Hydrogen Production from Unutilized Biomass by Anaerobic Bacteria

该研究是在爱知县-名古屋城区大学与社会合作资助计划（next）的支持下完成的，旨在利用几丁质分解厌氧菌副果状梭菌M-21将固体废物转化为氢气。通过遗传操作提高该菌的发酵能力，优化其培养条件，提高虾壳产气率。具体操作如下。从Mie大学校园土壤中分离到的paraputrificum M-21可以在虾壳的几丁质高分子上生长。在n -乙酰氨基葡萄糖2上培养时，其倍增时间约为30 min。克隆了几丁质酶和氢化酶的编码基因，并测定了它们的DNA序列。这些酶是由大肠杆菌转化体产生的。产气荚膜梭菌宿主-载体系统成功转化了副营养梭菌。通过电穿孔将副干酪根菌的几丁质酶和氢化酶基因整合到机体中，强化了它们的功能。转化菌株的甲壳素降解活性和产氢活性均高于野生菌株。产氢量提高到约3 mol H2/mol葡萄糖。转化后产生的沼气由氢气（65%）和二氧化碳（35%）组成，当气体被充电到燃料电池时，螺旋螺旋桨。构建了一个由两个发酵罐组成的双容器发酵罐，一个用于生产氢气，一个用于生产甲烷，并分别用于优化ph值为6和7。利用双容器发酵罐进行甲烷发酵，然后进行氢气发酵，提高了产气量，减少了固体废物。

The research was fulfilled under the support of the Grant-in-Aid for University and Society Collaboration (MEXT) in Aichi Prefecture-Nagoya City Area for microbial conversion of solid waste to hydrogen gas by using a chitinolytic anaerobe Clostridium paraputrificum M-21. The productivity of the gas from shrimp shell was increased by improving fermentation ability of the organism by genetic manipulation and by optimization of its cultivation conditions. The details are as follows :1. C. paraputrificum M-21 isolated from Mie University Campus soil can grow on chitin main high polymer in shrimp shell. Its doubling time was about 30 min when cultivated on N-acetylglucosamin.2. The genes encoding chitinases and hydrogenase were cloned and their DNA sequences were determined. These enzymes produced by the Escherichia coli transformants were characterized.3. The host-vector system for Clostridium perfringens was successfully applied to transform C.paraputrificum.4. Chitinases and hydrogenase of C.paraputrificum were fortified by integrating their genes into the organism by electroporation. The respective transformants revealed higher activities of chitin degradation and hydrogen gas production than wild ones. Hydrogen production was increased up to about 3 mol H2/mol glucose.5. The biogas produced by the transformant consisted of hydrogen (65%) and carbon dioxide (35%) and screwed propeller when the gas was charged to a fuel cell.6. A two-vessel fermentor consisting of two fermentors, one for hydrogen and one for methane production was constructed and used to optimize pHs at 6 and 7, respectively.7. Methane fermentation followed by hydrogen fermentation using the two-vessel fermentor enhanced gas production and reduction of solid waste.