Chlorinated Benzene 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 for the lower chlorinated benzenes, (2) Is contaminant biodegradation occurring?

Package 1  
QuantArray®-Chlor  
Stable Isotope Probing (SIP)  

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.

TARGETCODERELEVANCE / DATA INTERPRETATION
DehalococcoidesDHCAlthough 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.
DehalobacterDHBtWhile considered relatively recalcitrant under anaerobic conditions, recent work has implied Dehalobacter spp. in the reductive dechlorination of DCBs and CB.
DehalobiumDECODehalobium chlorocoercia DF-1 has been shown to be capable of reductive dechlorination of HCB, PeCB and 1,2,3,5-TeCB.
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, 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).

TARGETCODERELEVANCE / DATA INTERPRETATION
Phenol HydroxylasePHEPhenol 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 DioxygenaseTODToluene 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 MonooxygenaseRMOSimilar 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 DioxygenaseTCBOThe 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.

Compound Specific Isotope Analysis (CSIA)

Compound Specific Isotope Analysis (CSIA) of the carbon isotope ratios (δ13C) of chlorobenzene (CB) can provide conclusive evidence of anaerobic biodegradation:

    • Anaerobic Biodegradation:  Reductive dechlorination of chlorobenzene to benzene results in significant carbon isotope fractionation. For more information, please see the MI CSIA Database.

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.

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.