Stable Isotope Probing Identifies Novel m-Xylene Degraders in Soil Microcosms from Contaminated and Uncontaminated Sites

The remediation of groundwater contaminated with benzene, toluene, ethylbenzene, and the xylenes (BTEX) typically involves in situ biodegradation. Although the mechanisms of aerobic BTEX biodegradation in laboratory cultures have been well studied, less is known about the microorganisms responsible in mixed culture samples or at contaminated sites. In this study, the microorganisms responsible for in situ degradation within mixed culture samples were investigated using the molecular method stable isotope probing (SIP). For this, m-xylene was utilized as a model BTEX contaminant. Specifically, DNA-based SIP was utilized to identify active m-xylene degraders in microcosms constructed with soil from three sources (a gasoline-contaminated site and two agricultural sites). Replicate microcosms were amended with either labeled (¹³C) or unlabeled m-xylene, and the extracted DNA samples were ultracentrifuged, fractioned, and subjected to terminal restriction fragment length polymorphism (TRFLP). The dominant m-xylene degraders (responsible for ¹³C uptake) were determined by comparing relative abundance of TRFLP phylotypes in heavy fractions of labeled m-xylene (¹³C) amended samples to the controls (from unlabeled m-xylene amended samples). Four phylotypes were identified as the dominant m-xylene degrading species, falling within either the β Proteobacteria or the Bacilli. Of these, two 16S rRNA gene sequences were highly novel, displaying very limited similarity (94% and 90%) to any previously reported 16S rRNA gene sequence. Further, three of these phylotypes fell within genera with limited or no previous links to BTEX degradation, suggesting much information is still to be gained concerning the identity of microorganisms responsible for degradation within mixed culture samples.