【作者】 许继飞；

【导师】 任南琪；

【作者基本信息】 哈尔滨工业大学 ， 环境科学与工程， 2010， 博士

【摘要】 发酵法生物制氢技术能够将废弃物处理和能源回收有效地结合在一起,已经成为环境微生物技术领域研究的热点。为了实现生物制氢的工业化,降低制氢成本,研究者对生物制氢技术进行了广泛的研究,主要的研究方向之一是致力于寻找合适的产氢底物,扩大基质利用范围。自然界中存在大量廉价可再生的木质纤维类生物质资源,但是由于其是纤维素、半纤维素和木质素构成的复合体,很少有微生物能够直接将其降解,通常都是先经过预处理及酶解将其水解为以五碳糖和六碳糖为主的液态水解产物,之后再加以利用。目前的生物制氢研究一般都是以单一六碳糖为主,而对于利用五碳糖生物产氢的研究一直未有突破。因此为了提高对木质纤维素中半纤维素的利用,分离具有利用木糖等五碳糖高效产氢的细菌,对有效地利用木质纤维素产氢,降低产氢成本,实现发酵法生物制氢工业化具有重要的现实意义。基于此问题,本研究进行了同步代谢五碳糖和六碳糖中温发酵产氢菌的定向筛选。获得了能够同步代谢木糖和葡萄糖产氢的新菌种,并对其利用木糖、葡萄糖以及二者混合物的产氢特性进行了详细的研究,最后研究了产氢新菌利用秸秆产氢的潜力,分析了其实际应用价值。发现了同步代谢五碳糖和六碳糖中温发酵产氢新菌种。根据系统进化关系及生理生化特性分析,证实所分离的菌株T2为梭菌属（Clostridium）下的一个新种。分类名为Clostridium hydrogeniproducens sp. nov. ,产氢梭菌（Clostridium hydrogeniproducens）为其模式种,菌株T2^T （=NBRC 105657^T = CCTCC AB 209026^T）为模式株。优化了产氢菌株T2产氢的条件,获得了其利用五碳糖木糖和六碳糖葡萄糖的平均比产氢量分别为2.32 mol-H₂/mol-xylose和2.76 mol-H₂/mol-glucose,平均比产氢速率分别为10.07 mmol-H₂/g-cdw/h和9.47 mmol-H₂/g-cdw/h,其产氢能力居于现有产氢菌的前列。揭示了产氢菌株T2分别利用五碳糖和六碳糖产氢的特性,初步预测了其代谢木糖的产氢途径。用Logistic方程和修正的Gompertz模型描述产氢菌的生长过程;选用修正的Gompertz模型来描述产氢菌产氢的过程;成功的获得了相应过程的动力学参数。构建了描述产氢菌株T2对数生长期特性的潜在生长速率方程和同步性产氢指数方程,解决了用于描述产氢菌株T2在五碳糖和六碳糖基质中的对数期潜在生长速率及生长与产氢的同步性问题。初步推测出产氢菌株T2是通过木糖异构酶直接将木糖转化为木酮糖而后进入磷酸戊糖途径加以利用。分别采用批式、连续和补料分批发酵的方式,研究了产氢菌株T2利用木糖和葡萄糖二者混合基质的产氢规律。证实了产氢菌株T2能够有效的同步代谢木糖和葡萄糖产氢的特性,同时发现了补料分批发酵是产氢菌株T2同步代谢五、六碳混合糖产氢的较优工艺。通过基质浓度扩大法对产氢菌株T2利用酸化汽爆液中木糖和葡萄糖的产氢进行研究,获得其最大比产氢量为分别为1.37 mol-H₂/mol-xylose和2.17 mol-H₂/mol-glucose,分别为其正常条件下的60%和79%,表明其具有很强的适应和抗逆能力。同时采用分步酶解发酵和同步酶解发酵法,对产氢菌株T2利用处理秸秆发酵产氢能力进行研究,分别得到其最大比产氢量71 ml/g-秸秆和110 ml/g-秸秆,这是就目前来说纯菌利用秸秆所能获得的最高产氢量,这充分显示了产氢菌株T2能够利用天然生物质中五碳糖和六碳糖高效产氢的性能。更多还原

