CENSUS – Chlorinated Methanes

Detect and quantify bacteria responsible for biodegradation of Chlorinated Methanes

Chlorinated methanes including carbon tetrachloride (CT), chloroform (CF), and dichloromethane (DCM) were widely used as solvents, degreasers, fumigants, and chemical intermediates in industrial processes and now are common groundwater contaminants.   With potential adverse health effects including increased cancer risk, maximum contaminant levels (MCLs) have been esablished for carbon tetrachloride and dichloromethane.  In additiona, chlorinated methanes are also common co-contaminants at tetrachlorethene (PCE) and trichloroethene (TCE) impacted sites.   The presence of carbon tetrachloride and chloroform can be especially problematic at PCE/TCE sites due to inhibition of reductive dechlorination of chlorinated ethenes (Bagley et al. 2000; Duhamel et al. 2002)

The mechanisms for degradation of chlorinated methanes and consequently the assessment approaches differ considerably between compounds and depend on redox conditions. Below provides a brief summary of degradation mechanisms and applicable assessment tools.

Carbon Tetrachloride: Under anaerobic conditions, cometabolic degradation of carbon tetrachloride and chloroform by methanogens, acetogens, fermenters, sulfate-reducing bacteria, and iron-reducing bacteria has been widely reported (see Field and Sierra-Alvarez 2004 for a review). In these studies, CT is sequentially reduced to CF and DCM but CO2 and CS2 have also been reported as byproducts. The biotransformation of CT and CF is believed to be a result of reduced enzyme co-factors common in anaerobic microorganisms such as vitamin B12. CT is also susceptible to abiotic degradation by magnetite and other iron-bearing minerals including iron sulfides (He et al. 2009) formed as a result of iron- and sulfate-reducing bacteria.

Chloroform: Unlike CT, chloroform can serve as a growth supporting electron acceptor by some Dehalobacter and Desulfitobacterium spp. (Grostern et al. 2010; Tang et al. 2013; Ding et al. 2014). In these strains, reductive dechlorination of chloroform to DCM was linked to the cfrA and ctrA genes also implicated in reductive dechlorination of 1,1,1-trichloroethane. As with carbon tetrachloride, chloroform is susceptible to cometabolic degradation by methanogens and other anaerobic microorganisms. Under aerobic conditions, methanotrophs and other microorganisms producing oxygenase enzymes can co-oxidize chloroform.

Dichloromethane: Anaerobic degradation of DCM as a sole carbon and energy source has been demonstrated for methanogenic and aceotgenic cultures.  For example, Dehalobacterium formicoaceticum isolated from an acetogenic enrichment culture converts DCM and CO2 into formate and acetate (Magli et al. 1996).  More recently, a Dehalobacter isolate which had been thought to be an obligate halorespiring bacterium, has also been shown to ferment DCM producing acetate (Justicia-Leon et al. 2012).  Many aerobic methylotrophic bacteria, belonging to diverse genera (Hyphomicrobium, Methylobacterium, Methylophilus, Pseudomonas, Paracoccus, and Albibacter) have been isolated which are capable of utilizing DCM as a growth substrate.  The DCM metabolic pathway in methylotrophic bacteria is initiated by a DCM dehalogenase (dcmA) producing formaldehyde which is further oxidized (La Roche et al. 1990).  Finally, methanotrophs utilizing methane as a primary substrate can co-oxidize DCM (Oldenhuis et al. 1989).




MI Code

Relevance / Data Interpretation

Chloroform Reductase
CFRThe cfrA and ctrA genes encode the reductases which can catalyze reductive dechlorination of chloroform to dichloromethane by some strains of  Dehalobacter or Desulfitobacterium.
Dehlobacter spp. DCMDCMDichloromethane can support growth of a distinct group of Dehalobacter strains via fermentation (Justicia-Leon et al. 2012). The Dehalobacter DCM assays targets the 16S rRNA gene of these strains.
Dichloromethane DehalogenaseDCMATargets the dcmA gene responsible for aerobic biodegradation of dichloromethane by methylotrophs.
Soluble Methane MonooxygenasesMMOSoluble methane monooxygenase exhibits broad specificity and is capable of co-oxidation of a variety of chlorinated compounds including CF and DCM.
MethanogensMGNWhile not specific to chlorinated methanes, cometabolic CT and CF degradation has been observed in methanogenic cultures presumably due to reduced enzyme co-factors common in anaerobic microorganisms.
Sulfate Reducing BacteriaAPSTargets a key functional gene in sulfate reduction. Cometabolic degradation of CT and CF has been observed in sulfate reducing cultures. Moreover, ferrous sulfides formed in sulfate reducing environments can mediate abiotic CT degradation. 



Bagley, D. M., M. Lalonde, V. Kaseros, K. E. Stasiuk and B. E. Sleep (2000). “Acclimation of anaerobic systems to biodegrade tetrachloroethene in the presence of carbon tetrachloride and chloroform.” Water Research 34(1): 171-178.

Ding, C., S. Zhao and J. He (2014). “A Desulfitobacterium sp. strain PR reductively dechlorinates both 1,1,1-trichloroethane and chloroform.” Environmental Microbiology.

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.

Field, J. A. and R. Sierra-Alvarez (2004). “Biodegradability of chlorinated solvents and related chlorinated aliphatic compounds.” Reviews in Environmental Science and Biotechnology 3(3): 185-254.

Grostern, A., M. Duhamel, S. Dworatzek and E. A. Edwards (2010). “Chloroform respiration to dichloromethane by a Dehalobacter population.” Environmental Microbiology 12(4): 1053-1060.

He, Y., C. Su, J. T. Wilson, R. T. Wilkin, C. Adair, T. Lee, P. Bradley and M. Ferrey (2009). Identification and characterization of methods for reactive minerals respnsible for natural attenuation of chlorinated organic compounds in ground water, US EPA.

Justicia-Leon, S. D., K. M. Ritalahti, E. E. Mack and F. E. Löffler (2012). “Dichloromethane Fermentation by a Dehalobacter sp. in an Enrichment Culture Derived from Pristine River Sediment.” Applied and Environmental Microbiology 78(4): 1288-1291.

La Roche, S. D. and T. Leisinger (1990). “Sequence analysis and expression of the bacterial dichloromethane dehalogenase structural gene, a member of the glutathione S-transferase supergene family.” Journal of Bacteriology 172(1): 164-171.

Mägli, A., M. Wendt and T. Leisinger (1996). “Isolation and characterization of Dehalobacterium formicoaceticum gen. nov. sp. nov., a strictly anaerobic bacterium utilizing dichloromethane as source of carbon and energy.” Archives of Microbiology 166(2): 101-108.

Oldenhuis, R., R. L. Vink, D. B. Janssen and B. Witholt (1989). “Degradation of chlorinated aliphatic hydrocarbons by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase.” Applied and Environmental Microbiology 55(11): 2819-2826.