Gasoline (MTBE)

Aerobic biodegradation of MTBE and the intermediate TBA has been extensively studied in the laboratory and documented in the field. However, Methylibium petroleiphilum PM1 remains one of the few bacterial cultures isolated to date that utilizes MTBE and TBA as growth supporting substrates. The key steps in aerobic MTBE/TBA biodegradation by strain PM1 is the initial oxidation mediated by a monooxygenase and the continued biodegradation of TBA catalyzed by TBA hydroxylase.

Particularly outside the US, ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME) were also employed as fuel oxygenates. Aerobic ETBE biodegradation is initiated by a cytochrome P450 monooxygenase (ethB). As with many of the monooxygenases, ethB exhibits relaxed specificity and will co-oxidize MTBE and TAME to TBA and tert-amyl alcohol (TAA), respectively.

QuantArray®-Petro includes quantification of M. petroleiphilum PM1, TBA monooxygenase, and ethB as described below. In addition, QuantArray®-Petro includes a suite of assays targeting functional genes involved in aerobic and anaerobic pathways for biodegradation of BTEX and other petroleum hydrocarbons. Alternatively, CENSUS® qPCR can be performed to quantify a select subset of targets such as strain PM1 and TBA monooxygenase.

TARGET	CODE	RELEVANCE / DATA INTERPRETATION
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.
ethB	ETH	Targets the functional gene encoding a P450 cytochrome monooxygenase responsible for initiating aerobic biodegradation of ETBE. Also capable of co-oxidation of MTBE and TAME.

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.

At some gasoline impacted sites, MTBE biodegradation is a critical factor in the feasibility of monitored natural attenuation (MNA). Therefore, SIP studies with ¹³C MTBE are often performed as a key line of evidence when evaluating MNA as a remediation strategy.

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

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 like PM1 and TBA monooxygenase.

By comparing contaminant concentrations, geochemical conditions, and concentrations of functional genes responsible for BTEX and MTBE 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.

Compound Specific Isotope Analysis (CSIA)

As described above, Stable Isotope Probing (SIP) rather than Compound Specific Isotope Analysis (CSIA) is recommended to conclusively determine whether biodegradation of MTBE is occurring. While carbon and hydrogen isotopic fractionation have been observed during the biodegradation of MTBE, enrichment factors range from significant to minor to insignificant based on the redox conditions and the specific microorganisms and pathways involved. However, CSIA has been employed to investigate MTBE degradation.  In addition, 2D-CSIA (δ¹³C and δ²H) has important applications for contaminant source distinction/delineation at sites where multiple sources may be present.