PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS (ÁREA: MICROBIOLOGIA APLICADA) IFELOJU DAYO-OWOYEMI TAXONOMIC ASSESSEMENT AND BIOTECHNOLOGICAL POTENTIAL OF YEASTS HOLD AT THE UNESP - CENTRAL FOR MICROBIAL RESOURCES Rio Claro 2012 TAXONOMIC ASSESSEMENT AND BIOTECHNOLOGICAL POTENTIAL OF YEASTS HOLD AT THE UNESP –CENTRAL FOR MICROBIAL RESOURCES IFELOJU DAYO-OWOYEMI Thesis presented to the Institute of Biosciences, Universidade Estadual Paulista ´´Julio de Mesquita Filho``- Rio Claro, in fulfilment of requirements for the award of Doctor of Philosophy in Biological Sciences (Applied Microbiology) Supervisor: Prof. Dr. Fernando Carlos Pagnocca Co-supervisor: Prof. Dr. André Rodrigues Rio Claro 2012 DEDICATION To the memory of my loving father, Victor Adedayo Owoyemi ´If I have seen further it is by standing on the shoulders of giants`` Sir Isaac Newton Acknowledgement Completing my PhD was a long and challenging task. Many people supported and encouraged me in so many different ways during the process; it is therefore my pleasure to thank those who helped to see my dream come true. First, I thank the Almighty God, the true source of wisdom and knowledge, for his immense love and infinite mercy towards me. ´´A man has gotten nothing except he be given from above``; I appreciate the rare door of opportunity He opened for me and also for the strength and inspiration given to me for the successful completion of this work. I thank my supervisor Prof. Dr. Fernando Carlos Pagnocca for the wonderful opportunity he gave me in his laboratory. I am grateful to him for believing in me. I appreciate the fatherly role he also played during my stay and studies. I am also indebted to Dr Andre Rodrigues, who apart from being my co-supervisor also played the role of a colleague and brother. ``If I have seen further it is by standing on the shoulders of giants``, together, I thank him and my supervisor very much; for investing their time, resources and wealth of knowledge in me. Most importantly, I appreciate them for contributing to securing a promising future for me. I thank the Brazilian government through the National Council of Technological and Scientific Development (CNPq) as well as the Third World Academy of Science (TWAS) for the PhD fellowship awarded me and for the sponsorship of my education. I also aknowledge Universidade Estadual Paulista “Julio de Mesquita Filho” for the quality education I received and for all the material and infrastructural resources that were made available during the course of my work. I wish to equally use this opportunity to thank the good people of Brazil whose hard earned resources were used to give me an opportunity of quality education. I have been fortunate to have worked with kind and generous group of people whose emotional support and enthusiasm have contributed to the success of my work. Space will not permit me to mention all their names. However, I thank all of them for their generosity, especially, Virginia Elina Masiullionis, Silvio Lovato Arcuri (and his fiancée Clara), Thais Demarchi Mendes, Weilan Paixão Gomes (and her fiance Thiago Gazone), Paula Sanchez de Sousa, Tatiana de Carvalho, Aline da Silva Cruz, Aline Bartelolochi Pinto, Dr. Francisco Eduardo de Carvalho Costa, Samuel, Dirce, Fabio, Liu, Lydia (Berinjela), Sadala and Lucas and Rafael. Several other lecturers and non lectures as well as colleagues also contributed directly or indirectly to the success of my career and their contributions are well appreciated. They include Dra. Sandra Mara Martins Franchetti, Dr. Lara Durães Sette, Prof. Dr. Vanderlei Martins, Prof. Dr. Jonas Conteiro, Necis Lima, Rosemary D. Oliveira S. Cardoso, Josiele Fernanda Magri, Dr. Mauricio Bacci Jr., Joaquim Martim Jr., Mileni Ferro, Cynara, Lusiana, Alexandre, Alex and Paulo, Dr. Bolatito Boboye, Dr. Victor Oyetayo, Engineer and Mrs Taiwo Mogaji, Dr Mathias C. Ahii, Dr. Adebayo Adeyemo and Dr Olubunmi Adebayo. This note of acknowledgement would be incomplete if I fail to appreciate Dra. Derlene Attili de Angelis and her wonderful family. I am immensely grateful and specially thank them for being a family away from home. I appreciate Dra. Dejanira de Franceschi de Angelis, my Brazilian grandmother, for her constant love and concern for me. A special appreciation goes to my husband Olusegun Folaring Jonah. I would not have successfully completed this work without his support and encouragement. I greatly appreciate his unwavering love, trust, patience and understanding. I am grateful to him for standing solidly by me throughout the course of this work. Finally, my utmost appreciation goes to my parents Victor Adedayo Owoyemi (Late) and Philomena Dayo-Owoyemi. I am forever grateful to both of them for the sacrifice they made to give me a good moral and educational foundation. I thank them for their financial and spiritual supports, without which I would not have been able to attain this height. I am deeply grateful to my brother and sister Bolaji and Omolola respectively, and also to my uncle Kolade Ogunmolade for their love, care and encouragements. RESUMO Atualmente, existe um crescente interesse em explorar diversos habitats, a fim de revelar a biodiversidade microbiana, incluindo as leveduras. Tal diversidade ainda não acessada guarda a descoberta de novas espécies para ciência, provavelmente muitas das quais com potencial para aproveitamento em processos biotecnológicos. Com o objetivo de explorar e conservar a diversidade de fungos, o Central de Recursos Microbianos da UNESP (CRM – UNESP) mantém em seu acervo várias estirpes de leveduras isoladas de ecossistemas diversos, sendo alguns deles pouco explorados. No início deste trabalho sabíamos que muitas das leveduras depositadas no acervo do CRM – UNESP não estavam totalmente caracterizadas tanto em nível taxonômico, quanto em relação ao potencial biotecnológico que poderiam apresentar. Portanto, o presente estudo foi desenhado para caracterizar e identificar taxonomicamente leveduras depositadas no CRM – UNESP, bem como selecionar estirpes que produzem enzimas extracelulares degradadoras de polissacarídeos como amilase, celulase, xilanase, pectinase e ligninase. Usando uma abordagem polifásica, um total de 340 isolados de leveduras foi identificado, sendo que 71,2% compreendem 43 taxa de ascomicetos e os restantes 28,8% foram classificados em 27 taxa de basidiomicetos. O estudo também levou à descoberta de 8 prováveis novas espécies. Baseado nesta constatação, a classificação taxonômica e análise filogenética foi realizada para duas espécies anamórficas de ascomicetos e uma espécie teleomórfica de basidiomiceto. A descrição destas três espécies é apresentada neste estudo. Os resultados demonstraram que Wickerhamiella kiyanii FB1-1DASPT e W. pindamonhangabaensis H10YT pertencem à clade Wickerhamiella da ordem Saccharomycetales (Ascomycota: Saccharomycetes), enquanto que a espécie Bulleromyces texanaensis ATT 064T pertence à clade Bulleromyces / Papiliotrema / Auriculibuller da ordem Tremellales (Basidiomycota: Agaricomycotina). Num outro estudo, demonstramos a variabilidade intraespecífica em onze (11) isolados de Hannaella kunmingensis (incluindo a type strain CBS 8960T). Essas onze estirpes foram obtidas de substratos e locais diferentes e analisamos sua variabilidade fisiológica e genética. Ainda, usando uma combinação de análise filogenética e de rede parcimônia, demonstramos o grau da divergência genética (região espaçadora intergênica (ITS) e o gene do citocromo b) dentro desta espécie. Os nossos resultados revelaram variabilidade elevada das características morfológicas e bioquímicas, assim como a existência de três haplótipos genéticos em H. kunmingensis. Uma das estirpes (CBS 8356) apresentou uma divergência de 27,3% das outras linhagens no gene citocromo b, sugerindo a possibilidade de especiação desta estirpe. Este trabalho mostrou características que não foram previamente descritos em H. kunmingensis, e assim pudemos contribuir para a emenda referente à descrição desta espécie, procedimento necessário para acomodar as novas descobertas. Além disso, a partir da triagem das enzimas extracelulares de 312 estirpes, foi detectada a atividade de amilase em 28 estirpes (8,95% do total), celulase em 64 estirpes (20,51%), xilanase em 87 estirpes (27,88%), poligalacturonase em 45 estirpes (14,42%), pectina liase em 59 estirpes (18,91%) e ligninolítica em 2 estirpes (0,64%). As enzimas celulase, amilase, xilanase foram as mais encontradas entre as leveduras basidiomicetas; enquanto que as leveduras ascomicetas foram maiores produtores de pectinases. A determinação da produção de endoxilanase e β- xilosidase de 73 estirpes degradadoras de xilana levou à descoberta de três estirpes que demonstraram elevada produção de amilase, celulase, xilanase e pectinase na presença de bagaço de cana como substrato, indicando que elas são boas candidatas para as pesquisas envolvendo a produção de enzimas úteis na conversão de biomassa vegetal em bioetanol. No geral, este estudo revelou que o CRM – UNESP abriga um acervo de leveduras diversas, com capacidade de produzir várias enzimas industrialmente úteis. Estas leveduras poderiam ser aproveitadas para futuras aplicações biotecnológicas. Além disso, o acervo do CRM – UNESP também provou ser uma fonte de conservação de várias espécies novas para ciência, o que reflete a importância desse tipo de conservação ex-situ para o estudo da biodiversidade microbiana. Palavras chaves: biodiversidade, coleção de cultura, ascomicetos, basidiomicetos, polissacarídeos, enzimas. ABSTRACT In recent time, there has been an increasing interest in exploring diverse ecological habitats in order to reveal the yeast biodiversity. The increased awareness in the biotechnological potentials of yeasts has also spurred attempts to search for new species with novel biotechnological capabilities. Aiming to explore and conserve the fungal diversity from various ecosystems, the UNESP – Central for Microbial Resources (UNESP – CMR) harbors various strains of ecologically diverse yeasts isolates, some of which were yet to be identified. Therefore, this study was designed to identify and characterize some yeasts from the UNESP – MRC and to select strains possessing extracellular plant polysaccharide degrading enzymes namely amylase, cellulase, xylanase, pectinase and ligninase. Using a polyphasic approach, a total of 340 strains were identified. Taxonomic classification grouped 71.2% of these isolates into 43 ascomycetous taxa while the remaining 28.8% were classified in 27 basidiomycetous taxa. The study also led to the discovery of 8 putative new species. As a result, we classified two anamorphic species in the Ascomycota and one teleomorphic species in the Basidiomycota. In this study we provide the description of both species. Our results demonstrated that the two ascomycetous species proposed as Wickerhamiella kiyanii FB1-1DASPT and W. pindamonhangabaensis H10YT belong to the Wickerhamiella clade of the Saccharomycetales (Saccharomycetes) while the basidiomycetous species proposed as Bulleromyces texanaensis ATT064T belong to the Bulleromyces / Papiliotrema / Auriculibuller clade of the Tremellales (Agaricomycotina). In order to show the significance of intraspecific diversity in yeasts, in one of our studies, we subjected 11 strains, (including the type strain CBS 8960T) of Hannaella