Study On Ethanol Production Process By Stepwise Fermentation Of Corn Stover With Multiple Strains

Using agricultural waste stover to produce ethanol can not only reduce environmental pollution,but also alleviate energy shortage.Owing to the high cost of physical and chemical pretreatment and enzyme hydrolysis,this topic expect to produce ethanol by stepwise fermentation of corn stover with multiple strains without commercial enzymes in order to reduce the cost of ethanol conversion and environmental pollution.Firstly,in this paper,corn stover was used as raw material,and the pretreatment effects of different fungi on corn stover were studied,and the optimum strain and optimal time were determined.Secondly,the optimum conditions of producing ethanol by solid state fermentation of Pichia stipitis after the pretreatment were studied preliminarily.Finally,the optimum conditions for the production of cellulase by Trichoderma reesei were studied by fermenting pretreated stover,then yeasts were added to produce ethanol in different ways after the enzyme production in order to explore the feasibility of producing ethanol by stepwise fermentation of pretreated stover with multi-strains.The main results are as follows:1.The pretreatment effects of different fungi on corn stover by Pleurotus ostreatus,T.reesei and mixtures of the two strains pretreating corn stover 15 d,30 d,45 d,60 d respectively:(1)Results of enzyme activity in pretreatment process: During pretreatment,the Lac and MnP activities of P.ostreatus were the highest,and the activity of Lac and MnP reached the maximum at 30 d,respectively 6.57 U/g and 0.69 U/g.The Xyl and Cel activities of T.reesei were the highest,and Xyl activity reached the maximum of 3067.99 U/g at 45 d,and Cel activities reached the maximum of 129.14 U/g at 60 d.(2)Degradation results of stover components in the pretreatment process: The mixed fungus produced the largest dry matter degradation.The ability of degrade lignin was P.ostreatus > mixed fungus > T.reesei.The lignin degradation rate of 32.2% was the maximum caused by P.ostreatus at 60 d,but the degradation of cellulose and hemicellulose in the P.ostreatus group increased dramatically from 45 d to 60 d.The ability of degrade hemicellulose and cellulose was T.reesei > mixed fungus > P.ostreatus,and the degradation rate of hemicellulose and cellulose was the highest in 60 d,which was 45.17% and 33.72% respectively.During pretreatment,the selectivity index SI of P.ostreatus was larger than 1,and the degradation of lignin was preferentially selected.The time of cultivation was prolonged,and the mycelium was able to develop into the mushroom bud and the fruiting body.In addition to 15 d,the SI of T.reesei was less than 1,and it preferentially chose to degrade the fiber.In addition to 15 d,the SI of mixed fungus was around 1,and the degradation of cellulose and lignin was almost synchronous.(3)The surface structure of corn stover was observed by SEM: cell wall of unpretreated straw was intact,smooth,and the structure was compact.After the treatment of P.ostreatus,the vascular bundle and cell wall appeared various types of cracks,holes.After the treatment of T.reesei,the cell wall appeared obvious corrosion and dissolved areas,and the structure was badly damaged.(4)Effects of microbial pretreatment on enzyme hydrolysis and ethanol productivity by solid state fermentation: from 30 d to 60 d,the reducing sugar concentration and sugar conversion rate were significantly higher than untreated group and the other two kinds of microbial treatment(P<0.01).The reducing sugar concentration 13.36 g/L and sugar conversion rate 63.06% of 60 d pretreated samples by P.ostreatus were highest,but reducing sugar concentration was no significant difference with the 45 d samples.After solid state fermentation,the ethanol productivity of 45 d pretreated stover by P.ostreatus up to 0.035 g/g(per gram pretreated dry stover)was highest,and it was significantly higher than untreated sample and the other two groups(P<0.01).Overall consideration,the optimum pretreatment strain was P.ostreatus and the optimum pretreatment time was 45 d.2.Single factor experiment determined preliminarily the optimized condition of ethanol production by solid state fermentation of P.stipites after the pretreatment: the substrate was 45 d pretreated corn stover,yeast inoculation 10%,the initial moisture content 75%,cultivation temperature 28 ? for 7 d,the highest ethanol productivity was 0.049 g/g.3.Ethanol production by stepwise fermentation of pretreated corn stover with multi-strains: according to the results of single factor and orthogonal experiment,the optimal conditions of cellulose production with T.reesei by fermenting pretreated stover was concluded : the medium initial pH 4.8,ammonium sulfate concentration 3.5 g/L,stover substrate concentration 4%,T.reesei inoculation 5%,cultivation temperature 28 ? for 7 d under 150 r/min.After the enzyme production,the yeast was added in the optimal enzyme fermentation broth to produce ethanol.Ethanol productivity of NSSF was much higher than ethanol productivity of SSF.Ethanol productivity of Candida utilis was the highest and ethanol productivity was up to 0.123 g/g.This topic produced ethanol by stepwise fermentation of corn stover with multiple strains,and ethanol productivity was comparable to some physical and chemical pretreatment and enzyme hydrolysis technology,and the results of this study had certain guiding significance for low cost and low pollution utilization of corn stover to produce ethanol.