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RNA-Based Stable Isotope Probing and Isolation of Anaerobic Benzene-Degrading Bacteria from Gasoline-Contaminated Groundwater

Marine Biotechnology Institute, Heita, Kamaishi, Iwate 026-0001,¹ Taisei Corporation, Nase, Totsuka-ku, Yokohama 245-0051, Japan,² School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, Australia³

Received 29 November 2005/ Accepted 28 February 2006

Stable isotope probing (SIP) of benzene-degrading bacteria in gasoline-contaminated groundwater was coupled to denaturing gradient gel electrophoresis (DGGE) of DNA fragments amplified by reverse transcription-PCR from community 16S rRNA molecules. Supplementation of the groundwater with [¹³C₆]benzene together with an electron acceptor (nitrate, sulfate, or oxygen) showed that a phylotype affiliated with the genus Azoarcus specifically appeared in the ¹³C-RNA fraction only when nitrate was supplemented. This phylotype was also observed as the major band in DGGE analysis of bacterial 16S rRNA gene fragments amplified by PCR from the gasoline-contaminated groundwater. In order to isolate the Azoarcus strains, the groundwater sample was streaked on agar plates containing nonselective diluted CGY medium, and the DGGE analysis was used to screen colonies formed on the plates. This procedure identified five bacterial isolates (from 60 colonies) that corresponded to the SIP-identified Azoarcus phylotype, among which two strains (designated DN11 and AN9) degraded benzene under denitrifying conditions. Incubation of these strains with [¹⁴C]benzene showed that the labeled carbon was mostly incorporated into ¹⁴CO₂ within 14 days. These results indicate that the Azoarcus population was involved in benzene degradation in the gasoline-contaminated groundwater under denitrifying conditions. We suggest that RNA-based SIP identification coupled to phylogenetic screening of nonselective isolates facilitates the isolation of enrichment/isolation-resistant microorganisms with a specific function.

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