Produção de celulases por fungos endofíticos e aplicação das enzimas na sacarificação do bagaço de cana-de-açúcar
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Data
2017-09-22
Autores
Marques, Natália Paganini [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Atualmente, há um crescente interesse na produção de bioetanol celulósico como biocombustível de segunda geração, sendo uma alternativa promissora aos combustíveis fósseis. Fungos tem sido constantemente isolados na busca por novas fontes de celulases e os endofíticos são interessantes para esta finalidade, por serem potenciais produtores de enzimas de degradação de material vegetal, incluindo celulases, e pouco estudados neste sendido. O presente estudo teve por finalidade a avaliação da influência das condições de cultivo (isolado e co-cultivo) para a produção de celulases pelos fungos endofíticos Botryosphaeria sp. AM01 e Saccharicola sp. EJC04, por meio de metodologias estatísticas, além da utilização dos extratos enzimáticos na sacarificação do bagaço de cana-de-açúcar pré-tratado hidrotérmicamente com NaOH, otimizando-se também esta etapa empregando-se as ferramentas estatísticas citadas. Para a condição de co-cultivo dos fungos, a quantidade de substrato (5,7g) e a umidade inicial do substrato (60,6%) foram otimizadas visando maximizar a produção de endoglunacase (121,56 U/g). Para o cultivo isolado de Botryosphaeria sp. AM01, foi possível a produção de endoglunacase (168,99 U/g), empregando a concentração de NaNO3 a 6,0 g/L e 5,0 g como quantidade de substrato. Para o cultivo isolado de Saccharicola sp. EJC04, obteve-se a condição ótima para produção de β-glicosidase (750,37 U/g) na concentração de ureia de 0,0836 g/L, teor de umidade de 49,2% e quantidade de substrato de 3,84 g. Os extratos enzimáticos obtidos sob as condições ótimas de cultivo foram utilizados na sacarificação do bagaço pré-tratado hidrotérmicamente com NaOH. A caracterização do bagaço pré-tratado indicou 59,52, 31,62 e 11,32% como teores de celulose, hemicelulose e lignina, respectivamente. A sacarificação realizada com o extrato enzimático produzido por Botryosphaeria sp. AM01 foi mais eficiente quanto à obtenção de glicose, sendo selecionada para os experimentos envolvendo a otimização deste processo. Sob a condição otimizada de sacarificação (carga de bagaço a 18,82%, carga de enzima de 410,71 U totais de endoglucanase, 50 °C, 280 rpm e 24h) obteve-se a concentração máxima de glicose de 8,05 mg/mL. Quanto à presença de inibidores de fermentação no hidrolisado obtido sob condições otimizadas, observou-se a presença de ácido vanílico e ácido ferúlico nas concentrações de 0,111 e 0,054 g/L respectivamente. Não foi detectada a presença de 5-Hidroximetilfurfural, furfural, ácido siringico e ácido cumárico. Sendo baixas as concentrações dos inibidores presentes no hidrolisado, este apresenta potencial para ser utilizado em processos de fermentação alcoólica. Destaca-se a importância dos resultados obtidos como pesquisa básica, uma vez que não são relatados na literatura estudos quantitativos da produção de celulases pelos gêneros estudados.
