【作者】 刘冰峰；

【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】 目前,化石能源能源短缺,石油价格日益攀升,亟需寻求可再生、高效、清洁能源来替代。氢气作为清洁能源的首选,是未来理想的燃料之一。光发酵生物制氢技术能将有机废水净化处理、氢能开发和太阳能利用三者有机耦合,是一种低成本、低能耗的绿色能源生产技术。暗-光发酵细菌耦合生物制氢技术能够提高底物转化效率,实现氢气产量的最大化,深度产氢,具有更为广阔的应用前景,对于加快生物制氢技术的产业化步伐也具有重要的意义。本文从淡水鱼塘底泥中分离获得一株产氢光发酵细菌菌株RLD-53,经细胞形态学、生理生化特征以及系统进化发育分析,将其鉴定为Rhodopseudomonas faecalis的新菌株RLD-53,并采用间歇培养试验确定了其最佳生长和产氢条件。菌株RLD-53对乙酸具有高效的氢转化率,其比产氢量为2.64-2.835 mol H2/mol乙酸钠,底物转化效率为66%-71%,最大产氢速率可达32.62 ml H2/l/h,平均氢气含量约81%。菌株RLD-53不能利用丁酸和乙醇进行产氢,而丁酸和乙醇分别是丁酸型发酵细菌和乙醇型发酵细菌代谢物的主要成分,它们和乙酸的浓度比决定着耦合系统的产氢能力。因此,本文研究了双碳源作为底物时光发酵的氢气生产效能,结果表明丁酸钠添加到乙酸钠培养基中对菌株的生长和产氢影响显著,高浓度的丁酸钠抑制细菌的生长和产氢,同时确定了二者最佳产氢的比例关系。当丁酸钠对乙酸钠比例1:2时,比产氢量为3.425 mol H₂/mol乙酸钠,最大氢气产率32.53 ml H2/l/h,氢气含量高达84.27%。乙醇的添加对细菌的生长和产氢有微弱促进作用。Ni²⁺、Fe²⁺和Mg²⁺三种金属离子对光发酵细菌的产氢和生长有明显的影响。其中Ni²⁺和Fe²⁺是构成产氢关键酶的主要成分,在一定的浓度范围内能够促进细菌的生长和产氢,过高或过低的浓度对产氢和生长有抑制作用。Mg²⁺对产氢无明显影响,但在试验浓度范围内却明显的促进细胞的生长,是培养基中不可或缺的成分。不同的气体作为气相对产氢有显著影响,其中高浓度的二氧化碳作为气相明显抑制光发酵细菌的生长和产氢。据此,提出了二氧化碳和反应体系分离的试验方法,成功的促进了R.faecalis RLD-53的氢气生产,降低了二氧化碳的抑制作用。采用间歇重复补料的方法进行光发酵产氢试验,通过控制补料浓度和pH实现高效产氢,平均氢气产量达3.17 mol H₂/mol乙酸钠,同间歇试验产氢能力相比有较大的优越性。为了增加R. faecalis RLD-53对酸性环境的耐受力,提高基质的氢转化率,试验采用琼脂固定化RLD-53,从琼脂颗粒粒径、菌龄、琼脂颗粒浓度、包埋生物量、耐酸性和光照强度等方面展开固定化光发酵细菌产氢研究,结果表明:固定化细菌能够明显促进氢气生产,延长产氢时间,增加底物利用率和转化效率,而且能够耐受一定程度的酸性环境,甚至在pH 5.0时也有氢气产生。在暗光发酵细菌耦合产氢过程中,首次使用了游离的暗发酵细菌和固定化的光发酵细菌,更重要的是利用磷酸缓冲液来维持反应体系不同的pH,使暗发酵代谢物成分发生了转变,成功的增加了乙酸在代谢物中的含量,同时使用琼脂包埋RLD-53,增加了其对酸的耐受力和氢气产量。研究发现,在暗光两步法产氢过程中暗发酵代谢物的稀释率扮演着重要角色,影响着光发酵细菌对代谢物的有效转化和利用;在混合培养间歇产氢试验中,控制系统的初始pH和暗光细菌的比例也是一个关键因素。初始pH控制在7.5时,整个反应过程中pH在6.5-7.5,并适当的增加光发酵细菌的数量,以寻求暗光细菌在生长和产氢速率上的相对匹配,有助于暗光发酵细菌协同作用、高效产氢。同时对暗-光发酵耦合产氢机制和产氢关键因素进行了初步分析。更多还原

【Abstract】 At present, energy substitute for fossil fuels need urgently because the lack of fossil energy source and the increase of international petrolic price. Hydrogen is to replace fossil fuels in the future, will become world’s“clean energy choice”, it has to be produced renewably and in large scale, through environmentally benign processes. Among bio-hydrogen production processes, photo-fermentation hydrogen production can combining solar energy, wastewater treatment and cleaning energy source production, the cost was decreased, considered as a potential hydrogen production technology in engineering application. The combination of dark and photo-fermentation can improve hydrogen yield and substrate conversion efficiency, and it is significant for scale bio-hydrogen production in future.In this study, a photo-fermentation bacterium strain RLD-53 was isolated from freshwater pond sludge. According to their morphology, physiological and biochemical characteristics, blast of the sequences of 16S rDNA and analysis of phylogenetic tree, we characterized it as a new strain within the species R. faecalis and designated as R. faecalis strain RLD-53. Optimal culture and H₂ production conditions of strain RLD-53 were determined in batch tests. Strain RLD-53 can convert acetate into H₂ with high efficiency. The hydrogen yields were 2.64-2.84 mol H₂/mol acetate and the maximum hydrogen production rate was 32.62 ml-H₂/l/h and average hydrogen content was about 81%。