Study On The Conditions Of Cellulase Production By Trichoderma Reesei

Liquid fuels and bio-based chemicals produced from biomass resources such as lignocellulose are of great significance for sustainable economic and social development.The production of cellulosic ethanol contains several processes,including cellulase synthesis,lignocellulose saccharification etc.The cost of cellulase is one of the vital factors for the technical feasibility of biomass conversion to ethanol.Generally,it can be reduced by improving the composition of the enzyme system,using cheap culture medium,and increasing the productivity of the enzyme.Trichoderma reesei has been used as an industrial strain for many years,which is currently the most widely used enzyme-producing strain for enzymatic hydrolysis of cellulose.However,it has a significant disadvantage.It does not produce enough?-glucosidase,therefore its ability to break down cellobiose is insufficient.T.reesei SCB-18 is a high-yielding cellulase strain obtained from restructuring the transcriptional regulatory network by two genetic operations(Acrel,OEbgl1),which reduced carbon catabolite repression and overcame the problem of insufficient?-glucosidase.In this thesis,T.reesei SCB-18 was used as an enzyme-producing strain,and the conditions affecting the enzyme production of SCB-18 were studied.The experiment was scaled up to 5 L fermentor.The main studies and results of this thesis are as follows:1.Effects of Microcrystalline Cellulose(MCC)on Cellulase Production by T.reesei SCB-18The effects of different MCC concentrations on enzyme production were compared.The results showed that:Taking 2%MCC as the basis,adding MCC appropriately could induce enzyme production,and excessive addition could make the fermentation broth too viscous and affect enzyme production.This suggests that the concentration of MCC is as important as the viscosity of the fermentation broth to cellulase production.It was also found that the higher the concentration of MCC is,the slower the rate of enzyme production in the early stage of fermentation is.After the comparison,we found that the optimal concentration of MCC was about 4%.The effects of different feeding methods of MCC on enzyme production were also compared.The results showed that the feeding of MCC did not induce the enzyme production by SCB-18,but restrain it.The filter paper activity of the supernatant from three feeding methods was significantly lower than that of the 4%and 2%MCC medium controls.Regarding the feeding times,we found that the more frequent the feeding is,the more obvious the reduction is.Similar trend was also observed for the feeding amount2.Effect of Surfactant Tween-80 on Cellulase Production by T.reesei SCB-1 8By adding a fixed amount of 0.1%Tween-80 to shake flasks with different concentrations of MCC,we found that adding Tween-80 can significantly improve the enzyme production capacity of T.reesei.Compared with the control,the treatment with 2%MCC concentration increased by 45.8%,and the treatment with 4%MCC concentration increased by 24.5%.By varying Tween-80 concentration with the same carbon source concentration,it was found that although all tested concentrations of Tween-80 can increase the filter paper activity,it is not that the more Tween-80 is added,the better.The higher the substrate concentration,the more Tween-80 is needed3.Optimization of Fermentation Conditions for Cellulase Production by Trichoderma reesei SCB-18 and Addition of Transglycosyl OligosaccharidesThe fermentation medium was optimized,and a fermentation medium formula suitable for T.reesei SCB-18 strain was found.Flask-shaking experiments showed that its enzyme activity of filter paper reached 18.5 u/ml at 120 h,which was 3.04 times higher than that before optimization.The results of partial replacement of MCC with alkali treated straw on the basis of the results above showed that the treatment after optimization was 2.57 times higher than the control,which showed a better industrial application prospect.The results of adding transglycosyl oligosaccharides showed that rather than inducing enzyme production,it caused glucose repression.