降解甲基叔丁基醚（MTBE）的混合菌群的驯化和降解机理研究

Methyl tert-butyl ether (MTBE) is widely used as a gasoline oxygenate to reduce automobile emissions of carbon monoxide and compounds involved in photochemical smog formation. Because it is highly aoluble in water and recalcitrant to biodegradation, it moves rapidly in groundwater and causes the contamination. In this study, we will investigate the degradation of MTBE by consortia RS, which were isolated from the soil of a gas station in Riverside, USA. We will study the biodegradation of MTBE by cometabolism with short chain hydrocarbon (C3-C8), BTEX, and determine the optimized cometabolic carbon source. The pure culture of the strain with high degradation rate of MTBE, which was isolated from consortia RS, will be added into the consortia RS to increase the quantity of MTBE-degrading strains in the mixed culture. Cytochrome P450 enzyme has been identified as the key enzymes for biodegradtion of MTBE, and catalyzes the first step of MTBE metabolism. We will investigate the present cytochrome P450 enzyme in consortia RS, and try to determine the gene of cytochrome P450 enzyme, which will provide basic theory for the construction of recombinant bacteria in furture. Then it will be transferred into E. coli to study the expression, purification and characterization of this cytochrome P450 enzyme. Isotopic tracer method and SPDE-GC-MS will be applied to determine the degradation pathway of MTBE. The lab prepared highly active MTBE degrading bacteria will be employed to enhance the performance of an ineffective biological activated carbon (BAC) column. It will provide consortia for future studies and applications of the BAC process to remove MTBE from groundwater of the contaminated sites in China.

MTBE是目前国内普遍使用的汽油抗氧化剂，其进入环境后由于较高的水溶性及抗降解性，随地下水的流动而扩散，严重威胁饮用水的安全。本课题拟以MTBE降解混合菌群RS为对象，研究烷烃、苯系化合物等共代谢物等对菌群生长及降解MTBE能力的影响，确定最优共代谢物及MTBE降解能力最强的菌株；并将其扩增后投入到原始菌群RS中以增加高效降解菌的剂量，得到生物强化的菌群RSE。首次调查了混合菌群RS中MTBE降解关键酶细胞色素P450酶的存在，并对其酶学性质进行研究。同时，通过同位素示踪和SPDE-GC-MS方法，确定MTBE降解过程中的代谢产物，探询MTBE的降解途径。最后，探索建立长期有效去除水相中MTBE的生物活性炭体系。本研究为国内首次对高效MTBE降解混合菌群的共代谢物，及MTBE降解途径进行深入探讨，为以后国内MTBE污染的治理提供高效MTBE降解菌源，为基因工程菌株的构建提供理论基础。

甲基叔丁基醚（methyl tert-butyl ether, MTBE）是一种无色透明液体，且具有醚类气味的小分子有机化合物，是目前国内普遍使用的无铅汽油的抗爆剂。但由于其高水溶性、迁移性、难降解性和毒性，使它成为地下水的一种潜在污染物，对以地下水为水源水的地区造成了严重威胁。. 本课题以MTBE降解混合菌群RS为研究对象，定向驯化后对混合菌群的组成进行了分析，并筛选得到了3株高效降解MTBE的单菌，再将高效降解菌以不同比例添加到原始菌群中进行生物强化作用。提高混合菌群RS中高效降解MTBE的单菌株比例，来获得更高效降解MTBE的混合菌群。并对混合菌群降解MTBE的共代谢物进行了分析，确定了MTBE降解的最优共代谢物为正辛烷，其与MTBE的最优添加比例为5 mg/L和500 mg/L。随后，经不同时间点取样，通过使用固相动态微萃取结合气相质谱（SPDE-GC-MS）分析方法检测降解过程中生成的不同中间产物，推测MTBE先被转化为TBA，再被转变为HIBA后被进一步降解。. 在对MTBE降解工艺的研究中，我们主要对活性炭载体进行了改性，合成了新型生物载体，PPy负载的活性炭，并对其进行了表征。其对MTBE的吸附符合Langmuir等温吸附模型和准二级动力学方程。新载体PPy/GAC对混合菌群的负载能力是GAC的近两倍。在稳定运行60 d后，仍能去除水中98%以上的MTBE，出水中的MTBE浓度维持在5 μg/L以下，低于美国环保局制定的15 μg/L限值。. 综合上述结果，本研究为后国内MTBE 污染的治理提供高效MTBE 降解菌源，为基因工程菌株的构建提供了坚实的工作基础。

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