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1、国内图书分类号：T172 国际图书分类号：168.18理学硕士学位论文发酵木糖产乙醇细菌的筛选及特性研究硕士 研 究 生： 郑聪导师： 冯玉杰 教授申 请 学 位： 理学硕士学 科 、 专 业： 微生物学所 在 单 位： 市政环境工程学院答 辩 日 期： 2008 年 6 月授予学位单位： 哈尔滨工业大学Classified Index：T172U.D.C.:168.18Dissertation for a Masters Degree in ScienceISOLATION OF XYLOSEFERMENTATION BACTERIA ANDRESEARCH ON ITS CHARACTER
2、ISTICSCandidate：3BZheng Cong Supervisor：Academic Degree Applied for：Specialty：AffiliationDate of Defence：Degree-conferring-institution：Prof. Feng YujieMaster of scienceMicrobiology School of Muni.& Env. Eng.June, 2008Harbin Institute of Technology哈尔滨工业大学理学硕士学位论文摘要随着世界人口的迅猛增长以及世界经济的发展、生活水平的提高，能源 危机、环
3、境问题以及粮食短缺问题日益困扰着整个世界。寻求新的可再生清 洁能源，缓解环境危机和粮食危机则显得越发重要。燃料乙醇是公认的最有 发展前景的可再生清洁能源之一。以木质纤维素类生物质生产燃料乙醇不仅 能够降低燃料乙醇生产的原料成本，同时在废物处理、环境保护等方面起到 一定的作用。木糖是木质纤维素水解物中含量仅次于葡萄糖的一种单糖。木 糖的有效利用是木质纤维素生物转化生产乙醇的关键环节之一。因此，木糖 利用率和高耐抑制物能力的木糖发酵菌种的选育一直是该领域的热门课题。 本论文以微生物学的理论为基础，根据木糖发酵菌的生活环境特点，从大熊猫粪便中分离筛选出一株发酵木糖产乙醇的菌株 FZ12-N，应用菌株
4、的生理生化特性、微生物细胞膜短链脂肪酸特异性分析和 16S rDNA 分子生物 学 手 段 对 该 菌 株 进 行 了 鉴 定 ， 结 果 为 多 粘 芽 孢 杆 菌 (Paenibacillus polymyxa)。该菌有广泛的碳源，能以多种戊糖、己糖为碳源，并且能以木 糖为惟一碳源乙醇发酵，且有较高的产率。对影响 FZ12-N 发酵的营养物质和发酵条件进行了研究。将氧对发酵的影响进行简单对比研究发现，FZ12-N 在限氧条件下乙醇产量明显高于好氧 发酵和厌氧发酵，所以本研究采用限氧性发酵。确定发酵培养基中的氮源， 结果表明，有机氮源酵母浸膏和蛋白胨有利于发酵，其效果明显优于(NH4)2SO
5、4 等无机氮源。确定 FZ12-N 液体发酵条件是：温度 35， pH 6.3，摇床转速 130 r/m，发酵时间 72 h。在此最优条件下 FZ12-N 发酵液中 的乙醇浓度可达 45.26 mmol/L，是木糖发酵理论产率的 27.39%。 对 FZ12-N 木糖发酵产物进行了分析，并恒算了发酵的碳平衡。以木糖 为惟一碳源液体发酵，以 12 h 为时间单位取样，测定发酵液中的小分子产 物产量，分析发酵规律，简单解析发酵途径。结果显示 FZ12-N 木糖发酵有 多种副产物，主要有乙酸，2,3-丁二醇，乙偶姻等，所以在 FZ12-N 代谢过 程中有多种代谢支路存在。FZ12-N 主要代谢支路有
6、 PPP 途径，TCA 循环， 乙醇支路，乙酸支路，乙偶姻支路，2,3-丁二醇支路，甲酸支路，乳酸支路 等，并建立碳流向分配模型。对发酵过程的碳平衡恒算，碳回收率达到81.75，其中合成乙醇的碳素量占碳消耗量的 21.10。以葡萄糖和木糖 2:1 的比例进行混合糖发酵研究，结果表明，FZ12-NI哈尔滨工业大学理学硕士学位论文有明显的葡萄糖效应，葡萄糖更易于被菌体利用，但是乙醇产率没有明显升 高。 通过木糖发酵细菌菌株的筛选，鉴定，确定了发酵条件及对发酵产物进 行了分析，建立了木糖乙醇发酵的碳流向数学模型，为细菌乙醇发酵提供了 大量的基础数据，对未来工作有重要的积极意义。关键词木糖；乙醇；发酵
7、II哈尔滨工业大学理学硕士学位论文AbstractWith the rapid growth of the worlds population and world economic development, improvement of living standards, the energy crisis, environmental problems and food shortages increasingly troubled the world as a whole. To seek new and renewable sources of clean energy, environ
8、mental crisis and ease thefood crisis are becoming increasingly important. Fuel ethanol can not only reduce production cost of raw materials using wood cellulose, but waste disposal, environmental protection can become better. D-Xylose is the most abundant monosaccharide in lignocellulose hydrolysat
9、e after glucose. Transformation xylose to ethanol is one of the key problems of wood cellulose into ethanol production. Therefore, xylose utilization of capacity and high-inhibitor-resistant strains of the fermentation of xylose breeding has been a hot topic in the field. This study was based on mic
10、robiology theory. According to habitation of existing rich xylose, a bacterium FZ12-N, which can ferment xylose to produce ethanol，was filtered from natural environment. It was identified by analyzing its physiological biochemicalcharacteristics, MIDISherlock Microbial Identification System and appl
11、ying the molecule biological means 16S rDNA. It is classfied as Paenibacillus polymyxa. This bacteria can use a variety of e sugar, including many pentoses and hexoses, and can utilize xylose for unique carbonsource to and ethanol fermentation. Nutrients and fermentation conditions were studied that
12、 can impact FZ12-N fermentation. After studying the oxygen impact on fermentation, it found that the yield in the limited oxygen was significantly higher than that of aerobic fermentation and anaerobic digestion, so this study used limited oxygen to ferment. Studying the influence of nitrogen source
13、, the results showed that the organic source nitrogen yeast extract and peptone is superior to inorganicnitrogen eg. (NH4) 2SO4, et al. The optimum conditions of FZ12-N liquid fermentation is: temperature 35 , pH 6.3, rotate speed of 130 r / m, fermentation time 72 h. Under this optimum conditions,t
14、he alcohol concentration is 27.39 percent of the xylose theorical yield, up to 45.26 mmol / L.III哈尔滨工业大学理学硕士学位论文The FZ12-N xylose fermentation product and carbon balance was analyzed. Utilized xylose for unique carbon source, sampled per 12 h to meter the concentration of small molecule production i
15、n the fermentation fluid, analyzed the fermentation pathway. The results showed that FZ12-N Xylose ferment to generate a number of by-products, mainly including acetic acid, 2,3-butanediol, acetoin, and so on, so many fermention pathway exist in FZ12-N. The main fermentation pathway include PPP, TCA
16、 cycle, ethanol pathway,acetic acid pathway, acetoin pathway, 2,3-butanediol pathway, formic acid pathway, lactic acid pathway, and so on. Carbon balance was analyzed. The result showed that the carbon recovery rate reached 81.75 percent, of which the carbon of ethanol accounts for 21.10 percent of the carbon consumption. Utilize mixed sugar with the ratio 2:1 of Glucose and xylose to ferment, and the results show that, FZ12-N has obvious Glucose Effect. Although glucose can more easily
