Microbial Insights

Detect and quantify Dehalococcoides and other bacteria capable of reductive dechlorination

Characterization of sites impacted by chlorinated solvents such as tetrachloroethene (PCE) and trichloroethene (TCE) initially emphasizes determination of contaminants of concern (COCs) and evaluation of site geologic and hydrogeologic conditions. The primary goals of this first phase of site characterization are to determine whether dense non-aqueous phase liquids (DNAPL) are present, establish baseline COC concentrations, delineate the dissolved plume, evaluate groundwater flow direction and velocity, and perform risk assessment. Following this first phase, additional site characterization often focuses on evaluation of attenuation mechanisms including biodegradation to ultimately lead to an effective corrective action.

Chemical and geochemical data including the concentrations of contaminants, daughter products, and terminal electron acceptors (dissolved oxygen, nitrate, iron, sulfate, etc.) obtained during site characterization provide the first two lines of evidence to evaluate the feasibility of bioremediation as a remedial alternative. While providing valuable information, both are somewhat indirect approaches to assess biological activity. The most direct avenue to evaluate biodegradation as a treatment mechanism is to directly quantify the microorganisms or biological processes responsible for biodegradation of the contaminants of concern.

Under anaerobic conditions, PCE can be sequentially dehalogenated through TCE, cis-dichloroethene (cis-DCE), and vinyl chloride (VC) to ethene via microbially mediated reductive dechlorination. Because ethene is an innocuous end product, reductive dechlorination is an attractive treatment mechanism for PCE/TCE-impacted sites. However, practical application of the process can be hindered by a few site-specific factors which must be considered during site characterization.

The following sections describe individual CENSUS assays, their importance in evaluating reductive dechlorination as a treatment mechanism, and provide guidelines for integrating CENSUS results into routine groundwater monitoring for common corrective actions.

CENSUS Targets for Reductive Dechlorination

Dehalococcoides (qDHC): While other bacteria are capable of utilizing PCE and TCE, Dehalococcoides are the only bacterial group that has been isolated to date which is capable of complete reductive dechlorination of PCE to ethene.  In fact, the presence of Dehalococcoides spp. has been associated with the full dechlorination to ethene at sites across North America and Europe.

TCE Reductase (qTCE): Targets the functional gene responsible for reductive dechlorination of TCE by Dehalococcoides.

VC Reductase (qVC): Targets the functional genes responsible for reductive dechlorination of vinyl chloride to ethene by Dehalococcoides.  The accumulation of the daughter products cis-DCE and VC termed “DCE stall” is relatively common at PCE/TCE sites especially under MNA conditions.  DCE stall is particularly problematic because VC is generally considered more carcinogenic than the parent compounds. Within the Dehalococcoides genus, the range of chlorinated ethenes metabolized and cometabolized varies by species and strain. For example, Dehalococcoides ethenogenes str. 195 metabolizes PCE, TCE, and cis-DCE and cometabolizes VC to produce ethene.  Conversely, Dehalococcoides sp. CBDB1 utilizes PCE and TCE but does not cometabolize additional chloroethenes. Therefore, additional CENSUS assays targeting vinyl chloride reductase genes (bvcA and vcrA) were developed to more definitively confirm the potential for biodegradation of VC.

Dehalobacter (qDHB): Dehalobacter is another genus of anaerobic bacteria capable of reductive dechlorination of chlorinated compounds including PCE and TCE. Unlike most Dehalococcoides isolates that have been characterized, Dehalobacter pure cultures studied to date can utilize PCE and TCE but cannot metabolize or cometabolize the daughter products cis-DCE and VC. While the range of chlorinated ethenes utilized appears relatively limited, members of the genus Dehalobacter have the somewhat unique ability to utilize chlorinated ethanes including 1,1,1-trichloroethane (1,1,1-TCA) and 1,1,2-trichloroethane (1,1,2-TCA). TCAs were extensively used in industrial applications as degreasers and are therefore common co-contaminants at PCE/TCE impacted sites. The presence of 1,1,1-TCA is especially problematic at PCE impacted sites due to inhibition of reductive dechlorination of chlorinated ethenes particularly VC.

Desulfuromonas (qDSM): Like Dehalobacter, some members of the Desulfuromonas genus are capable of reductive dechlorination of PCE and TCE to cis-DCE. What makes Desulfuromonas somewhat unique among dechlorinating bacteria is not the range of electron acceptors which can be utilized (PCE and TCE) but rather the electron donors that support reductive dechlorination. Unlike most dechlorinators which use hydrogen as an electron donor, Desulfuromonas species can often utilize more complex electron donors including acetate, lactate, pyruvate, and succinate.

Methanogens (qMGN): Methanogens utilize hydrogen to produce methane and thus can compete with dechlorinating bacteria including Dehalococcoides for available hydrogen.

