Quantifying the Presence and Activity of Aerobic, Vinyl Chloride-Degrading Microorganisms in Dilute Groundwater Plumes by Using Real-Time PCR

Abstract : Vinyl chloride (VC), a known human carcinogen with a USEPA maximum contaminant level of 2 ppb, is a significant contaminant of concern present as dilute groundwater plumes at many DoD sites. The objective of this project is to develop quantitative real-time PCR (qPCR) and reverse-transcription qPCR (RT-qPCR) techniques to estimate the abundance and activity of aerobic, VC-oxidizing bacteria (including methanotrophs and etheneotrophs/VCassimilating bacteria) in groundwater. Two functional genes known to participate in VC oxidation in aerobic, ethene- and VC-assimilating bacteria (the alkene monooxygenase subunit gene etnC and the epoxyalkane:CoM transferase gene etnE) were targeted for quantification. Using known etnC and etnE sequences from isolates, enrichment cultures and environmental samples, two sets of degenerate qPCR primers were developed for each functional gene. After extensive testing of primer specificity, we developed a SYBR green-based qPCR method for quantification of etnC and etnE. The qPCR method for etheneotrophs was extended to incorporate mRNA analysis (i.e. RT-qPCR), which entailed selecting appropriate reference nucleic acids (ref mRNA or ref DNA) and adding known amounts of these reference material into samples following the RNA and DNA extraction steps, respectively. Following conversion of RNA to cDNA by reverse transcription (RT), the abundance of ref nucleic acids was quantified alongside the etnC and etnE genes (on the same qPCR plate). This facilitated calculation of the percent ref nucleic acid recovery The ref nucleic acid recovery allows assessment of RNA (and DNA) losses in the sample during several steps in the protocol (e.g. during the RT step). The qPCR method for etheneotrophs was successfully applied to 9 different samples from three different VC-contaminated sites, in some cases over a 3 year period.