Chlorinated Ethene Biodegradation Packages 1 and 2 answer 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 Package 2
CENSUS qPCR for Dehalococcoides, Functional Genes QuantArray®-Chlor
CSIA for 4 compounds CSIA for 5 or more compounds

Chlorinated Ethene MNA Packages 1 and 2 provide multiple lines of evidence to evaluate the contributions of aerobic cometabolism and abiotic degradation to MNA of chlorinated ethenes.

Package 1 Package 2
CENSUS qPCR for sMMO, PHE, RMO, and TOD QuantArray®-Chlor
Magnetic Susceptibility Magnetic Susceptibility
14C TCE Rate Study 14C TCE Rate Study
CSIA for 4 compounds

Abiotic Packages 1 and 2 provide multiple lines of evidence to evaluate to potential for abiotic degradation of chlorinated ethenes.

Package 1 Package 2
Magnetic Susceptibility Magnetic Susceptibility
X-Ray Diffraction (XRD) X-Ray Diffraction (XRD)
AMIBA 14C TCE Rate Study
CSIA for 4 compounds

Under anaerobic conditions, PCE can be reductively dechlorinated through TCE, cis-DCE, and vinyl chloride to ethene by halorespiring bacteria (e.g. Dehalococcoides).  Because ethene is a non-toxic product, anaerobic bioremediation is a common treatment strategy at chlorinated ethene sites.

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

TARGETCODERELEVANCE / DATA INTERPRETATION
DehalococcoidesDHCDehalococcoides (DHC) is the only known bacterial group capable of complete reductive dechlorination of PCE to ethene. Lu et al. have proposed a Dehalococcoides concentration of 1 x 10^4 cells/mL as a screening criterion to identify sites where biological reductive dechlorination is predicted to proceed at “generally useful” rates.
Functional GenesTCE, BVC, VCRThree functional genes encoding reductive dehalogenases for TCE, DCE and VC to evaluate the potential for complete reductive dechlorination to ethene.
DehalobacterDHBCapable of reductive dechlorination of PCE and TCE to cis-DCE but also utilize chlorinated ethanes, common co-contaminants at TCE sites.
DesulfuromonasDSMReductive dechlorination of PCE and TCE to cis-DCE using acetate as an electron donor.
DesulfitobacteriumDSBReductive dechlorination of PCE and TCE to cis-DCE.
PCE-1 ReductasePCE-1Targets the pceA reductase genes for the sequential reductive dechlorination of PCE to cis-DCE by Sulfurospirillum species. In mixed cultures, partial dechlorinators like Sulfurospirillum and Geobacter may be responsible for the majority of reductive dechlorination of PCE to cis-DCE with Dehalococcoides functioning as cis-DCE and vinyl chloride reducing specialists.
PCE-2 ReductasePCE-2Targets the pceA reductase genes responsible for the sequential reductive dechlorination of PCE to cis-DCE by Geobacter species.
DehalogenimonasDHGDehalogenimonas spp. are best known for dichloroelimination of chlorinated alkanes. However, the Dehalogenimonas WBC-2 culture and Dehalogenimonas strain GP have been shown to be capable of reductive dechlorination of trans-1,2-dichloroethene and vinyl chloride, respectively.
Vinyl chloride reductase from Dehalogenimonas strain GPCERTargets the vinyl chloride reductase gene from Dehalogenimonas strain GP, the only known organism other than Dehalococcoides capable of vinyl chloride reduction.
Trans-1,2 dichloroethene reductive dehalogenase TDRTargets the gene for trans-1,2-dichloroethene reductive dehalogenase (TdrA) from Dehalogenimonas sp. WBC-2 involved in the dechlorination of trans-DCE to vinyl chloride.
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.

Under aerobic conditions, several different types of bacteria including methane oxidizing bacteria (methanotrophs) and some toluene/phenol-utilizing bacteria can cometabolize or co-oxidize TCE, DCE, and vinyl chloride. In general, cometabolism of chlorinated ethenes is mediated by monooxygenase enzymes with “relaxed” specificity that oxidize a primary, growth supporting substrate such as methane and co-oxidize the chlorinated compound.

QuantArray®-Chlor includes quantification of all of the gene Targets listed in the table below except BMO and PPO.  CENSUS qPCR can be performed to quantify a select subset of functional genes such as soluble methane monooxygenase.

