharomyces biodiversity is high as recorded previously by
Briones et al. (1996). In three different cellars, selected at random a large number of S.
cerevisiae strains appeared with either the same, or a very similar, karyotype, indicating that
they are strains highly characteristic of these wineries.
4.3.2 Distillery plants
Thirty-three distilleries in Spain are licensed to produce ethanol from winemaking byproducts. Thirteen of these are located in the region of La Mancha.
During harvest, fermented and “fresh” pomace (from white-wine vinification) is transported
to the ethanol plant, where it is mixed and stored (generally outdoors) for between 10 and
15 days; during this period, “fresh” pomaces start to ferment. After, pomaces are washed to
extract ethanol. The liquid produced by this process is known as “piquette”, a mixture of
alcohol (3º-4º), water and sugar; the piquette is mixed with the liquid drained off during
outdoor storage-fermentation.
The piquette is fermented in stainless-steel or iron tanks for two or three days, attaining an
alcohol content of between 4º and 5º (V/V). Although a few ethanol plants use active dry
yeasts, fermentation is mostly spontaneous; this gives rise to a highly- varied Saccharomyces
and non- Saccharomyces biota, as discussed below.
To date, little research has been carried out on the yeast biodiversity found in Spanish
grape-based ethanol plants. A study of yeast populations in ethanol plants and distilleries in
La Mancha sought to determine yeast biodiversity at various sites, with six different ethanol
plants studied (subsequently referred to as plants A-F). Saccharomyces strains predominated
in all ethanol plants studied; the proportion of non-Saccharomyces strains ranged from 14%
to almost 47%. The 144 Saccharomyces sp. isolates matched 105 different genetic profiles; 46
profiles were from fresh piquettes, 43 from fermented piquettes and 16 from lees. In all samples
and all plants, variability exceeded 50%; in five cases, variability was higher than 80%.
Fresh piquettes displayed considerable strain diversity; variability was almost 90% at plant
B, and 81% at plant C. A total of 46 genetic profiles were found, 45 of which were different,
while one – although infrequent (4%) – was isolated at plants A and C. Only one majority
profile accounted for 22% of the yeasts isolated at plant C. Most strains were Saccharomyces
cerevisiae, with only a small number of S. paradoxus and S. bayanus strains.
In fermented piquettes, biodiversity was greater than in fresh piquettes, and at three plants
(B, D and E) different strains accounted for over 85% of the total. Plant A displayed the least
genetic diversity. Piquettes at plants B and C contained negligible amounts of S. paradoxus
and S. bayanus, respectively.
Lees obtained from piquette fermentation displayed less Saccharomyces strain diversity than
either fresh or fermented piquettes. Isolates fitted 15 different genetic patterns. While a 78%
strain variability was observed at plant C, lees sampled at plants A and B displayed only
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71
50% and 57% diversity, respectively. Although patterns tended to be typical of each plant,
majority profiles accounted for 57% of isolates at plant B, 33% at plant C and 30% at plant A.
The greatest degree of Saccharomyces variability was found for fermented piquettes,
although several strains co-existed in both lees and fresh piquettes. These results confirm
that, whilst genetic diversity in wineries has declined considerably due to the increasingly
widespread use of commercial starter cultures, Saccharomyces variability in ethanol plants
remains considerable.
With respect to non-Saccharomyces yeasts, the largest percentage (49%) was found in
fermented piquettes, even though the ethanol concentration varied between 4º - 5º (V/V). A
total of 41% of non-Saccharomyces strains were isolated in fresh piquettes, and only 10% in
lees. The greatest species diversity was observed in fresh and fermented piquettes, the most
frequently- isolated species being T. delbrueckii and C. silvae, respectively.
