TAILORED YEAST STRAINS FOR ETHANOL PRODUCTION: PROCESS-DRIVEN SELECTION

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2 Mario Lucio Lopes, Silene Cristina de Lima Paulillo, Rudimar Antonio Cherubin, Alexandre Godoy, Henrique Berbert de Amorim Neto, Henrique Vianna de Amorim TAILORED YEAST STRAINS FOR ETHANOL PRODUCTION: PROCESS-DRIVEN SELECTION 1 st st Edition Piracicaba Fermentec Tecnologias em Açúcar e Álcool Ltda 2015

3 PREFACE Science and technology do not always walk hand in hand, however, for the medium- and long-term, they complement each other very well. Sometimes it takes a while to turn knowledge into technology. The reason lies in difficulties to scale up production or economic unviability. On the other hand, some processes known for thousands of years have taken many years to be explained scientifically, as in the case of alcoholic fermentation. Humans have produced alcoholic beverages for at least for eight thousands years and only 130 years ago, the French, Germans and British discovered that the yeast transforms sugar into alcohol (ethanol). In the next 30 years, another area of science arises: biochemistry, the chemistry of life, trying to explain how yeast transforms sugar into ethanol. Even though a great deal of knowledge has been acquired in scientific and technological (practical) aspects, we still do not know many characteristics of wild and industrial yeast strains, chiefly in terms of adaptation to changes in the environment where they are inserted. Working in ethanol production with a team of biochemists, geneticists, biologists, chemists and agriculture scientists aiming to improve fermentation yield under industrial conditions for the last 40 years, Fermentec has been successfully selecting yeasts for the industry. In Brazil, the ethanol fermentation process uses continuous centrifuges with yeast recycling (Melle Boinot process). This process has many advantages if properly carried out, such as fast fermentation, high yields and low bacterial contamination, however, it is susceptible to yeast contamination if asepsis is not adequately performed. In Brazil, baker yeast or some strains for beer have been used to as starters in fermentation. However, new methods for yeast strain selection have shown that these yeast strains disappear from fermenters and other yeasts dominate fermentation. New methods developed represented the turning point in the knowledge to understand industrial fermentation: yeast behaviour and yeast selection. Yeast produced in laboratory does not dominate nor have a permanence in the fermenter in industrial scale. For this reason, the Fermentec team started selecting yeast strains that dominate fermentation that ensure good ethanol production in addition to having characteristics for good performance and low costs. Therefore, improvement of fermentation yield and cost reduction were obtained through improvements to the process itself (fermenters, piping, asepsis, automation, etc.) and yeast selection. Yeast selection was only possible with the use of methodologies that differentiate yeast (Saccharomyces cerevisiae) strains. The two analyses that represented the turning point were karyotyping of nuclear chromosomes and length restriction of polymorphisms of mitochondrial DNA. Currently, Fermentec is capable of selecting customized yeast strains for each distillery, because we observe that a yeast may be effective for one, but not necessarily for another plant. This selection process that will revolutionize fermentation in Brazil and in the world, the processdriven selection is possible due to yeast adaptation to a given process. The adaptation process is still unknown, but it deserves further investigation. Variants of original strains that accumulate mutations and chromosomal rearrangements arise in the yeast population and are continuously selected through 2

4 the process due to successive recycling of cells. In addition, epigenetics may play a key role in this process. These yeasts have already been selected for 13 distilleries that accounted for more than five billion litres of ethanol produced in the last seven years. These personalized yeasts dominate and avoid contamination of other yeasts, besides providing a better control of bacterial contamination. This is a new frontier in knowledge and in this publication, you, dear reader, will be able to understand how this frontier was achieved as well as visualise the economical return on the investment in selecting personalized yeasts developed by Fermentec. Good reading. Henrique Vianna de Amorim 3

5 ACKNOWLEDGEMENTS We would like to thank all Brazilian mills and distilleries that started their processes with selected yeasts and customized yeast strains. These distilleries allowed us to disclose data and information about their industrial processes and we are very honoured to have them as partners in several research projects and transfer of technologies. We would like to thank Professors Graeme Walker (Abertay University United Kingdom), Johan Thevelein (Katholieke Universiteit Leuven - Belgium) and Marc-André Lachance (University of Western Ontario Canada) for revisions, corrections and suggestions that improved this work and the language used to share our results and insights. We are thankful to Ariane Mendes Ferreira, Bruna Buch, Crisla Serra Souza, Flávia Piacentini Romano, Luciana Piccoli, Milene Bianchini Antonio, Thaise Freiberger and Vanessa Moreira Costa Diana for performing karyotyping and mitochondrial analyses as well as preserving yeast strains in our Bank of Microorganisms. Thanks to Marta Moraes by the front cover and Antonio Bianchi for English revision and tips. Our thanks to all Fermentec team that directly or indirectly contributed to success of this work. For all contributors, we would like to express our sincere acknowledgments. 4

6 SUMMARY Abstract... 6 Introduction... 6 Review... 8 Description of the Brazilian process of ethanol production... 8 Industrial yeast strains: methodology to assess diversity... 8 Contaminating yeast and genome complexity... 9 Mechanisms of interaction, selection and evolution Selection methods: classic and new Process-driven selection: tailored yeast strains Aluminium tolerance of industrial yeast strains Tailored yeast and bacterial contamination Conclusions Final remarks References

7 Tailored yeast strains for ethanol production: Process-driven selection Abstract Industrial processes of ethanol production in Brazil are characterized by large-volume fermenters, short fermentation times, recycling of yeast cells and stressful conditions that can affect yeast cell viability and fermentation yields of distilleries. Moreover, there are several distinct characteristics from one distillery to another, making each one unique. Industrial fermentation processes are subject to contamination by wild yeasts (Saccharomyces and non-saccharomyces species) due to difficulties to sterilize large volumes of sugar cane juice, molasses and water. Wild yeast strains may compete with selected yeast strains causing serious operational difficulties destabilizing the process and affecting fermentation yield. Besides contamination by wild yeasts, bacteria compete for fermentable sugar and produce compounds that are toxic to yeast cells. However, tailored yeast strains are more robust, resistant to stressful conditions and compete better with contaminants than traditional strains do. These tailored strains have shown a higher rate of dominance and persistence in industrial fermentations in comparison with traditional strains and baker yeast. As a result, the fermentation process remains more stable during the ethanol production season. In 2015, 13 distilleries started using tailored yeast strains in their fermentation processes. Here, we present the results from the last eight years of monitoring and use of tailored yeast strains for the Brazilian ethanol industry: a processdriven selection. Introduction Since ancient times, fermented foods and alcoholic beverages have been consumed in all regions of the world. However, for centuries, humans have adopted fermentation practices without knowing their chemical and microbiological bases. Empirical observations and knowledge have been handed down from generation to generation and have led to improved fermentation processes (ALBA-LOIS; SEGAL-KISCHINEVZKY, 2010). In the history of fermentation, Joseph Louis Gay-Lussac wrote the first scientific report in 1810, describing general reaction showing that ethanol and CO2 were the main products of sugar decomposition during alcoholic fermentation. However, fermentation remained a mysterious and misunderstood phenomenon for ages due to lack of information regarding microbiological transformations. Between 1837 and 1838, Charles Cagniard de la Tour, Theodor Schwann and Friedrich Traugott Kützing reported solid evidence of the living nature of yeasts. These authors published in three independent reports that yeast is a living organism that reproduces by budding and they demonstrated that yeast cells were closely associated to fermentation. Based on these works and microscopic observations, yeast was recognized as a living microorganism and not merely an inanimate thing. Moreover, these results provided strong evidence that metabolic activity of living cells was responsible for fermentation and demonstrated that sugar was used for multiplication of yeast cells. However, this vision contradicted chemical theories on fermentation that, unfortunately, was opposed by prominent researchers at that time who deeply criticized it and refuted it. Forty years later, knowledge about fermentation received a very important contribution from Louis Pasteur, who showed that fermentation is a process carried out by living microorganisms. He defined 6

