Chlorinated Ethenes

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.

TARGET	CODE	RELEVANCE / DATA INTERPRETATION
Dehalococcoides	DHC	Dehalococcoides (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 Genes	TCE, BVC, VCR	Three functional genes encoding reductive dehalogenases for TCE, DCE and VC to evaluate the potential for complete reductive dechlorination to ethene.
Dehalobacter	DHB	Capable of reductive dechlorination of PCE and TCE to cis-DCE but also utilize chlorinated ethanes, common co-contaminants at TCE sites.
Desulfuromonas	DSM	Reductive dechlorination of PCE and TCE to cis-DCE using acetate as an electron donor.
Desulfitobacterium	DSB	Reductive dechlorination of PCE and TCE to cis-DCE.
PCE-1 Reductase	PCE-1	Targets 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 Reductase	PCE-2	Targets the pceA reductase genes responsible for the sequential reductive dechlorination of PCE to cis-DCE by Geobacter species.
Dehalogenimonas	DHG	Dehalogenimonas 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 GP	CER	Targets 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	TDR	Targets 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.
MB Reductase	MBR	Targets the MB reductive dehalogenase gene found in Dehalococcoides spp. which may serve as a biomarker for the production of trans-DCE at sites contaminated with chlorinated ethenes.
Methanogens	MGN	Methanogens utilize hydrogen and can compete with halorespiring bacteria for available electron donor.
Sulfate Reducing Bacteria	APS	Sulfate reducing bacteria can compete with halorespiring bacteria for available hydrogen.
Total Bacteria	EBAC	Index of total bacterial biomass.

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.

TARGET	CODE	RELEVANCE / DATA INTERPRETATION
Soluble Methane Monooxygenase	sMMO	Targets 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 Monooxygenase	RMO	Targets 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 Monooxygenase	RDEG	Also targets the ring-hydroxylating toluene monooxygenase genes (toluene-2-monooxygenase). As with RMO, toluene-2-monooxygenases are capable of cometabolism of TCE.
Phenol Hydroxylase	PHE	While 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 Dioxygenase	TOD	Although reports of induction by TCE differ, toluene dioxygenases are also capable of cometabolism of TCE when expressed.
Ethene Monooxygenase	ETN	Enumerates 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 Monooxygenase	PPO	Propane can be added as a primary substrate to promote cometabolism of TCE.
Butane Monooxygenase	BMO	BMO 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).

TARGET	RELEVANCE / DATA INTERPRETATION
Magnetic Susceptibility	Abiotic 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 Clay	While 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.
AMIBA	Aqueous and Mineral Intrinsic Bioremediation Assessment (AMIBA). Quantify iron and sulfur availability in various redox states to allow assessment of the microbial/mineral/contaminant interactions.

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.