CENSUS – Gasoline – BTEX

Detect and quantify bacteria responsible for biodegradation of Gasoline – BTEX

At gasoline impacted sites, the monoaromatic hydrocarbons benzene, toluene, ethylbenzene, and xylenes (BTEX) are often the contaminants of concern due to their carcinogenicity, toxicity, and high water solubilities. Fortunately, BTEX-utilizing bacteria are common in the subsurface and BTEX biodegradation has been documented under both aerobic and anaerobic conditions.

Monitored Natural Attenuation:  Depending on site conditions (concentrations of DO and other electron acceptors, BTEX levels, proximity to sensitive receptors, and groundwater flowrates), monitored natural attenuation (MNA) can be an effective corrective action plan at gasoline impacted sites. Typically, evaluating MNA as a site management strategy will involve assessing trends in contaminant concentrations and geochemistry.  CENSUS and CENSUS-Expression can provide a complimentary line of evidence – quantification of the concentrations and expression of specific functional genes responsible for BTEX biodegradation.  Since aerobic and anaerobic biodegradation processes may be contributing under MNA conditions, consider CENSUS or CENSUS-Expression analysis for aerobic oxygenase genes (e.g. TOD, RMO, RDEG, and PHE) and anaerobic catabolic genes (e.g. bssA).

Aerobic Bioremediation: Aerobic bioremediation is often the most attractive treatment technology for gasoline impacted sites due to relatively high biodegradation rates.  Often however, the oxygen demand exerted by petroleum hydrocarbon degradation exceeds the natural oxygen recharge rate and DO becomes rate limiting.  Under these conditions, enhanced bioremediation either by injection of oxygen releasing materials (e.g. ORC® or PermeOx®) or engineered approaches such as bioventing/biosparging and in situ oxygen diffusion (e.g. iSOC®) are necessary to promote aerobic conditions and stimulate aerobic biodegradation. Regardless of the aeration approach, conclusive demonstration of the feasibility and effectiveness of bioremediation relies on converging lines of evidence from chemical and geochemical analysis coupled with microbiological evidence demonstrating stimulation of aerobic BTEX utilizing bacteria.  CENSUS and CENSUS-Expression analyses are used to quantify increases in the concentrations and activity of aerobic BTEX utilizing bacteria in response to site activities.

Anaerobic Bioremediation:  Although BTEX biodegradation rates are generally considered to be greater under aerobic conditions, enhanced anaerobic bioremediation through the addition of highly soluble, alternative electron acceptors like sulfate can also be an effective strategy.  The first step in the anaerobic biodegradation of toluene and other methyl-substituted benzenes is mediated by benzylsuccinate synthase (bssA) which catalyzes the addition of fumarate onto the toluene methyl group to form benzylsuccinate.  Three mechanisms (methylation, hydroxylation and carboxylation) have been proposed for the initial activation step in anaerobic biodegradation of benzene.  Of those, only carboxylation mediated by benzene carboxylase (abcA) has been fully elucidated.  However, Zhang et al. (2013) reported that benzene metabolism in Geobacter metallireducens proceeds via phenol confirming that there are multiple pathways for anaerobic benzene biodegradation.  More recently, the group identified two genes specificially required for anaerobic biodegradation of benzene (Zhang et al. 2014).

CENSUS Targets for Aerobic Gasoline – BTEX

The following table describes the individual CENSUS targets, their importance in evaluating aerobic BTEX biodegradation, and provides guidelines for integrating CENSUS results into routine groundwater monitoring for common corrective actions.

 

Target

MI Code 

  Relevance / Data Interpretation

Ring-hydroxylating Toluene Monooxygenases RMO

RDEG

Catalyzes the initial (and sometimes second) hydroxylation of BTEX compounds.  Presence indicates the potential for aerobic BTEX biodegradation.
Phenol Hydroxylase PHE Catalyzes further oxidation of BTEX compounds.  Presence indicates the potential for aerobic BTEX biodegradation.
Toluene/Xylene Monoxygenase TOL Attacks toluene and xylenes at the methyl group.
Naphthalene Dioxygenase NAH Catalyzes aerobic biodegradation of naphthalene and other PAHs by incorporation of oxygen into the aromatic ring.

CENSUS Target for Anaerobic Gasoline – BTEX

The following table describes the individual CENSUS target, its importance in evaluating anaerobic BTEX biodegradation, and provides guidelines for integrating CENSUS results into routine groundwater monitoring for common corrective actions.

 

Target

MI Code 

  Relevance / Data Interpretation

Benzyl Succinate Synthase bssA Targets gene encoding enzyme in anaerobic biodegradation of toluene.  Indicative of the potential for anaerobic TEX biodegradation.
Benzene Carboxylase abcA Targets the only known gene encoding an enzyme responsible for the initial activation step in anaerobic benzene biodegradation.
G. Metallireducens functional genes GMET Targets functional genes including a predicted oxidoreductase specifically required for anaerobic benzene metabolism by G. metallireducens.
Benzyl Coenzyme A reductase
BCR Anaerobic biodegradation of many aromatic hydrocarbons proceeds through a common intermediate called benzyl-CoA. The assay targets the benzoyl-CoA Reductase (BCR) gene which encodes the essential enzyme responsible for reducing the aromatic ring structure of this common intermediate.


References

Baldwin, B.R., C. Nakatsu, and L. Nies. 2008. “Enumeration of aromatic oxygenase genes to evaluate monitored natural attenuation at gasoline-contaminated sites”. Water Research 42: 723-731.

Baldwin, B.R., C.H. Nakatsu, and L. Nies. 2003. Detection and enumeration of aromatic oxygenase genes by multiplex and real-time PCR. Applied and Environmental Microbiology 69: 3350-3358.

Dominguez, R.F., M.L.B. da Silva, T.M. McGuire, D. Adamson, C.J. Newell, and P.J.J. Alvarez. 2008. “Aerobic bioremediation of chlorobenzene source-zone soil in flow-through columns: performance assessment using quantitative PCR”. Biodegradation 19: 545-553.

Nebe, J., B.R. Baldwin, R.L. Kassab, L. Nies, and C.H. Nakatsu. 2009. “Quantification of aromatic oxygenase genes to evaluate enhanced bioremediation by oxygen releasing materials at a gasoline-contaminated site”. Environmental Science & Technology.

Zhang, T., P.L. Tremblay, A.K. Charurasia, J.A. Smith, T.S. Bain and D.R. Lovley (2013).  “Anaerobic Benzene Oxidation via Phenol in Geobacter metallireducens.” Applied and Environmental Microbiology 79(24): 7800-7806.

Zhang, T., P.L. Tremblay, A.K. Charurasia, J.A. Smith, T.S. Bain and D.R. Lovley (2014).  “Identification of genes specifically required for the anaerobic metabolism of benzene in Geobacter metallireducens.” Frontiers in Microbiology 5.