Reductive dechlorination plays a major role in the transformation and detoxification of chloroorganic pollutants including chlorinated ethenes, and the application of molecular biological tools (MBTs) has already impacted site assessment and bioremediation monitoring at many Department of Defense (DoD) sites. Unfortunately, limitations in current tools provide an incomplete picture of the reductively dechlorinating bacterial community, thus limiting the value of the analysis. To overcome the current limitations and more accurately assess, predict, monitor, and manage reductive dechlorination processes at contaminated DoD sites, this research effort identified novel reductive dechlorination biomarker genes and developed MBTs and approaches that improve the understanding of target gene presence, abundance, and expression, and thus, contaminant detoxification.
The specific research objectives focused on (1) elucidating co-contaminant effects on the reductive dechlorination of chlorinated ethenes; (2) applying microarrays as tools to discover novel reductive dechlorination biomarker genes; (3) developing specific, sensitive, and economic quantitative real-time PCR (qPCR) assays for the enumeration of the most promising biomarker genes and their transcripts; (4) evaluating whether biomarker transcript quantification and transcript-to-gene abundance ratios correlate with reductive dechlorination activity; (5) testing fluorescence in situ hybridization (FISH) approaches for visualizing relevant microorganisms; and (6) applying the new biomarkers and procedures to field samples.
Several reductively dechlorinating consortia maintained with chlorinated ethenes, chlorinated ethanes, and chlorinated methanes as electron acceptors provided biomass for whole cell and cell-free extract enzyme assays to assess inhibition caused by other chlorinated solvents. Three complementary microarray approaches identified reductive dechlorination biomarker genes, for which qPCR assays were designed and validated. Dehalococcoides pure cultures and Dehalococcoides-containing consortia were used to interrogate three complementary microarray systems to elucidate genes that could serve as reductive dechlorination biomarkers.
This project (1) elucidated the underpinning mechanisms of reciprocal inhibition of cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC) reductive dechlorination by chloroform (CF) and 1,1,1-trichloroethane (1,1,1-TCA); (2) determined inhibition constants (Ki values) that are helpful to predict if co-contaminant inhibition will occur; (3) demonstrated that culture blends can relieve inhibition of cDCE and/or VC reductive dechlorination caused by co-contaminants such as CF and 1,1,1-TCA; (4) identified new biomarker genes encoding specific RDases including cfrA [CF to dichloromethane (DCM) and 1,1,1-TCA to 1,1-dichloroethane (1,1-DCA)], dcrA [1,1-DCA to chloroethane (CA)], and dcpA [1,2-dichloropropane (1,2-DCP) to propene]; (5) designed and validated qPCR assays for Dhc biomarker gene (and transcript) quantification; (6) validated that Sterivex cartridges are useful for on-site biomass collection (in collaboration with SERDP project ER-1561); (7) confirmed that the new qPCR tools can be applied to biomass collected from contaminated site samples; and (8) demonstrated that Dhc-to-total bacteria 16S rRNA gene ratios greater than 0.0005 (0.05%) correlate with ethene formation (i.e., detoxification).
A broader suite of reductive dechlorination biomarkers and enhanced tools for quantitative enumeration allows for a more efficient allocation of resources to sites amenable to bioremediation technologies, promotes science-based site management decisions, facilitates implementation of monitored natural attenuation (MNA) and enhanced bioremediation, facilitates regulatory acceptance, promotes site closures, and ultimately provides significant cost savings to DoD. Further, the tools and knowledge generated benefit the scientific community exploring the distribution and ecology of reductively dechlorinating organisms, unravel the specific requirements of keystone reductive dechlorinators (e.g., Dehalococcoides), and shed light on the relevance of lateral gene transfer for the dissemination of reductive dehalogenase genes.