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Tássio Brito De Oliveira Fungos Na Compostagem Da UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” unesp INSTITUTO DE BIOCIÊNCIAS – RIO CLARO PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS (ÁREA: MICROBIOLOGIA APLICADA) TÁSSIO BRITO DE OLIVEIRA FUNGOS NA COMPOSTAGEM DA TORTA DE FILTRO: DIVERSIDADE, GENÔMICA E POTENCIAL BIOTECNOLÓGICO Tese apresentada ao Instituto de Biociências, do Câmpus de Rio Claro, Universidade Estadual Paulista, como parte dos requisitos para obtenção do título de Doutor em Ciências Biológicas (Área: Microbiologia Aplicada). Rio Claro Dezembro – 2016 TÁSSIO BRITO DE OLIVEIRA FUNGOS NA COMPOSTAGEM DA TORTA DE FILTRO: DIVERSIDADE, GENÔMICA E POTENCIAL BIOTECNOLÓGICO Tese apresentada ao Instituto de Biociências, do Câmpus de Rio Claro, Universidade Estadual Paulista, como parte dos requisitos para obtenção do título de Doutor em Ciências Biológicas (Área: Microbiologia Aplicada). Orientador: Prof. Dr. André Rodrigues Co-orientadora: Profª. Drª. Eleni Gomes Rio Claro Dezembro – 2016 589.2 Oliveira, Tássio Brito de O48f Fungos na compostagem da torta de filtro : diversidade, genômica e potencial biotecnológico / Tássio Brito de Oliveira. - Rio Claro, 2016 127 f. : il., figs., gráfs., tabs., fots. Tese (doutorado) - Universidade Estadual Paulista, Instituto de Biociências de Rio Claro Orientador: André Rodrigues Coorientadora: Eleni Gomes 1. Fungos. 2. Pirosequenciamento. 3. Cana-de-açúcar. 4. Enzimas lignocelulolíticas. 5. Tratamento de resíduo. 6. Fungos termofílicos. I. Título. Ficha Catalográfica elaborada pela STATI - Biblioteca da UNESP Campus de Rio Claro/SP AGRADECIMENTOS Ao meu orientador, Dr. André Rodrigues, quem muito admiro pela competência profissional e genialidade, pela oportunidade, dedicação e incentivo à pesquisa durante sua orientação, que me abriu caminhos e diversas colaborações, permitindo meu crescimento profissional e pessoal. À minha co-orientadora, Drª. Eleni Gomes, pela contribuição na idealização do projeto, e disponibilização do seu laboratório, desde infraestrutura até material necessário para realização de experimentos. À minha supervisora no exterior, Drª. Nina Gunde-Cimerman, por abrir as portas do seu laboratório e por todo suporte oferecido, desde infraestrutura, até contato e todo material necessário para a realização da pesquisa. À UNESP e ao Programa de Pós-Graduação em Ciências Biológicas (Área: Microbiologia Aplicada) pela oportunidade de estudo e à FAPESP pela bolsa regular de doutorado e a Bolsa Estágio de Pesquisa no Exterior (BEPE) concedidas (processos FAPESP #2012/14594-7 e #2015/25252-8, respectivamente). Ao engenheiro agrônomo Haroldo Castilho pelos ensinamentos sobre compostagem e à Usina Santa Lúcia (Araras, SP) por conceder a torta de filtro para a montagem das leiras. Ao Dr. Adrian Tsang, pela disponibilização dos genomas de fungos sequenciados em seu projeto Genozymes for Bioproducts and Bioprocesses Development. À toda a equipe do Labortório de Ecologia e Sistemática de Fungos, aos novos e aos antigos membros, que de forma direta ou indireta contribuíram com o meu trabalho. Foram quatro anos de muitas conversas, discussões, ajudas, aprendizado, diversão, experiências compartilhadas, tanto dentro quanto fora do laboratório. À toda equipe do Laboratório de Biologia de Micro-organismos da Universidade de Ljubljana, Eslovênia. Agradeço à Polona Zalar e Cene Gostinčar, pelo apoio direto no desenvolvimento da pesquisa. Em especial, à Monika Novak Babič e Monika Kos, minhas companheiras de escritório, por toda amizade, carinho e os momentos de diversão que tivemos. À minha família por todo apoio, incentivo, por sempre acreditarem em mim e que mesmo estando distante não pouparam esforços para realização dos meus sonhos. Em especial à minha Mãe que é um exemplo de força, honra, coragem, companheirismo e determinação que tento sempre seguir. Aos meus amigos de república Ana Maria, Rafael Rodrigues e Gustavo Pedroso. Agradeço pela amizade, cumplicidade, por todo carinho e por não me deixarem desanimar em nenhum instante. Aos que colocaram obstáculos no caminho, pois despertaram em mim a força e a determinação que ainda não conhecia, me fizeram crescer para ser capaz de ultrapassá- los. Sem vocês eu não seria o que eu sou hoje. À todos aqueles, mesmo que o nome não esteja aqui, que de forma direta ou indireta colaboraram para a realização desse trabalho. Muito obrigado! RESUMO A torta de filtro é gerada após o processo de filtração do caldo de cana (cerca de 26 a 40 kg por t de cana) e geralmente é utilizada como fertilizante nas lavouras sem qualquer tratamento prévio. A diversidade de fungos presentes na torta de filtro fresca e no processo de compostagem desse substrato foi acessada utilizando sequenciamento em larga escala. Além disso, fungos tolerantes ao calor foram isolados e avaliados