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Graciete Mary Dos Santos GRACIETE MARY DOS SANTOS EFEITO DA VINHAÇA NA PRODUÇÃO BIOLÓGICA DE ÁLCOOIS E ÁCIDOS ORGÂNICOS VOLÁTEIS POR MEIO DE CONSÓRCIO MICROBIANO CAMPINAS 2015 UNIVERSIDADE ESTADUAL DE CAMPINAS Faculdade de Engenharia Agrícola GRACIETE MARY DOS SANTOS EFEITO DA VINHAÇA NA PRODUÇÃO BIOLÓGICA DE ÁLCOOIS E ÁCIDOS ORGÂNICOS VOLÁTEIS POR MEIO DE CONSÓRCIO MICROBIANO Dissertação de Mestrado apresentado ao Programa de Pós-Graduação da Faculdade de Engenharia Agrícola da Universidade Estadual de Campinas como parte dos requisitos exigidos para obtenção do título de Mestra em Engenharia Agrícola, na Área de Concentração de Água e Solo. Orientador: Prof. Dr. ARIOVALDO JOSÉ DA SILVA Co-Orientadora: Dra. BRUNA DE SOUZA MORAES ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA DISSERTAÇÃO DEFENDIDA PELA ALUNA GRACIETE MARY DOS SANTOS, ORIENTADA PELO PROF. DR. ARIOVALDO JOSÉ DA SILVA. _______________________________________ CAMPINAS 2015 RESUMO No Brasil, o efluente industrial produzido em maior quantidade é a vinhaça, caracterizada por altos níveis de ácidos orgânicos, fósforo, cálcio, potássio e magnésio. O reaproveitamento energético da vinhaça mostra-se como uma alternativa interessante para produção de biocombustíveis ou sub- produtos. Este trabalho avaliou o potencial da vinhaça como fonte de substrato e nutrientes para produção de álcoois e ácidos orgânicos voláteis (AOV) por meio fermentação em batelada utilizando consórcio anaeróbio (lodo de bovinocultura) pré-tratados com choque térmico (TT) e choque ácido- térmico (AT). Foram utilizados dois meios diferentes, de sacarose (S) e de vinhaça (V), sendo a sacarose a principal fonte de carbono. A vinhaça provou ser uma excelente fonte de nutrientes para os microrganismos envolvidos na fermentação butírica, uma vez que a adição de vinhaça melhora significativamente a produção de ácido butírico em comparação com meio de cultura sintético. As máximas concentrações de ácido butírico, iso-butírico e acético foram de 14,13 ± 0,77 g L-1 na amostra ATV B3; 10,34 ± 0,43 g L-1na amostra ATV B2 e; 4,13 ± 0,06 g L-1na amostra TTV B3, respectivamente. O rendimento dos AOV acético, iso-butírico e butírico e de etanol foi mais elevado nas amostras ATV B3 e TTV B3, atingindo valores máximos de 0,14; 0,28; 0,69 e; 0,26 g g-1 carboidratos totais, respectivamente. Não foram encontradas diferenças significativas entre métodos de pré-tratamento e enriquecimento de inóculo, AT e TT no que diz respeito a produção de ácido butírico e etanol. Em escala maior, operando em reator de 1,5 L, a fermentação de vinhaça bruta e melado de cana por consórcio microbiano AU mostrou potencial para produção de solventes como o butanol, uma vez que concentrações elevadas de ácido butírico foram produzidas, com concentração máxima, rendimento e produtividade de 13,85 g L-1; 0,64 g g-1 e; 199,98 mg L h-1, respectivamente. A caracterização microbiológica, pirosequenciamento, revelou a ocorrência em maior abundância de bactérias do gênero Clostridium, principalmente no consórcio AU e Lactobacillus mais abundante nos consórcios TT e AT. Foi identificada uma espécie conhecida pela produção de butanol, o C. pasteurianum no consórcio AU. Contudo, o presente trabalho representa um passo importante no desenvolvimento de um processo industrial para reutilização da vinhaça. A exploração de novos microrganismos e estudo dos fatores que interferem no processo de fermentação como pH, temperatura, nutrientes, densidade da cultura, cargas aplicadas e características do substrato, são fundamentais para o entendimento dos efeitos sinérgicos e antagônicos da associação de culturas. Palavras-chave: cultura mista de microrganismos; ácido butírico; etanol, fermentação em batelada. ABSTRACT In Brazil, industrial waste produced in the greatest amount is vinasse, characterized by high levels of organic acids, phosphorus, calcium, potassium and magnesium. The energy reuse of vinasse shows up as an interesting alternative for the production of biofuels or byproducts. This study evaluated the potential of vinasse as a source of substrate and nutrients for the production of alcohols and volatile fatty acids (VFA) through fermentation batch using anaerobic consortium (cattle sludge) pre-treated with heat shock (TT) and acid-shock thermal (AT). We used two different media, sucrose (S) and vinasse (V), with sucrose being the main source of carbon. The vinasse proved to be an excellent source of nutrients for microorganisms involved in the butyric fermentation, since the addition of vinasse significantly improves the production of butyric acid as compared to synthetic culture medium. The maximum concentrations of butyric acid, iso-butyric and acetic acid were 14.13 ± 0.77 g L-1 in the sample ATV B3; 10.34 ± 0.43 g L-1 in ATV B2 and 4.13 ± 0.06 g L-1 in TTV