Only Pichia kudriavzevii (formerly Issatchenkia orientalis) was isolated in all three types of
source. Kluyveromyces thermotolerans and Wickerhamomyces anomalus (formerly Pichia anomala)
were isolated in both fresh and fermented piquettes, while Candida ethanolica was isolated in
fresh piquettes and lees. The remaining species were isolated in only one type of source at
the various ethanol plants.
A number of species, including Hanseniaspora. vineae, P ichia kudriavzevii and Torulaspora
delbrueckii, have been reported in white-wine pomace or marc used for the production of
grappa (Bovo et al., 2009) while Zygosaccharomyces bailii and Saccharomycodes ludgiwii have
been identified, in smaller numbers, in agave fermentation for tequila production (Lachance,
1995).
4.3.3 Olive oil mills
Olive-fruit spontaneous microbiota comprises non-Saccharomyces yeasts, lactic acid bacteria
(LAB) and filamentous fungi. From other research it is known that during the olives
fermentation the presence of yeasts may produce compounds with suitable organoleptic
attributes determining the quality and flavour of the final product (Arroyo-López et al.,
2008). However the olive oil production is also important and there are few references in the
literature about yeast biodiversity present in both fresh olives intended for oil production
and their sub-products. Giannoutsou et al. (2004) suggested that “alpeorujo” is a good
substrate for yeast growth which could be used as a feed additive, as a fertilizer in crops or
as a substrate for the growth of edible mushrooms.
La Mancha is the second largest olive growing region in Spain (350,000 ha) and a major olive
oil producer. No previous studies have dealt with yeast populations in local olives, nor in
the by-products of olive processing, i.e. paste and pomace. Olive fruits from two varieties of
Olea europaea L. (Arbequina and Cornicabra) were randomly picked at various olive groves;
likewise, olive paste and olive pomace were also collected from different oil mills.
Fourteen different species of yeasts were identified, belonging to eight different genera
(Zygotorulaspora, Nakazawaea, Pichia, Lachancea, Kluyveromyces, Saccharomyces, Candida and
Torulaspora), thus demonstrating considerable species diversity. In fresh olive fruits, yeasts
were largely outnumbered by moulds and bacteria probably due to the fact that they had
not been processed (i.e. were collected straight off the tree), so the only contribution was the
environmental biota. Although a similar number of isolates were obtained from paste and
pomace, the latter displayed greater species diversity, with 11 different species identified.
Some species were typically found in olive paste (Nakazawaea holstii, Pichia. mississippiensis
and Lachancea sp.), whilst S. cerevisiae, Kazachstania rosinii (formerly Saccharomyces rosinii),
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Changing Diversity in Changing Environment
Candida sp. and C. diddensiae, Zygotorulaspora florentinus (formerly Zygosaccharomyces
florentinus) and Torulaspora delbrueckii were found only in pomace.
The species most commonly isolated in the Cornicabra variety was Pichia holstii (39%),
followed by Lachancea fermentati (25%), whilst the predominant species in Arbequina was
Pichia caribica (59%) followed by Lachancea fermentati (23%). The remaining 11 species did not
exceed 8% in either variety. Candida diddensiae was found in Arbequina olive variety, and
similar results were obtained by Hurtado et al. (2008) who also isolated this species in
Arbequina fruits. Nakazawaea holstii (formerly Pichia holstii) and Lachancea fermentati are
yeasts associated with wastewater from continuous olive mills in Southern Italy and Spain
(Barnett et al., 2000), while Meyerozyma caribbica (formely Pichia caribbica; anamorph: Candida
fermentati) is involved in artisanal cachaca fermentation in Brazil and is found in soils in
China (Barnett et al., 2000). The other species isolated are found chiefly in soils, although C.
diddensiae has also been reported in olives in Italy.
All species isolated were fermentative to a varying degree. S. cerevisiae was a striking finding
in this respect, and might represent a potential spoilage organism during olive oil storage.
However, this should not be a problem since none of these yeasts have lipolytic activity.
