Induction Effect Of Lignocellulose To Cellulase Synthesis By Trichoderma Reesei

Under the circumstance of the contradiction between the shortage of fossil fuel and theincreasing demand for energy, as well as environmental problems become increasingly seriousbring by the fossil energy, using lignocellulose to prepare fuel ethanol instead of traditional fossilenergy has become one the hottest topics for research in today’s world. Cellulase can effectivelyhydrolyze cellulose-rich straws into monosaccharide, which can be fermented to produce fuelethanol. It is very meaningful in the sustaining development of our society and economy that howto produce the costless and effective cellulase. The process of cellulase induction fromTrichoderma reesei RUT C30using different carbon sources was studied.In this dissertation, straws were used as the raw materials pretreated by dilute acid, alkaliand green liquor. Effects of pretreatment conditions for different carbon source on cellulaseinduction were compared. Cheap carbon source and pretreatment method for enzyme productionwas optimized. Response Surface Analysis was applied to optimize the enzyme productionmedium when dilute-acid treated pulp was used as the carbon sourse. To extend enzymeproduction cycle and to improve the efficiency of enzyme production, pretreated wheat straw anddilute-acid pretreated pulp were used separately or together in batch fermentation or fed-batchfermentation. SEM and X-ray diffraction were used to examine the structure changes oflignocellulose pretreated with different conditions and to investigate the effect on the increase ofthe enzyme production ability. The main results are as follows.The capability of enzyme induction by different straws pretreated with dilute acid, alkali,and green liquor was compared. Results indicated that green liquor pretreatment was preferredbecause of mild conditions, simple processing, less material loss, and high enzyme inducingability. The effects of green liquor pretreatment of rice straw, corn stalk, cotton stalk and wheatstraw on cellulase production with Trichoderma reesei were studied. Data showed that wheatstraw pretreated by green liquor was the more suitable carbon source for enzyme production.Analysis of pulp yield, cellulase activity and yield of enzymatic hydrolysis with different greenliquid pretreated lignocellulose indicated that higher weight loss, lower glycan recovery, andhigher level of delignification were observed when more severe condition of pretreatment was applied. The order of influence of green liquor pretreatment on wheat straw was total alkalicharge (Na2O, on oven dry material, TTA), cooking temperature, and sulfidity.For enzyme production with wheat straw, the best green liquor pretreatment condition wascooking temperature140°C, total alkali charge4%, and sulfidity20%. The pretreated wheatstraw was used as carbon source for enzyme production. When12g/L of cellulose was used forcellulase synthesis, batch fermentation could reach a filter paper activity of4.23FPIU/mL at thesixth day. Increasing the carbon source concentration to20g/L of cellulose resulted a filterpaper activity of5.89FPIU/mL.For enzymatic hydrolysis of wheat straw, the optimum condition of green liquorpretreatment was cooking temperature150℃, total alkali charge8%, and sulfidity40%.Hydrolysis was conducted at substrate concentration of10%for72h with a cellulase loading of15FPIU/g cellulose. Compared to un-pretreated wheat straw, the green liquor pretreated whatstraw was hydrolyzed more efficiently. When commercial cellulose was used, hydrolysis yield ofglucan was increase from10.50%to61.41%, and hydrolysis yield of xylan was increase from5.43%to55.65%. When lab-produced cellulase was used, however, hydrolysis was furtherimproved. Hydrolysis yield of glucan and hydrolysis yield of xylan were increased to81.07%and80.02%, respectively.Pulp pretreated with1.5%H2SO4was the most efficient carbon source in inducingTrichoderma reesei for cellulase production. Plackett-Burman (PB) experiments indicated thatthe three main factors of pretreatment affecting enzyme synthesis were pulp concentration, C/Nand MgSO4concentration. Response Surface Analysis experiments optimized these three factors.When the dilute acid-hydrolyzed pulp（AHP）concentration was30.94g/L, C/N was4.06, andMgSO4concentration was0.36g/L, after120h growth, the filter paper activity reached it’smaximum of10.35FPIU/mL, with β-glucosidase activity, protein content, cellulase productionrate and cellulase yield being4.98IU/mL,3.5g/L,86.25IU/L·h and334.52IU/g cellulose,respectively.Fed-batch culture could increase cellulase production significantly and was optimized fordifferent carbon sources. When green liquor pretreated wheat straw was used as the carbonsource in fed-batch enzyme production, the optimal feeding condition was addition of5.5g/L·dcellulose starting at the fifth day. At the tenth day filter paper activity reached the highest valueof9.56FPIU/mL; pH, β-glucosidase activity and cellulase production rate were6.56,2.44IU/mL and39.83IU/L·h, respectively. In fed-batch enzyme production with dilute-acid pretreatedpulp as the single carbon source, the best feeding mode was adding4g/L·d cellulose every dayfrom the first day. At the tenth day filter paper activity reached the highest value of17.58FPIU/mL, whereas pH, β-glucosidase activity, cellulase production rate and cellulose yield were6.57,7.01IU/mL,73.25IU/L·h and399.55IU/g cellulose, respectively. When green liquidpretreated wheat straw and dilute-acid pretreated pulp were used as mixed carbon source for fed-batch enzyme production, the best way of carbon source feeding was started with greenliquid pretreated wheat straw at8.0g/L of cellulose, followed by addition of dilute-acidpretreated pulp, from the first day and on, at a concentration of4g/L·d cellulose. The highestvalue of filter paper activity,15.33FPIU/mL, was determined at the ninth day. At the meantimecellulase production rate and cellulose yield were70.97g/L·h and638.75IU/g cellulose,respectively, higher than that when single carbon source was used.