kunmingensis, obtained from different substrates and geographic locations, to detailed physiological and genetic characterization. Using a combination of phylogenetic and parsimony network analysis, we demonstrated the extent of genetic (internal transcribed spacer region (ITS), D1/D2 domains of the large subunit rDNA (LSU), and cytochrome b gene) divergence within this species. Our findings revealed the high variability of morphological and biochemical characteristic as well as the existence of 3 genetic haplotypes in H. kunmingensis. One of the strains (CBS 8356T) exhibited a 27.3 % divergence from the other strains in the cytochrome b gene; hence, we concluded the possibility of speciation of this strain. This work led to the discovery of additional strains and characteristics not previously reported in H. kunmingensis, therefore, the emendation of H. Kunmingensis was done to accommodate the new discoveries. Furthermore, from the screening of 312 yeast strains for secreting extracellular enzymes, amylase activity was detected in 28 strains representing 8.97% of the total isolates screened; cellulase activity in 64 strains (20.51%), xylanase activity in 87 strains (27.88%), polygalacturonase activity in 45 strains (14.42%), pectin lyase activity in 59 strains (18.91%) and lignolytic activity in 2 strains (0.64%). This study further revealed that amylase, cellulase and xylanase are the major enzymes found among the basidiomycetous yeasts while ascomycetous yeasts are producers of pectinases. Determination of extracellular endoxylanase and β-xylosidase activities in culture supernatants of 73 xylanase positive strains led to the discovery of three strains which demonstrated high amylase (endo- and exomylase), cellulase, xylanase and pectinase activities in presence of sugar cane bagasse; therefore are good candidates for research involving production of enzymes useful in biomass conversion. Overall, this study revealed that UNESP – MRC possess metabolically diverse yeasts with ability to produce various industrially useful enzymes. Such strains could be harnessed for future biotechnological applications. In addition, the UNESP – MRC proved to harbors new species to science that are now preserved ex-situ of long-term maintenance. Keywords: biodiversity, culture collection, ascomycetes, basidiomycetes, polysaccharides, enzyme TABLE OF CONTENTS INTRODUCTION 19 Aims and objectives 21 STUDY OUTLINE 23 CHAPTER 1 25 BACKGROUND OF STUDIES AND LITERATURE REVIEW 1 BACKGROUND OF STUDIES AND LITERATURE REVIEW 26 1.1 UNESP – Central for Microbial Resource (UNESP-CMR) 26 1.2 Importance of culture collections to microbiology and biotechnology 30 1.3 Yeast: introduction and definition 32 1.4 Evolution of yeast identification methods 32 1.5 Yeast classification 36 1.6 Yeast ecology and diversity 42 1.7 Biotechnological importance of yeasts 47 1.8 Biodegradation of starch, lignocelluloses and pectin 47 REFERENCES 57 CHAPTER 2 69 TAXONOMIC STUDIES OF YEASTS HOLD AT UNESP – CENTRAL FOR MICROBIAL RESOURCES 2.1 Abstract 70 2.2 Introduction 71 2.3 Material and methods 72 2.3.1 Cultural Characterization 72 2.3.2 Molecular Identification 74 2.4 Results 76 2.5 Discussion 79 REFERENCES 85 CHAPTER 3 88 SCREENING FOR AMYLOLYTIC, LIGNOCELLOLYTIC AND PECTINOLYTIC YEASTS 3.1 Abstract 89 3.2 Introduction 90 3.3 Materials and methods 91 3.3.1 Screening procedures for extracellular enzymatic activities 91 3.3.2 Amylase activity 93 3.3.3 Cellulases 93 3.3.4 Xylanases 93 3.3.5 Pectinases 94 3.3.6 Ligninase 94 3.3.7 Xylanase enzymes assays 94 3.3.8 Fermentation of sugarcane bagasse 96 3.3.9 Enzyme assays 96 3.3.10 Statistical analysis 98 3.4 Results 98 3.4.1 Screening for enzymatic activities 98 3.4.2 Xylanase (endoxylanase and β-xylosidase) assays 106 3.4.3 Extracellular enzyme production from sugar cane bagasse fermentation 110 3.5 Discussion 112 3.5.1 Enzymatic activity profile 112 3.5.2 Enzyme production from sugar cane baggase 113 REFERENCES 115 CHAPTER 4 118 WICKERHAMIELLA KIYANII SP. NOV. AND W. PINDAMONHANGABAENSIS SP. NOV., TWO ANAMORPHIC YEASTS ISOLATED FROM NATIVE PLANTS OF THE SOUTH EASTERN ATLANTIC RAINFOREST OF BRAZIL 4.1 Abstract 119 4.2 Introduction 120 4.3 Material and methods 121 4.4 Results and discussion 123 4.5 Description of Candida kiyanii Pagnocca, Rosa, Dayo-Owoyemi and Rodrigues sp. nov. 128 4.6 Description of Candida pindamonhangabaensis Pagnocca, Rosa, Dayo-Owoyemi and Rodrigues sp. nov. 129 REFERENCES 131 CHAPTER 5 134 DESCRIPTION OF BULLEROMYCES TEXANAENSIS SP. NOV., ISOLATED FROM FUNGUS GARDEN OF THE LEAFCUTTER ANT ATTA TEXANA AND LEAVES OF BROMELIAD NEOREGELIA CRUENTA (BROMELIACEAE) 5.1 Abstract 135 5.2 Introduction 136 5.3 Materials and methods 137 5.3.1 Strain information 137 5.3.2. Morphological and phenotypic characterization 138 5.3.3 DNA extraction, Sequence and phylogenetic analyses 138 5.4 RESULTS AND DISCUSSION 139 5.4.1 DNA sequence and phylogenetic analysis 139 5.5 Description of Bulleromyces texanaensis Dayo-Owoyemi, Rodrigues, Garcia, Hagler and Pagnocca sp. nov. 144 5.5.1 Growth on YM broth 144 5.5.2 Growth on YM agar 144 5.5.4 Dalmau plate culture on corn meal agar 144 5.5.5 Formation of ballistoconidia 144 5.5.6 Sexual reproduction 144 5.6 PHENOTYPIC DESCRIPTION 147 5.7 Origin of the strains studied 147 5.8 Systematics and Ecology of Bulleromyces texanaensis 147 References 151 CHAPTER 6 154 Intraspecific variation and emendation of Hannaella kunmingensis CONCLUSIONS AND PERSPECTIVES 164 ABBREVIATIONS °C – Degree centigrade CBS – Centraalbureau voor Schimmelcultures CMCase – Carboxymethyl cellulase DBB – Diazonium blue B DNA – Deoxyribo nucleic acid g. – Gravitational force g.L – Gram per Liter gm – gram ITS – internal transcribed spacers LSU – Large subunit MCase- Microcrystalline cellulase M – Molar min – minutes mM – Milimolar MSP-PCR – Microsatellite Primed Polymerase chain reaction. NCBI – National Center for Biotechnology Information SNA – Synthetic nutrient agar TCS – Parsimony network analysis UNESP – CMR – Universidade Estadual Paulista – Central for Microbial Resources UV – Ultraviolet YMA – Yeast malt agar LIST OF FIGURES Figure 1.1 Map of fungi rRNA gene showing the internal transcribed spacer (ITS) region, intergenic spacer (IGS) regions,18S small subunit (SSU) and 25-28S large subunit (LSU) 34 Figure 1.2 Phylogeny of the phylum Ascomyceota showing the classification of Ascomycetous yeasts . 38 Figure 1.3 Phylogeny of the phylum Basidiomycota showing the classification of Basidiomycetous yeasts 40 Figure 1.4 Plant cell wall structure. 48 Figure 1.5 Action of major cellulase enzymes 51 Figure 1.6 Action of major enzymes involved in the depolymerization of Xylan 52 Figure 1.7 Action of major enzymes involved in the deconstruction of pectin. 53 Figure 1.8 Scheme showing the actions of lignin degrading enzymes 55 Figure 1.9 Scheme showing the actions of starch degrading enzymes 56 Figure 2.1 Growth (assimilation) test on carbon compounds 77 Figure 2.2 PCR fingerprinting patterns of some identified strains 77 Figure 3.1 Degradation halos around yeast strains producing 100 amylase (A), cellulose (B), xylanase (C), polygalacturonase (D), pectin lyase (E) and ligninase(F). Figure 3.2 Enzymatic activity profiles of the ascomycetous and basidiomycetous 103 yeasts screened Figure 3.3 Comparisons of enzyme activity profiles of ascomycetous and basidiomycetous yeasts respectively 104 Figure 3.4 Extracellular enzyme production from sugar cane bagasse fermentation by Aureobasidium pullulans strain CG5-5BY, Aureobasidium pullulans strain PBM1and Pseudozyma hubeiensis strain MP2-2CB 111 Figure 4.1 Phylogenetic placement of Candida kiyanii and C. pindamonhangabaensis in the Wickerhamiella clade (Saccharomycetes, Saccharomycetales) determined from Neighbor-joining analysis of sequences from LSU rRNA gene. Bootstrap values are from 1000 replicates. T = type species. 131 Figure 4.2 Candida kiyanii (A) and Candida pindamonhangabaensis (B and C). Phase contrast micrograph showing budding cells (A and B) with pseudomycelium after 3 days at 25 °C on YM agar (A) and on corn meal agar 25 °C (C). 126 Figure 4.3 Lipase activity test of Cpindamonhangabaensis (upper colonies) and C. kiyanii (lower colonies). 128 Figure 5.1 Evolutionary tree showing the relationships of Bulleromyces texanaensis and related species based on combined LSU and ITS sequences. 141 Figure 5.2 Phylogenetic relationships of strain Bulleromyces texanaensis and other closely related species based on LSU D1/D2 rRNA gene sequences. 142 Figure 5.3 Phylogenetic relationships of strain Bulleromyces texanaensis and other closely related species based on ITS sequences. 143 Figure - 5.4 Growth phases of Bulleromyces texanaensis 145 LIST OF TABLES Table 1.1 Some major international microbial culture collections, their acronyms and type of culture holding 27 Table 1.2 Quality control procedures recommended for microorganisms (OECD, 2007) 29 Table 1.3 Industrial applications of some enzymes 49 Table 3.1 Number and origin of yeasts and dimorphic fungi profiled for enzymatic activity 92 Table 3.2 Ascomycetous yeasts screened for amylolytic and lignocellulolytic and pectinolytic activities 100 Table 3.3 Basidiomycetous yeasts screened for amylolytic and lignocellulolytic and pectinolytic activities 102 Table 3.4 Xylanase activities in extracellular and cell wall associated cell free supernatant of selected strains 107 Table 3.5 Enzyme yield per gram of substrate 112 Table 4.1 Extent of D1/D2 LSU rDNA and ITS sequence divergences of C. kiyanii and close relatives pairwise some based on alignment. 125 Table 4.2 Physiological characteristics differentiating C. kiyanii from closely related strains 125 Table 5.1 Phenotypic characteristics of strain Bulleromyces texanaensis CBS 11955T 148 LIST OF APPENDIX Appendix 1 Identities of yeasts and dimorphic fungi maintained at UNESP – CMR 166 Appendix 2 Result of extracellular enzyme screening with some yeasts in the UNESP - Central for Microbial Resources 179 Appendix 3 Statisitical (one-way anova) analysis of reducing sugars (RS) produced from sugar cane bagasse fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM 1 and Aureobasidium pullulans strain CG5-5BY 192 Appendix 4 Statisitical (one way anova) analysis of activitiy of endoamylase produced through sugarcane baggase fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM 1and Aureobasidium pullulans strain CG5-5BY (using 0.5% starch) 194 Appendix 5 - Statisitical (one way anova) analysis of activitiy of exoamylase produced through sugarcane baggase fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM 1 and Aureobasidium pullulans strain CG5-5BY (using 1.0% starch) 196 Appendix 6 Statisitical (one way anova) analysis of activitiy of pectinase produced through sugarcane baggase fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM 1and Aureobasidium pullulans strain CG5-5BY 198 Appendix 7 Statisitical (one way anova) analysis of activitiy of xylanase produced through sugarcane baggase fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM1and Aureobasidium pullulans strain CG5-5BY 200 Appendix 8 Statisitical (one way anova) analysis of activitiy of carboxymethyl cellulase (CMCase) produced through sugarcane baggase fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM 1 and Aureobasidium pullulans strain CG5-5BY 202 Appendix 9 Statisitical (one way anova) analysis of activitiy of