Fundamental Study Of Free And Immobilized Cell Technology Preparation For Bioethanol Production

Lignocellulosic biomass is a promising and low-cost renewable bioethanol source. The second-generation bioethanol from agricultural residues of cellulose category can greatly reduce waste, environmental pollution and the reliance on fossil fuels. Due to the hemicellulose and lignocellulose covered up the crystallinity structure of cellulose, cellulosic part of lignocellulosic biomass has a difficulty in direct conversion into ethanol. It is necessary to conduct the pretreatment to deprive the lignocellulose and hemicellulose content, destruct crystallinity structure of cellulose, and enhance cellulase enzyme accessibility; thus, could increase the cellulose hydrolysis rate.In this thesis, comprehensive research of pretreatment, cellulase production, enzymatic hydrolysis and saccharification, and ethanol fermentation had been accomplished based on lignocellulosic agricultural wastes.Lignocellulosic biomass was subjected to microwave-alkali pretreatment in order to reduce lignin content in the lignocellulosic biomass significantly and make it advantageous to the subsequent enzymatic hydrolysis and fermentation experiments. The results showed that the optimal conditions of pretreatment were as followed:incubation in 2.5% NaOH for 30 minutes under 540 W microwave irradiation power with a liquid-solid ratio of 10:1 (V:m). Under these conditions, the hydrolysis of rice straw showed the highest reducing sugar yield of 74.09%.Production of cellulase enzymes from Aspergillus niger CICC 41125 were investigated, and the results showed that SSF have the highest yield,2-fold times higher, compared to SF and SmF in a maximum endocellulase of 315.7 U/gds and exocellulase of 79.28 U/gds. The optimum temperature for the maximum activity of exocellulase and endocellulase were 60℃.The use of immobilized yeast cell, which were prepared from sawdust and Na alginate, were investigated to produce ethanol using different concentrations of sodium alginate and sawdust to encapsulate yeast cells. The result indicated that the optimal strengthening process of yeast cell immobilization was 2.5% alginate concentration and alginate:DCM ratio of (1:1.5). In addition, pretreated rice straw was used for ethanol production using free and immobilized yeast cell. A maximal ethanol yield of 0.37g/(g theoretical ethanol) was obtained after 96h of incubation from immobilized cells. The immobilized yeast (Saccharomyces cerevisiae) cells were used up after nine cycles for its performance on repeated batch fermentation. The maximal ethanol yield can respectively reach 0.35g/(g theoretical ethanol) yield in six sequential fermentation cycles.The optimization of process parameters affecting cellulase production using pretreated lignocellulosic biomass, enzymatic hydrolysis and saccharification, and ethanol production were examined using the Box-Behnken design (BBD) of response surface methodology. The response surface methodology of cellulase fermentation condition via SSF by A.niger was investigated to enhance the enzymatic activities. From examining the findings, inoculum size (% w/w), moisture content (% v/w), and rice straw loading (% w/w) had significant effect on cellulase enzyme production yield. The optimal level predicted were 10.5% inoculum size,79% moisture content, and 15g biomass loading. The BBD model of optimal condition predicted that the design experiment could theoretically reach cellulase yield of 96.44 U/g DS, while in the validation experiment, actual amount of cellulose enzyme production was 95.86 U/g DS. This actual value is in close agreement with the predicted value, with a difference of only 0.61%.Response surface methodology was applied in enzymatic hydrolysis and saccharification analysis. Substrate concentration, saccharification time and crude enzyme loading were optimized for enzymatic saccharification and their predicted optimum values were 11.09% w/v,48.78 h,89.95 U/g DS, and percent reducing sugar yield of 55.54% respectively. Both of fitted models of predicted cellulase production and enzymatic saccharification were in good agreement with the experimental results. In addition, response surface methodology was utilized for analysis of ethanol production. The pH (4-5.5), fermentation time (24-72 h), and solid content loading (15-20% w/v) were tested to maximize bioethanol yield in the SSF process. The predicted optimum conditions were pH 4.5,56.42 h fermentation time, and 15.16% w/v solid content loading. Under optimized hydrolysis conditions, 0.4451 g/g theoretical ethanol yield was produced.