【作者】 陈明；

【导师】 夏黎明；

【作者基本信息】 浙江大学 ， 生物化工， 2007， 博士

【摘要】 利用丰富而廉价的木质纤维素资源代替粮食生产燃料乙醇，对我国经济和社会的可持续发展具有十分重大的意义。本文对木质纤维原料的预处理、纤维素和半纤维素的水解糖化、利用己糖、戊糖发酵乙醇、以及木质纤维原料的综合利用等关键技术进行了系统研究。针对玉米秸秆结构致密、难于直接被酶水解的特点，分别采用稀酸、氨水／稀酸、石灰、氢氧化钠等方法对玉米秸秆进行预处理。结果表明，采用2％NaOH于85℃对玉米秸秆处理75 min，木质素的脱除率达到73.9％，处理后的纤维残渣易于被酶降解，酶解得率达到81.2％；固液分离后的碱液可以重复利用3～4次，从而降低预处理成本；氢氧化钠预处理条件温和，能耗低，并可有效脱除了原料中的乙酸等发酵抑制物，有利于玉米秸秆水解液的乙醇发酵。采用纤维素酶水解玉米秸秆，对纤维素的糖化进程及纤维素酶的作用机制进行了研究。结果表明，底物的酶解得率和预处理过程中木质素的脱除率表现出正向相关性，而与半纤维素的脱除程度关系并不大；纤维素酶系组成对酶解进程有重要影响，补加纤维二糖酶可以改善里氏木霉纤维素酶的酶系组成，大大减弱纤维二糖累积引起的反馈抑制；纤维素水解过程中底物中的半纤维素同时被酶制剂中的木聚糖酶水解成木糖等单糖；针对纤维底物比容较大的特点，采用分批补料酶解工艺提高底物浓度，从而有效地提高了酶解液中的还原糖浓度，而且维持了较高的酶解得率，该工艺为提高后续发酵的乙醇浓度奠定了基础。木糖的乙醇发酵是利用木质纤维原料生产乙醇的关键环节。常用的酿酒酵母只能发酵葡萄糖，不能利用来自半纤维素的木糖。少数微生物如Pichia stipitis、Candida shehatae等虽然能够利用木糖，但由于要求“半好氧”的发酵条件及较差的乙醇耐受性而不适合工业化应用。对已构建的一株基因重组酵母(Saccharomyces cerevisiae ZU-10)的研究表明，重组酵母具有乙醇耐受性强、遗传性能稳定、可在厌氧条件下发酵葡萄糖和木糖产乙醇等优良性能，48 h内50 g／L葡萄糖和30 g／L木糖发酵生成32.8 g／L乙醇，对葡萄糖和木糖的乙醇得率达到0.41。但重组酵母生长缓慢、对乙酸和糠醛等抑制物较为敏感，浓度高于0.25 g／L乙酸或者高于0.08 g／L糠醛都会对其发酵木糖产生较大影响。利用重组酵母S. cerevisiae ZU-10在厌氧条件下对玉米秸秆纤维素、半纤维素水解液中的葡萄糖和木糖进行共发酵，66.9 g／L葡萄糖和32.1 g／L木糖发酵生成41.4 g／L乙醇，对葡萄糖和木糖的乙醇得率达到0.418。由于重组酵母S.cerevisiae ZU-10对半纤维素水解液中木糖的有效转化，从而使得乙醇产量相比普通酿酒酵母发酵增加了24.2％。为消除糖化过程中产物的反馈抑制，采用同步糖化发酵工艺，将纤维素和半纤维素的糖化与重组酵母对水解液中葡萄糖和木糖的共发酵相结合。研究表明，纤维素和半纤维素水解产生的葡萄糖和木糖被重组酵母S. cerevisiae ZU-10快速利用，有效解除了葡萄糖对纤维素酶的反馈抑制，从而提高了底物的乙醇转化率。鉴于重组酵母生长缓慢，生长阶段好氧、发酵阶段厌氧的特性，采用海藻酸钙凝胶包埋法固定重组酵母。利用固定化细胞发酵木糖，同游离细胞相比，固定化细胞的发酵周期由96 h缩短至60 h，对乙酸的耐受性由0.25 g／L提高到1.2 g／L，对糠醛的耐受性由0.08 g／L提高到0.12 g／L，而且细胞易于重复利用。利用固定化细胞重复12批发酵玉米秸秆水解液，葡萄糖利用率均为100％，木糖利用率均在90％以上，平均乙醇浓度为40.4 g／L，对葡萄糖和木糖的平均乙醇得率为0.41。将氢氧化钠预处理后残渣中纤维素和半纤维素的乙醇转化、碱处理液中木质素的回收、碱溶性半纤维素糖类的回收利用进行有机整合，从而实现玉米秸秆各组分的综合利用。对利用玉米秸秆生产燃料乙醇的关键技术进行集成优化，现已在河南天冠燃料乙醇有限公司建成年产300吨纤维乙醇的示范生产线，为进一步实现纤维乙醇的产业化生产奠定了扎实的基础。本文在基因重组酵母对葡萄糖和木糖的共发酵、利用基因重组酵母厌氧条件下发酵玉米秸秆纤维素和半纤维素水解液生产乙醇、以及利用木质纤维原料生产乙醇的关键技术集成等方面的研究具有明显的特色与创新，相关研究结果不仅有一定的学术价值，而且在促进可再生木质纤维素资源的转化利用、缓解人类的粮食危机、降低燃料乙醇的生产成本等方面具有重要的现实意义。更多还原