microcrystaline cellulase (MCase) produced through sugarcane baggase fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM 1 and Aureobasidium pullulans strain CG5-5BY 204 19 INTRODUCTION 20 INTRODUCTION Since the last decade, the destruction of the natural ecosystem by human activities and changing global climatic conditions has raised concerns to microbiologists and ecologist as to the endangering of microorganisms. Such events are leading to gradual microbial species extinction, hence reduction of microbial diversity. According to Hibbett et al. (2011) more than 100,000 fungal species have been described to date. Although, the exact number of extant fungal species is not known, several estimatives were raised by mycologists including an an estimate of 712,000 extant species elaborated by Schmit and Mueller (2007). However, recent estimates based on high-throughput sequencing methods suggest that as many as 5.1 million fungal species exist (BLACKWELL, 2011). Authors trying to estimate the number of extant yeast species usually conclude that more than 98% of yeasts are yet undiscovered. Given the average number of fungal species described each year since 1999 to be about 1200, Hibbett et al. (2011) predicted that at the current rate of discovery, it may take up to 4000 years to describe all fungal species. Since, local extinctions (i.e. strong reductions in the abundance of microbial species) occur quite frequently due to clearing of forests, agricultural activity or erupting volcanoes, it was predicted that microbial extinction rate might soon surpass the recovery rate of extant undescribed species. Effort towards microbial diversity recovery has included sampling of microorganisms from various ecological habitats, especially those whose biodiversity are been endangered and development of culture collections by private laboratories, institutions and universities for ex situ conservation. The urgent need therefore arises for biodiversity and taxonomic studies with emphasis on the discovery, classification and description of novel species. Due to their wide application in various industrial processes, the demands for plant polysaccharide degrading enzymes including amylases, cellulases, xylanases, pectinases, and ligninases, have greatly increased in recent times. For example, the primary enzymes used in animal feed are xylanases, β-glucanases (and phytases) because they aid in digestion of polysaccharides in monogastric animals. Hydrolysis of carbohydrate polymers such as cellulose, xylan and starch is used to produce fermentable sugars for bio-ethanol production. Ethanol production from lignocellulosic has been identified as a cheaper alternative for the sustainable fuel ethanol. The complete degradation of the cell wall of lignocellulosic materials to fermentable sugars requires the contribution of lignocellulolytic enzymes namely cellulases, hemicellulases and ligninase enzymes. Furthermore, in the fermentative production of ethanol from starch, amylolytic enzymes are routinely added as pretreatment of the 21 starch to convert it to linear oligomers and ultimately to glucose units, that are then fermented by yeasts. Xylan is the most abundant hemicellulose and xylanases are one of the major hemicellulases which hydrolyse the β-1,4 bond in the xylan to short xylooligomers which are further hydrolysed into single xylose units by β-xylosidase. Microbial xylanases are becoming more demanding due to their wide application in various industrial sectors. In the energy sector, one area of considerable importance is the enhanced production of ethanol through the release of substantial amount of fermentable feedstock (PÉREZ et al., 2002; ALMEIDA et al., 2007). In the step involving the conversion of hemicelluloses to fermentable sugars, some of the hemicelluloses, mostly xylan, remain associated with the cellulosic-rich water insoluble fraction (CHANDRA et al., 2007). Because, effective enzymes capable of digesting these woody materials are still lacking, in order to improve cellulose accessibility, hence, enhance substrate digestibility, cellulose enzymes are often supplemented with xylanases as ‘accessory enzymes’ (KUMAR; WYMAN, 2009). Hence, the demand for xylanase producing microorganisms has increased. One of the main areas of research in enzyme biotechnology has been driven by the need to isolate and identify organisms which are good producers of plant polysaccharides (lignocelluloses) degrading enzymes. In contrast to fungi and bacteria, few types of yeast are known to be capable of degrading lignocelluloses. Ability of yeasts to utilize plant polysaccharides is important because such information could be useful for taxonomic classification as well as biotechnological applications. Therefore, more information is needed about yeasts possessing these characteristics. However, it is known that yeasts are highly diverse in terms of nutrition, exploitation of ecological niches and secondary metabolism (this diversity reflects in their wide biotechnological applications in various industrial sectors such as food, beverages, chemicals, industrial enzymes, pharmaceuticals agriculture and environment) and that microbial diversity is the foundation for biotechnology; the basis for the discovery of new products, secondary metabolites and genes. Microbial culture collection serves as a pool where metabolically and genetically diverse yeast strains with unique properties and applications could be discovered. Aims and objectives This study was designed to meet the general objective of providing accurate taxonomic identification to some unidentified yeasts hold at the UNESP – Central for Microbial Resources (UNESP – CMR) situated at the Institute of Biosciences, Rio Claro, 22 State of São Paulo as well as to predicting biotechnological utility of newly discovered taxa and already existing taxa making up this collection. The specific objectives of this research therefore were: (i) to identify and characterize some yeast and yeast-like organisms at the UNESP – Central for Microbial Resources of the Institute of Biosciences, Rio Claro. (ii) to describe novel species in the culture collection (iii) to identify yeast and yeast-like organisms with ability to produce lignocellulose degrading enzymes namely: cellulases, hemicellulases and ligninase enzymes. (iv) to quantitatively determine the endo-xylanase and β-xylosidase activities of selected xylanase producing strains discovered in this study and determination afterwards of polysaccharide degrading enzymes produced from sugar cane bagasse fermentation. 23 STUDY OUTLINE The primary objectives of this study were to provide accurate identification of some yeasts hold at the UNESP – CMR with emphasis on classifying and describing new strains; as well as to provide information about the applicability in enzyme technology of some yeasts in this collection, particularly plant polysaccharide degrading enzymes namely amylases, cellulases, xylanases and pectinases. Here we organized the study into chapters with the first one being a brief background while the subsequent chapters present investigations and findings relating to the theme of the study. The chapter opener begins with a brief introduction of the UNESP – CMR and how its activities have contributed to broadening the knowledge of fungal diversity. It also present reviews of literature about the definition of yeasts and how advancements in molecular biology have shaped the methods used for yeast identification. Because the yeasts examined in this work were isolated from different habitats during different ecological studies, effort was laid on briefly reviewing some roles played by yeasts in these habitats. Furthermore, this chapter highlights the enzyme systems involved in the degrading of plant polysaccharides and yeasts found from previous studies to possess these enzymes. Chapter 2 presents taxonomic studies of yeasts hold at UNESP – CMR. Basically, this study involved the identification and classification of 340 yeast isolates obtained from various ecological studies embarked upon by the UNESP – CMR research team using a polyphasic approach. The identification process led to the discovery of several undescribed fungal species including two ascomycetous species whose novel status were proposed in the genus Candida (n = 2) and a basidiomycetous species proposed in the genus Bulleromyces. The findings of this study also revealed that the UNESP – CMR collection holds many metabolically and genetically diverse types of yeasts that could be harnessed for further biotechnological studies. Chapter 3 reports screening of the UNESP – CMR for yeasts possessing enzyme systems for the degradation of plant polysaccharides specifically starch, cellulose, xylan, pectin and lignin. These enzymes are useful in various industrial applications including the production of biofuels. Because few types of yeast are still known to produce plant polysaccharidases, the study aimed at screening to cover a wide taxonomic range (93 taxa) of yeasts. Three hundred and twelve strains were screened out of which 192 were strains identified in the previous study (Chapter 2). This study led to the discovery of many types of yeast possessing enzyme systems for the degradation of plant polymers. It also revealed 24 among other things that xylanase and cellulase activities are characteristics more expressed by basidiomycetous yeasts whereas, pectin degrading activities is more linked to ascomycetous yeasts. In addition, this study revealed three strains capable of producing amylases, celluloses, and pectinases at high levels from sugarcane bagasse fermentation. Chapter 4 and 5 presents the description of three novel species discovered in this study. Two anamorphic ascomycetous species namely Wickerhamiella kiyanii (strain FB1- 1DASPT) and Wickerhamiella pindamonhangabaensis (strains H10YT and H10-10AY) were proposed and their taxonomic descriptions as well as systematic classifications are provided in chapter 4. The presence of lipase enzyme systems was demonstrated in the latter species. These two species were found to phylogenetically belong to the Wickerhamiella clade (Saccharomycetes, Saccharomycetales). On the other hand, chapter 5 presents the description of a teleomorphic species proposed in the genus Bulleromyces. These two chapters contribute to our knowledge of yeast diversity. The description of these species will permit the deposition of their holotypes as well as their nomenclatural information in internationally recognized and publicly accessible culture collections as well as the official publication of their names. Finally, chapter 6 presents a study on the intraspecific variation and emendation of Hannaella kunmingensis. Our study so far with yeasts has been revealing the existence of intraspecific genetic and phenotypic variations among different strains of yeasts belonging to the same species. In one of such scenarios, three strains of yeasts from the UNESP – CMR were found to differ by 11 nucleotide substitutions in the internal transcribed spacer (ITS) region from H. kunmingensis (CBS 8960T), but based on their conspecificity in the D1D2 domains of the large subunit ribosomal DNA gene, the 3 strains were considered as H. kunmingensis species. The later discovery of sequences of 7 other similar strains deposited in the GenBank offered the opportunity to intensively study the intraspecific variation in genetic as well as phenotypic properties of this species. Using parsimony network analysis, the presence of three genetic haplotypes in H. kunmingensis was demonstrated. The study also revealed variations in morphological characteristics as well as biochemical characteristics among the 11 strains studied. Based on these findings, an emendation of H. kunmingensis species was carried out. Besides contributing to the knowledge of the intraspecific diversity, the study contributed to increasing the number of strains of H. kunmingensis, which was formely described based on a single strain. 