Chlorinated Ethane 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  
QuantArray®-Chlor  
CSIA for 5 or more compounds  

Under anaerobic conditions, chlorinated ethanes are susceptible to reductive dechlorination by several groups of halorespiring bacteria, most notably Dehalobacter and Dehalogenimonas spp., and functional genes for reductive dehalogenases continue to be identified.  Anaerobic biodegradation of 1,1,2-TCA and 1,2-DCA proceeds via dichloroelimination producing vinyl chloride and ethene, respectively.  Dehalobacter spp. have also been isolated that are capable of sequential reductive dechlorination of 1,1,1-TCA through 1,1-DCA to chloroethane.

QuantArray®-Chlor includes quantification of all Targets listed in the table below including Dehalobacter, Dehalococcoides, additional halorespiring bacteria, functional genes, and competing microorganisms.  Alternatively, CENSUS qPCR can be performed to quantify a select subset such as Dehalobacter and Dehalogenimonas.

TARGETCODERELEVANCE / DATA INTERPRETATION
DehalobacterDHBDehalobacter spp. and Dehalobacter-containing cultures have been shown to be responsible for sequential reductive dechlorination of 1,1,1-TCA through 1,1-DCA to chloroethane. Moreover, Dehalobacter spp. perform the dichloroelimination of 1,1,2-TCA and 1,2-DCA to vinyl chloride and ethene, respectively.
DehalogenimonasDHGDehalogenimonas spp. utilize a broad spectrum of vincinally chlorinated alkanes including 1,1,2,2-TeCA, 1,1,2-TCA, and 1,2-DCA as growth-supporting electron acceptors.
DehalococcoidesDHCDehalococcoides are capable of dichloroelimination of 1,2-DCA. However, perhaps the most important reason to quantify Dehalococcoides at chlorinated ethane sites is to assess the potential for reductive dechlorination of vinyl chloride produced by biodegradation of 1,1,2-TCA by Dehalobacter and Dehalogenimonas spp.
DesulfitobacteriumDSBThe range of electron acceptors and pathways utilized varies considerably between Desulfitobacterium isolates. For example, D. dichloroeliminans strain DCA1 utilizes 1,1,2-TCA and 1,2-DCA producing vinyl chloride and ethene while Desulfitobacterium sp. PR sequentially dechlorinates 1,1,2-TCA to 1,2-DCA which is cometabolized to chloroethane. In addition, strain PR sequentially dechlorinates 1,1,1-TCA to 1,1-DCA and chloroethane and D. hafniense Y51 transforms hexachloro-, pentachloro-, and tetrachloroethanes but not 1,1,2-TCA.
1,2-Dichloroethane Reductive DehalogenaseDCARTargets the 1,2-dichloroethane reductive dehalogenase gene from members of Desulfitobacterium and Dehalobacter , which dechlorinate 1,2-DCA to ethene.
1,1-Dichloroethane Reductive Dehalogenase (DCA)DCATargets the 1,1-dichloroethane reductive dehalogenase gene found in some strains of Dehalobacter.
Chloroform/1,1,1-TCA Reductase (CFR)CFRTargets the cfrA gene of Dehalobacter spp. that encodes a reductase enzyme responsible for dechlorination of 1,1,1-TCA.
Functional GenesTCE, BVC, VCRAs mentioned previously, anaerobic biodegradation of 1,1,2-TCA leads to the production of vinyl chloride. Quantification of vinyl chloride reductase genes (BVC, VCR) along with Dehalococcoides is used to assess whether vinyl chloride will accumulate or be further dechlorinated to ethene.
Total BacteriaEBACIndex of total bacterial biomass.
MethanogensMGNMethanogens utilize hydrogen and can compete with halorespiring bacteria for available electron donor.
Sulfate Reducing BacteriaAPSSulfate reducing bacteria can compete with halorespiring bacteria for available hydrogen.