8 fermentation as life without air. Pasteur was able to show that microorganisms, such as yeast and bacteria, carry out fermentations of beer and wine. Despite evidence showing the action of living microorganisms, Pasteur s research did not explain the basic nature of alcoholic fermentation. Nevertheless, he rejected the claims of Berzelius, Liebig and Traube that fermentation resulted from chemical agents or catalysts within the cells. Pasteur concluded that fermentation was a vital action dependent on cells and that it could not be carried out without a living microorganism. These different views gave rise to long and heated discussions about fermentation. However, this dispute was solved with elegance in 1897 when the German chemist Eduard Buchner extracted the content of yeast cells and reported that the liquid extract could be used to ferment a sugar solution, forming CO2 and alcohol, similar to living yeasts. Buchner observed that fermentation occurred without living cells. His work joined the biological and chemical theories, and gave rise to a new scientific discipline: biochemistry. Buchner s work opened a new field of investigation reconciling microbiological and chemical points of view about fermentation (SCHLENK, 1997). Later on, the investigation and understanding of fermentation contributed to progress in medicine, nutrition, technology and production of fermented and distilled beverages, biofuels and development in several other biotechnological fields. Regarding industrial fermentations for ethanol production, Firmin Boinot gave a very important contribution. He worked with distilleries in the Melle region, France, in the 1930s. Boinot patented a fermentation process based on the recycling of yeast cells, using a treatment with diluted sulphuric acid to kill bacteria (BOINOT, 1937). Because of the high concentration of living cells recycled by the process, Boinot was able to reduce fermentation time and increase yield. This fermentation process showed several advantages compared to fermentations without cell recycling and later Brazilian distilleries decided to adopt it. The process, known as Melle-Boinot Fermentation was improved over several years and is characterized by recycling of tons and tons of yeast cells several times during the sugar cane harvesting season (6-8 months). The continuous recycling of yeast cells allows the selection of new yeast strains that are more adapted to specific industrial conditions of each distillery in Brazil. In other words, it means that tailored and high-performance yeast strains could be selected for industrial fermentations according to characteristics of each distillery, an approach known as process-driven selection. Currently, tailored yeast strains have ensured a production of more than four billion litres of ethanol in 13 Brazilian distilleries. However, yeast biodiversity remains a largely unexplored and underreported subject facing scientific challenges (AMORIM et al., 2011). The present work aims to introduce the principles of process-driven selection and discusses results obtained from the use of tailored yeast strains for industrial processes of alcoholic fermentation in Brazil. 7

9 Review Description of the Brazilian process of ethanol production Essentially, ethanol is produced through alcoholic fermentation processes and distillation. The main characteristic that distinguishes the Brazilian process of ethanol production from other bioprocesses worldwide is the recycling of yeast cells (WHEALS et al., 1999). Industrial fermentations are carried out with high density of cells (8-12% w/v) inside fermenters and with short duration (6-12 hours). Approximately, 85% of all distilleries use the fed-batch fermentation process while the remaining 15% adopts the process of continuous fermentations (GODOY et al. 2008). In addition, sugarcane juice, water-diluted molasses and a mix of both are the main substrates for fermentation. Several distilleries work in partnership with sugar mill and ferment a mix of molasses and juice at different rates. For this reason, there is a wide variation in substrate compositions among distilleries (AMORIM; BASSO; LOPES, 2009). At the end of the fermentation process, alcoholic concentrations reach 7-11% (v/v) while residual sugars represent for less than 0.1% (w/v). After fermentation, raw wine is centrifuged in order to separate the yeast cells in a cream that is diluted with water prior to treatment with sulphuric acid (ph for 2-3 hours), while the centrifuged wine goes to distillation. After treatment with diluted sulphuric acid, yeast cells return to fermenters to start a new fermentation cycle. In general, considering all the time required for the steps, it is possible to run two complete cycles per day. A typical sugarcane harvesting season lasts around 240 days meaning that as many as 480 recycling processes may be carried out during this period (AMORIM et al., 2011). Brazilian distilleries have a large variation in geometry, number and size of fermenters. In the last 20 years, several distilleries have introduced fermentation tanks with a conical bottom designed by Fermentec while old fermenters still have a concave bottom. The number and size also vary and in general there are 6-7 fermentation tanks with capacity for million litres each (AMORIM et al., 2011). These differences in size of fermenters, along with the distinct substrates, fermentation processes and process peculiarities, make each distillery unique. Because yeast cells are recycled several times, stressful conditions of industrial fermentations require a selection of optimal yeast population. These factors vary from one distillery to another as well as within the same distillery at different times during the sugarcane harvest. In addition, industrial or starter yeast strains are in constant competition with wild Saccharomyces and non-saccharomyces species (BASSO et al., 2008). Industrial yeast strains: methodology to assess diversity The predominant yeast in industrial processes of fermentation for ethanol production in Brazil is Saccharomyces cerevisiae, which encompasses both starter and wild strains (Figure 1). However, before 1990, it was difficult to monitor the yeast strain composition in fermentation processes due to lack of appropriate and feasible methodologies. At the beginning of the harvesting season, Brazilian distilleries used to start their fermentation processes with baker yeast or even a well-known beer strain, IZ1904, from Instituto Zimotécnico-Universidade de Sao Paulo (Piracicaba, Brazil). Heated debates arose concerning the permanence and performance of these strains in industrial fermentations employing cell recycling during sugar cane harvesting seasons. Some distilleries claimed that these strains were very good fermenters while others claimed they had a poor fermentation performance. However, no distilleries had appropriate methods to monitor and analyse these strains in their own fermentation processes. 8

10 How could it be affirmed that these strains remained in the fermentation process? Additionally, how can it be definitively established which yeast strain is responsible for good or poor fermentation performance? Answers to these questions were forthcoming when the karyotyping technique was used to identify and monitor yeast populations, similarly to the process used for wine strains by Vezinhet, Blondin and Hallet (1990). Thanks to Françoise Vezinhet and Pierre Barre from the Institut National de la Recherche Agronomique (INRA) (Montpelier, France), the karyotyping methodology was shared with Professor Luiz Carlos Basso from Escola Superior de Agricultura Luiz de Queiroz (ESALQ) (Piracicaba, Brazil). Professor Basso started to monitor and identify yeast strains from industrial fermentations in collaboration with Fermentec through a research project approved by Fundação Luiz de Queiroz (FEALQ) (Piracicaba, Brazil). Karyotyping revealed the succession of yeast strains during the sugar cane harvesting season (BASSO et al., 1994). In addition, it was demonstrated that baker and beer yeasts used as starter strains did not survive more than two or three weeks due to stress conditions of industrial fermentations. However, when these yeast strains were replaced by other contaminating ones (with a high fermentation performance), it seemed that the baker and beer yeasts were good fermenters, unless replaced by flocculent and low performance strains. Karyotyping has therefore allowed a greater understanding of yeast strain dynamics during the alcohol production season. Furthermore, it has facilitated the selection of new strains such as PE2 (1994), CAT1 (1998), FT858L (2007) and more recently Fermel (2014). Contaminating yeast and genome complexity Wild Saccharomyces species are the most abundant contaminating yeasts in the fermentation process, competing with starter strains (AMORIM; LEÃO, 2005). However, other contaminating non- Saccharomyces yeast species have been described that cause serious problems to fermentations, which include species Dekkera, Candida and Schizosaccharomyces (AMORIM et al., 2004). These yeasts may deleteriously affect ethanol yields when they find favourable conditions to multiply during the fermentation process, thus competing with Saccharomyces cells. These contaminating non- Saccharomyces wild yeasts may be easily distinguished from Saccharomyces by karyotyping due to distinctive chromosome patterns (Figure 2). Despite efforts of the industry to eliminate contaminating yeasts with thermal treatment of large volumes of sugarcane juice, contamination by wild Saccharomyces is still a predominant problem. Moreover, because yeast cells are recycled hundreds of times during the harvest season, there is a constant battle between industrial strains and contaminating Saccharomyces for fermented sugars. Furthermore, variations in the composition of juice and molasses, as well as errors in the process, may contribute to reducing the population of starter strains in relation to wild Saccharomyces species (Figures 3 and 4). 9