25 CHAPTER 1 BACKGROUND OF STUDIES AND LITERATURE REVIEW 26 1. BACKGROUND OF STUDIES AND LITERATURE REVIEW The increasing awareness of the importance of biodiversity and the hidden genetic potential has resulted in a rise in recognition of the value of microbial culture collections. From the early days of biodiversity surveys and fieldtrip collections, microbiologists have been gathering samples and evidences related to their discoveries. In order to identify and perform more advanced investigations and analyses on their collected specimens, they had to be kept alive and maintained in a condition as close as possible to their original states. When properly preserved, microbial strains can maintain the same properties found in nature and can therefore be reused in many different types of studies, such as physiology, genetics or applied biotechnology. This is where the idea of culture collections came into being. Microbial culture collections are living libraries and reference sources of microorganisms. In the past, due to the lack of adequately functioning and reliable culture banks, many microbial cultures were lost (MAHILUM-TAPAY, 2002). The first scientist to realize the importance of culture collection was Professor Frantisek Kral (1846-1911) who collected cultures and made it available for free to other researchers. His collection was later transferred to Vienna in 1915 (MALIC; CLAUSE, 1987). The Centraalburreau voor Schmmelcultures (CBS) culture collection, Netherlands, is the next oldest collection been founded in 1906. A list of some major culture collections in various countries is given in Table 1.1. 1.1 UNESP Central for Microbial Resources (UNESP- CMR), In year 2006, the Microbiology laboratory (LAM) of the Institute of Biosciences, Rio Claro, developed a private collection of microorganisms, mainly yeasts, filamentous fungi and actinomycetes formerly known as Center for the Study of Social Insects (CEIS) UNESP campus of Rio Claro. Recent institutionalization of this culture collection led to its renaming as “Central for Microbial Resources (UNESP- CMR)", according to the Ordinance of the Institute of Biosciences / São Paulo State University (IB / UNESP) No. 4/2013. The UNESP - CMR's mission is to act as a Centre of Biological Resources, as defined by the OECD (OECD 2007). Currently, the UNESP- CMR holds a total of approximately 5,100 cultures, consisting of 4000 yeasts and filamentous fungi derived from samples of nests of ants and 27 T ab le 1 .1 - So m e m aj or in te rn at io na l m ic ro bi al c ul tu re c ol le ct io ns , t he ir a cr on ym s a nd ty pe o f c ul tu re h ol di ng C ul tu re c ol le ct io n (a cr on ym ) Pa te nt in st itu te T yp e of c ul tu re L oc at io n W eb a dd re ss A gr ic ul tu ra l R es ea rc h Se rv ic e C ul tu re C ol le ct io n (N RR L) N at io na l C en te r fo r A gr ic ul tu ra l U til iz at io n Re se ar ch A ct in om yc et es , B ac te ria , F un gi Pe or ia , I lli no is U SA ht tp :// nr rl. nc au r.u sd a. go v A ll- Ru ss ia n C ul tu re C ol le ct io n (V K M ) Ru ss ia n A ca de m y of S ci en ce s Ba ct er ia , A rc ha ea , F un gi M os co w R eg io n, Pu sh ch in o, R us si a ht tp :// w w w .v km .ru A m er ic an T yp e C ul tu re C ol le ct io n (A TC C ) A m er ic an T yp e C ul tu re C ol le ct io n Ba ct er ia , A rc ha ea , F un gi , v iru se s M an as sa s, V irg in ia U SA ht tp :// w w w .a tc c. or g Br az ili an C ol le ct io n of E nv iro nm en ta l a nd In du str ia l M ic ro or ga ni sm s U ni ve rs ity o f C am pi na s, Sã o Pa ul o Ba ct er ia , F ila m en to us fu ng i a nd Y ea st C am pi na s, Br az il ht tp :// w eb dr m .c pq ba .u ni ca m p. br /c bm ai / Be lg ia n C oo rd in at ed C ol le ct io ns o f M ic ro - O rg an ism s ( BC C M ) Be lg ia n C oo rd in at ed C ol le ct io ns o f M ic ro -O rg an is m s Ba ct er ia a nd F un gi V ar io us c iti es , B el gi um ht tp :// bc cm .b el sp o. be /in de x/ ph p C en tra al bu re au v oo r S ch im m el cu ltu re s (C BS ) In sti tu te o f R oy al A ca de m y, A rt an d Sc ie nc e Fu ng i U tre ch t, Th e N et he rla nd s ht tp :// w w w .c bs .k na w .n l G er m an C ol le ct io n of M ic ro or ga ni sm s a nd C el l C ul ut e (D SM Z) Le ib ni z- In sti tu te Ba ct er ia , F un gi a nd P la nt V iru se s Br au ns ch w ei g, G er m an y ht tp :// w w w .d sm z. de /h om e. ht m l Ja pa n C ol le ct io n of M ic ro or ga ni sm s ( JC M ) RI K EN B io Re so ur ce C en te r Ba ct er ia , A rc ha ea , F un gi Sa ita m a, Ja pa n ht tp :// w w w .jc m .ri ke n. jp N at io na l C ol le ct io n of Y ea st C ul tu re s (N C Y C ) Fo od R es ea rc h In sti tu te , C ol ne y La ne , N or w ic h N R4 7U A , N or fo lk , U K Y ea st s a nd o th er k no w n pa th og en s N or w ic h, U ni te d K in gd om ht tp :// w w w .N C Y C .c o. uk N at io na l I ns tit ut e of T ec hn ol og y Ev al ua tio n _ Bi ol og ic al R es ou rc e C en te r ( N BR C ) N at io na l I ns tit ut e of T ec hn ol og y an d Ev al ua tio n A ct in om yc et es , a rc ha ea a nd Fu ng i C hi ba , J ap an ht tp :// w w w .n br c. ni te .g o. jp Ph af f Y ea st C ul tu re C ol le ct io n, U ni ve rs ity of C al ifo rn ia , U ni ve rs ity o f C ar lif on ia , D av is Y ea st s D av is , C al ifo rn ia , U SA ht tp :// w w w .p ha ffc ol le ct io n. or g Po rtu gu es e Y ea st C ul tu re C ol le ct io n (P Y C C ) Fa cu ld ad e de C iê nc ia s e T ec no lo gi a U ni ve rs id ad e N ov a de L is bo a Y ea st C ap ar ic a, P or tu ga l ht tp :// w w w .se c- bi ot @ fc t.u nl .p t 27 28 environments impacted with petroleum and its products, 1,000 fungi associated with marine environments (coastal Antarctica and Brazil), and about 100 bacteria used as reference strains; and the number of cultures is rapidly growing due to the many biodiversity studies embarked upon by researchers affiliated with this culture collection. The collection was formed from research projects or collaborative research coordinated with the participation of researechers of UNESP and has been used for diversity studies and prospecting compounds of biotechnological interest, such as, enzymes, antibiotics, biofuels and bioremediation agents. UNESP - CMR is divided into two main parts namely (i) the main collection and (ii) the research collection. The main collection comprises isolates with biotechnological potential or are representatives of the biodiversity of a given environment and the reference strains and which have been correctly identified based on molecular taxonomy (ribosomal DNA sequencing, genetic fingerprinting and phylogenetic analysis) and conventional taxonomy (morphological and biochemical) while the strains contained in the research collection comprises isolates recovered from different environmental samples still under study and are potential candidates to be incorporated into the main UNESP- CMR collection after being subjected to necessary preliminary bioprospective studies as well taxonomic characterization. All the microorganisms in the UNESP - CMR are stocked and preserved according to the recommended guidelines of the Organization for Economic co-operation and Development OECD Best Practice Guidelines for microbial recource centers (Table 1.2) for microorganisms. Before starting this study, some of the yeasts in this collection were conventionally identified using physiological, biochemical or phenotypic methods, while many were yet to be identified. This offered an opportunity to apply DNA sequencing and molecular taxonomic methods for their proper identification as well as exploitation for biotechnological applications. Initially, the culture collection was majorly focused on yeast biodiversity studies; this biodiversity assessment led to the discovery of many previously unidentified yeast species namely Cryptococcus haglerorum isolated from the floor of a nest of the leaf-cutting ant Atta sexdens (MIDDELHOVEN et al. 2003), Blastobotrys attinorum formerly Sympodiomyces attinorum from the nests of the leaf-cutting ants (CARREIRO et al. 2004), Candida leandrae from fruit of Leandra reversa (Melastomataceae) from Atlantic rainforest in Brazil (RUIVO et al. 2004), Candida bromeliacearum and Candida ubatubensis known from Canistropsis seidelii collected from the Atlantic rainforest of south-eastern Brazil (RUIVO et al. 2005), Candida 29 Table 1.2 Quality control procedures recommended for microorganisms (OECD, 2007) 30 heliconiae, Candida picinguabensis and Candida saopaulonensis from flowers of Heliconia (RUIVO et al. 2006) and Trichosporon chiarellii from a nest of the fungus-growing ant Myrmicocrypta (PAGNOCCA et al. 2010). In addition, the culture collection contributed to the description of a new yeast genus, Bandoniozyma and seven new species inside (VALENTE et al., 2012). Due to the knowledge accumulated over the years, from various physiological and taxonomic studies, about the vast metabolic activities of the microorganisms making up the UNESP – CMR, bioprospecting of the collection has also been extended to screening of yeasts for biotechnologically important enzymes. Furthermore, currently, a research involving the bioprospection of the yeasts from this collection for the conversion of lignocellulosic materials (sugarcane bagasse) to ethanol is being implemented. Hence, UNESP – CMR, not only serve as a biotechnology-based bank of valuable microorganisms that could be harnessed for biotechnological applications, teaching and research and other purposes, but also as microbial repository for the preservation of Brazilian microbial biodiversity which constitute the heritage of the country. 