【作者】 杨涛；

【导师】 马美湖；

【作者基本信息】 湖南农业大学 ， 园艺产品采后科学与技术， 2008， 博士

【摘要】 我国水稻秸秆资源丰富,年产量达3亿多吨。利用水稻秸秆生产燃料乙醇,对解决未来我国能源问题、实现节粮代粮和环保有着巨大的潜力和广阔的应用前景。水稻秸秆的主要成分是纤维素,对纤维素的利用最主要的限制性因素是将纤维素转化为可发酵还原糖。解决的办法主要有两类途径:（1）提高纤维素酶生产的经济性,主要涉及纤维素酶高产菌的获得及纤维素酶的生产技术,提高其合成效率以降低单位纤维素酶生产成本;（2）提高纤维素酶利用效率,主要涉及纤维素酶解催化过程,以降低单位可发酵还原糖生产成本。因此,本研究从菌种的选育着手,研究了菌株的产酶特性,用响应面策略优化发酵培养基,形成了5L发酵罐分批发酵生产高活力纤维素酶技术;分离纯化了纤维素酶;构建了代谢纤维二糖的酿酒酵母工程菌;对酿酒酵母工程菌细胞固定化发酵进行了研究,利用二级串联式生物反应器耦合系统生物协同酶解水稻秸秆发酵生产燃料乙醇等。主要研究结果如下:1.筛选到一株纤维素酶高产菌株（Penicillium YT01）,原生质体紫外诱变后得到突变株YT02,YT02以水稻秸秆为碳源,豆饼粉和硫酸铵为氮源,在29℃,初始pH6.0发酵120 h,纤维素酶活力达到最高,摇瓶发酵滤纸酶活（FPA）、CMC酶活（CMCase）和β-葡萄糖苷酶活（CB）分别达3.86 IU/mL、207.41 IU/mL和1.40 IU/mL。2.用响应面方法（RSM）优化的发酵培养基组成为:水稻秸秆为41.95g/L,豆饼粉为24.83g/L,麸皮为22.16 g/L,（NH₄）₂SO₄、KH₂PO₄为4g/L,MgSO₄为0.5g/L;起始pH6.0。以优化的培养基发酵120 h,滤纸酶活、CMC酶活和β-葡萄糖苷酶活分别达到8.8967IU/mL、357.41 IU/mL and 3.704 IU/mL。远高于优化前的纤维素酶活水平。3.在5L发酵罐中研究了温度、pH值和溶氧对菌体生长和产酶的影响,确定了分批发酵的工艺条件为:0-32 h时发酵温度32℃,溶氧70%;32 h至120 h发酵结果发酵温度29℃,溶氧50%,发酵液初始pH值6.0,发酵96 h滤纸酶活、CMC酶活和β-葡萄糖苷酶活分别达到11.13 IU/mL、465.24 IU/mL and 4.08 IU/mL,均高于摇瓶发酵水平,分批发酵动力学过程显示,突变菌YT02菌体生长和纤维素酶各组分均为部分耦联。4.利用DEAE Sephadex A-25和Sephadex G-75分离纯化了二个内切葡聚糖酶（CMCase）和一个β-葡萄糖苷酶,CMCase纯化倍数为13.48,回收率为10.54%,β-葡萄糖苷酶纯化倍数为18.62,回收率为8.62%,经SDS-PAGE得到单蛋白分子条带,经分子量测定分别为73 kDa、43 kDa和57.8 kDa,并对其进行了N端测序和质谱分析。5.以生产乙醇性能优良的酿酒酵母菌株NAN-27作为工程菌株的受体菌。利用稳定性能良好的多拷贝整合型载体pYMIKP,使纤维二糖代谢基因BGL1整合到酿酒酵母的染色体上。从而在酿酒酵母工业菌株中建立了稳定的纤维二糖代谢途径,拓展了酒精生产的底物利用范围,降低了纤维二糖对纤维素酶解的抑制作用。采用海藻酸钙凝胶包埋固定代谢纤维二糖酿酒酵母工程菌,固定化细胞与游离细胞相比,发酵时间缩短,乙醇产率提高20%以上,并能有效地利用水稻秸秆水解液进行酒精发酵。6.对水稻秸秆酶解过程中底物性质、酶解温度、酶解pH、底物浓度及纤维素酶用量等关键因子进行了研究。由于YT02纤维素酶系中纤维二糖酶活力较低（CB/FPA为0.38）,经稀酸稀碱预处理后的水稻秸秆纤维素对乙醇转化率仅为18%。采用代谢纤维二糖酿酒酵母工程菌游离细胞发酵,可部分去除纤维二糖对酶解的抑制,水稻秸秆纤维素对乙醇转化率可提高至20%。进一步利用采用海藻酸钙凝胶包埋固定代谢纤维二糖酿酒酵母工程菌发酵,水稻秸秆纤维素对乙醇转化率可达26%。这方面的研究结果有助于深入了解纤维素酶的协同降解机制。7.将纤维原料的酶解、固定化代谢纤维二糖酿酒酵母工程菌的作用有机耦联,构建成新型的二级串联式生物反应器,在该反应器体系的协同作用下,可有效解除纤维二糖和葡萄糖对纤维素酶的反馈抑制作用,促进纤维原料水稻秸秆的酶水解,发酵40 h,乙醇浓度达25.5g/L,纤维素对乙醇的转化率达43.0%（纤维素对乙醇的理论转化率为56.61%）,是游离细胞同时糖化发酵（SSF）的1.65倍,生产效率达0.64g/（L·h）。采用分批添料式协同酶解发酵工艺,可提高纤维底物的终浓度达250g/L,产物乙醇的终浓度66.51g/L,有效提高了纤维素酶的利用率和乙醇生产效率,降低乙醇的生产成本。该反应器性能稳定,反应效率高,固定化细胞可以重复使用,便于自动化控制。更多还原

【Abstract】 Cellulosic material is the most abundant renewable carbon source in the world. The annual output of rice straw is mor than three hundreds million tons in China. Rice straw cellulose may be hydrolyzed by cellulase to produce glucose, and then glucose can be used for the production of fuel ethanol. The utilization of renewable biomass can not only save the foodstuff but also reduce the environmental pollution. Based on the screening and breeding of strains, the researches investigated the characters of the production of cellulase by the strains and the kinetic process of the fermentation, built the batch fermentation technologies, separated and purified the cellulase, constructed the engineering yeast strain with the cellobiose metabolizing pathway, studied the fermentation with immobilized engineering yeast