The Research Of Key Points On Fuel Ethanol Production Using Corn Stover

Lignocellulose is one of the most abundant and inexpensive renewable resources on the earth. Thus far, although the mechanisms and technologies that using lignocellulose in ethanol production have been recognized, many aspects including the pretreatment of raw material, decrease of enzyme costs and utilization of xylose still need to be further improved. Due to the high cost, the ethanol produced from Lignocellulose is less competitive with that produced from corn. So, it is widely realized that significant effort should be driven into the development of low-cost cellulosic ethanol.In this study, three factors including pretreatment of ligncellulose, utilization of cellulosic degradation community instead of enzyme and utilization of xylose addressed below were investigated.Based on mechanism of microwave, microwave is introduced into corn stover pretreatment. Several microwave absorbers were used and compared. Water is determined to be the best microwave absorber in pretreatment. By Design Expert software showed that the optimal operational parameters were: liquid to solid ratio 58:1, power of microwave 630 W, pretreatment time 5.5 min. Under this condition, the enzyme degradation percentage can be reached to 49.12%. Images obtained by AFM showed that micropores were formed on the surface of corn stover after pretreatment. Considered with results obtained above, Microwave pretreatment enlarged the interface between solids and enzyme due to the micro pores water molecule drilled, which also facilitates enzyme entrance into interiors of solids.Cellulosic degradation strains FLZ6 and FLZ10 were isolated from natural environment. Morphologic analysis, physiological-biochemical characterization and cell membrane fatty acid analysis revealed that the FLZ6 belongs to Bacillus-licheniformis, and the FLZ10 belongs to Chaetomium spp. FLZ6 and FLZ10 were chosed in Simultaneous sacchariferous fermentation (SSF) tests. By testing the ethanol production, the optimal parameters of SSF process were obtained as: temperature 37°C, original pH 6.0, the ratio of cellulose decomposing strains and yeast 1:1, retention time 36 h. Under this condition, the production rate was 13.1 g ethanol per 100 g corn stover.A xylose fermentive yeast FL-20-2 were isolated from humus soil.Using morphologic analysis, physiological-biochemical characterization and molecular biological methods, the yeast was identified as Candida tropicalis. The ethanol production rate was 32.4% of the theoretical percentage, accompanied with 31.05 g/L xylitol production with pH 5.5, temperature 35°C, inoculation percentage 10% and fermentation time 96 h. When xylose and glucose were mixed as carbon sources, the ethanol production rates were 37.7% and 79.2% of their theoretical rates respectively, with 30.2 g/L xylitol produced.A degenerate primers and other primers ware designed according to the result of homologous analysis of nucleotide sequences encoding XR of Candida sp. Gene of xylose reductase (XR) from Candida parapsilosis was amplified by RACE technology from the total RNA of Candida parapsilosis. The open reading frame of Candida parapsilosis xyl1 gene is 954bp. A BLAST search showed that this sequence contained homology with Candida albicans aldose reductase XM₇15658 and the homologous rate was 80%. This gene should be a new XR gene(Genebank accession:EF033247). The open reading frame of xyl1 from Candida parapsilosis was inserted into the downstream of the alpha-mating factor signal of the P.pastoris expression vector pGAPZαA .Under the control of promoter pGAP (glyceraldehydes 3-phosphate dehydrogenase)the recombinant protein can secreted express into culture medium. The products were purified using nickel affinity chromatography and an approximate 36-kDa protein band was obtained. Enzyme character analysis indicated that the recombinant XR showed high catalytic efficiency (kcat/Km =61 mM-1 min-1) for D-xylose and showed unusual coenzyme specificity with greater catalytic efficiency with NADH-dependent (kcat/Km=41.5μM-1 min-1 ) than with NADPH-dependent (kcat/Km=2.2μM-1 min-1).Based on the research above, ethanol production process using corn stover was determined. Using this technology, a ethanol yield of 17.9 g ethanol/100 g corn stover had been achieved. Simultaneously, based on the research on the coenzyme characteristics of xylose reducase (key enzyme), it is promising that the problem of coenzyme imbalance during xylose fermentation can be solved, which could further increase the ethanol output from corn stover.