Vinyl chloride (VC) plumes are present at many Department of Defense (DoD) sites, and there are significant costs associated with their cleanup. Microbial vinyl chloride (VC) oxidation could be occurring at many of these contaminated sites and contribute significantly to VC natural attenuation. However, there are few options available to site managers for documenting these aerobic processes.
The overall objective of this project was to demonstrate and validate quantitative, real-time PCR (qPCR) and reverse transcription technologies for enumerating the abundance and functionality of two major groups of VC-oxidizing bacteria (the etheneotrophs and methanotrophs) at several VC-contaminated sites. A biological data set that describes how the abundance and functionality of VC-oxidizing bacteria change both spatially and temporally within six different VC plumes was developed, which included associated geochemical parameters and contaminant concentrations in the groundwater. This demonstration is expected to show that application of environmental molecular diagnostic tools such as qPCR during long-term monitoring of contaminated groundwater will yield useful and cost-effective information for site managers who are interested in better understanding and documenting aerobic VC natural attenuation processes.
The qPCR technology, developed under SERDP project ER-1683, yields rapid information concerning presence, abundance, and functionality of VC-oxidizing etheneotrophs. This method employs protocols for DNA and RNA extraction, use of internal nucleic acid controls, and reverse transcription of RNA to complementary DNA (cDNA) so that the qPCR technology can be applied to DNA and RNA extraction from groundwater and aquifer sediment samples. Degenerate oligonucleotide primers that target the functional genes etnC and etnE, both of which are known to be involved in the aerobic VC and ethene biodegradation pathways of etheneotrophs, are used during qPCR. To provide context for interpreting the etheneotroph qPCR results, the technology also includes qPCR analysis for the additional functional genes that may participate in VC biodegradation. This includes the genes mmoX and pmoA, which target methanotrophic bacteria (potential cometabolic VC-oxidizers) and the genes bvcA and vcrA, which target VC reductive dehalogenase genes.
During the demonstration, the team collected over 100 distinct groundwater samples from VC plumes located at six different contaminated DoD sites. Both DNA and RNA were extracted from these samples. The resulting DNA and cDNA was subjected to qPCR estimation of etnC, etnE, pmoA, mmoX, bvcA, and vcrA gene and transcript abundance. The team also measured an array of geochemical parameters from the same groundwater samples during the groundwater sampling campaign. A statistical analysis of the gene and transcript data and the contaminant and geochemical parameters was performed. Functional genes from the etheneotrophs, methanotrophs and anaerobic VC-dechlorinators were present in 99% and expressed in 59% of the samples. Etheneotroph functional genes (etnC and etnE) and VC reductive dehalogenase genes (bvcA and vcrA) were strongly related to VC concentrations (p < 0.001). Methanotroph functional genes (mmoX and pmoA) were not related to VC concentration (p > 0.05). Samples from sites with bulk VC attenuation rates >0.08 year-1 contained higher levels of etheneotroph and anaerobic VC-dechlorinator functional genes and transcripts than those with bulk VC attenuation rates <0.004 year-1.
The team used cryogenic soil coring to collect 134 high-resolution samples from a chlorinated ethane contaminated aquifer at Parris Island Site 45, characterized aquifer geochemical conditions, and investigated the spatial relationships between functional genes of aerobic and anaerobic VC-degrading bacteria and chlorinated ethene concentrations. Functional genes for etheneotrophs (etnC and/or etnE), methanotrophs (mmoX and/or pmoA), and anaerobic VC dechlorinators (bvcA and/or vcrA) coexisted in 48% of soil samples, most of which appeared to be experiencing anaerobic conditions. Etheneotrophs and VC-dechlorinators were correlated to VC concentrations in the lower surficial aquifer (p<0.05), while methanotrophs were not related to VC concentrations. Cryogenic soil coring was a powerful tool for capturing high-spatial resolution trends in geochemical and nucleic acid data. Both the groundwater and aquifer sediment sampling campaigns indicated that aerobic etheneotrophs could play a significant role in VC biodegradation in aquifers that have little dissolved oxygen.
Implementation of qPCR technology for etheneotrophs at chlorinated ethene contaminated sites is already feasible in real-world situations as many long-term groundwater monitoring programs currently prescribe DNA extraction and qPCR analysis for anaerobic chlorinated ethene degrading bacterial biomarkers. However, RNA is not typically used in qPCR practice, as DNA is more readily obtained and easier to work with than RNA. While it may be possible to obtain meaningful relationships between attenuation (or biodegradation rates) and qPCR data obtained with DNA. But DNA is not necessarily a reliable indicator of active biomass, qPCR analysis of RNA may yield stronger correlations with rates than DNA. When using RNA, technology performance is dependent on the efficiency of RNA extraction. The team recommends that when RNA is desired, that at least 3L of each groundwater sample should be filtered, if possible, to maximize RNA yields.
Liang, Y., L.J. Cook, and T.E. Mattes. 2017. Temporal Abundance and Activity Trends of Vinyl Chloride (VC)-degrading Bacteria in a Dilute VC Plume at Naval Air Station Oceana. Environmental Science Pollution Research, 24(15):13760-13774.
Liang, Y., X. Liu, M.A. Singletary, K. Wang, and T.E. Mattes. 2017. Relationships Between the Abundance and Expression of Functional Genes from Vinyl Chloride (VC)-degrading Bacteria and Geochemical Parameters at VC-contaminated Sites. Environmental Science & Technology, 51(21):12164-12174.
Mattes T.E., Jin Y.O., Livermore J., Pearl M. and Liu X., 2015. Abundance and Activity of Vinyl Chloride (VC)-oxidizing Bacteria in a Dilute Groundwater VC Plume Biostimulated with Oxygen and Ethene. Applied Microbiology and Biotechnology, 99(21):9267-9276.
Richards P.M., Y. Liang, R.L. Johnson. and T.E. Mattes. 2019. Cryogenic Soil Coring Reveals Coexistence of Aerobic and Anaerobic Vinyl Chloride Degrading Bacteria in a Chlorinated Ethene Contaminated Aquifer. Water Research, 157(15):281-291.