Process Configuration And Nutrition Evaluation For Ethanol Production From Corncob By Cellobiose-utilizing Yeast Strain

Ethanol production from lignocellulose is of great significance for the energycrisis and environment pollution problem in the world. Various factors, mainlysubstrate, enzymes and hydrolysis, fermenting strain and fermentation, influence theefficiency and cost of cellulosic ethanol production. They interact with each other andlead to the optimal process configuration. While β-glucosidase is an importantenzyme in the enzymatic hydrolysis of cellulose, a recombinant Saccharomycescerevisiae strain efficiently expressing β-glucosidase was first constructed in order todecrease the cost of enzymes used. Then process configurations of ethanol productionfrom acid-and alkali-pretreated corncob were compared between the recombinant andnon-recombinant strains at optimal enzyme dosages and with urea addition. Last, theeffect of different nutrient components on the ethanol production was evaluated.First, the optimal expressing cassette for the β-glucosidase gene from Aspergillusaculeatus was integrated into the chromosome of S.cerevisiae W303-1A by using δsequence as integration sites, which has hundreds of copies in yeast chromosome. Theintegrating strain45#was obtained by large scale transformation and screening.Itconsumed2%(w/w) cellobiose in24h and the maximal ethanol concentration was0.79%, corresponding to the yield73.7%(expressed as a percentage of the theoreticalyield). Under the same conditions, it consumed2%glucose in24h and the ethanolyield was78.2%.Then the optimal enzyme dosages, cellulose10FPU/g biomass andβ-glucosidase5U/g biomass, were determined by enzymatic hydrolysisexperimentation. Further, process configurations were compared under conditions of10%solid content, optimized enzyme doses (10FPU cellulase/g biomass,5Uβ-glucosidase/g biomass),1g/kg yeast loading and0.33g/kg urea supplementationwith the strains45#(no β-glucosidase added), W303-1A and industrial strain Angel.The results indiceted that separate hydrolysis and fermentation (SHF) was optimal forW303-1A and Angel, and the ethanol titer reached3.22%and3.69%，with75.6%and86.8%of the theoretical yield, respectively. While simultaneous saccharification andfermentation (SSF) was optimal for strain45#and the ethanol titer and yield reached3.31%and77.7%, respectively. The effects of nutrient components other than urea on the ethanol productionwere evaluated. The results showed that the effects of nutrient addition greatlydepended on the inoculum size and strains used. Under the inoculum size of5g/kg,urea and corn steep liquor (CSL) all had no effect. Under the inoculum size of0.1g/kg,CSL could singnifantly improve ethanol production, while urea didn’t show positiveeffect. Under the inoculum size of1g/kg, urea and CSL to some extent increased theethanol titer and yield. The evalution of nine kinds of the nutrient components withthe inoculum size of1g/kg indicated that the organic nitrogens (urea, CSL, yeastextract and amino acids) all improved the ethanol production. In addition, theinorganic nitrogen (NH4)2HPO4also showed positive effect for Angel.The effect of pre-cultivating in the enzymatic hydrolyzate depended on the thestrains used. Pre-cultivation could improve the rate and yield of ethanol production bystrain45#, while it had no significant effect on Angel.