cells, discussed the cellulase hydrolysis technology of rice straw, and finally investigated the production of fuel ethanol using the two-step coupling bio-reactor. The research results are as follows:1. A strain named YT01 （Penicillium） with high cellulase activity was screened, and then it was mutated by ultraviolet with protoplast and optimized by liquid fermentation, which is named as YT02. The medium with rice straw powder as carbon source, bean powder and （NH₄）₂SO₄ as nitrogen source was optimal and the maximum cellulase activity was reached in the conditions of 29℃and origin pH 6.0 when cultivated for 120 h. The CMCase activity, filter paper activity andβ-glucosidase activity were 3.86 IU/mL, 207.41 IU/mL and 1.40 IU/mL respectively in shaking flask.2. Response surface methodology was used to optimize the medium for cellulase production by YT02. The optimized composition of fermentation medium was （g/L）: rice straw powder, 41.95; bean powder, 24.83; bran powder, 22.16; （NH₄）₂SO₄, 4; KH₂PO₄, 4; MgSO₄, 0.5. The CMCase activity, filter paper activity andβ-glucosidase activity were 8.8967 IU/mL, 357.41 IU/mL and 3.704 IU/mL respectively after the fermentation in the optimized medium for 120 h. All results are higher than before.3. Since the production of cellulase was the major contribution to the bioconversion process, the DO, temperature and pH value of submerged fermentation by YT02 was studied. The fermentation was scaled up in a 5 L stirred fermenter, and the technical conditions of batch fermentation are as follows: fermentation temperature at 0-32 h was 32℃, DO 70 %; fermentation temperature at 32-120 h was 29℃, DO 50 %; origin pH value of fermentation broth was 6.0. The CMCase activity , filter paper activity andβ-glucosidase activity were 11.13 IU/mL, 465.24 IU/mL and 4.08 IU/mL respectively after 4 days, which were higher than the values in shaking fermentation. The dynamic results of batch fermentation showed that the growth of YT02 and the cellulase activity were coupled partially.4. CMCase andβ-glucosidase were purified by DEAE Sephadex A-25 and Sephadex G-75. The purification multiple was 13.48-fold and 18.62-fold to homogeneity by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography with a recovery yield of 10.54 % and 8.62 % respectively. It appeared as a single protein band on SDS-PAGE gel with a molecular mass of approx. 73 kDa, 43 kDa and 57.8 kDa. N-end amino acid sequences and MALDI-TOF analysis of the cellulases were also performed.5. The strategy for direct integration of a cellobiase gene （BGL1） into the Saccharomyces cerevisia chromosome is an effective method for the stable expression of cellobiase in the industrial strain of Saccharomyces cerevisia NAN-27, using an integrating vector pYMIKP which containing a rDNA portion as a homologous recombination sequence to obtain multicopy integrants and PGK1 promoter and