Chlorinated Benzenes

Under anaerobic conditions, reductive dechlorination of higher chlorinated benzenes including hexachlorobenzene (HCB), pentachlorobenzene (PeCB), tetrachlorobenzene (TeCB) isomers, and trichlorobenzene (TCB) isomers by halorespiring bacteria has been well documented.  While the dichlorobenzene (DCB) isomers and chlorobenzene (CB) are relatively recalcitrant under anaerobic conditions, recent evidence has demonstrated reductive dechlorination of DCBs to CB and CB to benzene can occur along with an increase in Dehalobacter concentrations.

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 Dehalococcoides and Dehalobium spp.

TARGET	CODE	RELEVANCE / DATA INTERPRETATION
Dehalococcoides	DHC	Although biodegradation of individual compounds and specific isomers does vary somewhat between isolates, Dehalococcoides such as strain CBDB1 have identified which reductively dechlorinate, hexachlorobenzene (HCB), pentachlorobenzene (PeCB), all three tetrachlorobenzene (TeCB) isomers, 1,2,3-TCB and 1,2,4-TCB.
Dehalobacter	DHBt	While considered relatively recalcitrant under anaerobic conditions, recent work has implied Dehalobacter spp. in the reductive dechlorination of DCBs and CB.
Dehalobium	DECO	Dehalobium chlorocoercia DF-1 has been shown to be capable of reductive dechlorination of HCB, PeCB and 1,2,3,5-TeCB.
Total Bacteria	EBAC	Index of total bacterial biomass.
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.

Under aerobic conditions, the lower chlorinated benzenes chlorobenzene (CB), dichlorobenzenes (DCB), and some trichlorobenzene (TCB) compounds are susceptible to aerobic biodegradation, serving as carbon and energy sources.  As with BTEX, aerobic biodegradation of lower chlorinated benzenes is initiated by oxygenase enzymes.

QuantArray®-Chlor includes quantification of all of the gene targets listed in the table below.  Alternatively, CENSUS® qPCR can be performed to quantify a select subset of functional genes such as toluene monooxygenase (RMO) or phenol hydroxylase (PHE).

TARGET	CODE	RELEVANCE / DATA INTERPRETATION
Phenol Hydroxylase	PHE	Phenol hydroxylase catalyzes the continued oxidation and in some cases, the initial oxidation of a variety of monoaromatic compounds. In an independent study, significant increases in numbers of bacteria containing PHE genes corresponded to increases in biodegradation of DCB isomers.
Toluene Dioxygenase	TOD	Toluene dioxygenase has relatively relaxed substrate specificity and mediates the incorporation of both atoms of oxygen into the aromatic ring of benzene and substituted benzenes (toluene and chlorobenzene). Comparison of TOD levels in background and source zone samples from a CB impacted site suggested that CBs promoted growth of TOD containing bacteria
Ring Hydroxylating Toluene Monooxygenase	RMO	Similar to PHE, ring hydroxylating monooxygenases (RMO) catalyze the initial and in some cases second oxidation of a variety of monoaromatic compounds including BTEX and CB.
Trichlorobenzene Dioxygenase	TCBO	The TCBO assay targets the genes encoding aromatic dioxygenases responsible for initiating aerobic biodegradation of a number of chlorinated benzenes including chlorobenzene, 1,2-dichlorobenzene, 1,2,4-trichlorobenzene, and 1,2,4,5-tetrachlorobenzene.

Stable Isotope Probing (SIP)

Stable isotope probing (SIP) is an innovative molecular biological tool that can conclusively determine whether in situ biodegradation of a specific contaminant has occurred.

With the SIP method, a Bio-Trap® amended with a ¹³C “labeled” contaminant (e.g., ¹³C chlorobenzene) is deployed in an impacted monitoring well for 30 to 60 days. The ¹³C label serves much like a tracer which can be detected in the end products of biodegradation – microbial biomass and CO2. Following in field deployment, the Bio-Trap® is shipped to MI for analysis:  Detection of ¹³C enriched phospholipid fatty acids (PLFA) following in field deployment, conclusively demonstrates in situ biodegradation and incorporation into microbial biomass. Detection of ¹³C enriched dissolved inorganic carbon demonstrates contaminant mineralization to CO2.

Since chlorobenzene (CB) can be used as a carbon and energy source, SIP studies are commonly performed to conclusively determine whether in situ biodegradation is occurring and to evaluate the feasibility of monitored natural attenuation (MNA) as a remediation strategy.

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.

To evaluate aerobic bioremediation of chlorinated benzenes, an ISM study typically includes:

• An unamended MNA unit to evaluate monitored natural attenuation
• A BioStim unit amended with an electron acceptor product (e.g., oxygen releasing material)

Site managers may also want to evaluate enhanced anaerobic bioremediation approaches especially for sites impacted by more highly chlorinated benzenes. To evaluate anaerobic bioremediation of chlorinated benzenes, 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 key contaminant degrading bacteria and functional genes.

By comparing contaminant concentrations, 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.