There is a great interest in the production of cellulosic bioethanol as second generation biofuel and it is a promising alternative to fossil fuels. Fungi have been constantly isolated in the search for new sources of cellulases and the endophytics interesting for this purpose, being potential producers of plant material degrading enzymes, including cellulases, and underexploited in this sense.The present study had as purpose the optimization of culture conditions (monoculture and co-cultivation) for the production of cellulases by the endophytic fungi Botryosphaeria sp. AM01 and Saccharicola sp. EJC04, by statistics methodology, and also the use of enzymatic extracts in saccharification of sugarcane bagasse submitted to hydrothermal pretreatment with NaOH, optimizing this step using those statistical tools. For the fungi co-cultivation, the amount of substrate (5.7g) and the substrate initial moisture (60.6%) were optimized to maximize the production of endoglunacase (121.56 U/g). For the monoculture of Botryosphaeria sp. AM01, it was possible to produce endoglunacase (168.99 U/g), using the concentration of NaNO3 at 6.0 g/L and 5.0 g as substrate amount. For the monoculture of Saccharicola sp. EJC04, the optimal condition for β- glycosidase production (750.37 U/g) was obtained using urea at 0.0836 g/L, initial substrate moisture at 49.2% and substrate amount of 3.84 g. The enzymatic extracts obtained under optimized conditions were used in the saccharification of sugarcane bagasse submitted to alkaline hydrothermal pretreatment with NaOH. The pre-treated bagasse characterization indicated 59.52, 31.62 and 11.32% as the contents of cellulose, hemicellulose and lignin, respectively. The saccharification performed with the enzymatic extract produced by Botryosphaeria sp. AM01 was more efficient in glucose obtainment, being selected for the experiments involving the optimization of this process. Under the optimized saccharification condition (18.828% bagasse load, enzyme loading of 410.71 total U of endoglucanase, at 50 °C, 280 rpm and 24h) the maximum glucose concentration of 8.05 mg/mL was obtained. Regarding the presence of fermentation inhibitors in the hydrolyzate, it was detected the presence of ferulic acid and vanillic acid at the concentrations of 0.111 and 0.054 g/L, respectively. The presence of 5-Hydroxymethylfurfural, furfural, syringic acid and coumaric acid was not detected. Since the concentrations of inhibitors present in the hydrolyzate are low, it has potential to be used in alcoholic fermentation processes. This study highlights the importance of the results obtained as basic research, since quantitative studies of cellulases production by the studied genus are not reported in literature.
There is a great interest in the production of cellulosic bioethanol as second generation biofuel and it is a promising alternative to fossil fuels. Fungi have been constantly isolated in the search for new sources of cellulases and the endophytics interesting for this purpose, being potential producers of plant material degrading enzymes, including cellulases, and underexploited in this sense.The present study had as purpose the optimization of culture conditions (monoculture and co-cultivation) for the production of cellulases by the endophytic fungi Botryosphaeria sp. AM01 and Saccharicola sp. EJC04, by statistics methodology, and also the use of enzymatic extracts in saccharification of sugarcane bagasse submitted to hydrothermal pretreatment with NaOH, optimizing this step using those statistical tools. For the fungi co-cultivation, the amount of substrate (5.7g) and the substrate initial moisture (60.6%) were optimized to maximize the production of endoglunacase (121.56 U/g). For the monoculture of Botryosphaeria sp. AM01, it was possible to produce endoglunacase (168.99 U/g), using the concentration of NaNO3 at 6.0 g/L and 5.0 g as substrate amount. For the monoculture of Saccharicola sp. EJC04, the optimal condition for β- glycosidase production (750.37 U/g) was obtained using urea at 0.0836 g/L, initial substrate moisture at 49.2% and substrate amount of 3.84 g. The enzymatic extracts obtained under optimized conditions were used in the saccharification of sugarcane bagasse submitted to alkaline hydrothermal pretreatment with NaOH. The pre-treated bagasse characterization indicated 59.52, 31.62 and 11.32% as the contents of cellulose, hemicellulose and lignin, respectively. The saccharification performed with the enzymatic extract produced by Botryosphaeria sp. AM01 was more efficient in glucose obtainment, being selected for the experiments involving the optimization of this process. Under the optimized saccharification condition (18.828% bagasse load, enzyme loading of 410.71 total U of endoglucanase, at 50 °C, 280 rpm and 24h) the maximum glucose concentration of 8.05 mg/mL was obtained. Regarding the presence of fermentation inhibitors in the hydrolyzate, it was detected the presence of ferulic acid and vanillic acid at the concentrations of 0.111 and 0.054 g/L, respectively. The presence of 5-Hydroxymethylfurfural, furfural, syringic acid and coumaric acid was not detected. Since the concentrations of inhibitors present in the hydrolyzate are low, it has potential to be used in alcoholic fermentation processes. This study highlights the importance of the results obtained as basic research, since quantitative studies of cellulases production by the studied genus are not reported in literature.
Descrição
Palavras-chave
Fungos endofíticos, Celulases, Biomassa vegetal, Bagaço de cana, Hidrólise, Endophytic fungi, Cellulases, Biomass, Sugarcane bagasse, Hydrolysis