【作者】 牛坤；

【导师】 谭文松；

【作者基本信息】 华东理工大学 ， 生物化工， 2010， 博士

【摘要】 能源危机和环境污染等问题迫使人们寻找可以替代传统化石能源的新型清洁可再生能源。氢能因其自身的高转化效率及清洁、无污染等优点而被公认为最有发展前景的能源形式之一。常规的制氢方法有热化学法、电化学法及生物转化法,相比而言,生物制氢技术具有可在常温常压下操作,并能利用可再生生物废弃物等优点而备受关注。生物制氢主要包括光发酵制氢、暗发酵制氢及两者联合制氢等,其中暗发酵制氢具有产氢速度快,设备简单、易实施,以及可利用可再生资源和废弃物等特点而最具产业化前景。但是该技术存在微生物产氢得率低、发酵底物成本高等缺陷,成为限制暗发酵制氢工业化的主要技术瓶颈。针对上述问题,本文分别从产氢菌种、培养条件及发酵底物等方面进行了相关研究。首先,开展了高效产氢菌株的筛选、鉴定,及发酵产氢条件的优化等研究工作,了解了所筛菌株的基本产氢特性,并在此基础上考察了不同发酵条件对其产氢代谢途径的影响,以期为该菌株的分子生物学改造提供理论依据。其次,在上述优化条件下进行了木质纤维素水解液发酵产氢实验研究,考察了所筛菌株在木质纤维素水解液中的发酵产氢特性,并进一步分析了木质纤维素水解液中影响产氢得率的主要原因,为后续利用木质纤维素水解液进行暗发酵制氢过程的开发提供了必要的基础数据。本文以污水处理厂厌氧污泥为基质进行产氢菌种的筛选,通过初筛与复筛得到54株产氢菌株,最终选定氢气产量与产氢得率相对较高的三株细菌进行了鉴定。经形态学观察、生理生化特性实验及16S rRNA序列分析后,发现三株被鉴菌株均为克雷伯氏肺炎杆菌(Klebsiella pneumoniae),将产氢效率最高的一株菌株命名为Klebsiella pneumoniae ECU-15,并进一步开展了后续实验研究。为了优化培养条件和培养基组分,首先对K.pneumoniae ECU-15菌株的产氢特性进行了考察。实验发现严格厌氧环境有利于提高产氢效率,其氢气产量比微好氧条件提高了17.97%。细胞生长与氢气合成的最佳温度均为37℃,细胞生长的最佳pH值为7.0,但产氢的最佳pH值为6.0。ECU-15菌株能够利用单糖、双糖等碳源为底物,而不能利用纤维素及淀粉等多糖,且葡萄糖最适于氢气的合成。产氢得率随初始葡萄糖浓度的提高而降低,当初始葡萄糖浓度为5g/L时产氢得率达到最大值(2.07mol/mol glucose)。单位体积的产氢速率则随初始葡萄糖浓度的增加而提高,当初始葡萄糖浓度为30g／L时产氢速率达到最大值482ml／(L·h)。然而,40g/L的初始葡萄糖浓度则会对细胞生长与氢气合成产生抑制作用。另外,实验结果表明磷酸盐缓冲液的最佳浓度为200mM,而金属离子的加入也能显著提高该微生物的产氢得率,添加300mg/L Fe2+可使产氢得率提高31.03%,120mg／L Mg2+可使产氢得率提高18.89%,35mg／L Ni2+可使产氢得率提高24.83%。通过数据拟合,发现Logistic方程能较好地描述菌体生长,修正的Gompertz方程也能较好地描述氢气合成与葡萄糖消耗过程。与其它产氢菌株相比,该菌株具有相对较高的体积产氢速率。为了进一步了解培养条件对微生物代谢产氢及吸氢途径的具体影响,本文利用各操作条件下获得的厌氧发酵代谢数据,并结合代谢通量分析(MFA)方法,对K. pneumoniaeECU-15菌株的厌氧产氢代谢通量进行了计算与分析,得到了温度、pH及初始葡萄糖浓度对胞内代谢途径整体通量分布的影响,特别是针对氢气合成与消耗途径流量的影响。研究结果表明,温度、pH和初始葡萄糖浓度均对该过程中的吸氢通量有较大影响,在26～37℃的温度范围内吸氢通量会随温度的升高而降低,而相对于中性或弱碱性发酵环境,弱酸性的pH环境也会降低吸氢通量,同时,限制葡萄糖底物浓度亦能降低吸氢通量,而有利于产氢发酵。上述各因素对产氢途径及相关节点流量分配的影响也不尽相同,温度升高,乙醇通量降低,说明乙醇脱氢酶活性降低,此时NADH出现大量残余,从而有利于通过NADH/NAD+平衡调节途径的产氢。而在pH≥7.0时,丙酮酸甲酸裂解酶系呈现较高通量,有利于甲酸裂解产氢。另外,初始葡萄糖浓度在底物限制的情况下也会提高甲酸裂解酶途径的产氢通量,有利于氢气的合成。就发酵制氢过程而言,发酵底物类型及其成本将是制约生物制氢实用化和工业化的主要因素,为此本文选择了来源丰富且廉价的木质纤维素水解液为底物进行了发酵制氢的实验研究。为了降低木质纤维素的生物学抗性,提高纤维素酶对木质纤维素的水解效率,本文首次采用纳米光催化辅助NaOH溶液的方法对水稻秸秆进行了预处理,结果表明在1.5% NaOH溶液中添加2g/L纳米Ti02,并经紫外光照射1h后,木质素的脱除率达到67.84%,半纤维素的脱除率达到53.03%,对预处理后的秸秆进行纤维素酶水解实验发现其酶解率达到73.96%,均高于高浓度碱液单独处理的效果。利用上述水解液发酵后得到0.78V／V的氢气产量,高于蒸汽爆破法预处理玉米秸秆酶解液的发酵产氢量(0.65V／V),但两者均显著低于以葡萄糖为底物的发酵产氢结果。为了探明木质纤维素水解液中降低产氢量的主要因素,本文通过实验对木质纤维素水解液的组成及其对微生物代谢产氢的影响进行了研究,发现木质纤维素水解液中影响微生物代谢产氢的关键组分为葡萄糖、木糖及纤维二糖,而降低水解液产氢效率的主要成分为木糖。然后,以一定比例的木糖和葡萄糖混合物为底物进行产氢发酵实验,结果说明K. pneumoniae ECU-15菌株对木糖的利用效率较低,虽然木糖的存在不会显著影响微生物对葡萄糖的利用率,但葡萄糖的代谢产氢途径则明显受到干扰,最终导致发酵产氢效率的降低。更多还原

【Abstract】 Serious global environment pollution and energy crisis have been caused by excessive use of fossil fuels. Thus, there is a pressing need to develop nonpolluting and renewable energy resource. Hydrogen is widely thought to be an ideal and efficient energy carrier in the future due to its high conversion efficiency, recyclability and nonpolluting nature. It could be produced by thermochemical process, electrochemical process and bioconversion process. Among them, bioconversion process has attracted more and more attentions for two reasons: utilization of renewable resource, and usually operated at ambient temperature and atmospheric pressure. It can be conducted by photosynthetic fermentation, dark fermentation and two-stage fermentation. Dark fermentation is a promising method for its high hydrogen-producing rate, simple fermentative equipment, and bioconversion feasibility from the