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。
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℃.
引文
[1]BP世界能源统计2005[Z]http://wenku.baidu.com/view/adcf5875f46527d3240ce021.html.
[2]古共伟.积极发展石油替代化学品技术[J].天然气化工：C1化学与化工,2007,32(5)：51-57.
[3]方新湘,白云,陈爱华,等.绿色可再生能源之生物质能源[J].现代化工,2008(S2).
[4]马晓建.燃料乙醇生产与应用技术[M].北京：化学工业出版社,2007.
[5]钱明.BP和杜邦加快生产生物丁醇[Z]http://www.qrx.cn/d.aspx?id=71645.
[6]宋安东.可再生能源的微生物转化技术[M].北京：科学出版社,2009.
[7]王鑫鑫,郭红东.国外生物燃料产业发展及其产生的影响[J].世界农业,2008(4)：18-21.
[8]张岚,庄会永.生物质能源潜力巨大工业化应用前途光明[J].高科技与产业化,2010(9)：55-56.
[9]第二代生物燃料发展新动向[Z]http://www.indaa.com.cn/ny/kzs/200903/t20090313_133471.html.
[10]Chiao JS, Sun ZH.History of the acetone-butanol-ethanol fermentation industry in China:development of continuous production technology[J].Microbiol Biotechnol 13:12-4.
[11]董晓玲,袁自强.正丁醇市场动态及生产工艺探讨[J].甘肃科技,2009,25(20)：42-44.
[12]沈兆兵,杜风光,史吉平,等.丙酮丁醇生产技术进展[J].广州化工,2007,35(5)：8-9.
[13]生物丁醇生产技术工艺流程[Z]http://www.zkty.com.cn/cn/tec.asp.
[14]SangYuPLee,JinHWanPark,SheHeeJang,et al.Fermentative Butanol Production by Clostrid[J]. Biotechnol Bioeng,2008,101:209-228.
[15]Ladisch MR.Fermentation-derived butanol and scenarios for its uses in energy-related applications[J]. Enzyme Microb.Technol,1991,13,280-283.
[16]Schwarz WH, Gapes R.Butanol-rediscovering a renewable fuel[J]. Bio World Europe.2006,1:16-19.
[17]李雅丽.国内外丙酮生产现状与市场分析[J].精细与专用化学品,2005(17).
[18]刘娅,刘宏娟,张建安,等.新型生物燃料—丁醇的研究进展[J].现代化工,2008,28(6)：28-31.
[19]王风芹,楚乐然,谢慧,等.纤维燃料丁醇研究进展[J].生物加工过程,2009,7(1)：1-6.
[20]刘玉娣.生物丁醇开发进展[J].石油化工技术经济,2009(2)：21.
[21]粱佐佐.什么是乙醇的替代品[J].轻型汽车技术,2006(7)：37.
[22]诸葛健,李华钟.微生物学(第二版)[M].北京：科学出版社,2009.
[23]Keis S, Shaheen R, Jones DT.Emended descriptions of Clostridium acetobutylicum and Clostridium beijerinckii,and descriptions of Clostridium saccharoperbutylacetonicum sp.nov. and Clostridium saccharobutylicum sp.nov[J].Int.J.Syst.Evol.Microbiol.2001,50(6):2095-2013.
[24]靳孝庆,王桂兰,何冰芳.丙酮丁醇发酵的研究进展及其高产策略[J].化工进展,2007,26(12)：1727-1732.
[25]林有胜,王旭明,王竞,等.生物燃料丁醇的研究与前景[J].现代化工,2008,28(4)：84-87.
[26]Yan Ning Zheng, Liang Zhi Li,Mo Xian. Problems with the microbial production of butanol[J].J Ind Microbiol Biotechnol(2009)36:1127-1138.
[27]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京：科学出版社，2001.
[28]杜连祥.工业微生物实验技术[M].天津：天津科学出版社，1992.
[29]张彩莹,肖连冬.生物化学实验[M].北京：化学工业出版社,2009.
[30]胡琼英,狄洌.生物化学实验[M].北京：化学工业出版社,2007.
[31]陈驹声,陆祖祺.发酵法丙酮和丁醇生产技术[M].北京：化学工业出版社,1991.
[32]Andersch W, Bahl H, Gottschalk G. Level of enzymes involved in acetate, butyrate, acetone and butanol formation by Clostridium acetobutylicum[J]. Eur.J.Appl, Microbiol,1983,18:327-332.
[33]Ramesh VN,George NB, Eleftherios TP.Molecular Characterization of an Aldehyde/Alcohol Dehydrogenase Gene from Clostridium acetobutylicum ATCC 824[J]. Journal of Bacteriology,1994, 173,(3):871-885.