11 Figure 1. Diversity of yeast strains found in industrial processes of alcoholic fermentation for ethanol production. Variants from starter strains are identified by karyotyping followed by mitochondrial DNA analysis. Figure 2. Electrophoretic karyotyping of different yeast species. Profiles 1 to 8 (Saccharomyces) and profiles 9 to 11 (non-saccharomyces). A single gel allows distinguishing between Saccharomyces and non-saccharomyces species. (Source: Fermentec) 10

12 Figure 3. Electrophoretic karyotyping of industrial yeast strain CAT 1 (profiles 1 to 8) and wild Saccharomyces (profiles 9 to 11). Contaminating yeast strains exhibit very distinct chromosomes profiles compared to CAT1 and other industrial strains. (Source: Fermentec) 100% 80% 60% 40% 20% 0% Days of production Starter strain Wild Saccharomyces Figure 4. Electrophoretic karyotyping and monitoring of yeast population dynamics in a distillery during 197 days of ethanol production season. After 108 days, the starter strain was completely replaced by contaminating Saccharomyces. (Source: Fermentec) 11

13 During eight consecutive seasons, we analysed 384 contaminating yeast strains from several distilleries and observed that 91% of the strains were flocculating, foaming, or bad fermenters. Some strains had a combination of two or three of these characteristics. Flocculation behaviour can result in higher residual sugar contents in wine at the end of fermentation in comparison with non-flocculent yeast cells. Therefore, contamination by flocculating and foaming wild Saccharomyces may seriously hinder fermentation and result in high costs due to the need to use antifoaming procedures (Figures 5 and 6). Until recently, our knowledge about genetic diversity of industrial yeast strains and contaminating Saccharomyces was based on data from chromosome profiles (BASSO et al., 1994). Yeast strains were identified, classified and monitored using electrophoretic karyotyping to establish their dominance and persistence in the fermentation process. Dominance means the abundance of a strain in relation to other strains in the sample, while persistence refers to the permanence of yeast strains in the processes where these strains were introduced. Lopes (2000) reported that the industrial yeast strain PE2 could accumulate chromosomal rearrangements during the season hindering identification through karyotyping (Figure 7). Later, the introduction of mitochondrial DNA analyses allowed the use of another molecular tool to investigate yeast diversity (Figure 8), as well as to distinguish between strains related to PE2 that accumulate chromosomal rearrangements from wild strains. The first results obtained in 2007 showed that mitochondrial DNA was more conserved than the chromosomal DNA of industrial yeast strains such as PE2, CAT1 and FT858L. Chromosomes are subject to Mendelian laws, may suffer mitotic and meiotic recombination and are subject to replication and segregation errors during mitosis and meiosis. On the other hand, mitochondrial DNA is more conservative, does not follow Mendelian laws of segregation and independent assortment and occurs at the rate of up to 50 copies per cell. In other words, the introduction of a specific mutation into mitochondrial DNA requires incorporating this change into several copies before it can be detected as a new polymorphism. Thus, the combination of data from chromosomal patterns and polymorphism of mitochondrial DNA allowed obtaining another source of genetic diversity related to variants of industrial strains. The first genome from a eukaryotic cell to be completely sequenced belonged to yeast S. cerevisiae and an international effort was required to carry out the sequencing (GOFFEAU et al., 1996). This work improved our knowledge of cellular gene function and genome architecture in eukaryotic cells. Nowadays, the sequencing of whole genomes of many yeast species and strains is available for investigation in comparative genomics and evolutionary studies on yeasts. The chromosomal profile of S. cerevisiae is very well characterized and distinct from that of most other yeast and fungal species due to its size of million bases distributed in 16 chromosomes per haploid genome. Each chromosome is a single DNA molecule that may vary in sizes from 200 to 2,200 kb long. Chromosomal DNA accounts for approximately 85% of the yeast genome while non-mendelian elements are present in the yeast cells at smaller rates such as mitochondrial DNA (10%) and 2 µm plasmid DNA (5%) (SHERMAN, 2002). Moreover, some strains harbour a virus-like double-stranded RNA related to the killer factor (MARQUINA; SANTOS; PEINADO, 2002). However, the killer factor has not been an important factor for substitution of starter strains by the contaminating yeast strains in the Brazilian alcohol industry (AMORIM et al., 1998) 12

14 A B Figure 5. Fermentation with non-flocculated cells of CAT1 (A) and contaminating Saccharomyces strain with pronounced cell flocculation (B).(Source: Fermentec) A B Figure 6. Fermentation with low foaming industrial yeast strain CAT1 (A) and contaminating Saccharomyces strain with pronounced foam production (B).(Source: Fermentec) 13

15 The genomes of three industrial yeast strains selected by Fermentec: PE2 (ARGUESO et al. 2009), CAT1 (BABRZADEH et al., 2012), and more recently FT858L (Johan Thevelein, personal communication) have been sequenced and some important features were observed for these strains. Despite the importance of these strains for the Brazilian industry, the molecular mechanisms responsible for their high performance in comparison to baker yeast and laboratorial strains are still little understood. However, new insights and results from DNA sequencing projects have revealed special features in the genome of these strains. A detailed molecular analysis of JAY 291 (a haploid derivative of PE-2) showed extensive structural differences and high level of single nucleotide polymorphisms (approximately 2 SNPs/kb) compared to other sequenced yeast strains, with major differences in the telomeric regions (ARGUESO et al., 2009). In addition, chromosomal rearrangements amplify the number of genes involved in environmental stress responses associated to telomeric sequences. We postulate that this feature may attribute to PE2 a more plastic or adaptable genome. Telomeric sequences are subjected to mitotic and meiotic recombination as well as to other rearrangements while genes close to centromeric regions of the chromosomes are more conserved (ANDY CHOO, 1998). In practice, we have found several strains with dominance and persistence in industrial fermentations that are closely related to PE2. As many as 18 strains have been recognised that show very similar profiles of chromosomes and mitochondrial DNA in comparison with PE2. We assume that these strains are derived from PE2 and arose in specific processes according to tolerance and adaptation to specific conditions of fermentation. Another important characteristic observed for these industrial strains is the metabolism of vitamins B1 (thiamin) and B6 (pyridoxine). Industrial yeast strains such as PE2, CAT1 and VR1 showed an increased number of copies for genes SNO and SNZ, related to biosynthesis of vitamins B1 and B6, respectively. Using DNA microarrays, Stambuck et al. (2009) showed that PE2 and CAT1 have a higher number of copies than baker yeast and laboratory strains of S. cerevisiae. In addition, the authors showed that the high number of copies of SNO and SNZ confers the strain the ability to grow more efficiently under repressing thiamine effects, especially in high sugar medium without pyridoxine. Moreover, the same study showed that, in the absence of vitamins, CAT1 has a lower lag phase than laboratory strains do, with a low number of SNO and SNZ copies. In terms of fitness, yeast strains with increased copy numbers show a high performance than the small number of copies found in baker yeast and laboratory strains. According to Benitez, Martínez and Codón (1996), genomes of industrial yeast strains are more complex, heterogeneous and distinct from laboratory strains in terms of DNA content, number and size of chromosomes, gene homologies and polymorphic restriction fragments of mitochondrial DNA. Large chromosomal polymorphisms, as well as gene amplification could be related to selective conditions imposed by industrial processes of alcoholic fermentation. Moreover, variations in the number of copies of specific genes can be observed due to chromosomal rearrangements during meiosis or even mitosis for different yeast and fungi species (ZOLAN, 1995). Chromosomal rearrangements, gene amplification and aneuploidy are related to resistance to aluminium in Saccharomyces (CHANG et al., 2013) and non-saccharomyces (HARRISON et al., 2014). According to Chang et al. (2013), chromosomal rearrangements may be mediated by transposons during early stages of adaptive evolution. 14

