Degradation And Genetic Characters Of A MTBE-degrading Consortium

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.