CENSUS – Integrating Results

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.


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.



Freedman, D. L. and J. M. Gossett. 1989. “Biological reductive dechlorination of tetrachloroethylene and trichloroethylene to ethylene under methanogenic conditions.” Applied and Environmental Microbiology 55(9): 2144-2151.

DiStefano, T. D., J.M. Gossett, and S.H. Zinder. 1991.  “Reductive dechlorination of high concentrations of tetrachlorethene to ethene by an anaerobic enrichment culture in the absence of methanogenesis.”  Applied and Environmental Microbiology 57(8): 2287-2292.

Gerritse, J., V. Renard, T. M. Pedro Gomes, P. A. Lawson, M. D. Collins, and J. C. Gottschal. 1996.  “Desulfitobacterium sp. Strain PCE1, an anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene or ortho-chlorinated phenols.”Archives of Microbiology 165(2): 132-140.

Gerritse, J., O. Drzyzga, G. Kloetstra, M. Keijmel, L.P. Wiersum, R. Hutson, M. D. Collins, and J. C. Gottschal. 1999. “Influence of different electron donors and acceptors on dehalorespiration of tetrachloroethene by Desulfitobacterium frappieri TCE1.”Applied and Environmental Microbiology 65(12): 5212-5221.

Holliger, C., G. Schraa, A.J.M. Stams, and A.J.B. Zehnder. 1993. “A highly purified enrichment culture couples the reductive dechlorination of tetrachloroethene to growth.” Applied and Environmental Microbiology 59 (9): 2991-2997.

Krumholz, L. R., R. Sharp, and S. S. Fishbain. 1996. “A freshwater anaerobe coupling acetate oxidation to tetrachloroethylene dehalogenation.” Applied and Environmental Microbiology 62(11):  4108-4113.

Löffler, F.E., R.A. Sanford, and J.M. Tiedje. 1996. “Initial characterization of a reductive dehalogenase from Desulfitobacterium chlororespirans Co23.” Applied and Environmental Microbiology 62(10): 3809–3813.

Maymó-Gatell, X., T. Anguish, and S.H. Zinder. 1999. “Reductive dechlorination of chlorinated ethenes and 1,2-dichloroethane by Dehalococcoides ethenogenes 195.” Applied and Environmental Microbiology 65(7): 3108–3113.

Hendrickson, E.R., J. Payne, R.M. Young, M.G. Starr, M.P. Perry, S. Fahnestock, D.E. Ellis, and R.C. Eversole. 2002. “Molecular analysis of Dehalococcoides 16S ribosomal DNA from chloroethene-contaminated sites throughout North America and Europe.” Applied and Environmental Microbiology 68(2): 485-495.

Adrian, L, U. Szewzyk, J. Wecke, and H. Görisch. 2000 “Bacterial dehalorespiration with chlorinated benzenes.” Nature 408(6812): 580-583.

Holmes, V.F., J. He, P.K.H. Lee, and L. Alvarez-Cohen. 2006. “Discrimination of multiple Dehalococcoides strains in a trichlorethene enrichment by quantification of their reductive dehalogenase genes.”  Applied and Environmental Microbiology 72(9): 5877-5883.

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.

Sung, Y., K.M. Ritalahti, R.A. Sanford, J.W. Urbance, S.J. Flynn, J.M. Tiedge, and F.E. Löffler.  2003.  “Characterization of two tetrachloroethene-reducing, acetate-oxidizing anaerobic bacteria and their description as Desulfuromonas michiganensis sp. nov.”  Applied and Environmental Microbiology 69(5): 2964-2974.

Truex, M.J., C.J. Newell, B.B. Looney, and K.M. Vangelas.  2006.  “Scenarios evaluation tool for chlorinated solvent MNA”.  Savannah River National Laboratory, US DOE, Report WSRC-STI-2006-00096, Rev. 1.