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1、华中科技大学硕士学位论文高效产氢菌株筛选及厌氧发酵条件优化姓名：刘合钦申请学位级别：硕士专业：生物化学与分子生物学指导教师：闫云君;徐莉20090518I华华 中中 科科 技技 大大 学学 硕硕 士士 学学 位位 论论 文文 摘摘 要要 氢能由于其高效、清洁和还原性等特点，被广泛运用于化工合成、航空航天及金属冶金、电子等领域。目前，氢气的开发和利用技术备受世人关注，已成为生物科学和工程科学研究的热点领域。厌氧发酵制氢技术在当前所有制氢技术中由于其相对简单、原料可再生、低耗能等优点得到了广泛的探索与研究，目前已有一些工艺进入了工程化阶段，但由于产氢效率低等原因仍然制约这些工艺的工业化进程。解决的
2、主要办法是通过获得更高效的产氢菌、优化产氢条件和进行代谢调控等技术手段进一步提高产氢效率。本研究从高效产氢菌株筛选、产氢条件优化和厌氧发酵途径等方面进行了有益探索，主要研究工作如下。 （1） 从华中科技大学内的伏羲河中采集河底污泥样品， 经平板粗筛、 摇瓶复筛，获得两株高效产氢菌。 通过 16Sr DNA 序列分析和系统发育分析， 鉴定为克雷伯式菌属（Klebsiella sp.）和沙雷式菌属（Serratia sp），分别将克雷伯式菌属产氢菌命名为克雷伯氏菌 HQ-3（Klebsiella sp. HQ-3）、沙雷式菌属产氢菌命名为沙雷式菌B-3(Serratia sp.B-3)。 （2）对
3、克雷伯氏菌 HQ-3 进行了单因素厌氧发酵试验，确定产氢的最佳碳和氮源分别为葡萄糖和蛋白胨，最适 pH 为 8.0，最适葡萄糖、蛋白胨、KH2PO4质量配比分别为 24.0 g/L、27.0 g/L 和 8.0 g/L。运用 Plackett-Burrman 对产氢培养基的Na2HPO412H20，KH2PO4，MgSO47H2O，葡萄糖，蛋白胨，酵母膏，pH 八个因素进行主效因子筛选，确定 pH、葡萄糖、蛋白胨、KH2PO4为产氢的主效因子。进一步利用爬坡实验确定主效因子的试验中心值， 利用中心值结合 MATLAB 建模进行响应面法优化厌氧发酵产氢培养基，确定 pH、KH2PO4、蛋白胨、葡
4、萄糖的最优化值，对预测值进行了验证，得到实际产氢量为 826.3 mL/L，与最佳预测值 806.0 mL/L 基本符合。然后，对其发酵条件：温度（） 、接种量（%） 、转速（r/min）进行了单因素实验确定最佳发酵温度、接种量、转速。经上述试验，确定最佳培养条件为：酵母膏 8.0 g/L、Na2HPO415.0 g/L、MgSO43.0 g/L、初始 pH 8.3、KH2PO4 7.7 g/L、蛋白胨 18.1 g/L、葡萄糖 21.4 g/L，温度 32，接种量 3%，转速 60 rpm，在此培养条II华华 中中 科科 技技 大大 学学 硕硕 士士 学学 位位 论论 文文 件下产氢量为 8
5、65.0 mL/L 比初始产氢量（621.0 mL/L）提高了 39%。 （3）确定了产氢厌氧发酵代谢类型。在最优发酵条件下，对发酵液相产物：乙醇、甲酸、乙酸、丙酸、正丁酸、异丁酸进行测定，发现乙醇、甲酸、乙酸含量占总液相产物的 93%，确定其发酵类型为混合酸途径。 关键词关键词： 生物制氢 厌氧发酵 Plackett-Burrman 响应面实验 MATLAB III华华 中中 科科 技技 大大 学学 硕硕 士士 学学 位位 论论 文文 Abstract Owing to its high efficiency, cleanness and renewability, hydrogen is
6、widely used in many fields, such as chemical synthesis, aerospace, metal metallurgy, electronics, etc. At present, production of hydrogen has attracted more and more attention in the world, and is becoming one of the hot spots in biological science and engineering. Hydrogen production from anaerobic
7、 fermentation technology, being relatively simple, renewable raw materials, and power saving, has been extensively explored. So far, a number of technologies have been put into pilot trial, but the low efficiency of hydrogen production and high cost still obstructs their industrialization.The main a
8、pproaches to further improve the production efficiency are to sieve more efficient hydrogen-producing bacteria, to optimize fermen tation conditions, and to innovate techniques, such as employing metabolic regulation. In this study, we have screened two efficient hydrogen-producing strains, optimize
9、d hydrog en-producing fermentation conditions, and further identified the metabolic pathways. The main work is listed below: (1) The riverbed sludge samples were collected from sewage channel in Huazhong University of Science and Technology. By primary plate screening and secondary flask assay, two
10、strains with highly efficient hydrogen production were obtained. They were identified using 16Sr DNA sequence and phylogency analysis, and termed as Klebsiella sp. and Serratia sp., and named the strain of Klebsiella sp. as Klebsiella sp. HQ-3 and the other one Serratia sp. B-3. (2) Through monofact
11、orial anaerobic fermentation experiments, it was found that the best carbon and nitrogen sources for hydrogen production were glucose, peptone; the opti mum pH was 8.0, and the optimal glucose, peptone, KH2PO4 were 24.0 g/L, 27.0 g/L and 8.0 g/L, respectively. In order to optimize the liquid hydroge
12、n-producing culture medium, Plackett-Burrman design was applied to screen the principal factors from Na2HPO412H2O, KH2PO4, MgSO47H2O, glucose, peptone, yeast extract, pH in hydrogen production medium. PH, glucose, peptone, KH2PO4 were determined as the principal factors. Then, the path of steepest a
13、scent experiments design was adopted to determine the experimental center factor value of the principal factors. The anaerobic fermentation liquid medium was further optimized with Response Surface Methodology IV华华 中中 科科 技技 大大 学学 硕硕 士士 学学 位位 论论 文文 by Modeling tools MATLAB. The optimal fermentation c
14、onditions and predicted maximal hydrogen-producing value were determined. In order to verify the pedicted value, the experimental was measured under the optimal fermentation medium, the obtained value of hydrogen production was 826.3 mL/L, coinciding perfectly with the predictive value of 806.0 mL/L
15、. Subsequently, we employed monofactorial experiments to determine the optimum fermentation temperature, inoculum size, and shaking speed. Thus, the optimum culture conditions were: yeast extract 8.0g/L, Na2HPO4 15.0 g/L, MgSO4 3.0 g/L, initial pH 8.3, KH2PO4 7.7 g/L, peptone 18.1 g/L, glucose 21.4
16、g/L, temperature 32, inoculum loading 3%, shaking speed 60 rpm. In the optimum culture conditions, the hydrogen production volume measured was up to 865.0 mL/L, 39% more than the initial volume of 621.0 mL/L. (3) In order to determine the type of anaerobic fermentation, under optimum fermentation condition, we measured the liquid phase of the fermentation products ethanol, formic acid, acetic acid, propionic acid. The results showed that ethanol, formic acid, acetic acid occupied