16 Figure 7. Karyotyping of PE2 colonies from industrial fermentations showing chromosomal rearrangements indicated by yellow arrows (A, C, E, G) in comparison to the original profile without rearrangements (B, D, F, H). (Source: LOPES, 2000) Figure 8. Yeast diversity based on polymorphism of mitochondrial DNA of different strains isolated from industrial processes of alcoholic fermentation. M= molecular marker of DNA size.(source: Fermentec) 15

17 The genome architecture may contribute to fitness of industrial strains as reported by Argueso et al. (2009). In a competitive environment like the alcoholic fermentation process, diploid or aneuploid cells may rearrange their genes for stress tolerance or adjust the number of copies in new generations. These gene rearrangements in telomeric and subtelomeric chromosomal regions may explain the plasticity of PE2 and its capacity of adaptation in different distilleries. On the other hand, not all structural and physical changes in chromosomes can improve fitness in comparison to other strains once as some chromosomal rearrangements may be neutral (ZOLAN, 1995). In addition, meiotic recombination, hybridization and chromosomal rearrangements are common mechanisms of genome evolution in S. cerevisiae. In general, most yeast strains isolated from the environment and spontaneous fermentations are homothallic and present low levels of heterozygosity. Mortimer et al. (1994) analysed 43 Saccharomyces strains isolated from natural fermentations of grape musts in Italy and showed that most strains were diploid and homozygous for homothallic genes (HO/HO), but heterozygous for 1-7 loci investigated. The authors argued that these strains originated from sporulation of heterozygotes that had accumulated recessive mutations in different loci during long periods of mitotic reproduction followed by mating between mother and daughter cells. This process, known as autodiploidization, would facilitate the loss of deleterious genes and selection of new diploid strains harbouring beneficial alleles in both loci, allowing yeast strains to improve fitness and replace the original heterozygous strain. Mortimer and colleagues called this process the genome renewal. However, DNA sequencing of whole genomes shows that most environmental isolates of S. cerevisiae display several loci with abundant polymorphisms consistent with the idea that strains are homothallic or highly homozygous. These results suggest that heterozygous strains may be isolated from different environments and could not be explained by Mortimer s theory of genome renewal. On the other hand, Magwene (2014) suggested that heterozygosity is related to human domestication of yeast strains followed by rare cycles of meiosis. The author proposed that heterozygosity in S. cerevisiae might arise from yeast populations under human domestication while homozygous strains would be less affected by domestication. In addition, rare meiotic cycles and self-mating would facilitate the rapid adaptation of these strains to new environmental conditions. Evidence of heterothallism in industrial yeast strains originated in 1999 when strains PE2, VR1 and CAT1 sporulated and spores showed a shmoo formation for haploid mating type strains (a and Alfa). In addition, these strains showed different patterns for homologous chromosomes with different sizes that are not consistent to homothallic strains. Moreover, according to Lopes (2000), these strains sporulate very well and fast (Figure 9). After some hours, it is possible to see four spored asci (Figure 10). Even a rich medium containing glucose as C source shows ascosporic formation in yeast colonies as reported by Piccirillo and Honigberg (2010). In addition, these authors showed a strong relationship between sporulation of colonies and invasive growth of yeast cells in the solid medium. After eight days of incubation in media containing glucose as C source, the authors found 5-16% of asci in the population of wild S. cerevisiae. These results highlight the importance of selecting the best conditions to avoid sporulation and prevent industrial yeast strains from entering the meiosis cycle as it affects the correct balance of genes in these superior strains. A simple step of plating in agar medium can generate diversity in the yeast population and loss of original genetic background of the strain. 16

18 Figure 9. Frequency of sporulation for five industrial yeast strains (PE-2, VR-1, BG-1, SA-1 and CR-1) and a baker yeast (PAN) after 48 h in medium of potassium acetate (1%). Industrial strains presented a high frequency of asci in comparison with the baker yeast. LC= living cells; DC= dead cells; AS= asci. (Source: LOPES, 2000) Figure 10. Scanning electron micrograph of vegetative cell (A) and four spored asci (B) of strain PE2. (Source: Fermentec) 17

19 Industrial yeast strains sporulate very well but do not produce only four-spored asci. A high frequency of asci with two and three spores can be observed. Mating with sister spores may occur when asci have two or four spores. However, for each three spored asci, one will be free to mate with another haploid cell of opposite mating type increasing heterozygosity of populations. Even in four spored-asci, PE2 strain may form inviable spores that probably harbour lethal genes. This characteristic may increase the chances of haploids to escape and cross with haploid cells from other strains. Once the cells reprogram their life cycle to enter meiosis, the first step after DNA replication is the crossing-over. According to Esposito and Esposito (1974), when yeast cells start meiosis and are transferred from sporulation to a sugar-rich medium, they can re-enter the mitotic cell cycle directly, while yeast cells from the last stages of meiosis complete the sporulation process even if they are shifted to a sugar-rich medium. This phenomenon is known as "commitment to meiosis". Both cells carry out new combinations of genes in relation to parental lines. Once these strains are heterothallic, new gene combinations and new variant strains may arise from the population even without spore formation. Although S. cerevisiae is considered a highly inbred microorganism, Magwene (2014) suggested that surprising levels of genomic heterozygosity might arise in yeast populations due to human domestication as well as rare meiosis and self-crossing cycles that allow yeast to adapt faster to new environments. In addition, for Paquin and Adams (1983), adaptive mutations have a higher fixation frequency in diploid rather than in haploid populations of S. cerevisiae. Thus, a diploid yeast may have an evolutionary advantage in relation to haploid. Chromosomal DNA is subject to mitotic and meiotic recombination and variations (CODON; BENÍTEZ; KORHOLA, 1997). However, the analysis of mitochondrial DNA is based on accumulation of occasional mutations, recombination without Mendelian segregation. Both techniques have been used to identify yeasts of industrial processes in order to confirm their origin, whether they are wild or yeast-derived, as well as assist in the monitoring of new strains (VEZINHET; BLONDIN; HALLET, 1990, VEZINHET, et al., 1992 and LOPEZ et al., 2001). Most laboratories have worked with haploid or diploid strains of S. cerevisiae whereas industrial strains are diploid, aneuploid, polyploid and even allopolyploid whose strains contain chromosomes derived from two or three different species. Chromosomal rearrangements and aneuploidy in the industrial yeast strain (PE2) was detected by Lopes (2000) in samples from ten distilleries analysed during three sugar cane harvesting seasons. In contrast to several strains of S. cerevisiae strains used in laboratory scale research worldwide having a constant chromosome pattern, industrial yeast strains show a high level of chromosome length polymorphisms. According to Codón, Benítez and Korhola (1998), analyses of DNA content suggest that several industrial yeast strains are aneuploid while polymorphisms in the chromosome size and number are so large that electrophoretic karyotyping could individually identify each strain. When SUC2 was used as a probe, the results showed a widespread presence in baker and industrial strains suggesting that the amplification of SUC genes in these strains could be considered a result of adaptive mechanisms conferring better fitness to strains in relation to specific industrial conditions. The widespread transposon elements (Ty1 and Ty2) and subtelomeric sequences can account for chromosomal rearrangements detected by electrophoretic karyotyping. In addition, Codon et al. (1998) analysed the genomic constitution of industrial yeast strains and showed chromosomal reorganization after meiosis. The new strains had chromosomal patterns that were distinct in comparison to their parental strains used as control. This pattern could be explained by trisomy of some chromosomes as well as segregation of homologous chromosomes of different 18

