Fundamental Research on Application of Genetic Recombination Technique Wastewater Treatment

本研究对利用环境微污染物作为细胞碳和能量来源并对其进行降解的微生物及其酶进行了研究。作为研究对象的2-氯丙烯酸（CAA）和偶氮染料分别作为环境重要微污染物中的卤化和难降解有机化合物。对这两种有机化合物的研究结果如下：从不同来源的土壤样品中分离到3种可以利用2-氯丙烯酸作为细胞碳和能量来源的微生物。这三种微生物均为假单胞菌。通过对提取液的纯化，研究了其中一种微生物的脱卤酶特性。结果表明，该酶的分子量约为50,000，对类似的卤化有机物具有较高的酶活性，含有3个或更少c的多酚化合物。（本部分研究在京都大学化学研究所SODA教授的指导下进行。）用活性红b考察了微生物对固定性有机化合物偶氮染料的降解能力，并对不同的脱色环境条件进行了评价。用含偶氮染料和葡萄糖的人工废水对细菌培养物进行长期驯化，分离出11种微生物。11种细菌均为革兰氏阴性，以芽孢杆菌为主。这些物种表现出不同的脱色能力，脱色能力与葡萄糖利用能力呈正相关。使用驯化和分离培养物的实验表明，在较低ORP条件下脱色速度更快，并且必须存在易于生物降解的底物（例如葡萄糖和糖）作为能量来源。这些结果表明，脱色过程是通过共代谢和还原转化进行的，并且这些微生物可以有效地脱色除活性红B. Less以外的其他偶氮染料

In this research, several investigation were made on the microorganisms and their enzymes which utilize environmental micropollutants as source of cell carbon and energy and/or degrade them. As the compounds of investigation, 2-chloroacrylic acid (CAA) and an azo dye were respectively selected as halogenated and recalcitrant organic compounds among environmentally important micropollutants. The research results for these two organic compounds are as follows:Three species of microorganisms which could utilize 2-chloroacrylic acid as a source of cell carbon and energy, were isolated from various soil samples of different origins. These three microorganisms were confirmed to be Pseudmonas. The characteristics of the dehalogenase of one of these three microorganisms was investigated through a purification of the extracted liquid. The result indicated that the molecular weight of the enzyme was about 50,000 and that the enzymatic activity was relatively high for the similar halogenated orga … More nic compounds with three or less C. With a cloning technique, the dehalogenase gene was introduced into E.coli, and a clonal culture was successfully obtained. (This part of research was carried out under the guidance of Prof. Kenji SODA of the Institute for Chemical Research in Kyoto University.)The microorganisms degradation capability of recalcitrant organic compounds, azo dye, was investigated using Reactive Red B. Also, various environmental conditions for decolourisation were evaluated. Eleven species of microorganisms were isolated from the bacterial culture which was acclimated for a long period of time with an artificial wastewater containing the azo dye and glucose. All eleven species were Gram-negative, and most of them were bacillus. These species showed different decolourisation capability, which positively related to the glucose utilization capability. The experiments using the acclimated and isolated cultures indicated that the decolourisation rate was faster under lower ORP condition, and that easily biodegradable substrates (e.g., glucose and sugar) must exist as energy source. These results suggested that the decolourisation process was carried out through co-metabolism with reductive transformation, and that these microorganisms could effectively decolourise other azo dyes than Reactive Red B. Less