【Abstract】 Lignocellulosic biomass is a cheap, renewable, abundantly available resource, and its bioconversion to fuel ethanol is very meaningful in the sustaining development of our society and economy. In this dissertation, key technologies in ethanol production from lignocellulosic biomass including pretreatment, enzymatic hydrolysis of cellulose and hemicellulose, ethanol fermentation from hexose and pentose, and comprehensive utilization of lignocellulosic feedstock were investigated.Corn stover was pretreated by dilute acid, aqueous ammonia /dilute acid, lime, and sodium hydroxide, respectively, for enhancing the enzymatic susceptibility of substrate. Pretreatment with 2% NaOH at 85 °C for 75 min removed 73.9% lignin and almost all the acetate from the raw material, and the deligified corn stover residues were readily to degrade by cellulase complex. The alkaline liquor separated from the reaction mixture could be recycled for 3-4 times in repeated pretreatemnt batches.Enzymatic hydrolysis of corn stover by cellulase enzymes was thoroughly investigated to understand the mechanism of cellulase. It was found that the lignin removal plays a crucial role in enhancing enzymatic digestibility of substrate, and that hemicellulose removal is not necessary for high hydrolysis yield. The enzymatic hydrolysis of corn stover was highly affected by the cellulase complex composition. Supplementing cellobiase greatly reduced the inhibitory effect caused by cellobiose due to low cellobiase activity in T. reesei cellulase. Xylose and arabinose were detected out during the enzymatic hydrolysis of corn stover, indicating the hydrolysis of hemicellulose by xylanase in cellulase complex. It is difficult to get high reducing sugar concentration in batch hydrolysis due to the porous property of cellulosic substrate. Therefore fed-batch hydrolysis was performed to increase the reducing sugar concentration, thus helping to obtain higher ethanol concentration in subsequent ethanol fermentation.Effective conversion of xylose to ethanol is a key step for ethanol production from lignocellulosic materials. Sacchromyces cerevisiae has traditionally been used for ethanol fermentation, but it can’t utilize xylose. Several yeasts, such as Pichia stipitis and Candida shehatae, are able to ferment xylose to ethanol. However, these strains are constrained by their low ethanol tolerance and the requirement of some aeration for optimal ethanol productivity. A recombinant yeast of Sacchromycescerevisiae ZU-10 was examined for its capability of fermenting xylose to ethanol. It was found that S. cerevisiae ZU-10 had the capability of high ethanol tolerance, stable genetics character and effective cofermentation of glucose and xylose to ethanol under anaerobic conditions. 50 g/L glucose and 30 g/L xylose were fermented to 32.8 g/L ethanol, giving a ethanol yield of 0.41. However, the growth rate of S. cerevisiae ZU-10 was slow and the strain was sensitive to inhibitors such as higher than 0.25 g/L acetic acid and higher than 0.08 g/L furfural.It was found that the recombinant yeast S. cerevisiae ZU-10 could effectively ferment glucose and xylose in corn stover enzymatic hydrolysate to ethanol under anaerobic conditions, and that 66.9 g/1 glucose and 32.1 g/1 xylose were fermented to 41.4 g/L ethanol, giving a ethanol yield of 0.418.. The ethanol yield was increased by 24.2% due to the conversion of xylose to ethanol by recombinant yeast S. cerevisiae ZU-10.In order to eliminate end-product feedback inhibition of glucose on cellulase, simultaneous saccharification and fermentation (SSF) of corn stover for ethanol production was performed with S. cerevisiae ZU-10. The results showed that glucose and xylose liberated from cellulose and hemicellulose in corn stover were rapidly fermented to ethanol by S. cerevisiae ZU-10. The bioconversion efficiency of corn stover was highly enhanced because of the elimination of end-product feedback inhibition.In view of low grow rate of recombinant S. cerevisiae ZU-10, the cells were immobilized by entrapping them into calcium alginate gels. Comparing with free cells, fermentation of corn stover hydrolysates by immobilized S. cerevisiae ZU-10 cells had the advantages of shorter period, enhanced tolerance to acetic acid and furfural, and convenient recycling of cells. During 12 repeated batches of ethanol fermentation of corn stover enzymatic hydrolysate by immobilized cells, glucose was completely utilized and more than 90% xylose was consumed during each batch, giving an average ethanol concentration of 40.4 g/L and ethanol yield of 0.41.During fuel ethanol production from corn stover, bioconversion of cellulose and hemicellulose in NaOH-pretreated corn stover to ethanol, lignin recovery and alkali-soluable hemicellulose sugar recovery could be integrated into a biorefinery scheme, thus realizing the comprehensive utilization of overall biomass. Based on integration of key technologies in ethanol production from corn stover, a model product line with the productivity of 300 tons ethanol per year has been set up inHenan Tianguan Fuel Ethanol Co. Lmt.The characteristics and novelty in this dissertation was obvious in the following aspects: cofermentation of glucose and xylose to ethanol by recombinant yeast S. cerevisiae ZU-10, ethanol production from hydrolysates of cellulose and hemicellulose in corn stover by recombinant yeast S. cerevisiae ZU-10, and integration of key technologies in ethanol production from lignocellulosic biomass. These results were meaningful not only in academic research, also in utilization of renewable lignocellulosic biomass, decreasing the production cost of fuel ethanol , and alleviating the world grain crisis.更多还原