| With the irreversible depletion of the fossil resource,exacerbating energy crisis and other environmental issues,there has been a world-wideinterest in exploiting alternative sources of energy.Lignocellulosic material is the most abundant renewable resource in the world.However,this renewable resource has not been sufficiently exploited,even causing environmental problems.Utilization of cellulases produced by microorganisms for bioconversion of cellulosic materials into fermentable sugars and then liquid fuels and chemicals has been taken into account as the most promising sustainable strategy for resolving the resource,energy and environmental problems.This research optimized the strategy of on-site cellulase production and proposed the technology for sodium gluconate production by enzymatic catalysis of delignified corn cob residue(DCCR)hydrolysate,in order to provide an economically competitive process for industrialization of sodium gluconate production from lignocellulosic materials.The main results of the research are as follows:1.The improvement of cellulase productivity of Penicillium oxalicum RE-10 by repeated fed-batch fermentation strategyNatural rawmaterials including wheat bran,corncob residue and soybean powder,which are cheap and easily available,were used as themain components of the medium for cellulase production.Plackett-Burman design(PBD)and central composite design(CCD)were used to optimize the medium.PBD demonstrated that wheat bran and NaNO3 had significant influences on cellulase production.The CCD results showed the maximum filter paper activity(FPA)production of 8.61 U/mL could be achieved in Erlenmeyer flasks.The maximal FPA reached 12.69 U/mL by submerged batch fermentation in a 7.5-L stirred tank,1.76-fold higher than that on the original medium.Then,the repeated fed-batch fermentation strategy was performed successfully for increasing the cellulase productivity from 105.75 U/L/h in batch fermentation to 158.38 U/L/h.The cellulase activity and the glucan conversion in delignified corncob residue hydrolysis had no significant difference between the enzymes sampled from different cycles of the repeated fed-batch fermentation and that from batch culture.The repeated fed-batch fermentation strategy significantly increased cellulase productivity,which demonstrated that the strategy has a great potential for the industrial production of cellulase by fungi.2.Spent ammonium sulphite liquor induced cellulase expression in P.oxalicum and be used for fed-batch fermentationThe composition of spent ammonium sulphite liquor(SASL)was analysised.SASL contains 90-120 g/L hemicellulose,30-50 g/L β-glucan,20-40 g/L monosaccharide and 8-10 g/L residual ammonium(NH4+)· Feeding proper amounts of SASL could improve cellulase yield of P.oxalicum RE-10 in Erlenmeyer flasks.However,SASL contains furfural,SO2,volatile organic acid and aromatic compounds from lignin which could inhibit the growth and cellulase production of P.oxalicum when excess of SASL was fed.The maximal FPA reached 16.12 U/mL by feeding SASL at 1 mL/L/h in a 7.5-L stirred tank,enhancing FPase production by 26.93%than that by batch fermentation on the original medium.Considering that the cellulase was produced after 48 h,varied flow rate fed-batch fermentation strategy was used for cellulase production.The maximal FPase reached 17.66 U/mL at 144 h,when the CMCase,pNPCase,pNPGase activities,and protein concentration were 40.39 U/mL,1.77 U/mL,2.31 U/mL,and 7.50 mg/mL,respectively.The new strategy significantly increased the FPase specific activity from 1.74 U/mg in batch fermentation to 2.40 U/mg.To further compare the cellulolytic performances of cellulases obtained by different strategies,the crude cellulases were used for the hydrolysis of DCCR.The glucose yield and glucan conversion in DCCR hydrolysis had no significant difference between the enzymes produced by different strategies when using equal FPase loadings..However,the hydrolysis effect of the cellulases produced by fed-batch fermentation was obviously higher than the one produced by batch culture when using equal protein loadings.In comparison with the cellulase produced by batch fermentation,there were 21 proteins up-regulated in the secretome of fed-batch fermentation,which mostly associated with lignocellulose degradation.The SASL fed-batch fermentation strategy was further performed by employing Trichoderma reesei SN1 for cellulase production.In comparison with batch fermentation,the FPase,CMCase,pNPCase and pNPGase activities raised by 44.98%,39.83,72.17%and 59.21%,respectively.This shows SASL could also induce cellulase expression in T.reesei.3.Production of sodium gluconate from DCCR hydrolysate using co-immobilized glucose oxidase and catalaseAddition of β-glucosidase is one of the most effective strategies to improve cellulose saccharification.P.oxalicum 11-13 was used as a cellulase production strain with higher P-glucosidase activity.A fed-batch saccharification process was developed to obtain high yields of glucose from DCCR.At the end of hydrolysis,the glucose concentration reached 145.80 g/L.Glucose oxidase and catalase were co-immobilized to catalyze DCCR hydrolysate.Under the optimum conditions,166.87 g/L sodium gluconate can be obtained after 56 h of reaction,and the conversion ratio was 98.24%.The immobilized enzymes can still maintain more than 60%of the activity after repeated use for 6 times.This study provides a potential route for the production of valuable chemicals by enzymatic conversion of lignocellulosic materials.4.Construction of consolidated bioprocessing strain for sodium gluconate production using P.oxalicum as chassis organiamGOD and CAT from Aspergillus niger were separately expressed in P.oxalicum A11△.The results of SDS-PAGE and enzyme assays showed that the two enzymes were correctly expressed in P.oxalicum.Then,a recombinant strain named z19 was obtained to simultaneously over-express GOD and CAT from A.niger in the P.oxalicum wild-type strain 114-2.The cellulolytic ability of z19 was similar with that of 114-2.Two-stage temperature control strategy(0-120 h,30°℃;120-194 h,45℃)was utilized for sodium gluconate production from filter paper power,with 13.54 g/L of sodium gluconate obtained at the end of the fermentation.The recombinant strain z19 succeeded in "one-pot reaction" for producing sodium gluconate from lignocellulose. |
