【作者】 孙超；

【导师】 谢达平；

【作者基本信息】 湖南农业大学 ， 生化与分子生物学， 2011， 硕士

【摘要】 化石能源的逐渐枯竭和环境污染的日益恶化已成为直接影响到人类社会可持续发展的两个重要问题。因此,寻找对环境污染小和可再生的能源对人们来说是十分必要的。丙酮丁醇发酵的主产物——生物丁醇,作为一种极具潜力的第二代生物燃料,以其独特的优势逐渐受到人们的重视。本文针对传统丙酮丁醇发酵的劣势,以优质发酵菌种为出发菌种,通过对代谢途径中相关酶的研究和发酵的工艺条件进行优化来提高溶剂的产量,期望为丙酮丁醇发酵的工业化生产提供一定的参考。本研究以实验室保藏的丙酮丁醇梭菌s1为出发菌株,通过热激方式对丙酮丁醇梭菌进行初步选择,并对得到的优质菌株S5的形态和生理生化特征进行了鉴定。所得到的菌株S5生成丙酮的浓度和淀粉利用率分别是1.76g/L和70.2%,生成丁醇的浓度约为3.52g/L,产溶剂的能力比S1高7.32%,淀粉利用率比S1提高了4.0%。研究了丙酮丁醇梭状芽孢杆菌(Clostridium acetobutylicum)中丁醇代谢途径的关键酶——依赖NADH的丁醇脱氢酶(Butanol Dehydrogenase,BDH),对该酶进行了分离纯化及酶学性质研究。结果表明,粗酶液分别经过饱和度为20%和60%的硫酸铵沉淀及SephadexG-75凝胶柱层析,丁醇脱氢酶的比活力达到256.7U/mg,纯化倍数提高至12.7倍,回收率为28.9%。丁醇脱氢酶的最适反应温度是35℃,最适反应pH是6.0。在单因素试验基础上,利用Plackett-Burman试验设计法对影响丙酮丁醇梭菌发酵的9个影响因子——装液量、温度、(NH4)2SO4、ZnSO4-7H2O、FeSO4·7H2O、MgSO4·7H2O、吐温80、CaCO3和Na2S2O3进行筛选,结果筛选到三个具有显著性的影响因子,即温度、CaCO3和(NH4)2SO4。然后采用响应面法优化法对发酵过程进行优化,由极值条件下的发酵结果可知,丙酮的预测最优值为3.24481g/L,丙酮实际产量为3.06g/L,丁醇实际产量为6.12g/L,预测值与实际值比较接近。丙酮和丁醇的产量分别由最初的1.76g/L和3.52g/L增加到优化后的3.06g/L和6.12g/L,丙酮丁醇产量提高了73.8%。因此响应面法所建立的模型可以很好地解释丁醇发酵与影响因素之间的关系,说明该方法用于丁醇发酵是可行的,并确定了最佳的发酵工艺条件：其接种量为5%,种龄约18h,装液量为100ml,玉米粉7%,CaCO30.55%,(NH4)2SO40.27%,初始pH为7.0,35.5℃静置培养。更多还原

【Abstract】 Gradual depletion of non-renewable energy resources and the increasingly serious environmental pollution had been two serious issues which had direct impact on sustainable development of human being. So it is necessary for us to find alternative energy source that are environmentally friendly and renewable. Biobutanol, the principal product of ABE fermentation, is a kind of potential biofuel which has been gradually paid attention to by men for its unique advantage. This paper focuses on the disadvantage of traditional ABE fermentation and increases the yields of ABE, the key enzymes of the butanol formation pathway and the conditions for producing ABE from a good strain of Clostridium acetobutylicum in order to provide useful reference for the industrial production of ABE fermentation.This study made a preliminary choose by heat shocking from the original strain of Clostridium acetobutylicum,what we got from this study was a good strain which named S5 The identifications of cell shape and the physiological characteristics on the strain of S5 were done.The yield of acetone, butanol and starch utilization rate was 1.76g/L,3.52 g/L and 70.2%, increased by 7.32%and 4.0%than the original strain of S1, respectively.A NADH dependent butanol dehydrogenase which is one of the key enzymes of the butanol formation pathway in Clostridium acetobutylicum has been separated and purified by the methods of ammonium sulfate precipitation with saturation 20%to 60%and Sephadex G-75 chromatograph. The results showed that the enzyme activity reached 256.7U/mg, and it has been purified 12.7-fold, with a yield of 28.9%. The optimal temperature for the enzyme was 35℃and optimal pH was 6.0, respectively.On the basis of single factor tests, Plackett-Burman design were adopted to filter crucial factors from liquid volume, temperature, (NH4)SO4,ZnSO4·7H2O, FeSO4·7H2O, MgSO4·7H2O, Tween 80, CaCO3, Na2S2O3 during ABE fermentation. Then temperature, CaCO3 and (NH4)2SO4 were filtered as the three crucial factors as a result. Then the Response Surface Analysis (RSA) was applied to enhance the production of ABE fermentation. Under a extreme value condition, the predicted value of acetone is 3.24481 g/L and its experimental value is 3.06g/L. The yield of acetone and butanol increased from 1.76 g/L to 3.06 g/L and 3.52 g/L to 6.12 g/L respectively, was increased by 73.8%. It indicated RSA can well explain the relationship between ABE fermentation and effect factors, and it is feasible to optimize the ABE fermentation. The optimal conditions for ABE fermentation were also confirmed as follows:inoculums size 5%, inoculums age 18h, liquid volume 100ml, corn concentration 7%, CaCO3 0.55%, (NH4)2SO40.27%, the initial pH 7.0, and cultivation temperature 35.5℃.更多还原