QuantArray®-Petro: Bioremediation Testing for Petroleum Hydrocarbons

Comprehensive Microbial Analysis for Hydrocarbon Degradation

There are many ways petroleum hydrocarbons can enter the environment, from pipeline ruptures and transportation spills to leaking underground storage tanks at retail gas stations. Regardless of the source, petroleum products are complex mixtures of hundreds of aliphatic, aromatic, cyclic and heterocyclic compounds.  Even for common classes of contaminants like benzene, toluene, ethylbenzene, and xylenes (BTEX), biodegradation can proceed by a multitude of pathways under both aerobic and anaerobic conditions.

QuantArray^®-Petro has been designed to cost-effectively address these challenges. QuantArray^®-Petro provides simultaneous quantification of twenty-two functional genes and microbial groups responsible for aerobic and anaerobic biodegradation of BTEX, polycyclic aromatic hydrocarbons (PAHs), fuel oxygenates, and a variety of short and long chain alkanes.

HOW TO USE QUANTARRAY^®-PETRO:

Along with contaminant concentrations and geochemical parameters, the concentrations of specific microorganisms and functional genes responsible for contaminant biodegradation are a key component of remedy selection and performance monitoring at sites impacted by petroleum hydrocarbons.

Use QuantArray^® to help answer…

• How feasible is MNA? Is enhanced bioremediation needed?
  • What are the concentrations of contaminant degraders under existing conditions?
  • Are degrader concentrations greater in impacted wells than background wells?
  • Based on the MI Database, are concentrations of contaminant degraders low, medium, or high in the plume?

 

• Is enhanced bioremediation effective?
  • Did concentrations of contaminant degraders and functional genes increase in response to treatment?
  • Are additional types of degraders and functional genes now detected that were previously below detection limits?

GENE TARGETS INCLUDED IN A SINGLE QUANTARRAY^®-PETRO qPCR ANALYSIS

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.
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.
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.
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.
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	BPH4	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.
Methylibium petroleiphilum PM1	PM1	Targets M. petroleiphilum PM1, one of the few organisms isolated to date which is capable of utilizing MTBE and TBA as growth supporting substrates.
TBA Monooxygenase	TBA	Targets the functional gene which catalyzes the continued biodegradation of TBA, an intermediate produced during aerobic MTBE biodegradation. TBA is also produced as a metabolite of aerobic biodegradation of ETBE by most known strains.
Naphthalene Dioxygenase	NAH	Initiates aerobic metabolism of naphthalene by incorporating both atoms of molecular oxygen into the ring. The broad substrate specificity of naphthalene dioxygenase has been widely noted. When expressed, naphthalene dioxygenase is capable of catalyzing the oxidation of larger PAHs like anthracene, phenanthrene, acenaphthylene, acenaphthene, and fluorine.
Naphthalene-Inducible Dioxygenase	NidA	Targets the naphthalene inducible dioxygenases found in Mycobacterium and Rhodococcus spp. which are capable of mineralizing naphthalene and degrading some higher molecular weight PAHs including pyrene and benzo[a]pyrene.
Phenanthrene Dioxygenase	PHN	The PHN assays quantify phenanthrene/naphthalene dioxygenase genes from a diverse collection of microorganisms including Pseudomonas, Burkholderia, Sphingomonas, and Acidovorax spp. As with other naphthalene dioxygenases, substrate specificity is relatively broad.
Alkane Monooxygenase	ALK	Initiates the aerobic biodegradation of n-alkanes with carbon lengths from C5 to C16.
Alkane Monooxygenase	ALMA	Catalyzes the aerobic biodegradation of C20-C32 alkanes by some Alcanivorax species considered dominant in marine systems.
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.
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.
Naphthyl-2-methyl-succinate synthase	MNSSA	Gene encoding the enzyme responsible for initiating anaerobic biodegradation of 2-methylnaphthalene by catalyzing the addition of fumarate onto the methyl group. MNSSA is analogous to the well-studied benzylsuccinate synthase (BSS) described for anaerobic biodegradation of toluene.
Anaerobic Naphthalene Carboxylase	ANC	To date, the only pathway that has been characterized for anaerobic biodegradation of naphthalene is initiated by a naphthalene carboxylase enzyme.
Alkylsuccinate Synthase	ASSA	Initiates anaerobic biodegradation of alkanes with chain lengths from C6 to at least C18.
Total Eubacteria	EBAC	Index of total bacterial biomass
Sulfate Reducing Bacteria	APS	Quantification of sulfate reducing bacteria provides an additional line of evidence when evaluating redox conditions and terminal electron accepting processes.