While less widely studied than cometabolism of TCE and other chlorinated ethenes, some chlorinated ethanes including 1,1,1-TCA and 1,2-DCA are also susceptible to cometabolism/co-oxidation.  Cometabolism of chlorinated ethanes is also mediated by monooxygenase enzymes with “relaxed” specificity that oxidize a primary, growth supporting substrate such as methane and co-oxidize the chlorinated compound.

CENSUS qPCR can be performed to quantify specific functional genes such as soluble methane monooxygenase.  For sites impacted by both chlorinated ethanes and ethenes, 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.

TARGETCODERELEVANCE / DATA INTERPRETATION
Soluble Methane MonooxygenasesMMOTargets the gene encoding soluble methane monooxygenases which can co-oxidize a broad range of chlorinated compounds. In laboratory studies, sMMO has been shown to co-oxidize a number of chlorinated ethanes including 1,1,1-TCA and 1,2-DCA as well as TCE, cis-DCE, and vinyl chloride.
Propane MonooxygenasePPOPropane can be added as a primary substrate to promote growth of propane utilizing bacteria capable of cometabolism of TCE and chlorinated ethanes including 1,1,2-TCA.
Butane MonooxygenaseBMOLike propane, butane can be added as a primary substrate to support cometabolism of chlorinated ethenes and chlorinated ethanes including 1,1,1-TCA.

Compound Specific Isotope Analysis (CSIA)

While less extensively studied than chlorinated ethenes, Compound Specific Isotope Analysis (CSIA) of the carbon isotope ratios (δ13C) of chlorinated ethanes can provide conclusive evidence of degradation by a number of mechanisms:

    • Anaerobic Biodegradation:  In a Dehalobacter-containing mixed culture, notable carbon isotope enrichment was observed during reductive dechlorination of 1,1-DCA. Similarly, Hunkeler et al. (2002) reported significant carbon isotope fractionation during anaerobic biodegradation (dichloroelimination) of 1,2-DCA.  Isotopic enrichment was also reported during anaerobic biodegradation of 1,1,2-TCA and 1,1,1-TCA but less substantial enrichment factors.  For more information, please see the MI CSIA Database.
    • Aerobic Biodegradation:  While enrichment factors differ based on the enzymes involved, carbon isotope fractionation has been observed during aerobic biodegradation of 1,2-DCA.
    • Abiotic Degradation: Abiotic degradation of 1,1,1-TCA mediated by zero valent iron (ZVI) and iron bearing minerals (e.g. FeS) results in carbon isotope fractionation. Chemical oxidation of 1,1,1-TCA by persulfate has also been reported to result in significant carbon isotope fractionation.

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

    • For chlorinated ethanes, 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, Dehalococcoides and functional genes 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.

In Situ Microcosms (ISMs)

In Situ Microcosms (ISMs) are field deployed microcosm units containing passive samplers that provide the microbial, chemical, and geochemical data for simultaneous, cost-effective evaluation of multiple remediation options.

For remedy selection at chlorinated solvent sites, an ISM study typically includes:

    • An unamended MNA unit to evaluate monitored natural attenuation
    • A BioStim unit amended with an electron donor
    • A BioAug unit amended with a commercial bioaugmentation culture and an electron donor

Each ISM unit contains passive samplers – passive diffusion bags (PDBs) for VOCs analysis of contaminant concentrations, passive geochem samplers for dissolved gases (ethene, ethane, methane) and anions like sulfate, and Bio-Traps® for QuantArray®-Chlor or CENSUS qPCR quantification of Dehalobacter, Dehalogenimonas, Dehalococcoides, functional genes, and other halorespiring bacteria.

By comparing contaminant concentrations, daughter product formation, geochemical conditions, and concentrations of halorespiring bacteria between the MNA, BioStim, and BioAug units, site managers can evaluate each remediation option at a fraction of the cost of a lab bench treatability study or pilot scale study.