title: Using DNA-Stable Isotope Probing to Identify MTBE- and TBA-Degrading Microorganisms in Contaminated Groundwater.
abstract: Although the anaerobic biodegradation of methyltert-butyl ether (MTBE) andtert-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 with13C5-MTBE,13C1-MTBE (only methoxy carbon labeled), or13C4-TBA.13C-DNA and12C-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 thetert-butyl group of MTBE and TBA. Results indicated that microorganisms were actively degrading13C-labeled MTBE and TBA in situ and the13C 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 three13C clone libraries and were likely to be primary degraders at the site. Sequences related to sulfate-reducing bacteria and iron-reducers, such asGeobacterandGeothrix, were only detected in the clone libraries where MTBE and TBA were fully labeled with13C, suggesting that they were involved in processing carbon from thetert-butyl group. Sequences similar to thePseudomonasgenus predominated in the clone library where only the methoxy carbon of MTBE was labeled with13C. It is likely that members of this genus were secondary degraders cross-feeding on13C-labeled metabolites such as acetate.
authors.affiliation: Department of Chemical Engineering, University of Tulsa, Tulsa, OK, USA.
authors.forename: Katherine C
authors.initials: KC
authors.lastname: Key
authors.affiliation: Department of Chemical Engineering, University of Tulsa, Tulsa, OK, USA.
authors.forename: Kerry L
authors.initials: KL
authors.lastname: Sublette
authors.affiliation: Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Duncan
authors.affiliation: Department of Land, Air, and Water Resources, University of California at Davis, Davis, CA.
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Mackay
authors.affiliation: Department of Land, Air, and Water Resources, University of California at Davis, Davis, CA.
authors.forename: Kate M
authors.initials: KM
authors.lastname: Scow
authors.affiliation: Microbial Insights Inc., Knoxville, TN.
authors.forename: Dora
authors.initials: D
authors.lastname: Ogles