Lachancea genus was isolated from olive paste of both varieties. This new genus was formed
on the basis of five species; L. thermotolerans was chosen as type species and has been
isolated from mushrooms, flowers, leaves and oil wastewaters (Naumova et al., 2007).
Biodiversity was greater in olive by-products than olive fruits, and greater in Cornicabra
than in Arbequina (11 species vs. 6). Three species were common to all olive fruits and both
by-products (Lachancea fermentati, M. caribbica, Lachancea sp.).
Candida spp. were isolated from olive paste (Torres-Vila et al., 2003), other authors have
isolated yeasts in olive fruits and brines during fermentation process, including T.
delbrueckii, Candida boidinii, Cryptococcus spp., Wickerhamomyces anomalus, Kluyveromyces
marxianus (Marquina et al., 1992; Coton et al., 2006; Hernández et al., 2007).
With regard to yeast biodiversity in oil mills, species distribution was very much dependent
upon the oil mill plant. In some mills, 4 to 5 different species were identified, whereas in
others (4 mills) only 2 species were isolated. N. holstii was isolated in all samples from
Cornicabra except in one, and was not detected in the oil mills from Arbequina.
Nevertheless Lachancea fermentati was present in the majority of mills.
Our work also shows the potential of these strains isolated from olive by-products, i.e. olive
paste and pomace, suggesting that these olive wastes can also be used for industrial
biotechnological purposes, for the production of enzymes, commercial preparations or
fermentative processes in different industry sectors.
Characterization of these resources can also contribute to the development of a microbial
bank, providing data on technological properties and enzyme characteristics for potential
industrial applications. On the other hand, the quality and yield in olive oil extraction may
be influenced by the presence of some yeasts with high or moderate enzymatic activities
such as lipases, glucanases, cellulases, glucosidases or polygalacturonases.
5. The role of yeast culture collections: Preservation, applications and
challenges
5.1 The Catholic University yeast Collection (CLQCA)
The CLQCA, or Catholic University Yeast Collection, was originally set up to carry out a
survey of environmental biodiversity of yeasts in Ecuador in 2006, as a pioneer
bioprospecting study. Over the last five years this collection has developed a diverse range
Yeasts Biodiversity Significance: Case Studies in Natural and
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73
of techniques in order to identify and characterize yeast biodiversity. This collection has had
agreements and tight collaboration with other collections such as the National Collection of
Yeast Cultures (NCYC) in the United Kingdom, and the UFMG yeast collection in Brazil.
Currently, this yeast collection is the only one of its kind in Ecuador, and one of the few in
South America. To date, more than 15 novel yeast species have been isolated and three of
them have already been formally described and published (Candida carvajalis, Saturnispora
quitensis) in collaboration with the NCYC (UK). More than 2000 yeast isolates from the 24
provinces of Ecuador have been preserved at the CLQCA. One of the most important
surveys related to biodiversity was carried out in 2008 in the Galápagos Islands, where more
than 800 isolates were collected from four different islands. Other Ecuadorian environments
such as the high Andes, the Amazonia, and the Pacific Coast have also been sampled.
Approximately 1/3 of the isolates are already identified by ribosomal DNA (rDNA)
sequencing and/or RFLP-ITS method. So far, the predominant species registered are Candida
tropicalis (142 strains) and Saccharomyces cerevisiae (100 strains). However, yeasts from
Hanseniaspora, Pichia, Rhodotorula and other genera are also well represented, as shown in
Figure 4.
The CLQCA is not only a bank for the yeast biodiversity, but a biotechnology exploitation
platform, where several projects are being carried out. Some of the most important ones are
focused on second generation bioethanol production, biocontrol of molds, microbial
archaeology and beer production.
