Study On Screening And Identification Of High-Cellulase Producing Strain,Fermentation Process Optimization And Developing OF Low Cost Inducer

Cellulase can hydrolyze cellulosic material to sugars that can be fermented to ethanol by microorganisms. However, the high cost of cellulase production is still a key barrier in ligncellulosic ethanol production. Therefore, reducing the cost of cellulase and increasing its activity are considered as crucial points for commercial biomass fuel ethanol production. Screening of high-cellulase productivity strains, fermentation medium optimization and development of slow cost inducers are efficient ways to reduce the cost of cellulose production.Industrial cellulose has been recognized as a promising substrate and inducer for cellulase production from Trichoderma. reesei Rut-C30with the advantage of its low cost. In this study, the five-level three-variable Central Composite Design implemented in the package Response Surface Methodology, was used to evaluate the effects of industrial cellulose, wheat bran and soy flour on filter paper activity (FPA). The optimal composition of fermentation medium was determined as follows:industrial cellulose35.62g/L, wheat bran19.37g/L and soy flour38.49g/L. Thus, FPA was reached to9.13IU/mL and1.72times than before, the glucosidase activity was also increased by88.97%.In order to further improve cellulose activity of Trichoderma. reesei Rut-C30, the strain of T. reesei Rut-C30was subjected to mutation involving treatment of Ethyl Methyl Sulfonate (EMS) for24h followed by plasma-irradiation for5min. Mutant T. reesei D-7was proved to be the most promising cellulase producer and mutant T. reesei E-3could produce cellulase in fermentation medium contained30g/L glucose by screening program in shake flask fermentation after different plates screening. The most potential carbon and nitrogen sources were microcrystalline cellulose and yeast extract were determined through fermentation experiment. Then the Response Surface Methodology was used to optimize fermentation medium of T. reesei D-7for cellulase production and the optimal fermentation medium contained microcrystalline cellulose39.48g/L, wheat bran20.83g/L and yeast extract29.27g/L. Thus, the FPA of T. reesei D-7was reached to11.85IU/mL and increased by29.79%compared with T. reesei Rut-C30under above fermentation conditions. In order to develop low cost inducer, The effect of low cost corn starch hydrolyzed by a-amylase on cellulase production of T. reesei D-7was evaluated. The results showed that the FPA of T. reesei D-7was achieved to4.71IU/mL when39.48g/L oligosaccharide was used as carbon source and inducer. Moreover, the FPA of T. reesei D-7was reached to15.62IU/mL when the mixture of20g/L oligosaccharide and30g/L microcrystalline cellulose was added into fermentation medium, the FPA was increased by31.81%compared to contain only microcrystalline cellulose.Finally, the crude cellulase solution was applied to hydrolyze corn stalks in order to evaluate performance of the crude cellulase, which were pretreated by2%(w/v) NaOH for45min under121℃, and obtained biomass with the cellulose content of64.94%. The concentration of cellobiose and glucose obtained was10.65and23.51g/L and the hydrolysis yield was reached to94.68%.