Petroleum Hydrocarbon Biodegradation Testing: TPH, Alkanes & Crude Oil

At sites impacted by petroleum products or crude, the aromatic hydrocarbons benzene, toluene, ethylbenzene, xylenes (BTEX) as well as naphthalene and other polycyclic aromatic hydrocarbons (PAHs) are often the contaminants of greatest concern. However, alkanes are typically among the most abundant fractions in crude and petroleum products like gasoline and therefore significant contributors to total petroleum hydrocarbons (TPH) at impacted sites.

In addition to an assays targeting the genes encoding enzymes responsible for biodegradation of BTEX, naphthalene, and other PAHs, QuantArray^®-Petro includes an assay targeting the functional gene in the pathway for anaerobic biodegradation of alkanes. Alternatively, CENSUS^® qPCR can be performed to quantify only the assA gene.

TARGET	CODE	RELEVANCE / DATA INTERPRETATION
Alkylsuccinate Synthase	assA	Initiates anaerobic biodegradation of alkanes with chain lengths from C6 to at least C18.

Aerobic biodegradation of alkanes has been intensively studied and multiple alkane monooxygenase genes have been identified. QuantArray^®-Petro includes assays targeting genes encoding two alkane monooxygenases with different organisms and with different branch length specificities. As mentioned previously, QuantArray^®-Petro also includes quantification of functional genes in pathways for biodegradation of a broad spectrum of other petroleum hydrocarbons. Alternatively, CENSUS^® qPCR can be performed to quantify only the alkane monooxygenase genes.

TARGET	CODE	RELEVANCE / DATA INTERPRETATION
Alkane Monooxygenase	alkB	Initiates the aerobic biodegradation of n-alkanes with carbon lengths from C5 to C16.
Monooxygenase	almA	Catalyzes the aerobic biodegradation of C20-C32 alkanes by some Alcanivorax species considered dominant in marine systems.
Carbazole Dioxygenase	CAR	Catalyzes the oxidation of carbazole and other high molecular weight aromatics such as dibenzofuran.

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 biodegradation of a risk driving contaminant 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 are commonly performed to conclusively determine whether biodegradation of a specific contaminant of concern 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, ¹³C naphthalene, or ¹³C n-hexane) 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.

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
Callaghan AV, Davidova IA, Savage-Ashlock K, Parisi VA, Gieg LM, Suflita JM, et al. Diversity of benzyl- and alkylsuccinate synthase genes in hydrocarbon-impacted environments and enrichment cultures. Environmental Science & Technology. 2010;44:7287–94. https://doi.org/10.1021/es1002023.
Guo X, Zhang J, Han L, Lee J, Williams SC, Forsberg A, Xu Y, Narehood Austin R, Feng L. Structure and mechanism of the alkane-oxidizing enzyme AlkB. Nature Communications. 2023;14:2180. https://doi.org/10.1038/s41467-023-37869-z.
Liu C, Wang W, Wu Y, Zhou Z, Lai Q, Shao Z. Multiple alkane hydroxylase systems in a marine alkane degrader, Alcanivorax dieselolei b-5. Environmental Microbiology. 2011;13:1168–78. https://doi.org/10.1111/j.1462-2920.2010.02416.x.
Nojiri H, Nam J, Kosaka M, Morii K, Takemura T, Furihata K, Yamane H, Omori T. Diverse oxygenations catalyzed by carbazole 1,9a-dioxygenase from Pseudomonas sp. Strain CA10. Journal of Bacteriology. 1999;181:3105-13. https://doi.org/10.1128/jb.181.10.3105-3113.1999.