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Campinas 2015 Samantha Gonçalves Da Fonseca Caracterização E SAMANTHA GONÇALVES DA FONSECA CARACTERIZAÇÃO E MONITORAMENTO DE POPULAÇÕES DE MICRO- ORGANISMOS NITRIFICANTES EM LODO SALINO PROVENIENTE DE TERMINAL PETROLÍFERO. CAMPINAS 2015 UNIVERSIDADE ESTADUAL DE CAMPINAS Instituto de Biologia SAMANTHA GONÇALVES DA FONSECA CARACTERIZAÇÃO E MONITORAMENTO DE POPULAÇÕES DE MICRO- ORGANISMOS NITRIFICANTES EM LODO SALINO PROVENIENTE DE TERMINAL PETROLÍFERO. Dissertação apresentada ao Instituto de Biologia da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Mestra em Genética e Biologia Molecular na área de Genética de Microorganismos. Orientadora: Dra. Valéria Maia Merzel Co-orientadora: Profa. Dra. Cynthia Canedo da Silva Este exemplar corresponde à versão final da dissertação defendida pela aluna Samantha Gonçalves da Fonseca e orientada pela Dra. Valéria Maia Merzel. CAMPINAS 2015 Campinas, 17 de Dezembro de 2015. COMISSÃO EXAMINADORA Profa. Dra. Valéria Maia Merzel Prof. Dr. Tiago Palladino Delforno Prof. Dr. Wellington Luiz de Araújo Os membros da Comissão Examinadora acima assinaram a Ata de Defesa, que se encontra no processo de vida acadêmica do aluno. "Desistir? Eu já pensei seriamente nisso, mas nunca me levei realmente a sério. É que tem mais chão nos meus olhos do que cansaço nas minhas pernas, mais esperança nos meus passos do que tristeza nos meus ombros, mais estrada no meu coração do que medo na minha cabeça". Geraldo Eustáquio de Souza Agradecimentos Agradeço imensamente à minha orientadora Dra. Valéria Maia Merzel por toda confiança em meu trabalho, além da amizade, compreensão, paciência e apoio nos momentos difíceis que não foram poucos durante essa trajetória. À Dra. Cynthia Canedo da Silva por sua co-orientação, processos realizados por sua equipe e compartilhamento de dados e instruções. À Petrobrás, representada pela Dra. Maíra Sousa, Dra. Ana Paula Torres e Dr. Rodrigo Souza, pela disponibilidade da amostra e esclarecimentos sobre os processos. À Secretaria de Pós-graduação do Programa em Genética e Biologia Molecular, em especial à Lourdes, pelas instruções e por todo trabalho desempenhado. Ao corpo administrativo e à equipe de manutenção do CPQBA por todas as contribuições, em especial à Sandra pela solicitude e pelo tratamento sempre tão gentil, ao Luiz por todos os atendimentos sempre simpático e amável, e ao Celso por toda disposição e boa vontade. Ao prestativo e sempre atencioso Dr. Armando Dias (ESALQ - Piracicaba), por toda paciência e ajuda com as padronizações do DGGE. Agradeço imensamente ao Dr. Tiago Palladino pelas análises dos dados obtidos com o sequenciamento massivo, cujo auxílio foi essencial para conclusão deste trabalho. Ao Dr. Rubens Duarte, pela atenção e disponibilidade no esclarecimento de dúvidas e na tentativa de contribuir para soluções dos problemas encontrados com o programa MoThur. Aos professores membros da banca de qualificação, Dr. Fernando Dini Andreotte, Dr. Welington Araújo e Dra. Suzan Pantarotto, e banca prévia Dra. Lucélia Cabral e Dr. Tiago Palladino, o meu muito obrigada pelas valiosas contribuições e sugestões que levaram esse trabalho à sua conclusão. À todos os amigos e colegas da Divisão de Recursos Microbianos, pela convivência harmoniosa, pelas ajudas essenciais, pelo coletivo, pelos encontros de almas, pelos laços sinceros que perdurarão, principalmente à Fabi, Sinhô, Dani, Jujuba, Mary, Naty, Bibis, Tulinho, Jaque, Dai, Babu, Editinha, Fer, Rafa, Paulete, Mi, Bru (Frozinha). Vocês foram e são fundamentais! Pra sempre no coração! À Paty, que me apresentou para uma nova possibilidade, mudando minha vida completamente! Obrigada! Desde os velhos tempos e pra sempre! Ao meu querido esposo Cris, por toda paciência e carinho, por ter segurado a barra comigo, me ajudando e apoiando a cada dificuldade encontrada! Muito obrigada, e que nós tenhamos muitas outras conquistas juntos! Resumo “Caracterização e monitoramento de populações de micro-organismos nitrificantes em lodo salino proveniente de terminal petrolífero”. A indústria petroleira, em seus diferentes processos e unidades, é grande consumidora de água. No processo de separação água-óleo que ocorre nos terminais marítimos aquaviários, é gerada água de produção com altas concentrações de amônia. Lodos ativados são utilizados para remoção do nitrogênio amoniacal e outros poluentes, para que a água retorne para os corpos receptores dentro dos limites estabelecidos pela legislação. O efluente pode chegar aos tanques de tratamento com altas concentrações salinas, o que compromete a nitrificação, principalmente quando está em questão o tratamento da água de produção de campos hipersalinos de exploração. Assim, é de grande importância que os micro-organismos responsáveis pela remoção de nitrogênio amoniacal, sejam