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Dissertação De Mestrado Diversidade De Fungos Decompositores De Universidade Federal de Minas Gerais Instituto de Ciências Biológicas Departamento de Microbiologia Dissertação de Mestrado Diversidade de fungos decompositores de madeira presentes em ecossistemas brasileiros: bioprospecção de espécies úteis em processo de biodegradação de poluentes aromáticos Barbara Muniz Soares Belo Horizonte 2013 Barbara Muniz Soares Diversidade de fungos decompositores de madeira presentes em ecossistemas brasileiros: bioprospecção de espécies úteis em processo de biodegradação de poluentes aromáticos Dissertação de mestrado apresentada ao Programa de Pós-Graduação em Microbiologia do Departamento de Microbiologia do Instituto de Ciências Biológicas da Universidade Federal de Minas Gerais como requisito parcial para obtenção do título de Mestre em Microbiologia. Orientador: Luiz Henrique Rosa Coorientador: Carlos Augusto Rosa 2013 Departamento de Microbiologia Instituto de Ciências Biológicas Universidade Federal de Minas Gerais Agradecimentos À Deus pela oportunidade de uma vida repleta de possibilidades e de aprendizado; Ao Prof. Carlos A. Rosa pela acolhida e ensinamentos. Agradeço as oportunidades que me foram concedidas, toda orientação profissional e cuidado dispensados a mim durante todos esses anos. É um grande privilégio fazer parte desta equipe de trabalho que tanto admiro; Ao Prof. Luiz H. Rosa pela orientação e confiança. Agradeço pela oportunidade de trabalhar com projetos extraordinários, pela paciência de ensinar e pelo apoio constante. Sua ajuda e solidez foram imprescindíveis para o desenvolvimento deste trabalho; Aos membros da banca examinadora, Dra. Viviane Alves Gouveia e Dra. Patrícia Faleiro Pimentel, a ao membro suplente Dra.Valéria Godinho por terem aceitado o convite e pelas contribuições, essenciais para a melhoria deste trabalho; Ao Programa de Pós-graduação em Microbiologia pela oportunidade; e ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) pelo apoio financeiro; A todos os funcionários do Departamento de Microbiologia, em especial à Fatinha; À Doutora Sônia Cláudia Queiroz pela colaboração e permissão para utilizar a infra- estrutura do Laboratório de Resíduos e Contaminantes (LRC) da Embrapa Meio Ambiente ; Aos amigos do Laboratório de Taxonomia, Biodiversidade e Biotecnologia de fungos e Laboratório de Sistemática e Biomoléculas de fungos pelos bons momentos que compartilhamos e pelo apoio que me deram: Alice, Adriana Lessa, Alexandre, Ana Raquel, Arthur, Camila Gontijo, Camila Rodrigues, Camila Silva, Carla Pataro, César Faria, Cristiane Bigatti, Débora Gonçalves, Denise Scoaris, Fran Pinheiro, Fernanda Piló, Gabriella Breder, Gabriella Montadon, Graziele Mendes, Hebert, Iara Santiago, Isis, Inayara, Jordana, Larissa (Té), Laura Esteves, Lília, Luciana Brandão, Mariana Costa (Zé), Mariana Rocha, Mariana Vieira, Mirna, Michelle, Monaliza, Natália Pereira, Priscila, Raquel Cadete, Renata Santos, Silvana Safar (Salim), Susana Johann, Vívian e Valéria Godinho. Agradeço a todos pela prontidão em ajudar e tirar dúvidas e pela força que sempre me deram. Agradeço em especial pelas agradáveis horas de almoço, pelos dias das meninas, pela melhor equipe de limpeza do universo e, com certeza, pelos melhores e mais divertidos anos de trabalho que já tive. Em especial, à minha dupla de trabalho Mariana Costa por toda ajuda, companheirismo e amizade. Sou grata pelo ter te conhecido, com você pude aprender o real significado de força e superação. Desejo a você muitas vitórias! As Batutas (Zé, Camila e Té) pelos conselhos preciosos e por me proporcionar as experiências de vida mais absurdas. É bom saber que eu tenho companheiras. A todos os professores e colegas do curso de Pós-graduação em Microbiologia pelos ensinamentos, incentivo e convivência agradável. A minha família querida, agradeço aos meus pais (Wagner e Luiza) por toda fé, dedicação, apoio e amor. Como em tudo que faço vocês são meus exemplos de vida e fazem parte desse projeto. Aos meus irmãos, Thiago e Juninho, por todos os momentos que passamos juntos e que me ajudaram a crescer. Ao Thiago pelo amor, companheirismo e paciência. A todos os familiares pela torcida. SUMÁRIO LISTA DE ABREVIATURAS .................................................................. Erro! Indicador não definido. LISTA DE FIGURAS ............................................................................................................................ III LISTA DE TABELAS ............................................................................................................................ 4 RESUMO ............................................................................................................................................. VIII SUMMARY ............................................................................................................................................ X 1. REVISÃO DA LITERATURA ..................................................................................................... 12 1.1. Xenobióticos .......................................................................................................................... 12 1.2. Corantes industriais ........................................................................................................... 13 1.3. Hidrocarbonetos derivados de petróleo ........................................................................... 17 1.4. Fungos ligninolíticos .......................................................................................................... 19 1.5. Fungos degradadores de corantes e hidrocarbonetos ......................................................... 24 2. RELEVÂNCIA E JUSTIFICATIVA ............................................................................................ 29 3. OBJETIVOS ................................................................................................................................ 32 3.1. Objetivo Geral ....................................................................................................................... 32 3.2. Objetivos Específicos ............................................................................................................. 32 4. MATERIAIS E MÉTODOS ......................................................................................................... 32 4.1. Áreas de coleta ...................................................................................................................... 32 4.2. Coleta e processamento das amostras ................................................................................. 33 4.3. Isolamento e preservação dos fungos .................................................................................. 34 4.4. Extração do DNA Total ...................................................................................................... 34 4.5. Amplificação utilizando o iniciador (GTG)5 ........................................................................ 35 4.6. Amplificação da região ITS ................................................................................................ 36 4.7. Amplificação de fragmento do gene do Fator de Alongamento – 1 alpha ....................... 36 4.8. Purificação dos amplicons ................................................................................................. 37 4.9. Reações de sequenciamento............................................................................................. 37 4.10. Análise computacional das sequências ......................................................................... 38 4.11. Diversidade da comunidade fúngica: cálculo dos índices de abundância, riqueza e dominância .................................................................................................................................... 39 4.12. Triagem dos Corantes.................................................................................................... 40 4.13. Análise da degradação de corantes por dosagem de carbono orgânico total (COT) .... 41 4.14. Triagem do Petróleo ...................................................................................................... 41 I 4.15. Análise da degradação de antraceno por Cromatografia Líquida de Alta Eficiência (CLAE) 42 5. RESULTADOS E DISCUSSÃO .................................................................................................. 44 5.1. Distribuição das espécies ...................................................................................................... 44 5.2. Diversidade de fungos potenciais degradadores .................................................................. 54 5.3. Ensaios de degradação .......................................................................................................... 59 5.2. Avaliação da biodegradação ................................................................................................. 87 6. CONCLUSÕES ............................................................................................................................. 91 7. PRODUÇÃO CIENTÍFICA .......................................................................................................... 92 8. REFERÊNCIAS BIBLIOGRÁFICAS .......................................................................................... 92 9. ANEXOS ..................................................................................................................................... 102 ANEXA A – Quadro contendo a formulação dos meios de cultura utilizados. ...............................
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