Integrating CENSUS Results

Site characterizations at PCE contaminated sites involve thorough examination of available chemical, geochemical, and microbiological data to assess the role of reductive dechlorination in an overall site management approach.  Analysis of trends in chlorinated ethene concentrations provides the first line of evidence supporting or refuting active reductive dechlorination at a site. Specifically, decreases in parent compound concentrations coupled with production of daughter products (cis-DCE, VC, and ethene) indicate active reductive dechlorination. Conversely, the accumulation of cis-DCE and VC without the production of ethene may indicate incomplete reductive dechlorination also called “DCE stall”. Review of chemical data is not restricted to contaminants of concern, however.  Evaluation of site geochemistry and concentrations of competing terminal electron acceptors provides a second, potentially converging, line of evidence for reductive dechlorination as a treatment mechanism. Under anaerobic conditions, concentrations of more oxidized electron acceptors like oxygen and nitrate are low. Instead, anaerobic bacteria utilize reduced iron (ferric iron), sulfate, and carbon dioxide as electron acceptors producing ferrous iron, sulfide, or methane, respectively. Under these conditions, biological reductive dechlorination is much more likely to be a significant attenuation mechanism.

Characterization of the site microbial community in terms of biodegradation potential provides the third and potentially most direct avenue to:

• assess the feasibility of monitored natural attenuation (MNA),
• evaluate enhanced bioremediation as a corrective action, and
• assess the need for bioaugmentation.

Monitored Natural Attenuation (MNA):

MNA is often viewed as a “do nothing” approach that relies more on physical processes such as dilution than on biodegradation as a treatment mechanism. Depending on site conditions, however, reductive dechlorination can be an important component of MNA. Evaluating MNA as a corrective action and reductive dechlorination as a treatment mechanism often depends upon integrating chemical, geochemical, and microbiological data to answer the following questions:

• Is reductive dechlorination occurring under MNA conditions?

Production of the daughter products VC and especially ethene suggests that complete reductive dechlorination is occurring whereas accumulation of DCE suggests that MNA alone may not meet treatment objectives.

• Are geochemical conditions conducive to reductive dechlorination?

As mentioned previously, reductive dechlorination is more likely under anaerobic conditions. Dissolved oxygen (DO) and nitrate concentrations less than 1 mg/L combined with the production of ferrous iron, sulfide, or methane are generally indicative of anaerobic conditions.

• Are electron donors (substrate or food) present to support growth of dechlorinating bacteria?

Microbial reductive dechlorination is basically a metabolic process in which electrons are transferred from the electron donor (substrate) to an electron acceptor, in this case, a chlorinated compound. Total organic carbon (TOC) and volatile fatty acids (VFAs) are commonly used to evaluate electron donor availability.

• Are organisms capable of reductive dechlorination of PCE/TCE and all daughter products including VC present under MNA conditions?

Unfortunately, organisms capable of reductive dechlorination of PCE and daughter products particularly VC are not ubiquitous in nature. The only way to address this question is to use the CENSUS assays.  High concentrations of dechlorinating bacteria and the presence of VC reductase genes suggest that MNA may be effective. If dechlorinating populations are detected but low and TOC or VFA concentrations are low, the addition of an electron donor such as EOS, HRC, lactate, or molasses may be needed to promote growth the dechlorinators and stimulate reductive dechlorination. For sites where dechlorinating organisms are not detected, bioaugmentation through the addition of commercial cultures may be necessary.

Enhanced Bioremediation (Biostimulation):

While monitored MNA can be an effective remedial approach, reductive dechlorination can be hindered by a lack of electron donors. Therefore, biostimulation by subsurface injection of a suitable electron donor such as EOS, HRC, or lactate is often performed to promote reductive dechlorination and enhance anaerobic bioremediation. Evaluating the effectiveness of biostimulation follows much the same rationale as described above for MNA. Biostimulation should result in an increase in dechlorinating bacteria as evidenced by CENUS results, an increase in TOC and VFAs, a decrease in redox state to anaerobic conditions, and an increase in daughter product formation.

Bioaugmentation:

As mentioned previously, dechlorinating bacteria are not ubiquitous in nature and will not be detected at all sites or all locations at a given site. When CENSUS results during site characterization reveal a lack of dechlorinating bacteria, particularly Dehalococcoides, bioaugmentation with commercial cultures may be needed. CENSUS analysis can be used to document an increase in dechlorinators following bioaugmentation as well as to monitor their survival at the site.

References

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Lee, P.K.H., D.R. Johnson, V.F. Holmes, J. He, and L. Alvarez-Cohen.  2006.  “Reductive dehalogoenase gene expression as a biomarker for physiological activity of Dehalococcoides spp.”  Applied and Environmental Microbiology 72(9): 6161-6168.

Müller, J.A., B.M. Rosner, G. von Avendroth, G. Meshulam-Simon, P.L. McCarty, and A.M. Spormann.  “Molecular identification of the catabolic vinyl chloride reductase from Dehalococcoides sp. strain VS and its environmental distribution.”  Applied and Environmental Microbiology 70(8): 4880-4888.

Ritalahti, K.M., B.K. Amos, Y. Sung, Q. Wu, S.S. Koenigsberg, and F.E. Löffler.  2006.  “Quantitative PCR targeting 16S rRNA and reductive dehalogenase genes simultaneously monitors multiple Dehalococcoides strains.”  Applied and Environmental Microbiology 72(4): 2765-2774.

ATSDR Website (http://www.atsdr.cdc.gov/tfacts70.html)

Duhamel, M., S.D. Wehr, L. Yu, H. Rizvi, D. Seepersad, S. Dworatzek, E.E. Cox, and E.A. Edwards.  2002.  “Comparison of anaerobic dechlorinating enrichment cultures maintained on tetrachloroethene, trichloroethene, cis-dichloroethene and vinyl chloride.” Water Research 36(17): 4193-4202.

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