Using DNA-Stable Isotope Probing to Identify MTBE- and TBA-Degrading Microorganisms in Contaminated Groundwater

摘要

Although the anaerobic biodegradation of methyl fert-butyl ether (MTBE) and fert-butyl alcohol (TBA) has been documented in the laboratory and the field, knowledge of the microorganisms and mechanisms involved is still lacking. In this study, DNA-stable isotope probing (SIP) was used to identify microorganisms involved in anaerobic fuel oxygenate biodegradation in a sulfate-reducing MTBE and TBA plume. Microorganisms were collected in the field using Bio-Sep® beads amended with ~(13)C_5-MTBE,~(13)C_1-MTBE (only methoxy carbon labeled), or ~(13)C_4-TBA. ~(13)C-DNA and ~(12)C-DNA extracted from the Bio-Sep beads were cloned and 16S rRNA gene sequences were used to identify the indigenous microorganisms involved in degrading the methoxy group of MTBE and the fert-butyl group of MTBE and TBA. Results indicated that microorganisms were actively degrading ~(13)C-labeled MTBE and TBA in situ and the ~(13)C was incorporated into their DNA. Several sequences related to known MTBE- and TBA-degraders in the Burkholderiales and the Sphingomonadales orders were detected in all three ~(13)C clone libraries and were likely to be primary degraders at the site. Sequences related to sulfate-reducing bacteria and iron-reducers, such as Geobacter and Geothrix, were only detected in the clone libraries where MTBE and TBA were fully labeled with ~(13)C, suggesting that they were involved in processing carbon from the fert-butyl group. Sequences similar to the Pseudomonas genus predominated in the clone library where only the methoxy carbon of MTBE was labeled with ~(13)C. It is likely that members of this genus were secondary degraders cross-feeding on ~(13)C-labeled metabolites such as acetate.