| In this thesis,the promoting effect of softwood bleached kraft pulp on the production of cellulase was studied with Trichoderma reesei RUT C-30 as the strain,and the techniques for industrial application of cellulase was preliminarily explored.The main research results are as follows:Cellulase was produced by batch fermentation with pulp and pulp pretreated with dilute acid as mixed carbon source.When the pulp concentration was 4 g cellulose/L and the dilute acid pretreated pulp concentration was 8 g cellulose/L,the produced cellulase had a filter paper activity of 2.78 FPU/mL and a ?-glucosidase activity of 1.26 U/mL.When fed-batch cultivation was conducted,the initial carbon source was pulp with concentration of 8 g cellulose/L,and the optimum daily feeding rate was diluted acid pretreated pulp with concentration of 6 g cellulose/L.Under this condition,the filter paper activity was 9.78 FPU/mL and the ?-glucosidase activity was 1.72 U/mL.A reasonable collocation of pulp and pulp pretreated with dilute acid could greatly increase the yield of cellulase.During the cellulase production by fed-batch fermentation,enzyme recovery was performed.The results showed that the harvest intervals and the amount of harvested enzymes had a significant effect on the cellulase production.When the periodic harvest of enzymes began at the fourth day of fed-batch culture,the amount of enzymes harvested at an interval of one day was 15% of the original proteins,the total obtained filter paper activity were 689.10 FPU and the total obtained ?-glucosidase activity were 170.38 U.Compared to the control,the total obtained filter paper activity and ?-glucosidase activity were increased by 40.9% and 98.1%,respectively.Mycelia could also be removed to avoid infection.When the same amount of enzyme solution and mycelia(10%,12.5%,15%,17.5% and 20%)were removed every day,the total obtained filter paper activities and ?-glucosidase activities increased by 37%-43% and 66%-100%,respectively.When 15% of enzyme solution and mycelia were removed every day,the total obtained filter paper activity and ?-glucosidase activity were 698.98 FPU and 171.03 U,respectively,with an increase rate of 42.9% for filter paper activity and 98.8% for ?-glucosidase activity compared to the control.The volumetric productivity of cellulase was 45.51 FPU/(L·h)and the yield of cellulase was 279.59 FPU/g cellulose.The concentrations of mycelia were kept at 4-5 g/L.This method could increase the efficiency of cellulase production and reduce the consumption of carbon source by redundant fungal cells.The obtained cellulase preparations were used to hydrolyze corn stover pretreated with dilute acid-catalyzed steam explosion.Enzymatic hydrolysis was conducted at a substrate concentration of 50 g/L for 48 hours.The cellulase produced from fed-batch cultivation obtained a hydrolysis yield of 85.92%.Enzyme recovery only and enzyme recovery coupled with mycelia removal resulted in a hydrolysis yield of 89.4% and 87.5%,respectively,indicating a high hydrolytic ability of the cellulase produced in the enzyme recovery process.Hollow fiber ultrafiltration membrane with a pore size of 10 kDa was successfully used to concentrate the produced cellulase solutions,although the filter paper activity and ?-glucosidase activity decreased gradually as the concentration ratio was increased.The obtained cellulase preparations could be easily stored at 4? or-18? for 80 days in the production site with more than 80% of filter paper activity and 87% of ?-glucosidase activity were preserved. |
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