【Abstract】 Fermentative biological hydrogen production technology has now become a hot research field, in view of its ability to combine effectively waste treatment with energy recovery. In order to realize the industrialization of biohydrogen production and reduce the cost of hydrogen production, the researchers extensively conduct biohydrogen research, and one of the major research directions is dedicated to find low-cost substrates of hydrogen production, and expand the scope of available substrates. There are a lot of cheap and microorganisms rarely directly degrade renewable lignocelluloses; however, which mainly composed of cellulose, hemicellulose and lignin. Usually it needs to be pretreated and enzymatic hydrolyzed to liquid product-based pentose and hexose, and then is decided to be used. Currently, the attention of biohydrogen production, however, is generally dominated to research the utilization of single hexose substrate. Pentose, a component part of hemicellulose, for biohydrogen production has been no breakthrough. Therefore, in order to improve lignocellulose utilization of hemicellulose, it is necessity to isolate hydrogen-producing bacterium that is ability to use efficiently pentose and hexose to produce hydrogen. Which can increase the efficiency of lignocellulose and reduce the cost for fermentative biohydrogen production, to achieve bio-hydrogen fermentation industrialization has important practical significance.Based on the above issues, this study carried through finding hydrogen-producing bacterium simultaneous using xylose and glucose. Firstly, samples, came from cow dung compost, are be enriched and cultured in xylose, glucose and both mixed substrates, and highly efficient hydrogen producing communities were given, followed by DGGE technique to analyze the structure of their communities. The results showed that the different hydrogen producing communities from different substrates were obtained to have only differences in structure, and similar in type of communities. The novel hydrogen-producing bacteria were isolated from hydrogen producing communities with mixed sugar substrates using Hungate anaerobic technique. According to the phylogenetic relationships and physiological and biochemical characteristics, the isolated strain T2 was verified as a new species under Clostridium, whose category name is called Clostridium hydrogeniproducens sp. nov.. Clostridium hydrogeniproducens was its type species, strain T2^T （= NBRC 105657T = CCTCC AB 209026T） as a model strain.When initial pH was 6.5 and the substrate concentration was under 60 mmol/l, the average yeild of hydrogen production of strain T2 using xylose and glucose was 2.32 mol-H₂/mol-xylose and 2.76 mol-H₂/mol-glucose, and the specific hydrogen production rate was 10.07 mmol-H₂/g-cdw/h and 9.47 mmol-H₂/g-cdw/h, respectively. Hydrogen production capacity of strain T2 is front rank.The characteristics and differences of hydrogen production bacteria T2, how to use xylose and glucose to produce hydrogen, were revealed by researching the dynamics of hydrogen production using xylose and glucose. When the substrate concentration is the 2-10 mmol/l, the hydrogen-producing bacteria T2 obtained parameters, such as the maximum biomass, the potential growth rate, substrate consumption rate and hydrogen production rate, which appeared a linear growth by using xylose and glucose. In comparison, the obtained parameters from glucose as substrate were larger than other ones. The potential growth rate equation is constructed and described characteristics of hydrogen-producing bacteria T2 in logarithmic growth phase. The synchronization of hydrogen index equation is constructed and described relationship of growth and hydrogen production of strain T2. It is speculated that xylose by hydrogen-producing bacteria T2 uptake through the xylose isomerized xylulose by xylose isomerase and then enter the pentose phosphate pathway to be used.Hydrogen production characteristics of the hydrogen producing bacteria T2 using the mixed substates of both xylose and glucose, were studied based on batch, continuous and fed-batch fermentation. The results show that strain T2 can metabolize simultaneously effectively xylose and glucose to produce hydrogen, although the increases of the proportion of glucose in the mixed sugar, to a certain extent, inhibited the xylose utilized to produce hydrogen by T2. This problem was be solved very well by fed-batch fermentation strategy, making xylose utilized in mixed sugar equivalent to the utilization of pure xylose.Liquid and solid products were be acquired by steam explosion of corn straws acidified by acetic acid. The hydrogen-producing study on xylose and glucose from steam explosion liquid acidated utilized by T2 through the expansion of substrate concentration. The maximum yeild of hydrogen production was 1.37 mol-H₂/mol-xylose and 2.17 mol-H₂/mol-glucose, respectively, for 60% and 79% under normal conditions, indicating that T2 had strong adaptation and resilience. The maximum yeild of hydrogen production of T2 with the solid substances of corn straws to produce hydrogen by the methods of SHF and SSF, were 71ml/g-CS and 110ml /g-CS, respectively, which was the highest hydrogen yield of available straw used by pure strains. Therefore, it was be demonstrated adequately that the hydrogen-producing bacteria T2 could utilize pentose and hexose hydrolysates of corn straw to produce efficiently hydrogen.更多还原