recycle resources. However, the low yield of hydrogen production and high cost of the substrate are the main constraints for industrialization of biohydrogen production. Based on these problems, the researches of the effect of hydrogen producing strains, culture conditions arid the fermentative substrates on biohydrogen process were carried out in this paper. Firstly, high-producing strains were isolated and identified with the optimal culture conditions obtained. The effect of culture conditions on metabolic pathway was investigated to provide the theoretical foundation for the microorganism recombination. Secondly, the characteristics of biohydrogen production from hydrolysate were investigated, and the key factors and its action rules that influenced the hydrogen production in hydrolysate were analyzed. These results offered the fundamental data for the development of hydrogen production from lignocellulosic hydrolysate.In this paper,54 hydrogen producing strains were isolated from the anaerobic sewage sludge. Among them, only three strains with the highest hydrogen producing efficiency were identified by morphological observation, physiological and biochemical experiments and 16S rRNA analysis. The results indicated that the all the three strains were belonging to the Klebsiella pneumoniae. A strain named Klebsiella pneumoniae ECU-15 with the highest hydrogen-producing capability was chosen as the target strain for the following investigations.In order to optimize the culture conditions and medium compositions of the biohydrogen fermentation, the hydrogen production characteristics of K. pneumoniae ECU-15 were firstly investigated. It was found that the anaerobic condition was in favor of hydrogen production, and the hydrogen production was improved by 17.97% at the anaerobic condition than that under the micro-aerobic condition. The optimum culture temperature and pH were 37℃and 6.0 for the hydrogen fermentation, but the optimal pH for cell growth was observed at pH7.0. The strain of ECU-15 could grow in several kinds of monosaccharide and disaccharide but starch and cellulose. Fermentation process through glucose exhibited the maximum hydrogen productivity and production among these carbon sources. The hydrogen yield was decreased with the increasing of initial glucose concentration. The maximum yield of 2.07mol/mol glucose was obtained at the initial glucose concentration of 5g/L. The hydrogen production rate was increased with the increasing of initial glucose concentration, and the maximum production rate of 482ml/l/h was obtained at the initial glucose concentration of 30g/L. However, the cell growth and hydrogen production would be inhibited at the initial glucose concentration of 40g/L. In addition, the concentration of phosphate buffer was optimized to be 200mM. The hydrogen yield was improved by 31.03%,18.89% and 24.83% by adding concentration of 300mg/L Fe2+,120mg/L Mg2+ and 35mg/L Ni2+, respectively. The processes of cell growth, hydrogen production and glucose consumption in batch fermentation were simulated by Logistic model and Gompertz model. It was indicated that the experimental data of these three processes could fit well with these models. From the above results, it could be seen that K. pneumoniae ECU-15 exhibited the highest hydrogen production