terminator, and with the G418 resistance gene （KanMX） as dominant selection marker. This integrating vector is an ideal vector for construction of the genetically engineered S. cerevisiae that used industrial strain as the host. The strategy expended the substrate for fuel ethanol production, and reduced the inhibitor of cellobiose to cellulase hydrolysis. It was found that the engineering Saccharomyces cerevisia NAN-28 cells were immobilized efficiently and rich in cellobiase entrapped into calcium alginate gels. Comparing with the traditional immobilization of pure enzyme protein, this new method was more convenient and economical. The activity of enzyme was not destroyed, and the immobilized cells were quite stable with a long half-life and could accelerate the synergetic hydrolysis process of cellulosic biamass. Comparing with free cells, the fermentation term of immobilized cells was shortened, and the yield of fuel ethanol was improved. The immobilized cells could utilize cellulosic hydrolysate to produce fuel ethanol efficiently.6. During the saccharification of cellulosic material, the key influence factors including character and concentration of substrate, enzyme dosage, temperature and pH were investigated. Since the cellulase system from YT02 was poor in cellobiase （CB/FPA was 0.38）, the yield of rice straw residue to ethanol was only 18 %. When under the synergetic reaction of YT02 cellulase and immobilized engineering Saccharomyces cerevisia NAN-28 cells, the yield of rice straw residue to ethanol was raised to 26 %, while the yield of rice straw residue to ethanol in free cells was just 20 %. All results were significant for the elucidation of synergistic degradation mechanism of cellulase.7. A two-step coupling bioreactor was set up by coupling the cellulose hydrolysis, the immobilized cells and the immobilized cells producing cellobiase together. In this coupling bioreactor, the feedback inhibition to cellulase reaction caused by the accumulation of cellobiose and glucose was eliminated, and the hydrolysis of cellulosic material was promoted. The yield of fuel ethanol from cellulose reached 25.5 g/L at 40 h for fermentation, the conversion rate was 43.0 % （the theoretical conversion rate was 56.61 %）, and the production efficiency was 0.64 g/（ L·h） which was the 1.65 times of SSF in free cells. The new reactor was stable and efficient, and the immobilized cells could be repeatedly used for a long time. Under fed-batch process, the final concentration of cellulosic substrate and ethanol were increased to 250 g/L and 66.51 g/L respectively. The utilization of cellulase and the productive efficiency of ethanol were both improved. The bioreactor showed a good performance in synergetic saccharification and fermentation. This research work simplified the equipment, facilitated the automatic operation, and was important for cost-saving.更多还原