quanto à capacidade de produzir enzimas de degradação da biomassa (celulase, xilanase, lacase e poligalactoronase). Considerando que esse grupo de fungos carece de uma revisão taxonômica atual, aproveitamos as recentes mudanças proporcionadas pelo Código de Nomenclatura para Algas, Fungos e Plantas para gerar uma revisão taxonômica do grupo. Uma gama de patógenos oportunistas foi encontrada entre os taxa mais abundantes na torta de filtro fresca, como Lomentospora prolificans (43,13%), Trichosporon sp. (10,07%), Candida tropicalis (7,91%) e Hormographiella aspergillata (8,19%). Isso indica que a torta de filtro pode ser uma potencial fonte de fungos patogênicos, apresentando riscos para a saúde humana se aplicado como fertilizante sem qualquer tratamento. No entanto, o processo de compostagem reduz efetivamente a carga desses fungos. Além disso, cria um ambiente interessante para fungos capazes de produzir enzimas com potencial aplicação biotecnológica, uma vez que todos os 110 isolados avaliados foram capazes de produzir, pelo menos, uma das enzimas avaliadas. Além disso, a análise comparativa de genes codificantes para peptidases presentes nos genomas de fungos termofílicos (encontrados em sistemas de compostagem) e mesofílicos mostrou que a termofilia levou à várias adaptações para a termoestablidade enzimática. Palavras-chave: Pirosequenciamento. Cana-de-açúcar. Enzimas lignocelulolíticas. Tratamento de resíduo. Fungos termofílicos. Fungos termotolerantes. ABSTRACT Pressmud is derived from sugarcane juice filtrate (around 26 to 40 kg per ton of sugarcane) and it is mainly used as fertilizer in crops without prior treatment. Here, the fungal diversity present in both fresh and composting pressmud was revealed by 454 pyrosequencing. In addition, heat-tolerant fungi were isolated and surveyed for their repertoire of biomass-degrading enzymes (cellulase, xylanase, laccase and polygalactoronase). The fact that the taxonomy of such organisms is still obscure, we revised their taxonomy in the light of the recent changes adopted in the Code of Nomenclature for Algae, Fungi and Plants. A wide range of opportunistic pathogens was found among the most abundant taxa in fresh pressmud, such as Lomentospora prolificans (43.13%), Trichosporon sp. (10.07%), Candida tropicalis (7.91%), and Hormographiella aspergillata (8.19%). This indicates that fresh pressmud may be a source of human pathogenic fungi, presenting a potential threat to human health if applied as fertilizer without treatment. Composting of the pressmud effectively reduces the load of such fungi. Furthermore, the composting system creates an interesting environment for fungi able to produce enzymes with biotechnological applications, since all the 110 isolates screened were able to produce at least one of the tested enzymes.Furthermore, comparative analysis of peptidases genes encoded by thermophilic (generally found in composting systems) and mesophilic fungi showed that thermophyly selected for thermostable enzymes. Keywords: Pyrosequencing. Sugarcane. Lignocellulolytic enzymes. Residue treatment. Thermophilic fungi. Thermotolerant fungi. SUMÁRIO 1 INTRODUÇÃO ............................................................................................................. 8 2 CHAPTER I ................................................................................................................. 12 Thermophilic fungi in the new age of fungal taxonomy ................................................ 12 Abstract ........................................................................................................................... 14 Introduction .................................................................................................................... 15 Origin and ecology ......................................................................................................... 16 Fungal adaptations to thermophily ................................................................................. 18 Taxonomy and name changes......................................................................................... 19 Classification based on environmental sequences .......................................................... 22 Amid the taxonomic disorder, why the thermophilic fungi are interesting? .................. 25 Conclusions .................................................................................................................... 26 References ...................................................................................................................... 27 SUPPLEMENTARY MATERIAL ................................................................................ 34 3 CHAPTER II ..............................................................................................................
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