caracterizados e monitorados quando submetidos a diferentes concentrações salinas, a fim de que continuem realizando o seu papel transformador neste ambiente. Metodologia: A caracterização da diversidade de bactérias totais e metabolicamente ativas e arqueias totais presente no lodo de sistemas de tratamento de efluentes salinos foi realizada através da construção de biblioteca de DNA que codifica para o gene de RNA ribossomal 16S e também sequenciamento em larga escala via plataforma 454 FLX Roche. Foi realizado também o monitoramento de consórcios microbianos enriquecidos e aclimatados em diferentes concentrações salinas, para os quais observou-se eficiência de nitrificação. O monitoramento foi realizado através da técnica de fingerprint DGGE e também foram feitas bibliotecas de DNA que codifica para o gene de RNA ribossomal 16S a partir dos consórcios aclimatados mais eficientes. Resultados: Tanto nas bibliotecas de DNA que codifica para o gene de RNA ribossomal 16S quanto para sequenciamento em larga escala via plataforma 454, não foram observadas predominância de populações de micro-organismos nitrificantes, porém, dentre os micro-organismos nitrificantes encontrados, há o predominio de bactérias nitrificantes heterotróficas. A maior parte dos micro-organismos encontrados estão relacionados a bactérias não cultiváveis. O monitoramento dos consórcios salinos que apresentaram maior eficiência na nitrificação revelaram a presença de bactérias nitrificantes heterotróficas. Conclusão: O lodo ativado, objeto deste estudo, realiza nitrificação, haja vista os micro-organismos nitrificantes quimioautotróficos e, principalmente, heterotróficos encontrados, associados à presença, em menor quantidade, de Plantomycetes. Os consórcios nitrificantes aclimatados à altas salinidades são dominados por bactérias nitrificantes heterotróficas. Os resultados combinados sugerem que a remoção eficiente da amônia pode ser alcançada quando o lodo ativado é aclimatado com o gradual aumento da concentração salina, fator limitante para a nitrificação, e enriquecido de modo a favorecer a microbiota nitrificante. Abstract "Characterization and monitoring of nitrifying microorganism populations in saline sludge from oil terminal". The oil industry, in its various processes and units, is a major consumer of water. The water-oil separation process that occurs in marine terminals generates produced water which contains high concentrations of ammonia. Activated sludge (a type of biological treatment) is used to remove ammonia and other pollutants, so that the water returns to nature within the limits set by law. The effluent can reach the treatment tanks with high salt concentrations, which undermines the process of nitrification, especially when it concerns the treatment of produced water from of hypersaline oil fields. Therefore, it is of great importance that the bacteria responsible for ammoniacal nitrogen removal are characterized and monitored when subjected to different salt concentrations in order to continue performing its transforming role in this environment. Methodology: The characterization of the diversity of total bacteria and metabolically active and characterization of the diversity of total archaea present in the sludge of saline wastewater treatment systems has been accomplished through the construction of DNA library encoding the 16S ribosomal RNA gene as well as through large-scale sequencing 454 FLX Roche platform. It was also conducted the monitoring of microbial consortia enriched and acclimated at different salt concentrations, for which the nitrification indices were observed. The monitoring was performed by fingerprint DGGE technique and DNA libraries encoding the 16S ribosomal RNA gene were also made from the acclimated consortia that showed the highest levels of nitrification. Results: In both the DNA library encoding the 16S ribosomal RNA gene and the large-scale sequencing through platform 454, there were no predominance of populations of nitrifying micro-organisms. Among the findings, there is a predominance of heterotrophic nitrifying bacteria. Most of the microorganisms that were found are related to uncultured bacteria. The monitoring of the saline consortia that presented the higher nitrification rates revealed the presence of heterotrophic nitrifying bacteria. Conclusion: The activated sludge, object of this study, presents nitrifying potential, given the nitrifying microorganisms found, chemoautotrophs and especially heterotrophic, associated with the presence, to a lesser extent, of Plantomycetes. The nitrifying consortia acclimated to high salinity are dominated by bacteria with potential heterotrophic nitrification. The combined results suggest that efficient removal of ammonia can be achieved when the
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