Vinyl chloride (VC), a known human carcinogen with a U.S. EPA maximum contaminant level of 2 ppb, is a significant contaminant of concern present as dilute groundwater plumes at many Department of Defense (DoD) sites. The objective of this project was 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/VC-assimilating 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, a SYBR green-based qPCR method for quantification of etnC and etnE was developed.
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 reference nucleic acids was quantified alongside the etnC and etnE genes (on the same qPCR plate). This facilitated calculation of the percent reference nucleic acid recovery. The reference 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 nine different samples from three different VC-contaminated sites, in some cases over a 3-year period. The abundance of etnC ranged from 1.3×103 - 1.0×105 genes per liter of groundwater (LGW). The abundance of etnE ranged from 1.9×103 - 6.3×105 genes per LGW. Because field application of this method is limited, conclusions regarding these gene abundances cannot be made other than etheneotrophs were found to be present at all three sites.
Methanotroph qPCR for methane monooxygenase functional genes pmoA and mmoX was also performed on many of the same samples. This revealed that pmoA abundance ranged from 1.6×104 - 4.1×107 genes per LGW and that mmoX abundance ranged from 2.5×102 - 6.5×106 genes per LGW. The pmoA qPCR data suggest that methanotrophs are relatively more abundant at VC-contaminated sites than etheneotrophs. This is expected since methane concentrations are typically much higher than ethene and VC concentrations at the sites examined.
RT-qPCR was applied to Nocardioides sp. strain JS614 cultures grown on acetate, ethene, and VC as well as to starved JS614 cultures. Both transcript and gene abundances were measured and the gene expression results reported as “transcripts per gene.” These experiments indicated that when JS614 was starved, transcript per gene ratios were low (0.1-0.2). Acetate-grown JS614 cultures displayed transcript per gene ratios of 0.5-0.6. In contrast, ethene- and VC-grown JS614 cultures featured transcript per gene ratios of 2-12, suggesting that when the transcript per gene ratio is greater than 1, the bacteria expressing etnC and/or etnE are active.
RT-qPCR was successfully applied to four different groundwater samples from a VC-contaminated site in Carver, Massachusetts. Transcript per gene ratios were estimated as follows: 0.4 for both etnC and etnE in RB46D, 0.7 for etnC and 0.8 for etnE in RB63I, 5.7 for etnC and 2.2 for etnE in RB64I, and 9.3 for etnC and 12.6 for etnE in RB73. These results suggest that RT-qPCR could be useful for interrogating the physiological status of etheneotrophs within the zone of influence of monitoring wells.
RT-qPCR for methanotrophs was also performed on the same samples from Carver wells RB46D, RB63I, and RB64I. Transcript per gene ratios were estimated as follows: 0.02 for pmoA and 0.24 for mmoX in RB46D, 0.02 for pmoA and 0.00 for mmoX in RB63I, and 0.11 for pmoA and 0.2 for mmoX in RB64I. These values are substantially lower than the etheneotroph transcript per gene ratios estimated in the same samples. This suggests that although methanotrophs were more abundant in these wells, they were not necessarily more active than the less abundant etheneotrophs. It is recommended that additional studies be conducted to better delineate the differences in methanotroph/etheneotroph abundance and gene expression in environmental samples.
Effective RNA extraction was not always possible from Sterivex filters that had been preserved with RNAlater and stored at -80°C for a period of time prior to analysis. This is currently a drawback to the RT-qPCR method. Based on anecdotal reports, it is possible that freezing filters preserved with RNAlater at -80°C could lead to rapid RNA degradation. Ideally, nucleic acids are to be extracted from filters as soon as possible after sampling and freezing should be avoided. It is recommended that more research be conducted to determine the appropriate sample preservation and handling procedures for filtered groundwater samples in combination with the RT-qPCR method.
In addition to the qPCR and RT-qPCR aspect of this project, groundwater, enrichment culture, and pure culture microcosm experiments were conducted to gain a better understanding of the factors that affect the rate of VC oxidation in groundwater. More specifically, microcosms were designed to address substrate interactions during VC cometabolism in groundwater scenarios with relatively low concentrations of ethene, methane, and VC (all less than 100 µg/L), which are typical for groundwater down gradient of sites having previously undergone reductive dechlorination of chlorinated ethenes.
These experiments suggest that in general when both methane and ethene are present, there is a mixture of positive and negative substrate interactions that lead to an overall enhanced potential for VC to be completely degraded by cometabolism. Methanotrophs can produce epoxyethane, a compound known to stimulate ethene and VC degradation by etheneotrophs, in methane enrichment cultures fed ethene. Conversely, ethene appears to inhibit methane and VC oxidation by methanotrophs. Substrate interactions during VC cometabolism in groundwater systems will be site-specific and depend highly on the abundance of methanotrophs and etheneotrophs as well as the concentrations of VC, ethene, and methane present (assuming oxygen is not limiting the process). These factors should be considered in the design of future VC bioremediation strategies involving cometabolism by applying the tools described here.
Molecular biology tools (MBTs) represent an innovative approach for providing direct lines of evidence for aerobic natural attenuation of VC and will thus support existing anaerobic bioremediation technologies that generate VC as a metabolic intermediate and facilitate improved decision-making in ongoing and future bioremediation studies. This work improves understanding of the normalized rate of VC oxidation in dilute plumes.