Assessment of methanotroph presence and activity in dilute vinyl chloride contaminated groundwater

The extensive use of tetrachloroethene (PCE) and trichloroethene (TCE) as cleaning solvents has resulted in widespread contamination of groundwater systems with vinyl chloride (VC). VC, a known human carcinogen, is primarily formed in groundwater via incomplete anaerobic reductive dechlorination of PCE and TCE. Aerobic, methane-degrading bacteria (methanotrophs), which are capable of VC cometabolism while growing on methane, could be important in natural attenuation of VC plumes that escape anaerobic treatment. Real-time PCR (qPCR) represents an innovative approach for detecting and quantifying the presence and activity of these VC-degrading microbes. Immediate applications of this technique include use in a laboratory setting to help elucidate the potential bacterial-substrate interactions occurring in the subsurface environments at these contaminated sites; interactions that could ultimately affect the role of methanotrophs in VC degradation. This technique could also provide lines of evidence for natural attenuation of VC, thus support existing anaerobic bioremediation technologies that generate VC as a metabolic intermediate.

In this work, we evaluated several PCR primer sets from the literature for use in methanotroph qPCR assays of groundwater samples. PCR primers targeting two functional genes involved in VC cometabolism, pmoA (sub-unit of particulate methane monooxygenase (pMMO)) and mmoX (sub-unit of soluble MMO (sMMO)), as well as 16S rRNA gene primers that targeted Bacteria, and Type I and Type II methanotrophs were tested. These assays were made quantitative by constructing standard curves with DNA from Methylococcus capsulatus (Type I) and Methylocystis sp. strain Rockwell (Type II). Primer sets were evaluated by comparing gene abundance estimated against known amounts of Type I and Type II methanotroph DNA. After primer validation, an effort to substantiate this methanotroph qPCR method was made by attempting to investigate methanotroph populations in groundwater samples from VC-contaminated sites. Some samples studied were also subjected to 16S rRNA gene pyrosequencing, allowing for relative abundance comparisons with qPCR analyses. Following our primer assessment experiments, effective primer sets were used to estimate the presence of methanotrophs at environmental sites in Soldotna, Alaska; Naval Air Station Oceana, Virginia Beach, Virginia; and Carver, Massachusetts. Results showed that methanotrophs were present in nearly all wells sampled from all environmental sites. Estimations of methanotroph relative abundance in environmental samples were determined by comparing the Type I and Type II primer estimates to those of the 16S universal primers. Methanotrophs in these groundwater samples ranged from 0.2% to 6.6% of the total bacterial population. Pyrosequencing analysis of the same samples showed methanotroph relative abundances that ranged from 1.7% to 54%. In groundwater samples where both DNA and RNA was extracted, the quantities of functional gene transcripts per gene copy was compared, revealing that the transcripts/gene ratio for both pmoA and mmoX was less than one, implying relatively low methanotroph activity. Analysis of mmoX environmental sample dissociation curves revealed a double peak, indicating possible non-specific PCR products. Our data suggests that most of the qPCR primer sets used in the environmental samples adequately detect methanotrophs, though the mmoX primers need to be further validated. These primer sets will be useful for supporting VC bioremediation strategies by providing a rapid, convincing, and cost effective alternative the enrichment culture technique currently employed. Comparison of qPCR and pyrosequencing analysis revealed biases in either one, or both techniques. Finally, our preliminary transcripts/gene data suggests that the methanotrophs at the Carver site are not actively expressing pMMO and sMMO genes above basal levels.