Microbial Insights

Detect and quantify bacteria responsible for biodegradation of BTEX and MTBE

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 biodegradation rates under aerobic conditions are relatively high, BTEX-utilizing bacteria are common in the subsurface, and the metabolic pathways leading to BTEX mineralization are well characterized.  Consequently, aerobic bioremediation is often the most attractive treatment technology for gasoline impacted sites.

Depending on site conditions (DO levels, BTEX concentrations, proximity to sensitive receptors, and groundwater flowrates), monitored naturation attenuation (MNA) can be an effective corrective action plan at gasoline impacted sites. At some sites 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 thorough characterization of the subsurface microbial community.

The following sections describe individual CENSUS assays, their importance in evaluating aerobic BTEX biodegradation, and provide guidelines for integrating CENSUS results into routine groundwater monitoring for common corrective actions.

CENSUS Targets for Aerobic BTEX and MTBE

Toluene Dioxygenase (qTOD): Toluene dioxygenase incorporates both atoms of molecular oxygen directly into the aromatic ring to initiate biodegradation of toluene.  Although named toluene dioxygenase, the substrate specificity of this enzyme is relatively relaxed and also catalyzes the biodegradation of benzene and ethylbenzene.   

Ring-Hydroxylating Monooxygenases (qRMO): The qRMO assay targets a group of genes encoding a class of enzymes which incorporate one atom of molecular oxygen into the aromatic ring.  This group includes toluene-2-monooxygenase, toluene-3-monooxygenase, and toluene-4-monooxygenase which attack the aromatic ring at the ortho, meta, and para positions, respectively. Although named toluene monooxygenases, members of this enzyme group are also responsible for biodegradation of benzene, ethylbenzene, and xylenes.

Phenol Hydroxylase (qPHE): Phenol hydroxylase catalyzes the contined oxidation of phenol and cresol intermediates produced by ring-hydroxylating monooxygenases and in some cases the initial oxidation as well. In single compound microcosm studies, PHE was detected following amendment with benzene, toluene, p-xylene, naphthalene and biphenyl suggesting a role in the biodegradation of numerous aromatic compounds or their metabolites. PHE is commonly detected in field samples obtained from gasoline-impacted sites1 and observed increases in PHE copies have corresponded to decreases in BTEX concentrations accompanying enhanced biodegradation.  Increases in PHE copies have also corresponded to aerobic biodegradation of dichlorobenzene.

Xylene/Toluene Monooxygenase (qTOL): Xylene/Toluene monooxygenase catalyzes the incorporation of one atom of molecular oxygen into the methyl group of toluene, m-xylene, and p-xylene.

Naphthalene Dioxygenase (qNAH): As the name suggests, naphthalene dioxygenase catalyzes the incorporation of both atoms of molecular oxygen into naphthalene, however, the broad substrate specificity of naphthalene dioxygenases has been widely noted. When expressed, naphthalene dioxygenase is capable of catalyzing the oxidation of larger PAHs (anthracene, phenanthrene, acenaphthylene, flurorene, and acenaphthene) and heterocyclic aromatic compounds (dibenzo-1,4-dioxin, dibenzothiophene, and dibenzofuran). The qNAH assay is most often used to evaluate aerobic biodegradation of PAHs at diesel fuel, wood preserving, and manufactured gas plant facilities.  However, NAH has also been frequently detected at gasoline-impacted sites where the monoaromatics BTEX were the contaminants of concern. Enrichment of bacteria harboring naphthalene dioxygenase at gasoline sites is most likely due to the rather broad specificity of the enzyme. In fact, naphthalene dioxygenases are similar to dioxygenases responsible for biodegradation of nitrotoluene and nitrobenzenes (nitrotoluene, 2,4-dinitrotoluene, and TNT). Thus, the NAH subfamily targeted by the qNAH assay will also provide insight into biodegradation of BTEX at gasoline impacted sites.

Biphenyl Dioxygenase (qBPH): Biphenyl dioxygenases catalyze aerobic metabolism of biphenyl and the cometabolism of some PCB congeners. In general aerobic biodegradation of PCBs containing one to three chlorines is common, however, congener specificity as well as the ability to cometabolize more heavily chlorinated PCBs (four to six chlorines) varies considerably. While developed to assess biodegradation of PCBs, quantification of one of the biphenyl dioxygenase subfamilies (BPH4) may also provide insight into the biodegradation of BTEX.  BPH4 has been detected at gasoline impacted sites1 although biphenyl is not a major component of gasoline. The likely reason for the detection of BPH4 at gasoline sites is that this group also contains cumene dixoygenases responsible for catabolism of isopropylbenzene, toluene, ethylbenzene, and biphenyl by some strains.

MTBE-utilizing PM1 (qPM1): With increased use in the 1990’s, the fuel oxygenate methyl tert-butyl ether (MTBE) has become one of the most commonly detected groundwater contaminants at gasoline impacted sites. The qPM1 assay quantifies Methylibium petroleiphilum PM1, one of the few organisms that have been isolated to date which is capable of using MTBE as a growth supporting substrate.

Integrating CENSUS Results

Monitored Natural Attenuation (MNA):

Depending on site conditions, aerobic biodegradation of BTEX can be an important component of MNA. Evaluating MNA as a corrective action and aerobic biodegradation as a treatment mechanism often depends upon integrating chemical, geochemical, and microbiological data:

Chemical Evidence

Analysis of BTEX concentration trends (e.g. Mann-Kendall Trend Analysis) can be used determine whether the dissolved plume is stable, shrinking or expanding.

Geochemical Evidence

Analysis of electron acceptor concentrations (dissolved oxygen, nitrate, ferric iron, and sulfate) or reduction products (ferrous iron, sulfide, and methane) can be used as indirect evidence of microbial activity. For example, consistently lower DO levels in impacted wells compared to non-impacted upgradient wells suggests oxygen consumption. Likewise, consumption of nitrate and sulfate suggest anoxic and anaerobic activity.  Production of ferrous iron and methane in impacted wells indicates iron reduction and methanogenesis.

Microbiological Evidence – CENSUS BTEX

Select representative wells for CENSUS analysis based site chemistry and geochemistry. Try to include at least one upgradient well, monitoring wells near the source area, perimeter of compliance wells, and downgradient wells outside the dissolved plume.  CENSUS analysis quantifies the bacteria specifically capable of biodegradation of BTEX compounds and is a strong indicator that aerobic biodegradation is a component of MNA.

Enhanced Bioremediation (Biostimulation):

Monitored natural attenuation can be an effective remediation strategy, however, a lack of available DO can hinder BTEX biodegradation and dictate a more aggressive approach to reach site objectives in an acceptable timeframe. CENSUS analysis can be used to evaluate the effectiveness of engineered approaches to stimulate aerobic BTEX bioremediation.  Simply put, increases in aromatic oxygenase genes in wells undergoing treatment demonstrates growth of bacteria capable of BTEX biodegradation.

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.

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 (in press).

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.

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.