Study On Cellulase Production By Mixed Fermentation Of Trichoderma Ressei And Aspergillus Niger

Lignocellulose raw materials derived from crop straw is rich and cheaprenewable resources on earth, Which is hydrolyzed to convet to mixed sugarcontaining glucose and xylose. The mixed sugar is one of the potential andimportant biological transformation materials. The effective development and useof these cellulosic feedstocks is the efficient path to relieve energy crisis anddecrease the grain for industry. In this study, as the original strain, Trichodermareesei is mutated by Co⁶⁰γ-ray. High-yield cellulase stain was breeded and screened;Then response surface method was used to optimize the conditions of mixedfermentation of Trichoderma reesei mutants and Aspergillus niger; Finally, theeffect of cellulose enzymatic hydrolysis of lignocellulosic raw materials wasstudied. The main conclusions are as follows:1) The mutant effect of Co⁶⁰γ-ray on Trichoderma ressei was very well. Afterrepeated mutagenesis under optimal mutagenic dose400Gy,34strains wereobtained. Through cellulose congo-red medium screening and shake flaskre-screening, a high-yield cellulase strain was obtained. The filter paper activity ofthe mutant was1.170IU/mL, increasing by10.58%than the original stain.Hereditary stability test demonstrated that the productivities of mutant from1st to5th serial passage were stable.2) The fermentation conditions for enzyme production by mixed fermentationwere optimized by Box-Behnken response surface methodology. The regressionmodel was established using Design Expert. The optimal enzyme productioncondition were: A.niger delay vaccination time36h, Trichoderma reesei andAspergillus niger inoculation ratio of5:1, the fermentation time6d, the solid-liquidratio2:50（g/mL）, the amount of Tween-804mL/L, pH5.0, liquid volume of250mLshake flask50mL. In this condition, the filter paper activity was1.224IU/mL andthe β-glucosidase activity was0.315IU/mL. The proof experiment proved that thefilter paper activity falled within the95%response prediction interval range, whichshowed that the regression model could describe the real enzeme productionsituation well.3) The factors which influenced the effect of crude enzyme enzymatichydrolysis were explored.The best enzymatic hydrolysis reaction conditions was:cellulase hydrolysis temperature50℃, the optimum pH5, the optimum hydrolysistime5h. In this condition, crude enzyme was used to enzymatic hydrolysis corn stover pretreaded by hydrogen peroxide.The reducing sugar of the hydrolysates was11.047mg/mL and the yield was16.571%.4) Corn stover, wheat straw, rape straw, etc were hydrolyzed by both crudeenzyme and commercial cellulase. The result showed that the content of reducingsugar in both was close. The best effect of enzymatic hydrolysis was corn stoverpretreated by alkaline hydrogen peroxide.5) The result of ammonium sulfate fractionation showed that ammoniumsulfate fractionation could effectively increase the enzyme activity. The cellulaseactivity was up to26.286IU/mg. This enzyme was used to hydrolyze corn stoverpretreated by alkaline hydrogen peroxide. In enzymatic hydrolysis liquid, thexylose was10.207mg/mL, the glucose was12.365mg/mL, the reducing sugarcontent was22.572mg/mL and the yield of reducing sugar was33.286%. There wasno cellobiose in the enzymatic hydrolysis liquid. Mixed fermentation can make thecellulase system more complete.