1.3 Importance of culture collections to microbiology and biotechnology i. Depository of Microbial Diversity The primary role of a culture collection is the preservation of biological diversity. This is achieved through providing repositories for microorganisms of scientific and industrial interests, as well as of regional and international interests. Microbial culture collections provide services, which include accession, maintenance, preservation, documentation and cataloguing of microorganisms. Such microorganisms include strains of newly discovered taxa; type strains, neotype, unique biotype and selected reference strains; strains utilized in patent and novel applications; genetically modified strains and other strains of special educational, agricultural, biotechnological and medical significance (MALIK; CLAUSE, 1987). The importance of these functions could be emphasized from examples of microbial species that have not been re-isolated since their first discovery but whose biotypes are still maintained in public collections. For example, Candida tanzawaensis was described 22 years ago by Nakase et al. (1988) and has not 31 been isolated since then. Furthermore, many recent species descriptions and publications are based on materials collected in the past but preserved in culture collections with the hope that new technologies that could facilitate their description would later be developed (LACHANCE, 2006). ii. Screening and exploitation of microbial diversity Culture collections play important roles in providing lage microbial resources for screening for important biotechnological products. Introducing screening programs to collections allow biodiversity rich countries to benefit from exploitation of the microbial diversity they have (SMITH, 2003). For scientists who are interested in screening for novel products, culture collections provide an opportunity of access to large numbers of authenticated microorganisms. For example, in a search for L-arabinose fermenting yeasts for the bioconversion of biomass to ethanol, Dien et al. (1996) carried out an extensive screening of 116 yeast strains, from the ARS Culture Collection (National Center for Agricultural Utilization Research, Peoria, Illinois) in which four species namely Candida auringiensis, Candida succiphila, Ambrosiozyma monospora, and Candida sp. (YB-2248) were found to be able to ferment the sugar. In the same vein, Hou and Johnston (1992) screened 1229 selected microbial cultures (including 508 bacteria, 479 yeasts, 230 actinomycetes and 12 fungi) obtained from the ARS Culture Collection for lipase activities in which 25 % were lipase positive. ii. Development and training of new preservation methods and skills Successful maintenance of microbial cultures in such a way as to ensure their long term viability, stability and accessibility is very crucial. Methods used for culture preservation should be those that cause little or no damage to cells while at the same time still retaining their genetic and phenotypic characteristics as well as viability over a long period of storage. In most cases these require personal experience and a good understanding of individual microorganism coupled with familiarity with modern methods of preservation (KIRSOP, 1983). Therefore, culture collections test and develop new methods of culture preservation suitable for individual and 32 group of microorganisms. They also provide specialized training in culture handling and taxonomic studies. In addition, the identification of organisms is generally a specialized activity; hence, many collections provide identification services for people who have do not have the required skills or facilities required. 1.3 Yeast: introduction and definition Yeasts are microscopic fungi, which reproduce asexually by budding or fission and by the production of forcefully ejected ballistoconidia on stalks termed sterigmata; resulting in growth made up of single cells and whose sexual states are not formed within or upon a fruiting body (SUH et al. 2006; KURTZMAN; FELL; BOEKHOUT, 2011a). These characteristics differentiate them from other filamentous fungi and mushrooms that are predominantly multicellular, and whose sexual structures are enclosed within complex fruiting bodies (KURTZMAN; FELL; BOEKHOUT, 2011a). In contrast to the yeasts, filamentous and dimorphic fungi grow by means of hyphae that extend at their apices while branching sub-apically, thereby resulting in an interconnected network of hypha known as mycelium. Several yeasts however exhibit pseudohyphae made up of chains of elongated buds that remain attached to the parent cell after formation. In addition to ascomycete and basidiomycete yeasts, some fungi are dimorphic and exhibit a yeast stage that shifts to mycelial growth under certain cultural conditions. The term ‘‘yeast-like’’ has also been used to represent the cellular phase of dimorphic members of the zygomycete genus Mucor (FLEGEL, 1977), the black yeasts (HOOG, 1999), which comprise diverse pigmented ascomycete genera such as Aureobasidium, Fonsecaea and Phaeococcomyces as well as certain achlorophyllous algae in the genus Prototheca (KURTZMAN; FELL; BOEKHOUT, 2011a). 1.4 Evolution of yeast identification methods The approaches used in yeast taxonomy have rapidly evolved over the years. Before the current era of species identification by DNA comparisons, yeasts were normally delineated based on differences in observable characteristics, i.e. morphological characteristics and phenotypic attributes such as presence or absence of a sexual state, type of cell division, presence or absence 33 of hyphae and pseudohyphae, fermentation of simple sugars and growth on various carbon and nitrogen compounds (KURTZMAN, 2011). Chemotaxonomic characteristics such as cell wall carbohydrate composition and coenzyme Q are also sometimes used in yeast taxonomy (PRILLINGER et al. 2011). The need for more accurate diagnostic tools for yeast species identification soon became apparent after various works began to reveal variations in phenotypic characteristics displayed by yeast strains of the same species (KURTZMAN; FELL, 2006; KURTZMAN, 2011). The shift from phenotypic identification of yeasts to molecular identification began with DNA reassociation techniques. In this technique, single-stranded DNAs of two isolates are mixed and allowed to repair as a double strand. According to Price, Fuson and Phaff (1978), strains that showed 80% or greater nuclear DNA relatedness on the basis of shared phenotype, as measured by reassociation, are members of the same species. Although, DNA reassociation technique provided the first opportunity of genetic based yeast species delimitation, it is limited by the fact that only closely related species can be resolved using the technique. The use of DNA sequence comparisons for yeast identification soon became widely embraced after Peterson and Kurtzman (1991) and Kurtzman and Robnett (1998), studied the variable domains (D1 and D2) of the large subunit (LSU) rRNA gene of ascomycetous yeasts (Figure 1.1) and revealed that these regions offer the opportunity to resolve most closely and distantly related species. Kurtzman and Robnett (1998) predicted that strains showing six or more nucleotides differences (1% substitution) in the D1/D2 nucleotide domains of the ribosomal DNA (Figure 1.1) represent different species. A complementary D1/D2 database was created for basidiomycetous yeasts by Fell et al. (2000). Molecular analysis of the small and large subunit of the rRNA gene led to significant progress in systematic of basidiomycetous yeasts while various works such as Sugita et al. (1999, 2000) and Scorzetti et al. (2002) contributed to the development of databases for the internal transcribed spacers (ITS) 1 and 2 regions of the rDNA (figure 1) for basidiomycetous yeasts. The ITS region is highly substituted and provides a higher resolution for some closely related species that could not be separated by D1/D2 region (SCORZETTI et al. 2002). Several other genes have been successfully used for yeast taxonomic classification and they include the intergenic spacer (IGS) region, translation elongation factor-1α (TEF-1α), actin-1, mitochondrial small subunit (MtSm 5) rRNA, Cytochrome b gene and the cytochrome oxidase II (COX II) gene (KURTZMAN; FELL; BOEKHOUT, 2011b). Other methods used for rapid identification of species and for detection of polymorphisms in nuclear 34 Figure 1.1 Map of fungi rRNA gene showing the internal transcribed spacer (ITS) region, intergenic spacer (IGS) regions, 18S small subunit (SSU) and 25-28S large subunit (LSU). Source: http://biology.duke.edu/fungi/mycolab/primers.htm DNA include restriction-enzyme fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified polymorphic length polymorphism (AFLP) and microsatellites (OLIVE; BEAN, 1999). The estimation of genetic and historical relationships among yeast species have relied mostly on trees derived from phylogenetic analyses of sequences of target genes. Some of the methods used for phylogenetic estimation of sequence data include distance based methods namely Neighbor-Joining and UPGMA (Unweighted Pair-Group Method with Arithmetic mean); in addition to the algorithms of Parsimony, Maximum likelihood and Bayesian (WEIß; GÖKER, 2011). According to Heath, Hedtke and Hillis (2008) accurate phylogenetic estimates of true historical relationships among species are determined by four main factors: (1) appropriate selection of target genes for analysis; (2) collection of enough sequence data to obtain a robust and repeatable estimate; (3) use of accurate analytical methods; and (4) sufficient taxon sampling. Although, single gene sequence offers the advantage of rapid species identification, nucleotide substitution rates of diagnostic genes often vary within lineages, hence, species inference using single gene analysis usually suffer limitations such as the inability to clearly defined species boundaries as well as weak support for basal branches. On the other hand, multigene analysis 35 offers the opportunity of detecting nucleotide substitution rates and basal lineages are generally well supported (KURTZMAN; ROBNETT, 2003; KURTZMAN; FELL, 2006). Scorzetti et al. (2000) proposed the analysis of combined sequences of the D1/D2 and ITS regions for species identification after examining the resolution provided by these rRNA regions for the identification of basidiomycetous yeasts in the Pucciniomycotina and Agaricomycotina. Using a multigene approach of combined sequences of SSU, LSU, ITS, mitochondrial small subunit rDNAs with elongation factor 1-α and cytochrome oxidase II, species of the Saccharomyces complex were phylogentically resolved into 14 clades including 4 (Saccharomyces sensu stricto, Zygosaccharomyces sensu stricto, Torulaspora and Eremothecium) well-supported monophyletic clades (KURTZMAN; ROBNETT, 2003). By analyzing the combined sequences of SSU, LSU, ITS region and mitochondrial cytochrome b gene, Wang and Bai (2008) clearly separated three monophylectic clades namely, Dioszegia clade, Derxomyces mrakii and Hannaella sinensis from Cryptococcus luteolus lineage of the Tremellales. A similar approach by Kurtzman, Robnett and Basehoar-Powers (2008) found species formerly assigned to Issatchenkia to belong to the Pichia membranifaciens clade after the analysis of concatenated gene sequences from EF-1α and the LSU and SSU rRNA genes, hence, were consequently transferred to the genus Pichia. Phylogenetic tree constructing methods do not accurately measure gene genealogies of haplotypes resulting from intraspecific polymorphisms (CLEMENT; POSADA; CRANDALL, 2000). The extent of intraspecific genetic and phenotypic (morphological and physiological) variations among yeast strains that share a common gene pool have been continuously revealed by several studies (LACHANCE et al., 2010, 2011; DAYO-OWOYEMI et al., 2012) and this phenomenon is more pronounced in basidiomycetous yeasts as was exemplified in the work of Scorzetti et al. (2002). The use of Parsimony network analysis (TCS) have been shown to offer accuracy for the circumscription of phylogenetic species in the light of correctly measuring genetic discontinuities between species while at the same time revealing where such discontinuities only represent alleles of a locus or haplotypes within a species (CLEMENT; POSADA; CRANDALL, 2000; POSADA; CRANDALL, 2001; HART; SUNDAY, 2007). The accuracy of the TCS analysis for differentiating species based on barcoding sequences in higher organisms was thoroughly reviewed by Hart and Sunday (2007) and they reported the method as having a high positive rate to identify known species boundaries as well as for discovering new species from sequence data 36 especially where such taxa were adequately sampled. The inter-specific sequence variation and high discriminative power of the ITS region have assisted in solving many taxonomic and systematic problems relating to separation of species, hence it has served the role of a DNA barcode marker for some fungi identification (BEGEROW, 2010). However, the ITS region is often highly variable within species (BEGEROW, 2010), consequently, members of the same species may be treated as being separate when pairwise ITS sequence divergence is used as a means of species delimitation. Nevertheless when combined with the D1/D2 region for parsimony network analysis, intraspecific discontinuities can be identified (LACHANCE et al. 2010, 2011). 