5.2 Yeast culture collection in Patagonia
The CRUB (Centro Regional Universitario Bariloche) yeast collection is a research culture
collection kept at the Applied Microbiology and Biotechnology Lab. which is held at the
Biodiversity and Environmental Research Institute (INIBIOMA, CONICET-UNComahue) in
Bariloche, Argentina (Northwestern Patagonia). Certainly is the most southern culture
collection devoted to the preservation of native yeasts. Its collection derive from studies of
yeast diversity in Patagonian natural substrates that have been mainly focused on
environments with extreme conditions which impose a selective pressure towards the
prevalence of adapted microorganisms with innovative physiological characteristics that can
be biotechnologically relevant. Extreme environments such as glacial ice and meltwater (de
Virginia et al., 2007), and acidic waterbodies of volcanic origin that have high concentrations
of toxic metals (Russo et al., 2008) are being studied. Many strains have been proved to be
interesting as producers of psycro-enzymes (de Virginia et al., 2007; Brizzio et al., 2007;
Brandao et al., 2011), poly-unsaturated fatty acids (Libkind et al., 2008b) or because of their
tolerance to heavy metals (Russo et al., 2010). However, the studies in Patagonia have been
mostly concentrated in environments exposed to increased UV radiation (UVR) such as
transparent mountain lakes or the phylloplane of high altitude forests. Yeasts adapted to
high UVR exposure have shown to produce large quantities of photoprotective compounds
(PPC) which are necessary to reduce the detrimental effect of the damaging wavelengths of
UVR (Libkind et al., 2006). The synthesis of metabolites that have antioxidant and/or UV
screening activities are among the strategies commonly seen in yeasts for photoprotection.
6. Biodiversity and biotechnology
Yeasts biotechnology is a growing field where novel species and its physiological abilities
are potentially useful in the search of new products by means of the metabolic engineering
74
Changing Diversity in Changing Environment
Fig. 4. Number of identified yeast isolates preserved at CLQCA up to 2011.
Yeasts Biodiversity Significance: Case Studies in Natural and
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75
approach and the application of novel species for industrial production, not only as
fermenting organisms or molecules producers, but as sources of molecules that can be
purified from the structures of the yeast cell. As an example we can talk about -glucans and
mannans from cell walls that are being used as food additives for animal feed. Other
examples are the partially hydrolysate yeast cells used in animal feed as well with desirable
results in terms of weight and health improvements.
Biodiversity of yeasts is being studied not only to catalogue life on Earth; one of the most
promising fields related to the characterization and identification of yeast diversity is related
to the potential use of them in producing novel enzymes and chemicals. Psycrophilic yeasts
from Antarctic substrates as well as those from high altitudes or glaciers are potential work
horses in biotechnology industry to produce the breakdown of xenobiotics and
pharmaceutical novel variations of molecules. Lipases from Pseudozyma antarctica have been
extensively studied to understand their unique thermal stability at 90°C and also because of
its use in the pharmaceutical, agriculture, food, cosmetics and chemical industry. Other
enzymes which have been studied include extracellular alpha-amylase and glucoamylase
from the yeast Pseudozyma antarctica (Candida antarctica), an extra-cellular protease from
Cryptococcus humicola, an aspartyl proteinase from Cryptococcus humicola, a novel
extracellular subtilase from Leucosporidium antarcticum, and a xylanase from Cryptococcus
adeliensis (Shivaji and Prasad, 2009).
Other common use of the yeast biodiversity—as part of microbial communities—is in the
bioremediation of oil spills. Yeasts are able to use various petroleum components as sole
carbon source, showed that their biodegradability decreases from n-alkanes to high
molecular weight aromatic and polar compounds. The alkanes are mainly degraded using
the monoterminal oxidation pathway through cytochrome P450 system, and transformed
into fatty acids with the same length of the carbon chain. Extensive studies showed that
there are more than 80 genes involved in obtaining the alkane specific phenotype. Up to
date, about 14 different genera of yeasts have been reported to consume hydrocarbons,
exhibiting bioremediation potential uses. The abovementioned genera are:
Candida,Clavispora, Debaryomyces, Leucosporidium, Lodderomyces, Metschnikowia, Pichia,
Rhodosporidium, Rhodotorula, Sporidiobolus, Sporobolomyces, Stephanoascus,Trichosporon and
Yarrowia (Mauersberger, 1996; Scheller, 1998).
