Bioremediation plays a crucial role in transforming and detoxifying chlorinated solvents and has been implemented as a stand-alone technology at sites undergoing monitored natural attenuation, biostimulation with or without bioaugmentation treatment, or as a polishing step when physical/chemical treatment serves as the primary remedy. Molecular biological tools (MBTs), foremost quantitative polymerase chain reaction (qPCR), provide information about the presence and abundance of keystone dechlorinating bacteria, and are instrumental for site assessment, bioremediation monitoring, and the implementation of adaptive site management strategies. Although the benefits of microbial analyses are indisputable, uncertainty about the interpretation of MBT data exists, and a consensus has not been reached regarding the value of MBTs for improved decision-making to accelerate paths toward site closure.

The overarching objective of this project was to advance MBTs and their application to minimize biases and to more effectively assess, predict, monitor, optimize, and manage reductive dechlorination processes at Department of Defense (DoD) sites impacted with chlorinated solvents. To further assist in the interpretation of MBT data, additional aims were to assess measurable parameters that correlate with the detoxification or incomplete (stalled) degradation of chlorinated ethenes, and to identify knowledge gaps that currently limit the efficient application of MBTs for decision-making at chlorinated solvent sites. Specifically, the following interrelated technical research objectives were addressed:

1. Advance the understanding of the diversity and ecophysiology of organohalide-respiring bacteria contributing to chlorinated solvent detoxification.
2. Identify additional reductive dechlorination biomarkers.
3. Develop a high-throughput qPCR tool for monitoring reductive dechlorination biomarker genes.
4. Explore the utility of environmental proteomics workflows for bioremediation monitoring.
5. Extract information from existing databases and integrate MBT data with site geochemical information to predict reductive dechlorination performance.
6. Apply new tools to DoD sites impacted with chlorinated solvents.
7. Disseminate and explain the value of MBT information for site assessment, bioremediation monitoring and optimization, adaptive site management, and performance prediction to Remedial Project Managers (RPMs), industry, and government for general acceptance and broad implementation.

Available pure cultures and consortia capable of using chlorinated solvents, including chlorinated ethenes, as electron acceptors were used to unravel specific nutritional requirements and the response to inhibitors that impact dechlorination activity. Enrichment cultures were used to discover microbes with novel biomarkers for detoxification of chlorinated solvents. To expand the qPCR approach to a broader suite of biomarker genes, an open array plate targeting 112 reductive dechlorination biomarker genes was designed and validated. Further, an environmental proteomics pipeline was developed to allow the measurement of biomarker proteins in laboratory cultures and contaminated groundwater. To develop predictive understanding of detoxification (i.e., ethene formation) in groundwater aquifers impacted with chlorinated solvents, existing databases were mined for microbial (i.e., qPCR) and geochemical data, including contaminant and ethene concentrations. The outcomes of these research efforts were pubslished in peer-reviewed journals, presented at technical conferences and webinars, and communicated to practitioners, including RPMs.

The project made a series of discoveries that have already impacted bioremediation practice at DoD sites. The major accomplishments include:

• A new dechlorinating bacterium, ‘Candidatus Dehalogenimonas etheniformans’ was discovered that dechlorinates trichloroethene to ethene. This finding indicates that detoxification of chlorinated ethenes is not limited to Dehalococcoides mccartyi (Dhc) strains.
• Strain GP harbors 52 putative reductive dehalogenase genes, including cerA, which serves as a new biomarker for vinyl chloride reductive dechlorination.
• Additional novel biomarkers for the degradation of chlorinated solvents were discovered.
• The RD-qChip targeting 102 reductive dechlorination biomarkers and enabling high-throughput qPCR applications was designed and validated.
• The analysis of 859 groundwater representing 62 sites impacted with chlorinated ethenes corroborated the value of quantitative DNA biomarker analysis. Normalized qPCR measurements predict ethene formation, which is likely when Dhc 16S rRNA gene and vinyl chloride RDase gene abundances exceed 10e7 and 10e6 copies L−1, respectively, and when the 16S rRNA- and bvcA + vcrA-to-total bacterial 16S rRNA gene ratios exceed 0.1%.
• Detailed study of streambed sediment linked qPCR data with in situ contaminant attenuation and identified the fractured bedrock-sediment interface as a critical zone for microbial activity.
• Organohalide-respiring Dehalococcoidia are corrinoid auxotrophs and rely on the microbial community to supply this essential nutrient.
• The corrinoid lower base controls Dhc reductive dechlorination rates and extents (i.e., detoxification), emphasizing the role of the microbial community for synthesizing the corrinoid and lower base structures.
• Targeted proteomic workflows can be applied to groundwater samples and provide protein level information about Dhc dechlorination activity.
• Machine learning-based data mining applied to geochemical and microbial (i.e., qPCR) data sets collected from sites impacted with chlorinated ethenes can predict ethene formation. A major need for realizing the predictive capabilities is a curated, open-access, up-to-date and comprehensive collection of biogeochemical groundwater monitoring data.
• In plumes with co-mingled chlorofluorocarbons such as 1,1,2-trichloro-1,2,2-trifluoroethane (CFC-113) inhibition of Dhc reductive dechlorination must be expected.
• Nitrous oxide (N₂O) is a potent inhibitor of bacterial reductive dechlorination and the presence of micromolar concentrations can lead to stalled dechlorination at sites impacted with chlorinated ethenes.
• Biostimulation with ammonium can enhance Dhc reductive dechlorination rates; however, a “do nothing” approach that relies on indigenous diazotrophs can achieve similar dechlorination end points and avoids the potential for stalled dechlorination due to inhibitory levels of N₂O.

High-throughput qPCR technology and environmental proteomics assist in the identification of parameters determining the feasibility and potential success of microbial remedies, so that non-productive investments can be avoided, realistic performance predictions can be established, and bioremediation sites can be efficiently managed to achieve cleanup goals and early site closures. More robust and comprehensive information about the microbiology and its activity will prevent and overcome suboptimal bioremediation performance (e.g., inhibition, nutritional limitations), and long-term performance predictions of bioremediation systems with and without intervention become feasible. (Project Completion - 2018)

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