Gasoline BTEX

Assessing the feasibility and performance of MNA and enhanced anaerobic bioremediation at petroleum hydrocarbon sites relies on chemical, geochemical, and microbial lines of evidence: trends in BTEX concentrations, redox conditions/electron acceptors, and concentrations of BTEX degrading bacteria.

While not as well characterized as aerobic biodegradation, each of the BTEX compounds (benzene, toluene, ethylbenzene, and xylenes) is susceptible to anaerobic biodegradation and several pathways have been elucidated. QuantArray®-Petro includes assays targeting a number of upper and lower pathway functional genes involved in the anaerobic catabolism of BTEX compounds for more complete evaluation of anaerobic biodegradation. Alternatively, CENSUS® qPCR can be performed to quantify a select subset such as benzylsuccinate synthase (BSS) and anaerobic benzene carboxylase (ABC).

TARGET	CODE	RELEVANCE / DATA INTERPRETATION
Benzylsuccinate Synthase	BSS	The first step in anaerobic biodegradation of toluene, mediated by benzylsuccinate synthase (bssA), is the addition of fumarate onto the toluene methyl group to form benzylsuccinate. Some bacterial isolates utilize this same metabolic approach for anaerobic biodegradation of ethylbenzene and xylenes.
Anaerobic Benzene Carboxylase	ABC	Although additional pathways are possible, the only pathway for anaerobic biodegradation of benzene elucidated to date is initiated by a benzene carboxylase enzyme.
Geobacter metallireducens Functional Genes	GMET	Targets functional genes including a predicted oxidoreductase specifically required for anaerobic benzene metabolism by G. metallireducens.
Benzoyl Coenzyme A Reductase	BCR	Benzyl-CoA is the central intermediate in the anaerobic biodegradation of many aromatic hydrocarbons. Benzoyl-CoA Reductase (BCR) is the essential enzyme for reducing the benzene ring structure.
Total Bacteria	EBAC	Index of total bacterial biomass
Sulfate Reducing Bacteria	APS	While not specific to BTEX biodegradation, quantification of sulfate reducing bacteria provides an additional line of evidence when evaluating redox conditions, terminal electron accepting processes, and impact of sulfate addition for enhanced anaerobic bioremediation strategies.

Enhanced aerobic bioremediation by injection of oxygen releasing materials (e.g., ORC® or PermeOx®) or engineered approaches can be an attractive treatment technology for gasoline impacted sites due to relatively high biodegradation rates. Furthermore, aromatic oxygenases have been shown to function even at low dissolved oxygen (DO) levels suggesting that aerobic biodegradation may also contribute to MNA.

Aerobic biodegradation of BTEX has been intensively studied and multiple catabolic pathways have been well characterized. The substrate specificity of each pathway (the range of compounds biodegraded via each pathway) is largely determined by the specificity of the initial oxygenase enzyme. QuantArray®-Petro includes a suite of assays targeting the initial oxygenase genes of the known pathways for aerobic BTEX biodegradation as shown 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
Toluene Dioxygenase	TOD	Toluene/benzene dioxygenase (TOD) incorporates both atoms of molecular oxygen directly into the aromatic ring. Although commonly called toluene dioxygenase, the substrate specificity of this enzyme is relaxed, allowing growth on toluene, benzene, and chlorobenzene along with co-oxidation of a variety of compounds including ethylbenzene, p-xylene, m-xylene, and TCE.
Ring Hydroxylating Toluene Monooxygenase	RMO	Catalyzes the initial and sometimes second oxidation steps in aerobic BTEX biodegradation. The ring hydroxylating monooxygenases can be further described based upon where they attack the aromatic ring. The RMO assay targets as toluene-3-monooxygenases and toluene-4-monooxygenases.
Ring Hydroxylating Toluene Monooxygenase	RDEG	Like RMO, catalyzes the initial oxidation and sometimes second oxidation steps in aerobic BTEX biodegradation. The RDEG assay quantifies toluene-2-monooxygenases.
Phenol Hydroxylase	PHE	In general, phenol hydroxylases (PHE) catalyze the continued oxidation of phenols produced by RMOs. However, the difference between toluene monooxygenases (RMOs) and phenol hydroxylases (PHEs) is not absolute in terms of substrate specificity and catabolic function.
Toluene/Xylene Monooxygenase	TOL	The final known pathway for aerobic toluene biodegradation involves an initial monooxygenase attack at the methyl group by a toluene/xylene monooxygenase.
Ethylbenzene Dioxygenase	EDO	Similar to TOD, this group of aromatic oxygenases exhibits relatively broad specificity and are responsible for aerobic biodegradation of alkylbenzenes including ethylbenzene and isopropylbenzene or cumene.
Biphenyl/ Isopropylbenzene Dioxygenase	TCBO	In environmental restoration, biphenyl dioxygenases are best known for cometabolism of polychlorinated biphenyls (PCBs). However, this subfamily includes benzene and isopropylbenzene dioxygenases from Rhodococcus spp.

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.

Demonstrating that benzene biodegradation is occurring under the predominantly anaerobic conditions observed at many petroleum hydrocarbon sites is often critical in gaining approval for monitored natural attenuation (MNA). Therefore, SIP studies with ¹³C benzene are commonly performed to conclusively determine whether benzene biodegradation is occurring in situ and to evaluate the feasibility of MNA as a remediation strategy.

With the SIP method, a Bio-Trap® amended with a ¹³C “labeled” contaminant (e.g., ¹³C benzene) 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.

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 MNA and enhanced bioremediation options at petroleum hydrocarbon sites, 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, sulfate, nitrate) and/or nutrients (e.g., N, P)

Each ISM unit contains passive samplers – passive diffusion bags (PDBs) for VOCs analysis of contaminant concentrations, passive geochem samplers for dissolved gases (methane) and anions like sulfate, and Bio-Traps® for QuantArray®-Petro or CENSUS® qPCR quantification of key contaminant degrading bacteria and functional genes.

By comparing contaminant concentrations, geochemical conditions, and concentrations of functional genes responsible for BTEX biodegradation between the MNA and BioStim units, site managers can evaluate each remediation option at a fraction of the cost of a lab bench treatability study or pilot scale study.

Next Generation Sequencing (NGS)

Multiple lines of evidence can provide a more complete picture. At petroleum hydrocarbon sites, CENSUS® qPCR or QuantArray®-Petro is routinely performed to quantify functional genes in known pathways for biodegradation of BTEX and other contaminants. For especially complex sites, next generation sequencing (NGS) may be performed in addition to QuantArray®-Petro to generate an overall profile of the microbial community composition which may provide additional insight into the types of microbial processes that may be occurring.