Yeast can be used in foods and chemical production as they were probably one of the first
organisms domesticated by humankind. Production of wine, beer and bread are three
examples of the importance of yeasts in human nutrition and culture. More and more
applications for yeast will arise in the next future. The diversity of yeast had been the
answer to fulfill human necessities in the early times of civilization, and, undoubtedly, it is
going to continue being a source of new solutions in the future.
6.1 From biodiversity to biotechnology: the case study of photoprotective compounds
Carotenoids are a group of valuable molecules for the pharmaceutical, chemical, food and
feedindustries, not only because they can act as vitamin A precursors, but also for their
antioxidant and possible tumour-inhibiting activity (Johnson & Schroeder, 1995). Many yeasts
accumulate a variety of carotenoid pigments intracellularly are commonly known as
carotenogenic or red yeast. Red yeasts were found to occur widely in aquatic environments in
Patagonia, and many pigmented strains of the carotenogenic genera Rhodotorula,
Rhodosporidium, Sporobolomyces, Sporidiobolus, Dioszegia, Cystofilobasidium and Xanthophyllomyces
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were isolated and are kept at the CRUB collection. These new yeast isolates from Patagonian
habitats were studied for the production of biomass and carotenoids as the first step
towards the selection of hyper-producing strains and the design of a process optimization
approach. Patagonian yeast isolates considered as potential biomass and carotenoid sources
were studied using conventional media or semi-synthetic medium employing agroindustrial byproducts (cane molasses, corn syrup, raw malt extract) as carbon sources
(Libkind et al., 2004a; Libkind & van Broock, 2006). Maximum pigment production (400 ug
g-1 cell dry weight) was achieved after optimization through a factorial design with the yeast
Cystofilobasidium lacus-mascardii. -carotene, torulene and torularhodin were the major
carotenoids found in most yeasts (Buzzini et al., 2006; Libkind & van Broock, 2006). The
exceptions were Phaffia rhodozyma strains which produced the biotechnologically relevant
pigment astaxanthin (Libkind et al., 2008a). Moreover, photobiological studies were
performed that demonstrated the photoprotective role of these carotenoid pigments in
yeasts (Moliné et al., 2009), in particular torularhodin in the ubiquitous yeast Rhodotorula
mucilaginosa (Moliné et al., 2010). Thus, the CRUB collection represents an interesting source
of carotenogenic yeast strains already characterized regarding their biomass and carotenoid
production performance at Lab scale and providing a variety of pigments for diverse
applications.
In contrast to carotenoid pigments, Mycosporines, are water soluble UV-absorbing (310–320
nm) and very less known. They are compounds containing an aminocyclohexenone unit
bound to an amino acid or amino alcohol group (Bandaranayake, 1998). Although
mycosporines were initially discovered in fungal sporulating mycelia (Leach et al., 1965), it
was not until recently that their synthesis was reported in yeasts by us (Libkind et al.,
2004b). A number of basidiomycetous carotenogenic yeasts were found to synthesize a UVabsorbing compound (peak absorption at 309–310 nm) when grown under
photosynthetically active radiation (PAR, 400–750 nm). The compound was afterwards
identified as mycosporine-glutaminol-glucoside (MGG) (Sommaruga et al., 2004). More
recently the MGG was confirmed as a photoprotective agent in yeasts (Moline et al., 2011)
and its possible use in human sunscreens has been tested (Libkind et al., 2009c). To date,
many yeast species have been detected as MGG producers (Libkind et al., 2005b; 2011b) and
the level of synthesis appear to be related to the solar exposition history in the habitat of
origin (Libkind et al., 2006). Thus, the diversity surveys in highly UV exposed habitats have
rendered valuable isolates able to accumulate large quantities of MGG with concentrations
above 5% of the dry weight (Libkind et al., 2005b, 2011a; Brandao et al., 2011). These MGG
producing strains are conserved in the CRUB collection and are used in studies related to
the elucidation of the genetic bases of MGG synthesis in yeasts and fungi in general.
