Knowledge about the rates of in situ contaminant degradation is crucial for optimizing remedial design and supporting site management decisions. Despite progress understanding the factors influencing microbial degradation of chlorinated ethenes, determining rates of microbial contaminant degradation at field sites remains challenging. Molecular biological tools (MBTs) for quantifying Dehalococcoides mccartyi (Dhc) nucleic biomarkers are available and guide site management decision making; however, these measurements have not been useful to generate good estimates of contaminant degradation rates. Quantification of reductive dehalogenases (RDases) may provide a more direct measure of activity (as these are the actual enzymes/proteins that catalyze biodegradation of chlorinated ethenes), and technological advances in mass spectrometry instrumentation allow the sensitive, quantitative determination of RDase proteins of interest in groundwater. This project explores if RDase gene and protein biomarker abundances, alone or in combination, may be used to estimate degradation rates.
This demonstration had three specific objectives. The first objective was to demonstrate the utility of quantitative proteomics (qProt) to measure the absolute abundance of Dhc reductive dechlorination biomarker proteins in laboratory-controlled microcosms with various Dhc cell titers. Contaminant concentration and ethene measurements over time were used to determine cis-DCE and vinyl chloride (VC) reductive dechlorination rates. The second objective was to correlate observed degradation rates with Dhc biomarker gene and protein abundances. The successful completion of objectives 1 and 2 led to a go/no-go decision point before conducting demonstration/validation efforts of the qProt approach at military sites impacted with chlorinated ethenes.
The sensitive and quantitative measurement of proteins in environmental matrices is now possible, and process-specific biomarker proteins such as the Dhc RDases TceA, BvcA and VcrA can be measured in groundwater samples. Since the abundances of the catalysts (i.e., the specific RDase enzymes) control the rate of cis-DCE and VC reductive dechlorination, the quantitative measurement of these catalysts may be useful for estimating in situ degradation rates. Accurate assessment of in situ degradation rates often requires in situ test design, execution and appropriate data interpretation, which can be costly and time consuming to complete. Demonstration/validation of this qProt tool has significant potential to establish (1) the predictive link between in situ RDase enzyme abundances and corresponding in situ reductive dechlorination rates at multiple Department of Defense (DoD) field sites, (2) a framework remediation project managers (RPMs) may use to convert RDase enzyme abundances directly into a rate estimates, and (3) enhanced/expedited site management decisions that can result in substantial cost savings to the DoD and even early site closure.
Performance Assessment: The quantitative and qualitative performance metrics were met through demonstration in defined laboratory microcosm systems prepared using DoD site aquifer materials and the development of a model that predicts chlorinated volatile organic compound (cVOC) degradation rates based on RDase biomarker abundances. Bioaugmentation with the SDC-9 consortium was used to obtain the desired range of Dhc cell abundances and reductive dechlorination rates. Correlation and regression analyses results confirmed that RDase biomarker abundances were significantly and positively correlated with rate coefficients. Regression analysis results were used to test the rate-predictive power of the RDase biomarker abundances. RDase proteins predicted rate constants kcisDCE and kVC values within one order of magnitude; using RDase proteins and genes combined further improved predictions.
Cost Assessment: Implementation of advanced MBTs such as metagenome sequencing or proteomics, during the long-term monitoring and assessment phase of the project are impacted by multitude of factors such as: the size of the site, proximity of the site to nearby receptors, regulatory requirements, and nature and diversity of contaminant of concern. Although there are currently no regulatory requirements that specifically mandate advanced MBTs be used to assess a site, the data provided by the MBTs are meant to supplement and possibly replace other forms of data that provide lines of evidence that monitored natural attenuation (MNA) is occurring and to estimate a removal rate. Hence, the total sampling and analytical cost is driven by number of sample locations at a site and total number of samples collected (i.e., a greater number of samples equates to a higher cost). It should be noted however that the individual cost per sample may decrease based on a greater number of total samples requiring analyses since the lab work is highly specialized and cost efficiencies generally can be realized for a larger quantity of analyses.
Many of the advanced MBTs such as qProt have only limited commercial availability and/or are available through a university or other research laboratory. As such, application costs remain relatively high. It is expected as these techniques mature, they will become more widely available and the analytical cost per sample will decrease substantially. For comparison purposes, the cost of the metagenomics and metaproteomic analyses based on cost data collected during the commencement of this project in 2017 were $300 and $1,500 per sample, respectively, assuming analysis of a batch of 10 samples. These costs decreased to $150 and $1,000 (for cVOCs) when evaluated in 2019. These costs are anticipated to decrease further as the technologies mature.
The primary end users of qProt are expected to be DoD site managers, consultants and their contractors. The general concerns of these end users are likely to include the following: (1) regulatory acceptance; (2) insufficient confidence in results and access to specialized laboratories; and (3) technology cost compared to other more conventional monitoring options. Proteomics is a new tool in environmental assessment and one that requires further validation. It is anticipated that, as for many technologies such as quantitative polymerase chain reaction, regulatory acceptance will occur as the technology is field-validated, its benefits over existing approaches (e.g., ability to predict cVOC degradation rates) are realized, and the regulatory community is educated regarding its field application. As noted in the previous section, the issues of limited commercial availability of the technique and relatively high cost are also likely to be improve over time (i.e., more availability and lower cost) as the qProt technology matures.
Kucharzyk, K. H., J. E. Meisel, F. Kara-Murdoch, R. W. Murdoch, S. A. Higgins, S. Vainberg, C. M. Bartling, L. Mullins, P. B. Hatzinger, and F. E. Löffler. 2020. Metagenome-Guided Proteomic Quantification of Reductive Dehalogenases in the Dehalococcoides mccartyi-Containing Consortium SDC-9. Journal of Proteome Research, 19(4):1812-1823.
Michalsen, M. M., F. K. Murdoch, F. E. Löffler, J. Wilson, P. B. Hatzinger, J. D. Istok, L. Mullins, A. Hill, R. W. Murdoch, C. Condee, and K. H. Kucharzyk. 2021. ACS ES&T Engineering, doi.org/10.1021/acsestengg.1c00207.