Chlorinated Methanes

CSIA can also be used to evaluate degradation of the chlorinated methanes, carbon tetrachloride, chloroform, and dichloromethane.

  • Anaerobic Biodegradation:  Under anaerobic conditions, cometabolic degradation of carbon tetrachloride by methanogens, acetogens, fermenters, sulfate-reducers, and iron-reducers has been widely demonstrated.  To date however, isotopic enrichment factors associated with biotic degradation of carbon tetrachloride have not reported.

Chloroform can serve as a growth supporting electron acceptor by some Dehalobacter and Desulfitobacterium spp. (Ding et al. 2014; Grostern et al. 2010; Tang and Edwards 2013).  Reductive dechlorination of chloroform to dichloromethane by Dehalobacter-containing cultures results in significant carbon isotope fractionation (ε = -27.5‰) that can be used to monitor chloroform biodegradation (Chan et al. 2012).

In addition to CSIA, CENSUS qPCR or QuantArray-Chlor can be used to quantify chloroform reductase genes and Dehalobacter DCM strains as a supporting line of evidence in assessing chloroform and dichloromethane biodegradation.

  • Aerobic Biodegradation:  Under aerobic conditions, methanotrophs can cometabolize chloroform and dichloromethane.  In addition, many methylotrophic bacteria belonging to diverse genera (Hyphomicrobium, Methylobacterium, Methylophilus, etc.) have been isolated which are capable of utilizing dichloromethane as a growth supporting substrate.  Significant carbon isotope enrichment has been observed during dichloromethane biodegradation by methylotrophic bacteria under aerobic (ε = -41 to -66‰) and denitrifying (ε = -46 to -61‰) conditions (Nikolausz et al. 2006).
  • Abiotic Degradation:   Abiotic transformations with iron containing minerals including iron oxides and iron sulfides may play a significant role in natural attenuation of carbon tetrachloride (McCormick et al. 2002; Zwank et al. 2005).

For iron (hydr)oxides (magnetite, goethite, hematite, and lepidocrocite) and siderite (FeCO3), carbon isotope enrichment factors during abiotic degradation of carbon tetrachloride are in the range of -29 ± 3‰ (Zwank et al. 2005).  Although lower, carbon isotope fractionation during abiotic degradation of carbon tetrachloride by FeS (ε=-15.9‰) was also significant.

Zero valent iron (ZVI) mediated degradation of carbon tetrachloride also results in significant carbon isotope fractionation (VanStone et al. 2008).

Consider 2D-CSIA:  Two dimensional compound specific isotope analysis (2D-CSIA) is simply the analysis of the isotope ratios of multiple elements (e.g. 13C/12C, 2H/1H, or 37Cl/35Cl).  For chlorinated hydrocarbons, 2D-CSIA typically combines δ13C with δ37Cl although 3D-CSIA with δ13C, δ37Cl, and δ2H has also been described (Kuder et al. 2013).

For the 2D-CSIA approach, δ37Cl vs δ13C plots are constructed.  The slope of the line is (Λ) which is often characteristic of specific degradation mechanisms.  Therefore, 2D-CSIA results have been used to aid in identifying and distinguishing active biotic and abiotic chloroform degradation (Rodríguez-Fernández et al. 2015).


Chan, C.C.H., S.O.C. Mundle, T. Eckert, X. Liang, S. Tang, G. Lacrampe-Couloume, E.A. Edwards, and B. Sherwood Lollar. 2012. Large Carbon Isotope Fractionation during Biodegradation of Chloroform by Dehalobacter Cultures. Environmental Science & Technology 46 no. 18: 10154-10160.

Ding, C., S. Zhao, and J. He. 2014. A Desulfitobacterium sp. strain PR reductively dechlorinates both 1,1,1-trichloroethane and chloroform. Environmental Microbiology: n/a-n/a.

Grostern, A., M. Duhamel, S. Dworatzek, and E.A. Edwards. 2010. Chloroform respiration to dichloromethane by a Dehalobacter population. Environmental Microbiology 12 no. 4: 1053-1060.

Kuder, T., B.M. van Breukelen, M. Vanderford, and P. Philp. 2013. 3D-CSIA: Carbon, Chlorine, and Hydrogen Isotope Fractionation in Transformation of TCE to Ethene by a Dehalococcoides Culture. Environmental Science & Technology 47 no. 17: 9668-9677.

McCormick, M.L., E.J. Bouwer, and P. Adriaens. 2002. Carbon Tetrachloride Transformation in a Model Iron-Reducing Culture:  Relative Kinetics of Biotic and Abiotic Reactions. Environmental Science & Technology 36 no. 3: 403-410.

Nikolausz, M., I. Nijenhuis, K. Ziller, H.-H. Richnow, and M. Kästner. 2006. Stable carbon isotope fractionation during degradation of dichloromethane by methylotrophic bacteria. Environmental Microbiology 8 no. 1: 156-164.

Rodríguez-Fernández, D., M. Rosell, C. Domènech, C. Torrentó, J. Palau, and A. Soler. 2015. C and Cl-CSIA for Elucidating Chlorinated Methanes Biotic and Abiotic Degradation at a Polluted Bedrock Aquifer. Procedia Earth and Planetary Science 13: 120-123.

Tang, S., and E.A. Edwards. 2013. Identification of Dehalobacter reductive dehalogenases that catalyse dechlorination of chloroform, 1,1,1-trichloroethane and 1,1-dichloroethane. Philosophical Transactions of the Royal Society B: Biological Sciences 368 no. 1616.

VanStone, N., M. Elsner, G. Lacrampe-Couloume, S. Mabury, and B. Sherwood Lollar. 2008. Potential for Identifying Abiotic Chloroalkane Degradation Mechanisms using Carbon Isotopic Fractionation. Environmental Science & Technology 42 no. 1: 126-132.

Zwank, L., M. Elsner, A. Aeberhard, R.P. Schwarzenbach, and S.B. Haderlein. 2005. Carbon Isotope Fractionation in the Reductive Dehalogenation of Carbon Tetrachloride at Iron (Hydr)Oxide and Iron Sulfide Minerals. Environmental Science & Technology 39 no. 15: 5634-5641.