Successful anaerobic bioremediation at chlorinated solvent sites relies on the presence of bacteria, such as Dehalococcoides (Dhc), capable of organohalide respiration (i.e., respiratory reductive dechlorination or [de]chlororespiration). Nucleic acid-based assays like the quantitative real-time polymerase chain reaction (qPCR) technique detect and enumerate Dhc in soil or groundwater samples by targeting Dhc-specific biomarker genes, including the 16S rRNA gene and the tceA, bvcA, and vcrA reductive dechlorinase (RDase) genes implicated in chlorinated ethene reductive dechlorination. The results of nucleic acid-based tests, like the qPCR approach, are expected to assist site managers and practitioners in identifying sites where implementation of long-term monitored natural attenuation (MNA) will be effective, where biostimulation will achieve complete dechlorination without dichloroethene (DCE)/vinyl chloride (VC) stall, and where bioaugmentation is required.

The objectives of this project were to (1) demonstrate correlations between dechlorination of chlorinated ethenes and the presence and abundance of Dhc biomarker genes; (2) define limitations of the DNA biomarker-based approach and specify conditions where the qPCR assay offers or fails to provide meaningful information; and (3) develop a guidance protocol for practitioners to apply this tool.

The project was conducted in two phases. In the first phase, a standard operating procedure (SOP) was developed for collecting groundwater samples. To avoid problems associated with contemporary procedures that rely on shipment of large volumes of contaminated groundwater, on-site biomass collection using Sterivex cartridges for Dhc biomarker quantification was developed and validated. In the second phase, this SOP was used to collect groundwater samples from a selection of chlorinated ethene-impacted sites, including sites undergoing MNA and enhanced bioremediation (biostimulation and/or bioaugmentation).

Data were evaluated using qualitative and quantitative (e.g., Spearman correlations) methods.

Validation of Use of RDase Gene Targets. To date, the four functional genes pceA (presumably encoding a tetrachloroethene [PCE]-to-trichloroethene [TCE] RDase), tceA (encoding a TCE-to-VC RDase), and bvcA and vcrA (both encoding VC-to-ethene RDases) have been identified in chlorinated ethene-dechlorinating Dhc strains. At each site included in this study, groundwater samples were collected for qPCR analysis of the RDase gene targets tceA, bvcA, and/or vcrA. The gene copy numbers were correlated to concentrations of PCE, TCE, dechlorination intermediates (cis-DCE and VC), and/or ethene, the nontoxic dechlorination end product, as well as contaminant/product ratios using the Spearman Correlation approach. Strong Spearman Correlations (greater than 0.66) were obtained consistently using vcrA as a predictor of ethene production. The vcrA and bvcA genes are both implicated in VC-to-ethene reductive dechlorination. Selection of an appropriate functional gene target (or targets) will be governed by site-specific conditions and objectives; however, the quantitative analysis of Dhc 16S rRNA genes and the VC RDase genes vcrA and bvcA at chlorinated solvent sites is anticipated to provide useful, reliable information describing complete reductive dechlorination to ethene.

RDase gene copy number correlations to daughter product concentration ratios or concentrations of individual dechlorination intermediates provided site-specific information about the relationship between these variables but were not consistent from site to site.

Minimum Number of Dhc Gene Copies Indicative of Ethene Formation. Groundwater samples were collected from all sites for Dhc 16S rRNA gene and/or RDase gene analysis, and results were correlated to ethene concentrations in the sample. Strong Spearman Correlations (greater than 0.66) were observed when Dhc cell titers or vcrA gene copies exceeded 10⁶ to 10⁷ per liter of groundwater.

Correlation of Dhc Cell Titers to Dechlorination Rates. Groundwater samples were collected from the NASA Mobile Launch Platform/Vehicle Assembly Building (MLP/VAB) site for Dhc 16S rRNA gene and RDase gene analysis. First-order dechlorination rates were calculated from chlorinated ethene data collected from wells inside the plume. The first-order dechlorination rates were correlated to Dhc and vcrA abundance using the Spearman Correlation. Strong correlations were established between TCE, cis-DCE, and VC dechlorination rates and Dhc cell titers, while medium correlations were observed between VC dechlorination rates and vcrA gene copy numbers. The analysis was limited by the use of only three monitoring well locations for rate calculations.

Influence of Alternative TEAPs on Dhc Abundance. Geochemical data for identifying terminal electron accepting processes (TEAPs) were obtained for monitoring locations where Dhc analyses were conducted. These data were reviewed qualitatively, since mixed TEAPs are typically observed in contaminated aquifers. Dhc cell titers above the detection limit of 10³ gene copies per liter were generally observed when conditions were reducing (anaerobic), as reflected in dissolved oxygen (DO) concentrations of less than 0.5 mg/L or redox potentials below -75 mV.

False Positive and False Negative Dhc Detections. Biomarker loss during sample handling may result in false negative results. This issue was addressed by improving sampling and handling procedures to obtain Dhc biomarker recoveries of greater than 90%. False positive results were eliminated by optimized qPCR protocols and appropriate controls. Further, the simultaneous quantification of Dhc 16S rRNA gene and RDase gene targets in undiluted and 10-fold diluted samples enabled the detection of PCR irregularities, including the presence of PCR inhibitors.

Sample Collection Methods. The groundwater sampling procedure was optimized and applied throughout this project to ensure sample consistency (i.e., minimize the effect of sampling procedures on the results) and quality (i.e., avoid biomarker loss). A study comparing off-site (in the lab) to on-site (in the field) groundwater filtration and biomass collection indicated that the Dhc yield of field-filtered samples exceeded 90% with high precision.

Analytical Sensitivity. A reliable limit for Dhc 16S rRNA or RDase gene detection is 10³ cells (i.e., gene copies) per L of groundwater. The quantification limit (i.e., the minimum gene target number that can be reliably quantified) is about five-fold greater than the method detection limit. Greater sensitivity is not needed, as reductive dechlorination is not observed in the field at gene copy abundances below 10³ per L.

Analytical Sample Reproducibility. The qPCR technique is highly reproducible. All qPCR data were generated with at least two replicate DNA extractions, each analyzed for at least two dilutions in triplicate qPCR runs. Differences between replicate samples analyzed in terms of DNA yields and biomarker gene quantification using the same biomass collection method were less than two-fold.

Ease of Using On-Site Filtration Methods. Groundwater sampling methods included attaching a Sterivex cartridge to low-flow discharge tubing, measuring the discharge volume during sampling, and packaging the cartridge. This method added 15 to 30 minutes to the time needed to sample a monitoring well for volatile organic compounds (VOCs).

A Guidance Protocol was developed that includes an SOP for groundwater sampling, as well as guidelines for sampling locations, sampling frequency, and data interpretation. Flowcharts are provided for use of Dhc biomarker gene data to support decision making at sites where MNA is being evaluated, to predict sites where biostimulation will be successful, and to identify sites where bioaugmentation is required. With the increased knowledge and understanding of the reductive dechlorination process, along with improved and rigorously tested assessment and monitoring tools, as well as guidance documents, site managers and regulators will have the means to convincingly argue that MNA and/or enhanced treatment are viable, cost-effective approaches for source zone remediation and plume control to achieve long-lasting risk reduction.