1.5 Yeast classification Yeasts are classified under two broad taxonomic groups, i.e. ascomycetes and basidiomycetes; each comprising anamorphic and teleomorphic states. These two groups of yeast differ in their cell wall composition and molecular structure and also in their mode of bud formation (i.e. asexual reproduction) and spore formation (sexual reproduction). Yeast cellular compositions vary with their phylogenetic diversity, as observed by the variety in the biochemical composition of the cell walls, ultrastructural organization and morphology of the septa (VAN DER KLEI et. al. 2011). However, cell walls of ascomycetous yeasts consist of two layers: an inner skeletal layer consisting of load-bearing polysaccharides and an outer layer consisting of glycoproteins that are covalently linked to the inner layer (YAMAGUCHI et al. 2002; SUH et al. 2006, VAN DER KLEI et al. 2011). In basidiomycetous yeasts, the walls are often, but not always, multilayered with alternating regions of electron-dense and electron- translucent material. While the cell walls of ascomycetous and basidiomycetous fungi contain a similar β-1,3-glucan, cell wall polysaccharide composition is dominated by chitin in the basidiomycetes (SUH, 2006, VAN DER KLEI et. al., 2011). In the ascomycetous yeasts, budding (vegetative reproduction) is holoblastic, in which budding results from the stretching out of the entire cell wall of the mother cell; the bud separates from the narrow base leaving a scar through which no further budding occurs. On the other hand, in the basidiomycetous yeasts, budding is enteroblastic, in which newly formed bud cell rupture the cell wall of the mother cell, resulting in the formation of collaret due 37 to recurrent formation and separation of a succession of buds (KURTZMAN; FELL; BOEKHOUT, 2011a). In ascomycetous yeasts, sexual reproduction occurs through spore formation whereas; sexual states of basidiomycetous yeasts are characterized by formation of septate dikaryotic hyphae with clamp connections. Basidiomycetous yeasts can also be differentiated from ascomycetous yeasts based on Diazonium Blue B (DBB) test. A dark red reaction is observed on the former when a buffered solution of Diazonium Blue B (DBB) is applied to cultures left overnight at 60 °C (BOEKHOUT et al., 2011). i. Phylogenetic classification of ascomycetous yeasts The relationships among the ascomycetes are becoming clearer since the introduction of multigene sequence analysis for the estimation of phylogeny; and consequently have resulted in changes in the classification of the ascomycetous yeasts. For instance, the reassignment of some formerly Pichia species (e.g P. anomala) to a new genus Wickerhamomyces after the polyphylectic nature of Pichia species was further confirmed by phylogenetic analyses (KURTZMAN; ROBNETT; BASEHOAR-POWERS, 2008; KURTZMAN, 2011). Another important change is the re-assignment of the saturn spored genus Williopsis into four distinct genera namely Barnettozyma, Lindnera, Ogatae and Wickerhamomyces. Based on the latest phylogenetic classification by Hibbett et al. (2007), the phylum Ascomycota is divided into three subphyla (their respective classes are provided in parenthesis): Subphylum Taphrinomycotina (classes Taphrinomycetes, Neolectomycetes, Pneumocystidomycetes, Schizosaccharomycetes) Subphylum Saccharomycotina (class Saccharomycetes), Subphylum Pezizomycotina (classes Arthoniomycetes, Dothideomycetes, Eurotiomycetes, Arthoniomycetes, Dothideomycetes, Eurotiomycetes, Laboulbeniomycetes, Lecanoromycetes, Leotiomycetes, Orbiliomycetes, Pezizomycetes, Sordariomycetes) (Figure 1.2). This phylogenetic classification was previously revealed by a single gene analysis based on 5S rRNA gene sequence (WALKER, 1985). Multigene sequence of concatenated data of SSU, LSU, 5.8S rRNA, elongation factor 1-α (EF1α), and two RNA polymerase II subunits (RPB1 and 38 Figure 1.2 Phylogeny of the phylum Ascomyceota showing the classification of Ascomycetous yeasts (Hibett et al. 2007). 39 RPB2) (JAMES et al., 2006) confirmed that ascomycetous yeasts belong to a single lineage, Saccharomycotina. Yeasts in the subphylum Taphrinomycotina are classified in the order Schizosaccharomycetales (Schizosaccharomycetes) while the subphylum Pezizomycotina consists majorly of filamentous fungi. The teleomorphic genus Schizosaccharomyces Linder is the only genus of yeasts currently known in the Schizosaccharomycetales and currently includes 3 species namely Schiz japonicas, Schiz. pombe and Schiz.octosporus. Some distinct characteristics of this genus is their cylindrical shape and asexual form of reproduction which is by fission (VAUGHAN-MARTINI; MARTINI, 2011). ii. Phylogenetic classification of basidiomycetous yeasts Basidiomycetous yeasts including sexual and asexual forms are currently classified into three subphyla namely (i) Subphylum Pucciniomycotina, (formerly class Urediniomycetes), (ii) Subphylum Ustilaginomycotina (formerly class Ustilaginomycetes) and (iii) Subphylum Agaricomycotina (formerly Hymenomycetes) (Figure 1.3) (HIBBETT et al. 2007). The presence of three main lineages within the Basidiomycota was earlier demonstrated by many phylogenetic studies including sequence analyses small-subunit SSU (FELL et al. 2000), LSU and ITS (SCORZETTI et al. 2002). Basidiomycetous yeasts accommodated in the Pucciniomycotina are distributed in the class Agaricostilbomycetes (e.g. Kondoa spp., Chionosphaera sp. and the asexual genera Bensingtonia, Kurtzmanomyces, Sporobolomyces and Sterigmatomyces); class Cystobasidiomycetes (e.g. some pink-colored asexual genera: Bannoa, Cyrenella, Rhodotorula, Sporobolomyces, Erythrobasidium and some sexual and dimorphic species such as Cystobasidium, Occultifur, Naohidea and Sakaguchia) and class Microbotryomycetes (e.g Rhodosporidium and Sporidiobolus and their asexual counterparts, Rhodotorula and Sporobolomyces, also the teliospore-forming genera Leucosporidium and Mastigobasidium, and their asexual state Leucosporidiella (BOEKHOUT et al. 2011a). The subphylum Ustilaginomycotina are the smut fungi and their relatives and whose sexual states are characterized by transversely septate auricularoid basidium. They are distributed in the class 40 Figure 1.3. Phylogeny of the phylum Basidiomycota showing the classification of Basidiomycetous yeasts (Hibbett et al. 2007). 41 Ustilaginomycetes and class Exobasidiomycetes. The Exobasidiomycetes include anamorphic species such as Tilletiopsis and its teleomorph Tilletiaria, Acaromyces, Meira, Malassezia and Sympodiomycopsis while only two species namely Farysizyma and Pseudozyma are currently classified in the Ustilaginomycetes. The subphylum Agaricomycotina formerly known as Hymenomycetes comprises three classes, namely Agaricomycetes, Dacrymycetes and Tremellomycetes (HIBBETT et al. 2007). Yeast states are found only in the Tremellomycetes and are classified in four distinct orders namely Tremellales, Trichosporonales, Filobasidiales and Cystofilobasidiales (SCORZETTI et al. 2002). While ballistoconidia may be produced by members of the Tremellales (e.g., Bullera, Dioszegia, Kockovaella) and the Cystofilobasidiales (e.g., Udeniomyces), distinct morphological trait of the Trichosporonales (e.g Trichosporon chiarellii, T. ashaii, T. insectorum) is the formation of arthroconidia. Some sexual states of the Tremellales such as Auricullibuller fuscus, Bulleribasidium and Papiliotrema are characterized by the formation of conspicuous basidiocarps and tremelloid haustoria branches that are adapted to their mycoparasitic mode of life (BOEKHOUT; FONSECA; BATENBURG-VAN DER VEGTE, 1991; SAMPAIO et al. 2002). The teleomorphic genus Tremella and the anamorphic genera Bullera and Cryptococcus are polyphyletic and latter is distributed in all the four lineages of the Tremellales (SCORZETTI et al. 2002). Three teleomorphic genera are dinstincted in the Cystofilobasidiales namely Mrakia, Cystofilobasidium and Xanthophyllomyces. The first two genera are characterized by the formation of teleospores. Examples of species found in this lineage include species of the cold tolerant genus Mrakia (e.g M. frigida and M. gelida) and the carotenoid pigmented species of Cystofilobasidium e. g Cystofilobasidium infirmominiatum. Only one species is recognized in the genus Xanthophyllomyces, namely X. dendrorhous (anamorph Phaffia rhodozyma). The Xanthophyllomyces have a unique mode of sexual reproducition involving cell to cell mating to produce basidiospores on the apex of an elongate basidium (GOLUBEV, 1995). They are important for their carotenoid pigment, astaxanthin (JOHNSON, 2003). The monotypic anamorphic species Guehomyces pullulans and arthroconidia forming genus Tausonia pamirica are also phylogenetically placed within the Cystofilobasidiales (BOEKHOUT et al. 2011). 