6.2 Innovative biotechnological method to resuscitate ancient yeasts from fermenters
useful in microbial archaeology
Fermenters from ancient cultures are suitable substrates to keep dormant yeasts within its
pores. Both in Ecuador and Spain, yeasts isolates were recovered from archaeological pieces
belonging to fermenters used by ancient cultures. The most remarkable ones were vessels
from about 2500 b.C. belonging to the Iberos culture (Spain), and Sierra Norte (Ecuador)
from about 200 a.C. Other remarkable yeast recovered from ancient fermenters belonged to
the first brewery founded in America in 1566 (Ecuador), where wooden vessels were
sampled as well.
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The method developed to recover these yeast strains is based upon the so-called “resuscitation
triangle”, where cell walls restoring and membranes fluidization is carried out firstable; after
that, a hydration step is performed; and, finally, a metabolic activation step is accomplished to
resuscitate ancient and valuable yeast strains. Underlying these techniques is the nascent
Microbial Archaeology that pursues an understanding of the ancient microflora and its
implications for human beings by using archaeological rests as sources of ancient
microorganisms. This method, allowed retrieving and isolating dozens of different yeasts,
most of them belonging to species such as Saccharomyces cerevisiae, Clavispora luisitaniae,
Cryptococcus saitoi, Rhodotorula mucilaginosa, Meyerozyma guillermondi, Cr. diffluens, Candida
parapsilosis, and C. tropicalis and other undescribed species.
The method used to recover these ancient yeasts is a trade secret belonging to the Pontificia
Universidad Católica del Ecuador, where the CLQCA is placed.
7. Conclusion
The yeast biodiversity study is currently being boosted by more and more groups that show
interest in discover novel yeasts species and understand the ecology, physiology, and
evolutionary aspects of yeasts.
As pointed out previously, there is still much to be knonwn about yeast biodiversity in vast
zones of the planet. In our understanding, this gap will take a long time to be closed taking
into account the still unexplored habitats occupied by these organism.
Moreover, taxonomy of yeasts faces challenges in terms of the accomodation of species
inasmuch as frequently yeasts are being re-clasified. The genus Candida is the more extended
in yeasts, nevertheless, this genus is only created to accommodate those yeasts that haven’t
shown teleomorphic (sexual) phase. But this characteristic can be reverted if an appropriate
medium allows the yeast sporulation.
On the other hand, molecular techniques developed during the last 30 years are very
valuable tools for research. Molecular approaches are fundamental in current studies of
yeast for its identification and classification. Concomitantly, chemotaxonomic methods are
complementary to characterize yeast strains. These methods allow the researchers find out
the metabolic abilities of yeast strains whose understanding is the first step to potential use
of in biotechnology and industry.
Yeast collections play a fundamental role in preservation, identification and characterization
of these microorganisms; represent safe repositories where biodiversity is preserved for the
future. The ex situ preservation of yeasts is a big effort not only in economical, but also in
technical terms. Qualified personnel are needed as well as economic sources to carry out the
Contamination and loss of viability are two main concerns of curators in yeast collections,
that is why the ex situ preservation of yeasts is a big endeavour, even though yeasts
themselves are quite small.
8. Acknowledgments
The authors would like to acknowledge the Pontificia Universidad Católica del Ecuador as
38 well as the SENESCYT and Conselho Nacional de Desenvolvimento Cientifico e
Tecnologico 39 (CNPq) for founding the research of yeast in Ecuador and Brazil,
respectively. INR and SAJ are supported by the BBSRC in the UK.
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