A Quantitative PCR Assay for Aerobic, Vinyl Chloride- and Ethene-Assimilating Microorganisms in Groundwater

Vinyl chloride (VC) is a known human carcinogen that is primarily formed in groundwater via incomplete anaerobic dechlorination of chloroethenes. Aerobic, ethene-degrading bacteria (etheneotrophs), which are capable of both fortuitous and growth-linked VC oxidation, could be important in natural attenuation of VC plume: that escape anaerobic treatment. In this work, we developed a quantitative, real-time PCR (qPCR) assay for etheneotrophs in groundwater. We designed and tested degenerate qPCR primers for two functional genes involved in aerobic, growth-coupled VC- and ethene-oxidation (etnC and etnE). Primer specificity to these target genes was tested by comparison to nucleotide sequence databases, PCR analysis of template DNA extracted from isolates and environmental samples, and sequencing of qPCR products obtained from VC-contaminated groundwater. The assay was made quantitative by constructing standard curves (threshold cycle vs log gene copy number) with DNA amplified from Mycobacterium strain JS60, an etheneotrophic isolate. Analysis of groundwater samples from three different VC-contaminated sites revealed that etnC abundance ranged from 1.6 x 10(3) - 1.0 x 10(5) copies/L groundwater while etnE abundance ranged from 4.3 x 10(3) - 6.3 x 10(5) copies/L groundwater. Our data suggest this novel environmental measurement method will be useful for supporting VC bioremediation strategies, assisting in site closure, and conducting mic robial ecology studies involving etheneotrophs.