R. faecalis RLD-53 can not utilize butyrate and ethanol as sole carbon source for hydrogen production under anaerobic light conditions. But, butyrate and ethanol are main byproducts in butyrate and ethanol fermentation, respectively. The ratio of butyrate and ethanol to acetate was key factors for hydrogen production of the combination of dark and photo-fermentation bacteria. Therefore, the capacity of hydrogen production was studied when dual carbon sources were used as substrate. Results indicate that butyrate added in acetate medium significantly affected on photo-hydrogen production and butyrate of high concentration evidently inhibited on the growth and hydrogen production of RLD-53, and the optimal ratio of butyrate to acetate for H₂ production was determined. When the ratio of butyrate to acetate at 1:2, the hydrogen yields and maximum hydrogen production rate were 3.43 mol H₂/mol acetate and 32.53 ml H₂/l/h, respectively, and average hydrogen content reached about 84.27%. Effect of ethanol on hydrogen production was slightly. Effects of Ni²⁺, Fe²⁺ and Mg²⁺ on photo-hydrogen production and cell growth were obviously. In an appropriate range, Ni²⁺ and Fe²⁺ can increase the hydrogen production and cell growth with increasing their concentration. A low or high Ni²⁺ and Fe²⁺ concentration will inhibit photo-hydrogen production. Mg²⁺ concentration did not obviously affect hydrogen production by R. faecalis RLD-53. However, Mg²⁺ of different concentration in culture medium just promoted cell growth.Different gases as gas phase evidently influenced on photo-hydrogen production. Observation indicates that CO2 of high concentration have inhibited effect on cell growth and H₂ production. Therefore, we proposed a method of separation of CO2 from reaction system and it successfully stimulated photo-H₂ production in the entire H₂ production process.In fed-batch culture hydrogen production process, hydrogen production was improved by the control of feeding acetate concentration and pH. The average hydrogen yield of 3.17 mol H₂/mol acetate in this study was substantially higher than that in other study by pure cultures of bacteria. So experiments demonstrated the repeated fed-batch mode compared with batch culture obtained higher efficiency for hydrogen production.In order to increase acid-resistant capacity of RLD-53 and efficiency of substrate conversion into hydrogen, immobilized cells were used. The effects of diameter of agar granule, inoculant age, agar concentration, biomass in agar and light intensty on immobilized RLD-53 were investigated and at pH 5.0 hydrogen was also produced.The combination of dissociate dark-fermentation bacteria and immobilized photo-fermentation bacteria for hydrogen production was adopted first time in this study. To important, phosphate buffer was used to maintain different pH of culture system and the change of component of metabolites from dark-fermentation, and the content of acetate was successfully increased. Results showed that the dilution ratio of metabolites from dark-fermentation play an important role in two step hydrogen production and the control of initial pH and the ratio of dark and photo-bacteria were key factors in mixed culture hydrogen production. At initial pH 7.5, pH of entire process at 6.5-7.5, and properly increased the concentration of strain RLD-53, these were favorable to dark and photo-fermentation bacteria. The mechanism and key factors of hydrogen production by the combination of dark and photo-fermentation was analyzed preliminary.更多还原