Chlorinated Methane Biodegradation Package 1 answers the key questions impacting the feasibility and performance of bioremediation as a treatment strategy: (1) What are the concentrations of contaminant degrading microorganisms and (2) Has contaminant degradation occurred?

Package 1  
CSIA for 3 compounds  

Under anaerobic conditions, chlorinated methanes are susceptible to biodegradation although not all pathways have been fully elucidated. Chloroform can be reductively dechlorinated to dichloromethane as an electron acceptor by some strains of Dehalobacter and Desulfitobacterium. Anaerobic cometabolic degradation of carbon tetrachloride, chloroform, and dichloromethane has been widely reported for methanogens, acetogens, and other anaerobes. For Dehalobacter spp. DCM, dichloromethane is fermented as a sole substrate.

QuantArray®-Chlor includes quantification of all Targets listed in the table below.  Alternatively, CENSUS® qPCR can be performed to quantify a select subset such as Dehalobacter DCM and chloroform reductase genes.

Chloroform ReductaseCFRThe cfrA and ctrA genes encode the reductases which can catalyze reductive dechlorination of chloroform to dichloromethane by some strains of Dehalobacter or Desulfitobacterium.
Dehalobacter spp. DCMDCMDichloromethane can support growth of a distinct group of Dehalobacter strains via fermentation. The Dehalobacter DCM assays targets the 16S rRNA gene of these strains.
Total BacteriaEBACIndex of total bacterial biomass.
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.

Under aerobic conditions, chloroform and dichloromethane are susceptible to cometabolism. In addition, some aerobic methylotrophic bacteria can utilize dichloromethane as a growth substrate.

CENSUS® qPCR can be performed to quantify specific functional genes such as soluble methane monooxygenase. For sites where chlorinated methanes are present as co-contaminants with other chlorinated solvents, consider QuantArray®-Chlor which includes quantification of soluble methane monooxygenase (sMMO) genes as well as toluene monooxygenase (RMO, RDEG), phenol hydroxylase (PHE), the toluene dioxygenase (TOD) which are capable of cometabolism of TCE.

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.

Abiotic transformations with iron containing minerals including iron oxides and iron sulfides may play a significant role in natural attenuation of carbon tetrachloride.

Magnetic SusceptibilityAbiotic degradation of carbon tetrachloride by magnetite has been documented. Magnetic susceptibility provides an inexpensive estimate of the quantity of magnetite in environmental samples.
X-Ray Diffraction (XRD)XRD can provide relative abundances of reactive iron bearing minerals including the crystalline form of mackinawite and pyrite. Both minerals will transform carbon tetrachloride. In addition, Fe(II) sorbed to the surfaces of iron oxides like goethite also mediate abiotic degradation of carbon tetrachloride.
Percent ClayWhile less well studied than the other iron-bearing minerals, various phyllosilicate clays have been shown to be capable of degradation of carbon tetrachloride.

Compound Specific Isotope Analysis (CSIA)

Compound Specific Isotope Analysis (CSIA) of the carbon isotope ratios (δ13C) of chlorinated methanes can provide conclusive evidence of degradation by a number of mechanisms:

    • Anaerobic Biodegradation:  Reductive dechlorination of chloroform to dichloromethane by Dehalobacter-containing cultures results in significant carbon isotope fractionation that can be used to monitor chloroform biodegradation. For more information, please see the MI CSIA Database.
    • Aerobic Biodegradation:  Significant carbon isotope enrichment has been observed during dichloromethane biodegradation by methylotrophic bacteria under aerobic and denitrifying conditions.
    • Abiotic Degradation: Abiotic degradation of carbon tetrachloride mediated by zero valent iron (ZVI), ferrous sulfides (e.g. FeS), and iron oxides (e.g., magnetite, goethite) results in carbon isotope fractionation.

CSIA is commonly used for contaminant source distinction/delineation at sites where multiple sources may be present.

    • 2D-CSIA based on contaminant δ13C and δ37Cl values can provide conclusive evidence of contaminant sources and insight into fate and transport.

Next Generation Sequencing (NGS)

Multiple lines of evidence can provide a more complete picture.  At complex sites, CENSUS® qPCR or QuantArray® is performed to quantify known contaminant degraders like Dehalobacter and functional genes such as chloroform reductase (CFR) while next generation sequencing (NGS) is often used for an overall profile of the microbial community composition.  Knowing which microorganisms are present and their relative abundances provides insight into the types of microbial processes might be occurring such as fermentation or metals reduction.