Applied Basic Research On Fuel Ethanol Production From Plant Cellulosic Materials

Bio-ethanol production from lignocellulosic materials is of great significance to solving serious problems such as energy shortage and environmental pollution people are faced with. By far, the technique still needs to be improved. The major limitations include:low productivity of cellulase, relatively high dosage and cost of cellulase in enzymatic hydrolysis of cellulose, low ethanol conversion from lignocellulosic materials and the difficulty in hemicellulose utilization.The aim of this work is to study the key techniques of fuel ethanol production from lignocellulosic materials. In the aspect of cellulase production, a soluble inducer for submerged fermentation of cellulase was produced by transglycosylation from glucose. The suitable condition for transglycosylation was as follows:400 g/L glucose as the substrate,50℃ and pH value 4.0. Sophorose reached 51.40 g/L within 5 d. Sorphorose is a strong inducer for cellulase and it’s very expensive. The results are important to improving cellulase productivity and reducing its production cost.Submerged fermentation of celluase was carried out in a 50 L fermentator. Fed-batch process was conducted with the compound sugar made by transglycosylation and microcrystalline cellulose (MCC) as the carbon sources. The feeding rate was controlled according to pH changes:feeding began when pH value reached 4.8 or above; feeding ceased when pH value reached 4.5 or below. Filter paper activity reached 94.12 IU/mL within 264 h in the fermentation broth. As Results indicated, fed-batch fermentation depending on pH value controlled strategy could maintain slight cell growth, which would be beneficial for continuous increase of cellulase activity.After acid pretreatment, cellulosic residue of corncob was hydrolyzed by cellulase. Ethanol production from cellulosic hydrolysate containing 52.60 g/L glucose was performed at 38℃ by a thermotolerant strain Saccharomyces cerevisiae HTR-11, 25.14 g/L ethanol was obtained within 18 h and ethanol yield on glucose was 0.48 g/g, reaching 94.12% of the theoretical yield.Simultaneous saccharification and fermentation (SSF) was carried out with cellulosic residue of corncob as the substrate. In batch SSF process, under the condition of initial substrate concentration 100 g/L,38℃,20 FPIU/g substrate, pH4.8 and innoculum 10%(v/v), ethanol concentration reached 17.61 g/L within 72 h. The dosage of 10 CBIU/g substrate could effectively reduce cellobiose accumulation and remove its feedback inhibition on cellulase system. Ethanol concentration reached 30.27 g/L with an ethanol yield on cellulose of 0.51 g/g. In fed-batch SSF process, the feeding strategy of 6 dosages within 24 h to reach a final substrate concentration 120 g/L from 60g/L initially was applied. Ethanol concentration reached 36.82 g/L within 90 h and ethanol yield on cellulose was 0.52 g/g.Hemicellulosic hydrolysate was used in ethanol production. Fermentation inhibitors in hemicellulosic hydrolysate and detoxification methods were studied. Inhibitory effects to recombinat yeast S. cerevisiae ZU-10 were found to be evident for sulfate ion, acetic acid and furfural above the concentration of 5g/L,0.25 g/L and 0.08 g/L respectively. Acid pretreatment temperature was important to formation of fermentation inhibitors. After cellulosic material was hydrolyzed for 12 h at 95℃ with 1% sulfate and detoxified with lime-neutralization; roto-evaporation and ion-exchange. Acetic acid was 0.080 g/L and furfural could not be detected out in the hydrolysate. Fermentation of the above hemicellulosic hydrolysate containing 80g/L reducing sugar was carried out at 30℃ with the inoculum 1.2g/L (calculated on dry weight of cells). At initial pH 5.5, ethanol concentration was 31.05 g/L and ethanol yield on glucose and xylose was 0.40 g/g within 96 h anaerobic fermentation. Xylose fermentation was the key technical difficulty in ethanol production from plant cellulosic materials. This study is of great significance in accelerating the industrialization process of fuel ethanol.To realize comprehensive utilization of cellulose and hemicellulose, dilute alkaline pretreatment of corn stover was studied. Under the condition of sodium hydroxide dosage 0.15 g/g substate,85℃,1 h,75.40% lignin could be effectively removed and 32.97% hemicellulose was removed. The corncob residue was easy to be hydrolyzed and converted to fermentable sugars. Synergetic hydrolysis proved that enzymatic hydrolysis yield could reach 89.24% when cellulase activity was 25.75 FPIU/g substrate, cellobiase activity was 8.20 IU/g substrate and xylanase activity was 369.63 U/g substate. In the corn stover enzymatic hydrolysate, glucose took up 65.63% and xylose took up 20.97% of the total reducing sugar.Cocentrated corn stover enzymatic hydrolysate was fermented with recombinant Saccharomyces cerevisiae ZU-10. Within 72 h,47.10 g/L ethanol was obtained from 76.51 g/L glucose and 36.64 g/L xylose. The ethanol yield on glucose and xylose was 0.42 g/g. Co-fermentation of hexose and pentose sugars was realized.Immobilized recombinant yeast cells were used in the fermentation of corn stover enzymatic hydrolysate. In 6 repeated batches, average ethanol concentration was 40.43 g/L. Glucose could be utilized quickly and completely, and utilization rates of xylose maintained above 90%. Utilization of immobilized cells could significantly shorten the fermentation cycle and improved ethanol productivity compared with free cells. It will be a promising technique for mass fuel ethanol production in the near future.