20 sizes. In addition, the presence and absence of bands in relation to parental strains were attributed to transposable elements and subtelomeric sequences that could cause asymmetrical recombination between chromosomes of different sizes. Moreover, the authors observed that chromosomal reorganization frequently occurs when cells enter meiosis. Similar results were obtained by Lopes (2000) after PE2 sporulation. A single cycle of meiosis and sporulation was enough to induce several chromosomal rearrangements. The same rearrangements (size change, loss and gain of chromosomes) observed after meiosis were also observed in variants of PE2 re-isolated from 10 distilleries where this strain was introduced as a starter during three successive years. Some chromosomes displayed rearrangements that are more frequent and rearrangements were cumulative during the season. After four chromosomal rearrangements, yeast strain identification by electrophoretic karyotyping was hindered, making it very difficult to distinguish between variants of PE2 and contaminating wild Saccharomyces. Although mitosis is less disruptive, it also gives rise to chromosomal recombination and other errors that could generate variants of industrial strains. Puig et al. (2000) reported that native strains of S. cerevisiae, homothallic and poorly sporulating, are frequently heterozygous and may be aneuploid. These characteristics may be advantageous to natural strains as they confer characteristics that are adaptive to some environments. After 30 mitotic divisions, chromosomal rearrangements were detected. The authors showed that meiotic rearrangements were not involved and suggested that mitotic recombination between homologous sequences could be responsible for changes in the genomes of starter yeasts during the vinification process. Because mitochondrial DNA is not subject to same Mendelian laws as chromosomes are, it has been used as a complementary technique to karyotyping. Mitochondrial DNA analyses have allowed identification whether strains arising in the process are closely related variants of selected yeast strains or contaminants. The combination of the two molecular techniques, electrophoretic karyotyping of chromosomes and polymorphism of mitochondrial DNA, allowed the monitoring of yeasts during the sugarcane harvest season, allowing to determine whether new strains are wild or closely related to industrial starter strains introduced at the beginning of the harvesting season. Mechanisms of interaction, selection and evolution Biodiversity among yeast species and within a yeast species has been a theme of increased importance in recent years, in particular after the Convention on Biological Diversity and Brazilian Laws of Biodiversity. In addition, there is a sense of urgency to study the mechanisms of yeast interaction, selection and evolution once only a small fraction of the biodiversity is known (LACHANCE, 2006). Industrial fermenters can be considered true islands of evolutionary adaptation, competition and selection of new yeast strains. They act as a large-scale laboratory where one can study evolution, interactions among yeast strains, population differentiation during the season and changes from one distillery to another. Each distillery has its own characteristics as to sugarcane must composition, installations and facilities, fermentation procedures, ethanol concentration in wine, temperature changes, acid treatment and several other parameters that make each distillery unique, like a typical island. Because yeast cells are recycled hundreds of times during the season, the best-adapted strains survive stressful conditions, dominate and prevail in the industrial process of alcoholic fermentation. This is correctly described as domestication of wine yeast (QUEROL et al., 2003). In particular, structural changes in yeast chromosomes have been related to adaptive evolution of wine yeasts (PEREZ-ORTÍN et al., 2002). 19

21 Furthermore, it is important to stress the need to discover and describe yeast biodiversity within a holistic view taking into consideration the context in which the yeast populations live in order to better understand their distribution, ecology and function within ecosystems as well as mechanisms of selection (LACHANCE, 2006 and VEGA et al., 2012). Natural selection is a continuous and gradual process where heritable characteristics become more or less common in a population due to differential effect of the environment on members of the population. Selected traits accumulate and fix in the population due to higher reproduction rate of favoured individuals compared to original strains. For example, FLO genes may be determinants for some fermentations, such as brewing, where yeast cells are recycled by flocculation, a rapidly evolving trait. However, several processes may affect yeast competition, dominance and persistence inside large fermenters. Another very interesting case may be observed in sherry wine where the formation of a vellum of yeast cells is common, also called Flor Yeast. This kind of yeast has been described for strains isolated in wines from Spain and other European countries. These strains are reproductively isolated and accumulate mutations and aneuploidies that may explain the adaptation of Flor yeasts to wine aging, high ethanol concentrations and velum formation when sugars are depleted from the fermentation medium (SANCHO; HERNANDEZ; RODRIGUES-NAVARRO, 1986). This characteristic of Flor yeast strains is attributed to their ability to aggregate due hydrophobic interactions. High hydrophobicity of Flor cells can be explained by modifications in lipid composition and expression of FLO11, which encodes a GPI anchored protein with a central region rich in serine and threonine. Legras, Erny and Charpentier (2014) presented the first large scale comparison of all European Flor strains involved in the process of biological maturation of wine. The authors analysed ploidia and microsatellite genotype diversity of yeast populations of Flor strains in Europe and showed that almost all Flor strains belong to the same cluster and are diploid, with the exception of some Spanish strains. Comparison of the array hybridization profiles of six Flor strains originating from four European countries with that of three wine strains did not reveal any large segmental amplification. Finally, they correlated Flo11p with thin velum formation in a cluster of strains. These results showed that combinations of different adaptive changes might increase yeast cell hydrophobicity and affect velum formation in very specific, geographically isolated populations. It is important to distinguish between natural selection and genetic drift (random genetic change). These processes can affect yeast evolution and lead to the development of new strains, natural selection works in a non-random way, while genetic drift drives evolution randomly, especially in small populations. Natural selection is guided towards heritable adaptations to current environmental conditions. When these conditions change, the direction of selection changes too. On the other hand, genetic drift has no direction and results from sampling and chance errors. In practical terms, natural selection or process driven-selection seems to be the dominant mechanism of yeast strain selection in very large fermentation tanks working with cell recycling. However, genetic drift can be observed when microbiologists work with pure cultures, single cell colonies, or even a small number of cells. Variants may occur in the offspring of the original strain and be randomly selected for propagation or inoculation. Under artificial conditions of yeast growth in the laboratory, fixation or loss of some genes for stress tolerance may not be observed but they will become evident when the yeast returns to industrial fermentations or when the cells are exposed to stressful conditions. Müller et al. (2014) reported a very interesting result that illustrates genetic drift and mutualism in a single yeast colony. Working with cross-feeding yeast strains (auxotrophic mutants for amino acids) the authors demonstrated theoretically and experimentally that mutualism selects species coexistence 20

22 and genetic diversity while colonization creates competition and regions of low diversity due to repeated founder effects. This may influence the dynamics of yeast populations as well as mutualistic interactions, which could be beneficial for both species of strains under specific conditions of available N sources. To explore the antagonism between mutualism and genetic drift, the authors inoculated two cross-feeding strains of the budding yeast S. cerevisiae on agar surfaces to study the effects during spatial expansion of the colonies. Auxotrophic strains exchanged amino acids, allowing them to control the intensity of mutualism as a function of amino acid concentrations in the growth medium. The authors showed that a strong mutualism is able to overshadow genetic drift, ensuring genetic diversity. However, weak mutualism is overcome by genetic drift, slowing yeast growth and genetic diversity in the colonies. Another promising subject in yeast interaction and competition is epigenetics, which is a process of gene regulation that can change the behaviour of yeast cells, eventually leading them to display features other than those expected for one strain. Epigenetics influences gene regulation and phenotypic expression due to changes in chromosomes or chromatin structure rather than differences in the DNA sequence. Epigenetic phenomena differ from classical genetic processes in that they can respond directly to environmental influences and they only persist over a small number of generations. Examples of epigenetics regulation have been reported in organisms as diverse as the yeast S. cerevisiae and human cells (HENDRICH; WILLARD, 1995). Moreover, epigenetics makes cellular lineages different from one another (TSANKOV et al., 2015). The mechanisms that cause heritable changes at the level of DNA methylation and histone modifications, which result in selective gene expression or repression, have been observed in laboratory strains of S. cerevisiae (BERNSTEIN et al., 2002). However, few cases have been reported for industrial yeast strains and processes recycling huge numbers of cells. Selection methods: classic and new The methodologies for selection of new yeast strains comprise two main strategies adopted by several laboratories worldwide for different applications (wine, beer, baking, bioethanol). The first approach is based on traditional methods for genetic modification followed by selection of new variants. The main processes are mutagenesis and hybridization. Mutagenesis can be induced at high rates by chemicals and physical treatments while hybridization may be carried out by conjugation of haploid cells with complementary mating-types, spore-cell mating, rare mating, cytoduction and spheroplast fusion (PRETORIUS, 2000). Hybridization procedures may affect large chromosomal regions or even entire genomes while isolated mutations occur in specific genes but without an effective control over the number of genes that could be affected. These methods do not specifically introduce new characteristics in a controlled manner. For this reason, although mutagenesis and hybridization may be used to create variability, selection strategies are necessary to identify the best strains without loss of superior characteristics. On the other hand, there is a risk of selecting a new strain regarding based on certain characteristics, while neglecting other traits of technical and economic importance. Nevertheless, traditional methods of yeast hybridization may be used to select new yeast strains, whose desired characteristics are polygenic (quantitative traits). In contrast, isolated mutagenesis can be used to delete single genes (qualitative trait). However, new mutated strains are not easily distinguished in simple screening tests when the original strain is a diploid, aneuploid or polyploid strain. Only if the mutation is dominant, the phenotypic effect could be detected without the need for additional modifications. In case of hybridization of haploid yeast strains of opposite mating type, the technique faces obstacles once several strains cannot sporulate or the strains are predominantly homothallic, as observed for several wine strains (MORTIMER et al., 1994). 21

