Diesel: Naphthalene and PAHs (Census)

At sites impacted by diesel or heavier petroleum products (e.g., jet fuel, heating oil, creosote), naphthalene and other polycyclic aromatic hydrocarbons (PAHs) are often the contaminants of greatest concern. Assessing MNA and enhanced bioremediation at these sites relies on chemical, geochemical, and microbial lines of evidence: trends in PAH concentrations, redox conditions/electron acceptors, and concentrations of bacteria capable of biodegradation of naphthalene and other PAHs.

While not as well characterized as aerobic biodegradation, naphthalene and 2-methylnaphthalene are susceptible to anaerobic biodegradation. QuantArray®-Petro includes assays targeting the genes encoding enzymes responsible for initiating anaerobic biodegradation of these contaminants. In addition, QuantArray®-Petro includes a suite of assays targeting functional genes involved in anaerobic and aerobic pathways for biodegradation of BTEX and other petroleum hydrocarbons. Alternatively, CENSUS® qPCR can be performed to quantify a select subset of targets such as anaerobic naphthalene carboxylase (ANC).

TARGET	CODE	RELEVANCE / DATA INTERPRETATION
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.

Enhanced aerobic bioremediation by injection of oxygen releasing materials (e.g., ORC® or PermeOx®) or engineered approaches can be an attractive treatment technology for diesel and other petroleum 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 naphthalene has been intensively studied and multiple catabolic pathways have been well characterized. QuantArray®-Petro includes assays targeting the initial oxygenase genes of the known pathways for aerobic biodegradation of naphthalene and some larger PAHs as shown below. As mentioned previously, QuantArray®-Petro also includes quantification of oxygenase genes in pathways for aerobic BTEX biodegradation. Alternatively, CENSUS® qPCR can be performed to quantify a select subset of functional genes.

TARGET	CODE	RELEVANCE / DATA INTERPRETATION
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.
Phenol Hydroxylase	PHE	Involved in aerobic biodegradation of benzene, toluene, ethylbenzene and xylenes (BTEX). In general, phenol hydroxylases (PHE) catalyze the continued oxidation of phenols produced by toluene monooxygenases and indicate the potential for aerobic BTEX biodegradation.

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 naphthalene 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 naphthalene are commonly performed to conclusively determine whether naphthalene 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 naphthalene) 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) 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 naphthalene and 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, PAHs, 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.

REFERENCE
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Khan AA, Wang R, Cao W, Doerge DR, Wennerstrom D, Cerniglia. Molecular cloning, nucleotide sequence, and expression of genes encoding a polycyclic aromatic ring dioxygenase from Mycobacterium sp. Strain PYR-1. Applied and Environmental Microbiology. 2001;67:3577-85. https://doi.org/10.1128/AEM.67.8.3577-3585.2001.
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Mouttaki H, Johannes J, Meckenstock RU. Identification of naphthalene carboxylase as a prototype for the anaerobic activation of non-substituted aromatic hydrocarbons. Environmental Microbiology. 2012;14:2770–4. https://doi.org/10.1111/j.1462-2920.2012.02768.x.
Pagnout C, Frache G, Poupin P, Maunit B, Muller J-F, Férard J-F. Isolation and characterization of a gene cluster involved in PAH degradation in Mycobacterium sp. strain SNP11: Expression in Mycobacterium smegmatis mc2155. Research in Microbiology. 2007;158:175-186. https://doi.org/10.1016/j.resmic.2006.11.002.
Selesi D, Jehmlich N, Bergen M von, Schmidt F, Rattei T, Tischler P, Lueders T, Meckenstock RU. Combined genomic and proteomic approaches identify gene clusters involved in anaerobic 2-methylnaphthalene degradation in the sulfate-reducing enrichment culture N47. Journal of Bacteriology. 2010;192:295–306. https://doi.org/10.1128/jb.00874-09.