B3, respectively. The yield of acetate, iso-butyric acid, butyrate and ethanol was higher in ATV B3 and TTV B3 samples, reaching maximum values of 0.14; 0.28; And 0.69; 0.26 g g-1 total carbohydrates, respectively. There were no significant differences between pretreatment and enrichment methods inoculum, TA and TT as regards the production of butyric acid and ethanol. On a larger scale, operating at 1.5 L reactor, crude fermentation vinasse and molasses of sugar cane from AU microbial consortium showed potential for producing butanol as the solvent, since high concentrations of butyric acid was produced, with maximum concentration, yield and productivity of 13.85 g L-1 0.64 g g-1 and 199.98 mg h L-1, respectively. Microbiological characterization, pyrosequencing, revealed the occurrence in greater abundance of the genus Clostridium bacteria, particularly the AU and most abundant Lactobacillus in consortium TT and AT consortia. C. pasteurianum, known for the production of butanol was identified in AU consortium. However, this study represents an important step in the development of an industrial process for reuse of vinasse. The exploration of new microorganisms and study of the factors that interfere in fermentation process such as pH, temperature, nutrients, cultures, applied loads and characteristics of the substrate are critical for understanding the synergistic and antagonistic effects of culture association. Key Word: mixed culture; butyric acid, ethanol, bath fermentation. SUMÁRIO 1. INTRODUÇÃO .................................................................................................................... 29 1.1 Justificativa ....................................................................................................................... 31 1.2 Objetivos ............................................................................................................................ 32 1.2.1 Objetivo Geral ............................................................................................................. 32 1.2.2 Objetivos Específicos .................................................................................................. 32 2. REVISÃO DA LITERATURA ........................................................................................... 35 2.1 Rotas Metabólicas da Fermentação ABE ....................................................................... 35 2.2 Culturas ............................................................................................................................. 38 2.2.1 Clostrídium sp ............................................................................................................. 38 2.2.2 Consórcio microbiano ................................................................................................. 40 2.2.3 Métodos de pré-tratamento de culturas ....................................................................... 42 2.2.4 Técnicas de biologia molecular para identificação da população microbiana ............ 44 2.3 Perspectivas da Produção de Biocombustíveis via Fermentação ................................. 46 2.3.1 Ácido butírico .............................................................................................................. 46 2.3.2 Butanol ........................................................................................................................ 48 2.3.1 Etanol .......................................................................................................................... 52 2.4 Fatores que afetam a solventogênese .............................................................................. 52 2.4.1 pH ................................................................................................................................ 53 2.4.2 Temperatura ................................................................................................................ 54 2.4.3 Pressão Parcial de gases .............................................................................................. 55 2.4.4 Concentração da carga orgânica .................................................................................. 56 2.4.5 Nutrientes no meio de cultura ..................................................................................... 57 2.4.6 Toxicidade dos metabólitos ......................................................................................... 59 2.5 Características da vinhaça ............................................................................................... 60 3. METODOLOGIA ................................................................................................................ 64 3.1 Caracterização da vinhaça ..............................................................................................
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