42 1.6 Yeast ecology and diversity Yeasts are typically known as decomposers of dead organic matter in which they bring about nutrient transformation. They can engage in intimate relationships with other organisms as mutualists, competitors, parasites, or pathogens (STARMER; LACHANCE, 2011). Yeasts are widely distributed in almost all biomes of the world and their ubiquity is complemented by their diversity (KURTZMAN; FELL; BOEKHOUT, 2011c). They grow well in moist environments where there is availability of simple sugars, amino acids. Some are however able to degrade complex polysaccharide such as starch, cellulose, hemicellulose and pectin (ALONSO et al. 2010; BIELY; KREMNICKÝ, 1998). The following discussions of diversity of yeasts in various habitats relate to some of the isolation sources of the yeasts used in this study. i. Yeasts associated with plants (flowers and fruits) Due to their rich content of easily utilizable carbon, fruits serve as natural habitats for a variety of yeasts. Yeasts particularly ascomycetes are distributed on surfaces of fruits, exudates of leaves, flowers and tree trunks. Prada and Pagnocca (1997) reported the isolation of two hundred and two strains of yeasts and yeast-like fungi from naturally occuring fruits in the tropical rain forest of Juréia-Itatins Ecological Reserve, with 38 species constituting 74 % of the total isolates been ascomycetous. The finding that naturally occurring apple yeasts can protect fruit against postharvest diseases spurred interest in the isolation of yeasts from various fruits with the aim of discovering new yeast antagonists against postharvest diseases (JANISIEWICZ, 1987). Certain yeasts associated with fruits can produce sugar-derived prebiotics such as fructooligosaccharides. According to Maugeri and Hernalsteens (2007) apud Johnson and Echavarri-Erasun (2011), yeasts from fruits and flowers in Brazilian tropical forests including Candida, Rhodotorula, and Cryptococcus produced substantial quantities of fructooligosaccharides. ii. Yeasts associated with leaf phylloplane Plant surfaces harbor numerous and diverse microbial communities in which yeasts, particularly basidiomycetous yeasts, are among the most frequently membered (INÁCIO, 2002; 43 FONSECA; INÁCIO, 2006). Basidiomycetous yeasts particularly species of the genera Sporobolomyces, Rhodotorula, Cryptococcus, Bensingtonia, Cystofilobasidium, Leucosporidium and Pseudozyma are dominant on leaf phylloplanes (MAKSIMOVA; CHERNOV, 2004; STAMER; LACHANCE, 2011). Species of Bullera, Sporobolomyces and Tilletiopsis are particularly adapted to this environment due to the production of forcibly ejected ballistospores (FONSECA; INÁCIO, 2006). A survey on the phylloplane yeasts from Mediterranean plants (Acer monspessulanum, Quercus faginea, Cistus albidus and Pistacia lentiscus) collected at the ‘Arrabida Natural Park,’ Portugal revealed the high frequency of occurrence of species of Taphrina and Lalaria including five previously unknown species of the latter (INÁCIO, 2004). In an investigation studying the yeasts community colonizing the leaf surfaces of various fruit trees in southwest Slovakia, Slávicová, Vadkertiová and Vránová (2009) isolated 150 strains belonging to seventeen yeast species out of which Aureobasidium pullulans, Cryptococcus laurentii, and Metschnikowia pulcherrima were the most abundant species isolated. Yeasts are also found living in fruiting bodies of mushrooms, which has also been the source of many ascomycetous and basidiomycetous yeast species (NAKASE et al. 1999; BABJEVA et al., 2000; MIDDELHOVEN, 2004). Yurkov et al. (2012) isolated various yeasts species of Rhodotorula, Rhodosporidium, Mastigobasidium, Cryptococcus, Cystofilobasidium, Holtermanniella, Trichosporon and the ascomycetous Kluyveromyces from Boletales fruiting bodies truffle ascocarps. Nakase et al. (1999) isolated three new species of yeasts namely Candida fungicola, C. sagamina and C. fukazawae from fruiting bodies of unidentified mushrooms collected from Tanzawa Mountains, Kanagawa Prefeitura. iii. Yeasts associated with insects Yeasts have been isolated from insects in many different families. While such insects serve the primary role of dispersing yeasts to new habitats, many of these yeasts have also been shown to improve insect nutrition and to detoxify plant chemicals to which insects are exposed (SUH et al., 2005; LACHNACE et al., 2001, ROSA et al., 2003). Nests of fungus-growing ants provide a suitable habitat for yeasts (CRAVEN et al. 1970; CARREIRO, 1997; 2000). Fungus-growing ants of the higher-attini lineages (Atta and 44 Acromyrmex) exclusively cut fresh leaves and plant material to cultivate their mutualistic fungus (WEBER, 1972; MUELLER; REHNER; SCHULTZ, 1998). Breakdown and transformation of the protein and starch rich plant materials by the cultivated fungus make nutrients available within the fungus garden matrix and this is a major target for exploitation by other microorganisms including yeasts (CARREIRO et al., 1997; RODRIGUES et al., 2009; SCOTT et al., 2010). Yeasts associated with fungus-growing ant nests contribute to the stability of the complex microbiota found in the leaf-cutting ant nests through nutrients generation and removal of potentially toxic compounds (MENDES et al., 2012). Carreiro (1997) identified yeasts species of the genera Candida, Pichia, Cryptococcus, Rhodotorula, Sporobolomyces, Tremella and Trichosporon from the nests of the leaf-cutting ant Atta sexdens. Rodrigues et al. (2009) investigated the diversity of yeasts in Atta texana gardens and isolated ascomycetous yeasts belonging to the genera Aureobasidium, Candida, Kodamaea, Saccharomyces and basidiomycetous yeasts of the genera Bullera, Bulleromyces, Cryptococcus, Pseudozyma, Rhodosporidium, Rhodotorula, Sporidiobolus and Trichosporon. The dispersal of yeasts by leaf-cutting ants was studied by Pagnocca et al. (2008) and it was revealed that these insects harbor various species of yeasts in their body including several opportunistic human pathogens e.g. Candida parapsilosis and C. metapsilosis, hence, may serve as vectors of these pathogens. Another groups of yeasts commonly found associated with fungus- growing ants are black yeasts in the genus Phialophora. Black yeast is the term used to refer to groups of yeasts characterized by melanized cell wall (STERFLINGER, 2006). These yeasts grown on the cuticle of the ants and are considered symbionts that play antagonistic roles in the fungus-growing ant mutualism (LITTLE; CURRIE, 2008). Although the association of yeasts with the wasps Polybia ignobilis has not been extensively studied, socials wasps were identified as vector and natural reservoir of S. cerevisiae (STEFANINI et al., 2012). Yeasts in Starmerella and neighbouring clades are mostly associated with bees (ROSA et al., 2003). Study involving the characterization of yeasts associated with the wasp Polybia ignobilis revealed the presence of various species including Candida, Cryptococcus, Hanseniaspora and Rhodotorula with Candida azyma, Candida chrysomelidarum, 45 Cryptococcus liquefaciens and Rhodotorula mucilaginosa being most frequently encountered (SANCHEZ DE SOUSA, 2011). Ascomycetous yeasts associated with flowers and exudates are usually found in specialized niches involving interactions with insects or other invertebrate animals that they rely upon for dispersal (MORAIS et al., 1992; SUH et al., 2005; LACHNACE et al., 2001; ROSA et al., 2003). Lachance et al. (2001) reported the affiliation of yeasts in the Metschnikowia, Kodamaea, Wickerhamiella, and Starmerella clades with these floricolous insects that visit Hibiscus flowers and some flowers in the families Convolvulaceae and Cactaceae. Beetles may have a yet unclear strong symbiotic relationship with yeasts; possibly, the yeasts may carry out the transformation of scarce and poorly digestible components of flowers into a richer diet for the insects (STARMER; LACHANCE, 2011). iv. Yeasts associated with honey Ability of some yeast to tolerate low temperature, low oxygen concentration, high acidic conditions, high osmotic pressure or high salinity are important adaptive properties that determine their ability to survive under restricted habitats. Zygosaccharomyces rouxii and Z. bailii are often implicated in the spoilage of honey and jam because of their unique abilities to tolerate the high osmotic stresses and low water activity (MARTORELL et al. 2007). Other yeasts such as Z. bisporus, Z. mellis, Schizosaccharomyces pombe, Torulaspora delbrueckii, Debaryomyces hansenii, and various Candida and Moniliella species are commonly associated with foods containing high concentrations (40-70%) of sugar such as honey (FLEET, 2011). v. Yeasts found in soil Many studies on the biological diversity of various soil types have shown that soil is a diverse habitat dominated by invertebrates, prokaryotes and fungi including yeasts (BOTHA, 2006, VISHNIAC, 2006a). In fact, majority of described yeasts species were isolated in the soil environment. Recently seven new yeast taxa including Clavispora reshetovae, Barnettozyma vustinii and Leucosporidium drummii were discovered during an investigation on yeast diversity in soils (YURKOV; SCHÄFER; BEGEROW, 2012). Soil yeasts play important roles, which include soil aggregation formation and maintenance of soil structure, mineralization of organic 46 materials and carbon cycling, whereby they serve as mutrient sources for bacteria and other predators in the soil (FITTER et al. 2005; BOTHA, 2006; 2011). Ecological soil surveys revealed the most abundant yeast soils to include Cryptococcus albidus, Cr. curvatus, Cr. gastricus, Cr. gilvescens, Cr. humicolus, Cr. laurentii, Cr. podzolicus. Cr. erreus, Filobasidium uniguttulatum, Cystofilobasidium capitatum, Leucosporidius scottii, Mrakia frigida, Rhodotorula aurantiaca, R. glutinis, R. mucilaginosa, Sporobolomyces roseus, Trichosporon cutaneum and Schizoblastosporion starkeyi-henricii (BOTHA, 2006). Recently, Yurkov; Schäfer and Begerow (2012b) investigated the diversity of cultivable yeasts in soils under different land use and isolated 40 yeast species, 11 of which had earlier been reported from soil, i.e. the basidiomycetous Cryptococcus aerius, Cr. laurentii, Cr. terreus, Cr. terricola, Cr. podzolicus, Geotrichum pullulans and the ascomycetous Barnettozyma californica, B. pratensis, Schwanniomyces (Debaryomyces) occidentalis, Lindnera (Williopsis) saturnus and Schizoblastosporion starkeyi-henricii. vi. Yeasts in Antarctic environments Many species of yeasts have been found to successfully colonize the Antarctic continent. While some of these yeasts are Antarctic indigenes and obligate psychrophiles that do not survive when subjected to temperatures different from those obtainable in their natural habitat (i.e., > 20 °C), several could be considered as non-indigenes that were brought in by wind and ocean currents as well as by birds, humans and other animals who occasionally visit this habitat, and became adapted to Antarctic habitat (VISHNIAC, 1996; 2006b). The latter groups are mostly psychroptrophs and mesophiles, which equally grow and multiply at room temperature (25 ± 2 °C), although may remain dormant for a long time at low temperatures. The ability to tolerate low temperature, high salinity, high radiation and other extreme conditions are fundamental adaptations of yeasts found in Antarctic environments (RAY et al., 1989; ROBINSON, 2001; SHIVAJI; PRASAD, 2009). Yeasts such as Candida psychrophila, Leucosporidium antarcticum, Cr. vishniacii, Mrakia frigida, Mrakia robertii and Mrakia blollopis are obligate psychrophiles are not able to grow at temperatures above 20 °C (VISHNIAC, 2006b; THOMAS-HALL et al., 2010). Other yeasts that have been isolated from various antartic samples include Candida sake, Cryptococcus antarcticus, Cr. victoriae, Cr. watticus, Cr adeliensis, Dioszegia hungarica and 47 Leucosporidium scottii (VISHNIAC, 2006b; VAZ et al., 2011). Some of them are mesophilic yeasts that became adapted to Antarctic habitat. The extreme environmental conditions obtainable in the Antartica means that microorganisms found there would have evolved unique characteristics for survival that could be exploited for biotechnological applications. Hence, yeast biodiversity of the Antarctica has raised interest for bioprospection for novel enzymes and biomolecules (SHIVAJI; PRASAD, 2009; VAZ et al., 2011). 