【作者】 张立宏；

【导师】 岑可法； 周俊虎； 曹欣玉；

【作者基本信息】 浙江大学 ， 工程热物理， 2008， 博士

【摘要】 随着能源的日益紧张以及环境污染的日益严重，作为清洁能源的氢能的研究逐渐受到重视。相对于传统的制氢方法，生物产氢由于低能耗、高效率、无污染以及可再生等优点而备受关注。其中光合生物产氢集太阳能利用、可再生资源利用、有机废弃物处理以及产氢相联合，将成为最有潜力的产氢技术。认识光合微生物产氢的规律，能动地利用这些客观规律指导能源工作，同时也是地球生态平衡、物质循环极为重要的一个环节。混合菌种是值得重视并需要加强研究和利用的微生物资源。混合菌种多种类细菌间发生协同效应，使代谢产物不易积累，相互创造有利的生存环境，使各菌种的代谢活性充分发挥，从而提高了混合菌种的产氢能力。混合菌种生物技术（MCB，mixed culturebiotechnology）可以成为超越基于传统单一纯菌株生产化学产物或者／和生物能源的生物技术的一种新的选择。与工业化生物技术的纯菌株相对比，利用混合菌种生物技术（MCB）光合产氢具有以下优点：不需要灭菌；由于微生物的多样性具有很强的适应能力；对于多种底物具有适应能力以及持续生产过程的可能性。本文对从沼气池活性污泥富集培养出以多种光合细菌为主的混合菌种进行了光合产氢的实验研究，同时以纯菌种沼泽红假单胞菌Rh．Palustris Z02作了相应的对比试验，并利用修正的Gompertz方程进行产氢动力学分析。结果表明，混合菌种比纯菌种的光合细菌表现出更好的产氢能力和更高的稳定性。尤其在对蔗糖和可溶性淀粉的利用上，混合菌种对这两种碳源的利用比较充分，产氢率分别为3．47 mol H₂／mol蔗糖和6．68 mmol H₂／g可溶性淀粉，氢气含量分别为83．30％和76．06％，且气相中没有甲烷气产生。此外，对各种参数，如pH、温度、接种量以及光强等对产氢率的影响进行了研究。表明混合菌种的产氢条件较Rh．Palustris Z02更为宽松。同时利用Arrhenius模型较好地反映混合菌种产氢速率与反应温度的关系。本文同时对影响光合细菌产氢的固氮酶的三大调控因素：气相条件、NH₄⁺、二价铁系金属离子对混合菌种生物技术（MCB）光合产氢影响进行了试验研究。结果表明气相中大量的O₂对固氮酶以及氢酶的毒害导致无法产氢，而微量O₂的存在却能对产氢有促进作用，尤其是在Ar与微量O₂的混合气相条件下，产氢率达到最高的2．38molH₂／mol乙酸钠，且产氢延迟期最短。同时发现气相条件对混合菌种产氢较纯菌种Rh．Palustris Z02的影响小。通过pH值的控制与不控制时不同NH₄⁺浓度的对比产氢试验，发现不控制pH值时，NH₄⁺对混合菌种光合产氢的抑制作用显著；而控制pH=7．0时，适当低浓度的NH₄⁺可以提高混合菌种的光合产氢率以及产氢速率。无论是否控制pH值，只有NH₄⁺被菌体的生长所消耗掉，才开始产氢，这表明NH₄⁺对产氢的抑制作用在一定范围内是可恢复的。同时通过混合菌种与纯菌株Rh．Palustris Z02的对比试验发现，无论对于生长还是产氢，NH₄⁺对混合菌种的影响均小于对纯菌株Rh．Palustris Z02的影响。二价铁系金属离子Fe²⁺、Co²⁺和Ni²⁺作为金属酶的辅基对光合细菌的生长和产氢有重要的作用。通过对不同浓度的Fe²⁺，Co²⁺和Ni²⁺对光合细菌的生长与产氢的影响实验研究发现，适当浓度的二价铁系离子对产氢具有一定的促进作用，Fe²⁺，Co²⁺和Ni²⁺最佳产氢浓度分别为9μmol／L、0．45μmoI／L和0．1μmol／L。对产氢的影响作用大小顺序为：Fe²⁺>Ni²⁺>Co²⁺。二价铁系离子在适当的浓度范围内可提高生长速率，其中Co²⁺对生长的促进作用最大。pH值影响二价铁系离子对固氮酶活性的作用，当pH=7．0时，Fe²⁺浓度的变化对产氢的影响最显著。随着pH值偏离7．0，Fe²⁺浓度的变化对产氢的影响逐渐减小。碳源与氮源的种类和数量也是影响产氢活性的重要因素。通过对混合菌种利用不同氮源和碳源进行的产氢试验，确定了适合混合菌种光合产氢的氮源和碳源，并进一步确定了产氢最佳的C／N浓度比。本文最后通过对模拟含淀粉废水进行光合产氢的试验研究，确定了淀粉产氢的影响因素。结果发现淀粉浓度是影响光合产氢的一个主要因素，虽然淀粉是混合菌种光合产氢的非抑制性底物，即提高淀粉浓度可以提高产氢量，但对于产氢率来说则是随着淀粉浓度的增高而逐渐减小的，所以产氢过程中如何确定淀粉浓度，还需要综合考虑产氢速率、产氢延迟期等相关参数。结果表明混合菌种利用含淀粉废水光合产氢是可行的。本文同时对反应的菌液进行了DNA提取、纯化以及PCR扩增，通过对DGGE图谱分析，发现混合菌种利用淀粉光反应和暗反应产氢的样品条带在数量和亮度上都存在一定的差异，说明暗反应条件下产氢的优势菌群要略多于光合反应的菌群。更多还原