[34]何景昌,张正波,裘娟萍.生物丁醇合成途径中关键酶及其基因的研究进展[J].食品与发酵工业,2009,35，,(2)：116-120.
[35]Peter D,Anita K,Matthias G,et al.Enzymatic investigations on butanol dehydrogenase and butyraldehyde dehydrogenase in extracts of Clostridium acetobutylicum[J]. Appl Microbiol Biotechnol,1987, (26):268-272.
[36]Welch RW, Rudolph FB, Papoutsakis ET. Purification and characterization of the NADH-dependent butanol dehydrogenase from Clostridium acetobutylicum(ATCC 824)[J]. Arch Biochem Biophys,1989,273(2):309-318.
[37]Karl AW, George NB, Eleftherios TP. Molecular Characterization of Two Clostridium acetobutylicum ATCC 824 Butanol Dehydrogenase Isozyme Genes[J]. Journal of Bacteriology,1992,174,(22):7149-7158.
[38]Daniel JP, Richard WW, Frederick BR. Molecular cloning of an alcohol (butanol) dehydrogenase gene cluster from Clostridium acetobutylicum ATCC 824[J]. Journal of Bacteriology,1991,173,(5):1831-1834.
[39]蒋宇,邵蔚蓝.嗜热厌氧产乙醇杆菌乙醇代谢途径的初步研究[J].南京师大学报(自然科学版),2005,28,(3)：69-73.
[40]黄志华,张延平,黄星,等.在Klebsiella pneumoniae醛脱氢酶失活菌中构建NADH再生系统[J]. 中国生物工程杂志,2006,26(12)：75-80.
[41]陈雅蕙,萧能赓,余瑞元,等.生物化学实验原理和方法(第二版)[M].北京：北京大学出版社,2005：240-242.
[42]赵永芳.生物化学技术原理及应用(第二版)[M].北京：科学出版社,2002：23-26.
[43]陈钧辉,李俊,张太平,等.生物化学实验(第四版)[M].北京：科学出版社,2008：101-103.
[44]颜真,张英起.蛋白质研究技术[M].西安：第四军医大学出版社,2007：240-244.
[45]熊宗贵.发酵工艺原理[M].北京：中国医药科技出版社,2006.
[46]周德庆.微生物学教程[M].北京：高等教育出版社,2002.
[47]Beres DL, Hawkins DM.Plaekett-Burman Technique for Sensitivity Analysis of Many-Parametered Models[J]. Ecological Modelling.2001,141:171-183
[48]诸景光,罗立新.基于响应面法的Epothilone发酵优化[J].现代食品科技,2009(07).
[49]张楠,夏尚远,刘训理.统计优化技术在微生物发酵中的应用[J].山东农业大学学报：自然科学版,2009,40(3)：465-468.
[50]杨冀艳,胡磊,许杨Plackett-Burman设计和响应面法优化荷叶总黄酮的提取工艺[J].食品科学,2009(06).
[51]Ahuja SK, Ferreira GM, Moreira AR. Application of Plackett-Burman design and response surface methodology to achieve exponential growth for aggregated shipworm bacterium[J]. Biotechnology Bioengineering,2004,85(6):666-675.
[52]施晓琴,吴松刚.工业微生物育种学(第三版)[M].北京：科学出版社,2009.
[53]林有胜.玉米秸秆的酶水解与丁醇发酵研究[D].大连理工大学,2009.
[54]杨承剑,黄兴国,李伟,等Plackett-Burman设计在益生菌生长主要影响因子筛选中的应用[J].饲料工业,2007(16).
[55]陈坚,李寅.发酵过程优化原理与实践[M].北京：化学工业出版社,2003.
[56]代志凯,张翠,阮征.试验设计和优化及其在发酵培养基优化中的应用[J].微生物学通报,2010(06).
[57]郝学财,余晓斌,刘志钰,等.响应面方法在优化微生物培养基中的应用[J].食品研究与开发,2006,27(1)：38-41.
[58]林有胜,王竞,王旭明,等.利用响应曲面法优化秸秆水解液的丁醇发酵条件研究[J].科学通报,2010(36).
[59]刘志祥,曾超珍.响应面法在发酵培养基优化中的应用[J].北方园艺,2009(2)：127-129.
[60]王永菲,王成国.响应面法的理论与应用[J].中央民族大学学报：自然科学版,2005,14(3)：236-240.
[61]Myers RH. Response Surface Methodology-Current Status and Future Directions[J]. Journal of Quality Technology,1999,31(1).
[62]薛薇.SPSS统计分析方法及应用[M].北京：电子工业出版社,2004.