1.7 Biotechnological importance of yeasts The biotechnological potentials of yeasts have been exploited by man in many industrial processes ranging from food industries to the biofuel industries where yeast is used for the production of bioethanol. Yeasts, especially Saccharomyces cerevisiae are used for making various fermented products such as beer, wine, bakery products, cheese etc. They are also used in the production of enzymes, biocatalysts, pigment, flavours, and pharmaceutical prouducts as well as as biocontrol agents (JOHNSON; ECHAVARRI-ERASUN, 2011). Meyerozyma (Pichia) guilliermondii is known as a hyper producer of riboflavin (SIBIRNY; BORETSKY, 2009). Due to the ease of growth and genetic manipulation, yeasts such as S. cerevisiae Schizosaccharomyces pombe are used as model organisms for genetic studies (TAKEGAWA, et al., 2009). Other biotechnological potentials been investigated in yeasts include phenol and alkane degradation by Candida maltosa and C. tropica, production of biosurfactants by Pseudozyma spp., production of heterologous protein by Schizosaccharomyces pombe, fermentation of xylose to ethanol by Scheffersomyces stipitis and production of lipids and single cell oil by Yarrowia lipolytica (SATYANARAYANA; KUNZE, 2009). 1.8 Biodegradation of starch, lignocelluloses and pectin Plant cell walls are the planet`s dominant form of lignocellulose biomass. The main structural polymers are: cellulose, a homopolymer of β-(1,4)-linked cellobiose residues which make up 40 %; hemicelluloses, a branched cross-linking heteropolymer of varied compositions comprising an average of 33 % and 23 % lignins and are strongly intermeshed and chemically linked by non-covalent forces and by covalent cross-linkages (Figure 1.4) (LODISH.; BERK; 48 Figure 1.4. Plant cell wall structure. Source: Lodish et al. (2000) ZIPURSKY et al., 2000; PÉREZ et al., 2002; HOWARD et al., 2003). Hemicelluloses are polysaccharides consisting of xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan present along with cellulose in plant cell walls (LODISH et al. 2000; SCHELLER; ULVSKOV, 2010). Ability of yeasts and yeast-like fungi to degrade plant polysaccharide is of great importance in ecology and physiology as well as in biotechnology. This is due to the ample information that can be obtained about their biotechnological potentials such as their usefulness in the production of industrially important enzymes as well as in the conversion of complex polymers to useful products e.g biological fuels. Table 1.3 gives a summary of industrial applications of some enzymes. 49 Table 1.3. Industrial applications of some enzymes (KIRK, BORCHERT, FUGISANG, 2002) 50 i. Cellulose degradation Cellulose is the most abundant renewable organic polymer in the biosphere and is highly crystalline, water insoluble and relatively resistance to depolymerization. The degradation of cellulose to glucose requires the synergistic action of three distinct classes of enzymes namely, (i) endoglucanases, (ii) exoglucanases and (iii) β-glucosidases (cellobiases) (EC 3.2.1.21) (KARMAKAR; RAY, 2011) (i) The ´´endo-1,4-β-glucanase`` or 1,4-β-D-glucan 4- glucanohydrolases (EC 3.2.1.4), breaks internal bonds of cellulose i.e., β(1→ 4) linkages, to disrupt the crystalline structure and expose individual cellulose polysaccharide chains. (ii) The ´´exo-1,4- β-D-glucanases`` include both the 1,4- β-D-glucan glucohydrolases (EC 3.2.1.74) which liberate D- glucose from 1,4-β-D-glucans and 1,4- β-D-glucan cellobiohydrolase (EC 3.2.1.91) which liberates D-cellobiose from 1,4- β-glucans; resulting in tetrasaccharides or disaccharides, such as cellobiose. (iii) The ´´β-D-glucosidases`` or β-D-glucosidase glucohydrolases (EC 3.2.1.21) hydrolyses the exoglucanase product into individual monosaccharides i.e. release of D-glucose units from cellobiose (Figure 1.5) (KARMAKAR; RAY, 2011). Cellulose activities have been found in yeasts such as Candida glabrata, C. stellata, C. sheatae, Kloeckera apiculata (STRAUSS et al., 2001) Aureobasidium pullulans (KUDANGA; MWENJE, 2005; THONGEKKAEW; KHUMSAP; CHATSA-NGA, 2012). Trichosporon cutaneum, T. pullulans (STEVENS; PAYNE, 1977) and Gueomyces pullulans (SONG et al. 2010). Cellulases have wide industrial applications including in the pulp and paper industry for enhancement of drainage and beatability of pulp, in the textile industry for finishing of cellulose- based textiles, biostoning of denim garments and softening and defribillation of garments; in the bioethanol industry for the fermentation of biomass into Biofuels. In the wine and brewry industry, cellulose is added to malt to reduce the viscosity of wort, hence, improve the filterability. Other industrial uses include the manufacture of detergents; extraction and clarification of vegetable and fruit juices as well as for improvement of digestibility of animal feed. 51 Figure 1.5. Action of major cellulase enzymes. Endoglucanases cleave internal β-1,4-linkages; exoglucanases cleaves two to four units from the ends of the exposed chains produced by endoglucanase while β-glucosidases cleave cellobiose to glucose units (KARMAKAR; RAY, 2011). ii. Xylan degradation The major enzymes responsible for the complete depolymerization of xylan are collectively known as xylanases and are composed of various hydrolases namely β-1,4- endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-glucuronidase, acetyl xylan esterase, and phenolic acid (ferulic and p-coumaric acid) esterase. Endoxylanases (EC3.2.1.8) act on the back bone of xylan specifically β-(1,4)-xylopyranose polymers to produce xygooligosaccharides which after the action of other debranching enzymes are finally converted by β-xylosidase (EC 3.2.1.37) to subunits of xylose (Figure 1.6) (BEG et al. 2001, JEFFRIES, 1994, SUN et al. 2012). Xylanases of yeasts such as Aureobasidium pullulans (LI et al. 1993), Pseudozyma hubeiensis (ADSUL; BASTAWDE; GOKHALE, 2009) and Trichosporon cutaneum (LIU et al. 1998) have been extensively studied while the cold adapted endoxylanase of Cryptococcus adeliensis (CBS 8351) was characterized by Petrescu et al. (2000). 52 Figure 1.6 - Action of major enzymes involved in the depolymerization of xylan (SUN et al., 2012). Xylanases are important in the bleaching of pulp in the paper industry. Potential applications of xylanases also include bioconversion of lignocellulosic material and agro-wastes to fermentative products, clarification of juices, improvement in consistency of beer and the digestibility of animal feed stock. Xylanases are also used in addition with proteases and cellulases directly or indirectly for improvement improve of the strength of the gluten network hence, the quality of bread. Application of xylanase in the saccharification of xylan in agrowastes and lignocellulose biomass for the production of bio-energy intensifies the need of exploiting the potential role of them in biotechnology (SUBRAMANIYAN; PREMA, 2002) iii. Pectin degradation Pectin, the major constituent of plant cell walls, is a complex heteropolysaccharide mainly composed of d-polygalacturonic acid residues (Figure 1.7) (VORAGEN et al. 2009). Pectin degrading enzymes are known as pectic enzymes, pectinases, or pectinolytic enzymes. Diverse enzymes are required for pectin hydrolysis and are classified in two main groups, namely 53 pectinesterases (PE) and depolymerases. Pectinesterases de-esterify pectin by removal of methoxyl residues while depolymerases split the main chain of pectin by hydrolysis of α-(1,4) linkages (BLANCO; SIEIRO; VILLA, 1999, JAYANI; SAXENA; GUPTA, 2005). The depolymerising enzymes are divided into polygalacturonases (PG) [Exo-PG (EC 3.2.1.67), Endo-PG (EC 3.2.1.15) and Polymethylgalacturonases (PMG) (EC 3.2.1.15)], enzymes that cleave the glycosidic bonds by hydrolysis, and lyases (PL) [Exo-PL (EC 4.2.2.9), Endo-PL (EC 4.2.2.2) and Pectin methyl-lyase (PML) (EC 4.2.2.10)] which break the glycosidic bonds by a β-elimination process (Figure 1.7) (BLANCO; SIEIRO; VILLA, 1999; JAYANI; SAXENA; GUPTA, 2005; HIMMEL et al. 2007). Figure 1.7. Action of major enzymes involved in the deconstruction of pectin. Arrows indicate the point of action of pectinase enzymes. PMG, polymethylgalacturonases; PG, polygalacturonases (EC 3.2.1.15); PE, pectinesterase (EC 3.1.1.11); PL, pectin lyase (EC-4.2.2.10) (JAYANI, SAXENA, GUPTA, 2005). 54 Pectinases find their application in the extraction and clarification of fruit juices, grape must, wine technology, maceration of vegetables and fruits, extraction of vegetable oils as well as in coffee and tea fermentation (JAYANI; SAXENA; GUPTA, 2005). Some yeasts are able to use pectin as carbon source hence, are pectin degrading enzyme producers, however, pectin degrading activity is reported in few genera of yeasts including Candida, Pichia, Zygosaccharomyces, Kluyveromyces, Rhodotorula, Cryptococcus and Trichosporon (BIELY; KREMNCKÝ, 1998). iv. Lignin degradation Structurally, lignin is a non-water soluble amorphous heteropolymer that consist of phenyl propane units joined together by different types of linkages (LODISH et al. 2000; SANCHÉZ, 2009). Coniferyl alcohol is the principal component of softwood lignins, whereas guaiacyl and syringyl alcohols are the main constituents of hardwood lignins. Enzymes that degrade lignin are named lignin-modifying enzymes (LMEs) (Figure 1.8). Four types of LMEs have been characterized namely lignin peroxidases (EC 1.11.1.14), manganese peroxidases (EC 1.11.1.13), laccases (EC 1.10.3.2) and versatile peroxidases (EC 1.11.1.16) (MARTINEZ et al. 2005; DOSORETZ; REDDY, 2007). Only few lignin degrading yeasts have been identified and they include Trichosporon cutaneum (GEORGIEVA et al. 2006), Trichosporon pullulans (SLÁVIKOVÁ et al. 2009) and Rhodotorula glutinis (GUPTA et al. 1990). Basidiomycetous fungi that cause white rot decay of wood are the most efficient degraders of ligninand show complete mineralization of lignin to carbon dioxide and water (DOSORETZ; REDDY, 2007). v. Starch degradation Starch which is a primary photosynthetic product in leaves of plants is a mixture of amylose (α-1,4-linked D-glucose residues) and amylopectin polysaccharide polymers containing both α-1,4-and α-1,6-linked D-glucose residues (HOSTINOVÁ, 2002). Enzymes that bring about the breakdown of the internal α-1,4-glycosidic linkages in starch to low molecular weight compounds, such as glucose, maltose and maltotriose are α-amylases. β-amylase catalyzes the 55 Figure 1.8. Scheme showing the actions of lignin degrading enzymes (MARTINEZ et al. 2005). 56 hydrolysis of the second α-1,4 glycosidic bond cleaving off two glucose units (maltose) at a time while γ-amylase cleaves α-(1-6) glycosidic linkages, in addition to cleaving the last α-(1-4) glycosidic linkages at the nonreducing end of amylose and amylopectin, yielding glucose (Figure 1.9). Yeasts possessing amylolytic systems are of biotechnological interest due to their application in food and energy industries. Some yeasts known to produce amylolytic enzymes include Schwanniomyces occidentalis, and species in the genera Cryptococcus, Candida, Dioszegia, Sporobolomyces, Pichia, Schwanniomyces, Saccharomycopsis, Aureobasidium, and Galactomyces (STRAUSS et al. 2001; BUZZINI; MARTINI, 2002; BRIZZIO et al. 2007). Amylases have potential application in a number of industrial processes such in bread making, manufacture of glucose and fructose syrups, detergents, fuel ethanol from starches, fruit juices, alcoholic beverages and sweeteners (HOSTINOVÁ, 2002; KIRK, BORCHERT, FUGISANG, 2002). Figure 1.9. Scheme showing the actions of starch degrading enzymes (SIGMA, 2007) 57 REFERENCES ADSUL, M. G,; BASTAWDE, K. B.; GOKHALE, D. V. Biochemical characterization of two xylanases from yeast Pseudozyma hubeiensis producing only xylooligosaccharides. Bioresource Technology, Essex, v. 100, n. 24, p. 6488-6495, dec. 2009. ALONSO, S; ARÉVALO-VILLENA, M; UBEDA, J; BRIONES, A. 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