TARGETCODERELEVANCE / DATA INTERPRETATION
Soluble Methane MonooxygenasesMMOTargets the gene encoding soluble methane monooxygenases which can co-oxidize a broad range of chlorinated compounds including TCE, cis-DCE, and vinyl chloride. Furthermore, soluble methane monooxygenases are generally believed to support greater rates of aerobic cometabolism.
Toluene MonooxygenaseRMOTargets a group of genes encoding ring-hydroxylating toluene monooxygenase (toluene-3- and toluene-4-monooxygenases) capable of co-oxidation of TCE. In some laboratory studies, TCE or a degradation product has been shown to induce expression of toluene monooxygenases, raising the possibility of TCE cometabolism with alternative (non-aromatic) growth substrates.
Toluene MonooxygenaseRDEGAlso targets the ring-hydroxylating toluene monooxygenase genes (toluene-2-monooxygenase). As with RMO, toluene-2-monooxygenases are capable of cometabolism of TCE.
Phenol HydroxylasePHEWhile degradation rates differ, phenol hydroxylases also co-oxidize TCE. As mentioned previously, TCE or a degradation product can induce expression of toluene monooxygenases and recent research has shown positive correlations between concentrations of monooxygenase genes (sMMO, RMO, PHE) and the rate of TCE degradation.
Toluene DioxygenaseTODAlthough reports of induction by TCE differ, toluene dioxygenases are also capable of cometabolism of TCE when expressed.
Ethene MonooxygenaseETNEnumerates functional genes (etnC and etnE) involved in ethene utilization and vinyl chloride (co)metabolism. The ethene monooxygenase (EtnABCD) converts ethene and vinyl chloride to their respective epoxyalkanes, while epoxyalkane:CoM transferase (EtnE) mediates conjugation and breaking of the epoxide.
Propane MonooxygenasePPOPropane can be added as a primary substrate to promote cometabolism of TCE.
Butane MonooxygenaseBMOBMO can be added as a primary substrate to promote cometabolism of TCE.

Abiotic degradation can be a substantial or even the primary attenuation process for TCE and other chlorinated hydrocarbons in long dilute plumes and at sites undergoing or transitioning to monitored natural attenuation (MNA).

TARGETRELEVANCE / DATA INTERPRETATION
Magnetic SusceptibilityAbiotic degradation of PCE, TCE, cis-DCE, and vinyl chloride by magnetite can be an important attenuation mechanism. 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 pyrite and the crystalline form of mackinawite. Both minerals will transform PCE and TCE.
Percent ClayWhile less well studied than the other iron-bearing minerals, various phyllosilicate clays have been shown to be capable of degradation of PCE, TCE, cis-DCE, vinyl chloride, and carbon tetrachloride.
14C TCE Rate ConstantProf. David Freedman from Clemson University has developed an innovative 14C assay to determine TCE degradation rate constants from environmental samples as part of a recent ESTCP project.
AMIBAAqueous and Mineral Intrinsic Bioremediation Assessment (AMIBA). Quantify iron and sulfur availability in various redox states to allow assessment of the microbial/mineral/contaminant interactions.

Compound Specific Isotope Analysis (CSIA)

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

    • Anaerobic Biodegradation:  Reductive dechlorination of PCE, TCE, DCE isomers and vinyl chloride results in significant carbon isotope fractionation making assessment of anaerobic biodegradation of chlorinated ethenes one of the most common applications for CSIA in environmental remediation.
    • Aerobic Biodegradation:  While enrichment factors differ based on the enzymes involved, carbon isotope fractionation has been observed during aerobic biodegradation of chlorinated ethenes and has been used as a line of evidence supporting assessment of aerobic biodegradation of TCE and cDCE in a fractured bedrock aquifer.
    • Abiotic Degradation: Abiotic degradation of chlorinated ethenes mediated by zero valent iron (ZVI) and iron bearing minerals (e.g. FeS) results in carbon isotope fractionation.

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

    • For chlorinated ethenes including TCE, 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 PCE and TCE sites, CENSUS qPCR or QuantArray® is performed to quantify known contaminant degraders like 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 PCE and TCE impacted 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 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.