23 The second strategy to modify and select new yeast strains is genetic engineering, which allows modifying an existing characteristic, introducing a new gene or group of genes and eliminating an unwanted trait without affecting other desirable properties. Several methodologies for yeast transformation and selection of genetically modified strains have been developed, such as plasmid vectors, artificial chromosomes, cassettes for expression and secretion of heterologous proteins encoded by S. cerevisiae and other yeast species. The main advantage of genetic engineering is the specificity and precision of genetic tools to make these changes in yeast cells regardless of ploidy and homothallism. However, auxotrophic markers that have been used to select yeast transformers in laboratory strains are not available to industrial strains. Another strategy that has been used is adaptive evolution of wild and modified yeast strains over successive generations. Adaptive changes are frequently associated with changes involving mobile elements (ADAMS; OELLER, 1986) as well as to chromosomal rearrangements (ADAMs et al., 1992). S. cerevisiae and other yeasts have been considered an ideal model of microorganism for adaptive evolution experiments at the laboratory scale because of their short generation times, as well as the ability to investigate haploid and diploid phases of its life cycle (GU; OLIVER, 2009). Tilloy, Ortiz-Julien and Dequin (2014) explored that strategy to select new wine yeast strains. These researchers selected new strains of S. cerevisiae after 200 generations under high osmotic pressure with enhanced glycerol production and reduced ethanol yield in relation to original strain. Yeast strains that maintained a good fermentation performance also presented a higher tolerance to osmotic stress and glucose starvation conditions in comparison to the original strain. Another approach was designed by Marlière et al. (2011). The model of operation was the conditional pulse-feed regime that is a simplified and generalized version of a methodology initially described by Brown and Oliver (1982) for continuous isolation of ethanol tolerant yeast. Bacterial or yeast cells are exposed to several alternating pulse-feed regimes that allow the microbial genome to accumulate mutations during successive generations. These mutations in the genome can confer a lower requirement for essential nutrients or a higher survival rate when the cells are under starvation. Demeke et al. (2015) demonstrated the amplification of exogenous genes for xylose fermentation by genetically modified Saccharomyces strains and showed that during the process of evolutionary adaptation an extrachromosomal circular DNA element was spontaneously created by the yeast. The circular DNA element had an ARS element (site where the DNA polymerase initiates DNA replication) adjacent to xylose isomerase gene. According to the authors, it was the first that a yeast plasmid created from genomic DNA in the absence of flanking repetitive sequences. Nowadays, molecular tools are used to improve industrial yeast strains and to introduce new features in traditional strains. Swinnen et al. (2012) developed a pooled-segregating whole-genome sequence analysis for the polygenic analysis of complex yeast traits of industrial interest such as tolerance to ethanol, acetic acid and temperature as well as for the production of low glycerol concentrations. The superior alleles identified in this way in natural yeast strains with superior properties can be used for the improvement of industrial yeast strains by natural self-cloning. 22

24 Process-driven selection: tailored yeast strains Process-driven selection is based on the selection of yeast strains that are best adapted to fermentation conditions of each plant and to changes in the industrial process throughout the sugarcane harvesting season. These conditions of fermentation may vary from one distillery to another and they are subject to human interference. Some years ago, a distillery with an oversized sugar cane milling capacity in relation to fermentation, started to increase the ethanol content and imposed a selection pressure during fermentations in order to meet market demand for ethanol. However, traditional strains did not survive the stressing conditions imposed by the industrial process and the more resistant contaminating Saccharomyces strains dominated the population and settled in the process. During the sugar cane harvesting season, a new yeast strain was identified and its population monitored by the karyotyping technique. This strain (FT859L) was selected, evaluated in laboratory scale and used as starter strain in the following harvesting season. This strain dominated the yeast community during 36 weeks (Figure 14) and the plant succeeded to increase the ethanol content in wine and reduced fermentation time, increasing ethanol production compared to the same period of the previous harvest. This example shows promising field of personalized yeast strains. Brazil has 360 distilleries, each with its own peculiarities. The second successful case of tailored yeast strains was the selection of a highly tolerant strain to ferment molasses and water to alcoholic concentrations above 10% (v/v). In the past, distilleries yielded 7.5 % (v/v) of ethanol in wine but traditional strains did not tolerate higher ethanol concentrations and low quality molasses. However, the selection of a new tailored strain, better adapted to ferment molasses, allowed an increase in ethanol content in wine to 10-11% (v/v). It is one of the Brazilian distilleries with the highest ethanol concentration in wine, despite of the use of lowquality molasses. This strain had excellent fermentation characteristics and very low residual sugars in wine. Moreover, for the first time, this distillery succeeded in maintaining a selected yeast strain dominant during entire the season of ethanol production (Figure 15). However, karyotyping and mitochondrial DNA showed that this strain is not related to PE2 or other industrial strains. Another case study to show process-driven selection was the detection of variants. The karyotyping technique and other molecular tools may indicate when contamination by wild strains of Saccharomyces occurs. However, the main industrial yeast strains used by Brazilian distilleries are diploid or aneuploid with homologous chromosomes of different sizes. Chromosomal rearrangements are commonly detected by karyotyping. Some strains accumulate several chromosomal rearrangements that hinder their identification. In addition, it hard to know if the new strain was originated from an industrial yeast or if it was a wild contaminating strain. However, some strains accumulate several chromosomal rearrangements that hinder identification whether the yeast was originated from an industrial strain or from a wild contaminating strain. After hundreds of karyotyping readings, the question remains open whether Saccharomyces variants are derived from the original strain or are they a contaminating strain? Without contamination by wild Saccharomyces, could the industrial process have generated new strains? In 2011, we followed the fermentation process of one distillery where new strains originated in the process (Figure 16). Through karyotyping, these strains could be classified as wild Saccharomyces. However, mitochondrial DNA revealed that these variants were derived from or closely related to PE2. After some weeks, new analyses showed that other variants developed in the fermentation process. These variants were more closely related to the first variants than to original strain (Figure 17). We interpreted these observations considering: i) the industrial process of alcoholic fermentation can 23

