【作者】 刘海舟；

【导师】 袁志明；

【作者基本信息】 中国科学院研究生院（武汉病毒研究所） ， 微生物学， 2007， 博士

【摘要】 甲基叔丁基醚(methyl tert-butyl ether，MTBE)是从20世纪90年代以来广泛使用的一种汽油添加剂，以提高汽油发动机运行效率、降低发动机废气造成空气污染和消除含铅汽油导致的铅污染。但由于MTBE在水中溶解度较高，而且很难生物降解，泄漏到环境中后，尤其在地下水中能够长期存留而蓄积，最终造成严重的地下水污染，影响人类健康和生活质量。随着我国MTBE的大量生产及其作为汽油添加剂的应用，大范围的地下水MTBE污染必将成为今后重要的环境生态问题。为了有效地开展地下水MTBE污染进行生物修复，我们在野外富集获得的MTBE降解混合物的基础上，开展了该混合物的MTBE降解效率及影响因素、混合物微生物组成类型及特性、关键酶基因序列及调控等方面的研究，为建立MTBE的生物降解技术提供了储备。首先建立了检测水溶液中MTBE含量稳定的环境顶空-气相色谱(ambient headspace-gas chromatography)检测方法，分别适用于检测MTBE浓度为1-1000μg／L和1-500mg／L的样品，并进行了环境样品检测验证，为实验室以及环境样品中MTBE含量的测定和MTBE污染监测奠定了基础。混合培养物降解分析特性表明，该培养物能够降解浓度达500mg／L的MTBE，降解速率为33.14mg／L／h。辅助碳源，如葡萄糖，能够提高培养物降解MTBE的速率。连续培养的培养物降解延迟期缩短，降解速率加快。在采用传统分离技术对混合培养物中微生物分离鉴定的基础上，通过对混合物中不同组分的16S rDNA PCR及RFLP分析，证明混合物主要是由Terrimonas ferruginea，以及酸杆菌门(Acidobacteria)的细菌组成，而后者是已知的在环境中广泛存在的未培养微生物。这也是首次在MTBE降解培养物中报道酸杆菌门微生物的存在。同时，本研究通过PCR的方法得到了一个长为260 bp的DNA片段，BLAST结果表明是与alk基因有很高的一致性。随后以此片段开始，应用基因组步行(genome walking)的方法获得了长度为2810 bp DNA片段，经过ORF预测软件分析，其中可能包含3个ORF，依次分别是TetR家族转录调节蛋白、烷烃单加氧酶和phyH双加氧酶基因。为下一步alk基因的克隆表达和活性检测，以及此混合培养物降解MTBE途径的研究提供了良好的基础。更多还原

【Abstract】 Methyl tert-butyl ether (MTBE) is a broadly used gasoline additive which wasused to replace the traditional gasoline additive tetra-ethyl lead for reducing gasolinelead pollution, improving gasoline engine operating efficiency and decreasing airpollution caused by engine. However MTBE caused serious groundwater pollutionbecause of its high resolution in water and resistance to biodegradation. AlthoughMTBE has been used in China since 1990s, the possible groundwater pollution is stillunknown. With the increasing usage of MTBE, the management of seriousgroundwater pollution by MTBE will be one of the greatest challenges in environmentmanagement program in China.Considering the future national need for the management of MTBE pollutant, weinitiated the study on biodegradation of MTBE. In this paper, the MTBE degradationability, bacterial composition, and affecting factors, as well as one key gene of MTBEdegrading consortium has been investigated. The data might provide solid informationfor the development of MTBE bioremediation technology. In the first part of thisthesis, an ambient headspace-gas chromatography method was established to measureMTBE in water, which could be used for water solution contained 1-1000μg/L or1-500 mg/L MTBE. This stable MTBE assay method can be used for monitoringMTBE pollution in environments.The consortium culture degraded MTBE concentration up to 500 mg/L and thedegrading speed was 33.14 mg/L/h. Extra supplement of low concentration of carbon sources, such as glucose, increased consortium degradation speed. After a continuouscultivation, the consortium had an increased MTBE degrading rate.Several isolates were isolated from the consortium, but none of them has beenproved to have MTBE degradation ability. 16S rDNA sequence and their RFLPanalysis revealed that the consortium, was composed of several bacteria and most ofthem belonged to Terrimonas ferruginea and Acidobacteria. The latter were knownuncultured bacteria. This was the first report on the existence of Acidobacteria inMTBE degradation cultures.The alkane-1-monooxygenase is the key enzyme in MTBE degradation pathway.A 260 bp DNA fragment was obtained by PCR from the total consortium DNA. Byusing a genome walking method, a 2810 bp DNA fragment was spliced. NCBI ORFprediction indicted there were 3 possible ORFs: a TetR family regulatory factor gene, an alk gene, and a pyhH dioxygenase gene. The data provide a basis for the futureresearch on degradation pathway and the MTBE management.更多还原