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Ifeloju Dayo-Owoyemi Taxonomic Assessement and Biotechnological Potential of Yeasts Hold at the Unesp

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Ifeloju Dayo-Owoyemi Taxonomic Assessement and Biotechnological Potential of Yeasts Hold at the Unesp 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.
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