25 induce errors or variability in yeast cell population; ii) the same process allows to eliminate less tolerant strains, favouring the most robust and adapted variants; iii) a new strain, more robust and adapted to the process can replace the original strain. When a new strain occurs and dominates the population of yeast in industrial fermenters, it has the potential to become a tailored yeast strain. Considering a fermentation tank with 1,000 m 3 of wine and 10% yeast cells, the number of cells in a reactor is 4 x (10% of fermentation volume). In addition, these cells multiply at a rate of 10% per cycle (6-12 hours). In other words, each fermentation cycle generates new 2 x cells. Taking into consideration a rate of mutation or chromosomal rearrangements of 3 x 10-6, we can expect 6 x spontaneous mutants per cycle of fermentation for just one tank. It is 10 6 times the frequency of spontaneous mutants that would be observed for 1-liter reactors under the same fermentation conditions (Table 1). Moreover, several Brazilian distilleries have six, seven or more fermentation tanks. These data illustrates the number of mutations that may accumulate in yeast populations because of cell recycling, very high-density cell population and large fermenters. Furthermore, other aspects should be considered such as yeast strains, fermentation conditions and stressful environments for the cells and genome hotspots. Gang (2007) showed that the mutation rate is robust to variation during the cell cycle. On the other hand, the mutation rate varies considerably among strains, environments and regions of the genome. Besides robustness, a tailored yeast strain should exhibit special features such as high fermentation yield, high fermentation speed, low residual sugars in wine, low foam formation, no cell flocculation, and other characteristics of industrial importance. A yeast strain with superior characteristics for industrial fermentations is a rare commodity. Generally, predominant strains of S. cerevisiae are foaming and flocculating. These characteristics are related to hydrophobicity of cell wall (FIGUEIREDO, 2008) and have been observed consistently year after year in several distilleries that monitor yeast populations (AMORIM et al., 2011). Gang (2007) showed that the yeast genome contains intergenic sequences repeated in tandem. The majority of these sequences showed length variation when different yeast strains were compared to each other. In addition, several intergenic sequences repeated in tandem have been observed in genes involved in the synthesis of cell wall proteins and cell wall maintenance. A special feature was a correlation between the number of sequences repeated in tandem and genes such as FLO1, which affect adhesion phenotypes. When the number of repetitions increased, adhesion strength also increased. Tailored yeast strains have been selected since Between 2008 and 2014, these tailored strains were introduced in the fermentation process of 12 Brazilian distilleries (Table 2) and have produced around four billion litres of ethanol during that time. All these examples show how the yeast cells may adapt and evolve in different conditions. The potential to generate new variant yeast strains in high-density populations as used by Brazilian distilleries during hundreds of recycles of yeast cells is enormous in comparison with the variability observed in small-scale tests. A large-scale fermenter is a true island of diversity for investigation of the yeast variability, competition, adaptation and selection of new strains that are better adapted to stressful conditions of each distillery. 24

26 Table 1. Number of spontaneous mutants that may develop in a large volume fermentation tank (1,000,000 liters) compared to laboratory reactor of small volume (1 litre). Fermentation tank (1,000,000 liters) Reactor (1L) Yeast cell biomass (% v/v) Total number of cells 4E+17 4E+11 Spontaneous mutation rate * 3 x x 10-6 Multiplication of yeast cells by cycle (%) Number of spontaneous mutants per cycle 120,000,000, ,000 Number of mutants per gene** 20,000, *Mutation per diploid genome according to Gang (2007) **Considering an equal probability for all genes (without hotspots in genome) Table 2. 2 Use of tailored yeast strains in 12 Brazilian distilleries. Since 2008, these distilleries have produced around four billion litres of ethanol. Distilleries Ipiranga Mococa 2 Mandu 3 Alta Mogiana 4 Rio do Cachimbo 5 Água Bonita 6 Colorado 7 CEMorrinhos 8 Tonon Bioenergia 9 Barrálcool 10 Aroeira 11 UMOE 12 Adecoagro - Angélica (Source: Fermentec) 25

27 100% 80% 60% 40% 20% 0% Weeks of etanol production PE2 FT859L Figure 14. Dominance and persistence of tailored yeast strain FT859L derived from or closely related to PE2 in distillery Ipiranga Mococa where karyotyping and mitochondrial DNA selected this strain. (Source: Fermentec) 100% 80% 60% 40% 20% 0% Weeks of etanol production PE2 CAT1 FT858L FT1255L Figure 15. Dominance and persistence of tailored yeast strain FT1255 in relation to industrial strains in distillery Alta Mogiana where this strain was selected. Karyotyping and mitochondrial DNA does not relate strain FT1255L to other industrial strains. (Source: Fermentec) 26

28 Figure 16. Dominance and persistence of tailored yeast strains derived from or closely related to PE2 in distillery Colorado where these strains were selected. Strains FT1628L, FT1756L and FT1756Lv are closely related to PE2, and FT1756Lv is a variant from FT1756L as shown by karyotyping and mitochondrial DNA. (Source: Fermentec) A B Figure 17. Chromosomal polymorphisms of tailored yeast strains derived from PE2 (A) and mitochondrial DNA profiles to the same strains (B) isolated from distillery Colorado. These variants arose in the industrial process of alcoholic fermentation. Red arrows indicate the major differences in the mitochondrial DNA profiles of new strains when compared to PE2. Similar profiles were first observed between PE2 and FT1628L. Later, other similar profiles of mitochondrial DNA were also found between FT1628L and FT1756L, as well as, between FT1756L and FT1756Lv (Source: Fermentec) 27

29 Aluminium tolerance of industrial yeast strains Additional evidence for process-driven selection concerns aluminium tolerance by industrial yeast strains. In Brazil, most distilleries are attached to sugar mills and use a mix of molasses and juice or pure molasses diluted with water for fermentation. In contrast, some autonomous distilleries produce only ethanol and ferment only sugarcane juice. We have observed that distilleries that work with a mix of molasses and juice do not experience problems of aluminium toxicity because of the attenuating effects of organic acids and other compounds in molasses (BASSO et al., 2004). Ma, Ryan and Delhaize (2001) showed how organic acids form complexes with aluminium, reducing its toxicity. However, sugarcane juice does not have such tempering properties and may contain aluminium concentrations as high as 130 mg.l -1, which are toxic to yeast cells (ARANHA, 2002). Aluminium concentrations of 300 mg.l-1 were observed in some juice samples analysed by Fermentec. These high concentrations of aluminium in sugar cane juice can affect the viability and morphology of yeast cells reducing fermentation rate and ethanol yield, increasing the content of residual sugars in wine. On the other hand, magnesium has a protective effect against aluminium toxicity (BASSO et al., 2004). The role of magnesium in yeast has been well documented. Magnesium is essential for yeast growth and fermentation and it plays an important role to physiological and structural functions of yeast cells, including tolerance to high ethanol concentrations (WALKER, 2000). Due to acidic conditions of fermentation and acid treatment of yeast cells, the effects of aluminium (Al 3+ ) toxicity are sufficiently important to affect productivity and industrial yield. Autonomous distilleries experience inhibition of fermentation due to aluminium toxicity at concentrations above 30 mg.l -1 while fermentations carried out with molasses are much less affected. Because yeast cells are subjected to a constant pressure of aluminium toxicity in autonomous distilleries, more tolerant contaminating yeasts are able to outcompete with starter strains. After some cycles, other strains replace less tolerant strains for strains with better fitness. We have observed that the baker yeast is the most sensitive strain to aluminium. PE2 shows intermediary tolerance, while CAT1 and FT858L are the most tolerant strains. Figure 18 shows the dominance of strain FT858L in an industrial fermentation of sugarcane juice from a distillery where high concentrations of aluminium in juice occur with frequency. Figure 18. Karyotyping of yeast cells from an autonomous distillery that shows dominance (100%) of the starter strain (FT858L) in fermentation of sugarcane juice. (Source: Fermentec) 28

