Resumo

Due to concerns on the scarcity of fossil fuels as well as air pollution derived from their use, the interest in biofuel production has increased in the last years. The most widely used substitute of fossil fuels is ethanol, and lignocellulosic biomass is now considered a very attractive alternative source for its production. However, efficient conversion of biomass to ethanol requires the development of technologies for low-cost thermochemical pretreatment, the production of highly efficient enzymes for cellulose and hemicellulose hydrolysis and effective microorganisms for fermentation. The present work aimed the optimization of cellulases and hemicellulases production by the hypercellulolytic mutant Trichoderma reesei RP98 using factorial design. The application of the enzymes for sugarcane bagasse hydrolysis was also investigated. The influence of lactose and yeast extract concentrations and culture time on enzyme production was evaluated using central composite rotational design (CCRD). A 23 factorial design was performed, with 6 axial points and 3 replicates at the central point, resulting in 17 assays. For each run, total protein and CMCase, FPase, avicelase, cellobiohidrolase, β-glucosidase, xylanase and β-xylosidase activities were estimated. Hydrolysis experiments were performed using 10 FPU/g of steam exploded milled bagasse, at 50°C for up to 72h under stirring. Bagasse hydrolysis was also conducted with the commercial enzyme cocktail Accellerase®1500 (Genencor, Inc. U.S. Danisco). Aliquots were taken at different times for determination of total reducing sugars (DNS method) and glucose (GOD method). The best culture conditions for enzymatic production were 0.29% (w/v) yeast extract, 0.25% (w/v) lactose and 144 h. Under these conditions, the production of FPase, CMCase, β-glucosidase and xylanase was increased by 4.6, 5.6, 237 and 140 fold, respectively. The enzymatic cocktail produced released 53% more reducing sugars and 26% more glucose than the commercial cocktail Accellerase®1500 in bagasse hydrolysis. These results clearly demonstrate the valuable aid of the factorial design methodology for the optimization of the production of important enzymes involved in the hydrolysis of biomass. Furthermore, the enzymatic cocktail produced under optimized conditions has potential for industrial application in sugarcane bagasse hydrolysis.