rate compared with the other strains.In order to find out the influence of culture conditions on the metabolic pathway of K. pneumoniae ECU-15, the network of glucose metabolism under anaerobic conditions was calculated and analyzed by metabolic flux analysis (MFA). The effects of temperature, pH and initial glucose concentration on the intracellular fluxes, as well as the fluxes of hydrogen producing and uptake were caculated and analyzed. The results indicated that the uptake flux of hydrogen was affected by temperature, pH and initial glucose concentration significantly. It was decreased with the increasing temperature from 26℃to 37℃. Compared with the weak acidity pH, the neutral or weak basicity pH was in favor of the uptake of hydrogen. Under the limited glucose concentration conditions, the uptake flux of hydrogen would also decrease. The hydrogen producing flux and the flux distribution of some key nodes in the whole metabolic networks could also be affected by these factors. The flux of the ethanol producing pathway was decreased with the temperature. Thus NADH would be remained for hydrogen production. And the fluxes of pyruvate dehydrogenase pathway and the pyruvate formate lyase metabolic pathway were higher at pH>7.0 than that of other pH values. Moreover, the flux of pyruvate formate lyase metabolic pathway was increased under the limited glucose concentration condition, which would in favor of the hydrogen production.As for the biohydrogen fermentation, both the types and the cost of fermentative substrates were important factors that restricted the practicality and industrialization of the process. Therefore, the lignocellulosic hydrolysate which had rich resource and lower cost was used for biohydrogen fermentation in this paper. In order to decrease the biological resistance and increase the enzymatic hydrolysis of lignocellulose, the pretreatment technology of rice straw through NaOH solution assisted with photocatalysis was investigated through experiments. The results showed that the degradation rates of the lignin and hemicellulose were 67.84% and 53.03%, respectively under the condition of 2g/L nano-TiO2 and 1h of photocatalysis time in 1.5% NaOH solution. The enzymatic hydrolysis rate of the pretreated straw was 73.96%, which was higher than that with the alkali procedure. The biohydrogen production with this hydrolysate was 0.78 V/V, which was higher than that of the corn stalk hydrolysate pretreated by steam-explosion process (0.65V/V). These results indicated that the hydrogen production of the hydrolysate was lower than that of glucose. In order to determine the main compositions in hydrolysate that influenced the biohydrogen production, the fermentation with various substrates of this bacterium was investigated. It was found that glucose, xylose and cellobiose were the main compositions in hydrolysate. The lower hydrogen production of the hydrolysate might be due to the existence of xylose. In addition, the fermentation results of K. pneumoniae ECU-15 with various proportions of glucose and xylose indicated that the consumption rate of xylose was lower than that of glucose. The biohydrogen metabolic pathway of glucose was seriouslly influenced by xylose, which resulted in the decrease the hydrogen efficiency of ECU-15 from hydrolysate.更多还原