30 Tailored yeast and bacterial contamination Another aspect of industrial importance is the effect of tailored yeast strains on bacterial contamination. Bacterial contamination is a serious problem for large-scale fermentation processes of ethanol production as it affects yield and increases costs due to the need to use antibiotics, sulphuric acid and other antibacterial products. Bacteria compete with yeast cells for sugar and produce high concentrations of organic acids, such as lactate and acetate, which affect yeast cell viability. For this reason, yeast strains that are tolerant to acidity are desirable for industrial fermentations. Fermentec has selected tailored yeast strains that are better adapted to specific conditions of each industrial fermentation process. These strains were compared to the baker yeast in terms of cell viability, fermentation speed, bacterial population, lactic and acetic acid production. Tailored yeast strains were more resistant to bacterial contamination and acidity in comparison to the baker yeast. After three fermentation cycles, the greatest difference in bacterial contamination between the first and third cycles was observed for the baker yeast (8.5 x 10 7 bacteria/ml). This bacterial count was correlated with the highest concentrations of lactic acid (> 3,000 mg/l) and acetic acid (>1,000 mg/l) in wine. On the other hand, tailored yeast strains showed the best results when compared to the baker yeast, but each strain presented different results in relation to bacterial contamination (Figures 19, 20 and 21). Some strains were able to inhibit bacterial population, production of lactic and acetic acids. Three strains showed a reduction of bacterial contamination in the third cycle when compared to initial cycle, as well as lower concentrations of lactic acid (<500 mg/l) and acetic acid (< 200 mg/l). These results showed that different industrial yeast strains could inhibit bacterial growth and reduce products of bacterial metabolism in fermentations that simulate industrial conditions. Moreover, all strains evaluated were better than the baker yeast regarding counts of living bacteria, lactic and acetic acids produced by the bacteria. Inhibition of bacteria by yeasts is attributed to the production of succinic acid. Basso, Alves and Amorim (1997) demonstrated that succinic acid and ethanol have synergistic effects in reducing the bacterial population. Later, Cherubin (2003) showed that reducing cell viability of the baker yeast increases the bacterial population in alcoholic fermentations with cell recycles, whereas an industrial yeast (PE2) retained their viability and lower bacterial contamination under the same fermentation conditions. Other researchers have reported the effect of yeast cell viability (GOMES, 2009) and antibacterial action of yeast strains (OLIVA NETO; FERREIRA; YOKOYA, 2004 and MENEGHIN; REIS; CECCATO-ANTONINI, 2010). According to Klerk (2010), several factors may affect production of succinic acid by yeast strains during alcoholic fermentation. These include genetic background, temperature, composition of the sugarcane juice and molasses, metabolically available N sources, unsaturated long-chain fatty acids and aeration. Nevertheless, strain differences may be a major determinant. Recently, a tailored yeast strain was evaluated for production of succinic acid in fermentations with a must of sugarcane molasses diluted with water. The results showed that the strain was able to produce almost twice as much dicarboxylic acids (succinate+malate) in relation to the mix of selected yeast strains traditionally used by distilleries (Table 3). In addition, a clear negative correlation was observed between succinate+malate by yeast and lactic acid by bacteria (Figure 22). These results can also explain why tailored yeast strains are able to outcompete traditional strains in the distillery where the strain was selected. Personalized strains are much more effective to compete and inhibit bacterial contamination than traditional strains. The more robust and adapted strains are, the more they prevail over other yeasts introduced at the beginning of the sugarcane harvesting season as demonstrated by Vicente (2015). 29

31 100 Bacterial rods x 10 6 /ml Baker's yeast PE2 FT858L Fermel FT859L FT1255L FT1416L FT2052L Figure 19. Inhibition of bacterial contamination (Lactobacillus fermentum) by industrial yeast strains (PE2, FT858L, Fermel) and tailored yeast strains (FT859L, FT1255L, FT1416L, FT2052L) in comparison to baker yeast. The results are differences in bacterial contamination between the first and third fermentation cycles. (Source: Fermentec) Lactic acid (mg.l -1 ) Baker's yeast PE2 FT858L Fermel FT859L FT1255L FT1416L FT2052L Figure 20. Inhibition of lactic acid production by Lactobacillus fermentum by industrial yeast strains (PE2, FT858L, Fermel) and tailored yeast strains (FT859L, FT1255L, FT1416L, FT2052L) in comparison to baker yeast. The results are differences of lactic acid production between the first and third fermentation cycles. (Source: Fermentec) 30

32 Acetic acid (mg.l -1 ) Baker's yeast PE2 FT858L Fermel FT859L FT1255L FT1416L FT2052L -400 Figure 21. Inhibition of acetic acid production by Lactobacillus fermentum by industrial yeast strains (PE2, FT858L, Fermel) and tailored yeast strains (FT859L, FT1255L, FT1416L, FT2052L) in comparison to baker yeast. The results are differences of acetic acid production between the first and third fermentation cycles. (Source: Fermentec) Table 3. 3 Comparison of a tailored yeast strain and a mix of industrial strains at the fifth fermentation cycle in their production of dicarboxylic acids (succinate+malate) and their inhibition of bacterial growth and metabolites. Average of three repetitions. Tailored yeast strain Mix of industrial strains Succinate + malate (mg.l -1 ) Ethanol concentration in wine (% v/v) Bacterial population (x 10 7 /ml) < Lactic acid (mg.l - 1 ) 649 5,886 Acetic acid (mg.l -1 ) 1,410 6,941 Manitol (mg.l -1 ) 1,170 6,492 (Source: Fermentec) 31

33 Figure 22. Negative correlation between dicarboxylic acids (succinic+malic) produced by yeast strains and lactate produced by bacterial contamination during alcoholic fermentations carried out at bench scale (five cycles). (Source: Fermentec) 32

34 Conclusions We can conclude that industrial processes of alcoholic fermentation for ethanol production drive the structure of yeast populations because each distillery has unique characteristics that impose a selective pressure on yeast populations. The most robust and adapted strains have a better chance of survival and are able to dominate the yeast population in fermentation tanks. These strains are tailored for each distillery and fermentation conditions adopted by the distillery. Tailored yeast strains showed greater permanence and dominance during the sugar cane harvesting season (more than 30 weeks). In 2013, nine distilleries started the process using tailored yeast strains and in 2014, another 10 distilleries followed suit. These strains are robust to the stressful conditions of industrial fermentations and represent a new opportunity for distilleries to reduce losses caused by contaminating yeast strains. Tailored yeast strains prevent the establishment of contaminating wild Saccharomyces, harmful to industrial fermentations. However, a strain that is well adapted to one distillery is not guaranteed to have success in another distillery. Thus, process-driven selection of yeast strains is a new way to select tailored strains for distilleries where yeast recycling is implemented. Tailored yeast strains will become the next genetic platform to receive new genes for new industrial applications. Final remarks We demonstrate how the industrial process drives the selection of more robust and better adapted strains in comparison with non-industrial yeast strains. However, several questions remain unexplored such as: why several yeast strains are heterotallic and aneuploids? Are there any relation between ploidy and tolerance to stressing conditions? Are chromosomal rearrangements induced by mitotic or meiotic recombination? Which factors can interfere in the transmission of chromosomes from mother to daughter cells? How these factors may affect the chromosomal constitution of industrial yeast strains? And how these traits evolve in the cell population? Another aspect to be explored in industrial yeast strains is related to gene expression and phenotypic changes. Like an orchestra, the yeast cells turn on and turn off several genes to adapt, compete and multiply in different conditions of fermentation. These cells are subject to stressing conditions and need to adjust their metabolism like an orchestra playing Mozart. Who are the main conductors? How many they are and how do they synchronize to the different music played? Of course, we have made huge progresses regarding laboratory strains but we have shown here how diverse industrial and wild strains of Saccharomyces may be considering the well-known laboratory strains or baker yeast. Finally, a new frontier of knowledge has been opened regarding the epigenetics of yeast cells. It has been suggested that yeast would be an extremely useful model for research investigation. Epigenetics of multicellular organisms has been investigated in several countries but the use of simple organisms like a yeast may speed up new discoveries and explain different questions that are still open or not well understood. DNA methylation, histone modification and chromatin remodelling would affect the gene expression of yeast, as well as short RNA molecules latching on to DNA. These RNA patterns may persist through multiple generations affecting important features for yeast performance and experiments on the role of non-coding mrna, which has gained importance among yeast researchers. We believe that the inheritance of genetic traits of industrial importance may extend beyond the yeast genome, but these mechanisms remain a puzzle for industrial yeast strains. 33

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