【Abstract】 Fossil fuel depletion and pollutant emission led to serious energy crisis and environmental problems. Hydrogen gas was considered as an ideal alternative energy because of high energy yield and nonpolluting. Compared with conventional hydrogen production methods, biohydrogen production has received considerable attention in recent years owing to the advantage of renewable, high efficiency and clean. And phototrophic biohydrogen production technology combined solar energy utilization, organic wastes treatment and energy production together, which will be the most competitive power technology of hydrogen production. Research on phototrophic hydrogen production was very important.Mixed culture is a kind of microorganism resource which deserves pay more attention and carefully studied. Mixed culture biotechnology （MCB） could become an attractive addition or alternative to traditional pure culture based biotechnology for the production of bioenergy. Compared with pure culture based industrial biotechnology, specific advantages of MCB include: no sterilization requirements, adaptive capacity owing to microbial diversity, the capacity to use mixed substrates, and the possibility of a continuous process.In this paper, phototrophic hydrogen production was carried out in batch experiment using mixed cultures extracted from active sludge of marsh gas tank, and the contrastive test was using pure culture Rh.Palustris Z02. The modified Gompertz model was used for kinetic analysis of hydrogen production. The experimental result showed that hydrogen yield of mixed cultures was higher than that of pure culture Rh.Palustris Z02, especially using sugar and soluble starch as substrate. Mixed cultures utilized these two carbon sources more sufficiently; hydrogen yield was 3.47mol H₂/mol sugar and 6.68mmol H₂/g soluble starch respectively, hydrogen content was 83.30% and 76.06% respectively, and no methane gas was detected. Moreover, effects of various parameters, initial pH, temperature, inoculums concentration, light intensity etc, were examined with respect to maximum hydrogen yield. The conditions of hydrogen production using mixed cultures are not strict as using pure cultures. Temperature affected the hydrogen production rate according to the Arrhenius equation.The effects of three influencing factors （gas phase, ammonia and iron group ion） on phototropic hydrogen production using MCB were studied. The results showed that a large amount of O₂ in gas phase was toxic for nitrogenase and hydrogenase, which resulted in no hydrogen gas produced. However, a small quantity of O₂ can promote hydrogen production. When Argon gas and a small quantity of O₂ mixed together, the highest hydrogen yield of 2.38mol/mol acetate was achieved, and the lag phase was shortest. Furthermore, the effects of various gas phases on hydrogen production of mixed cultures were fewer than that of pure culture. Contrast test of pH controlled and uncontrolled was carried out. The result showed that the inhibitory effect of NH₄⁺ was obvious when pH was uncontrolled. And appropriate low NH₄⁺ concentration could enhance hydrogen yield and hydrogen production rate when pH was controlled at 7.0. The depletion of NH₄⁺ triggered hydrogen production whenever pH controlled or uncontrolled. The effect of NH₄⁺ concentration on growth and hydrogen production of mixed cultures were lesser than those of pure culture Rh.Palustris Z02. Fe²⁺, Co²⁺ and Ni²⁺ as the prosthetic group of metalloenzyme are important for phototrophic bacterial growth and hydrogen production. The experimental results indicated that appropriate concentration of Fe²⁺, Co²⁺ and Ni²⁺ promoted hydrogen production and increased cell growth. The optimal concentration of Fe²⁺, Co²⁺ and Ni²⁺ for hydrogen production was 9μmol/L, 0.45μmol/L and 0.1μmol/L, respectively. The effect on hydrogen production of iron group ion followed the order of Fe²⁺ > Ni²⁺ > Co²⁺. For cell growth, Co²⁺ was the most important ion than the others two. The effect of Fe²⁺ concentrations on hydrogen production at various pH values was investigated. The result showed that the effect of Fe²⁺ concentration on hydrogen production was conspicuous when pH was controlled at 7.0. As pH value diverged from 7.0, the effect of Fe²⁺ concentration on hydrogen production was decreased gradually.The classes and amount of carbon source and nitrogen source are the important factors for hydrogen production. The effects of carbon and nitrogen on hydrogen phototropic production were examined by mixed cultures. Appropriate carbon source and nitrogen source of hydrogen production and the optimum C/N mass ratio of hydrogen production were determined. The experiments of phototrophic hydrogen production from simulated starch wastewater by mixed cultures were conducted. The results showed that starch concentration was the main influencing factor. The cumulative hydrogen production increased and hydrogen yield decreased with the starch concentration increasing. Therefore, the starch concentration, hydrogen production rate and lag time should be considered carefully. The results indicated that it is feasible for phototrophic hydrogen production from starch wastewater by mixed cultures. Finally, PCR-DGGE method was applied to determine the relative genetic complexity of microbial communities in mixed cultures. Analysis of DGGE profile showed that the quantity and brightness of bands were different under light and dark condition during hydrogen production from starch, predominant microbial population of dark condition was more in quantity.更多还原