Enumeration of aromatic oxygenase genes to evaluate monitored natural attenuation at gasoline-contaminated sites

Monitoring groundwater benzene, toluene, ethylbenzene, and xylene (BTEX) concentrations is the typical method to assess monitored natural attenuation (MNA) and bioremediation as corrective actions at gasoline-contaminated sites. Conclusive demonstration of bioremediation, however, relies on converging lines of chemical and biological evidence to support a decision. In this study, real-time PCR quantification of aromatic oxygenase genes was used to evaluate the feasibility of MNA at two gasoline-impacted sites. Phenol hydroxylase (PHE), ring-hydroxylating toluene monooxygenase (RMO), naphthalene dioxygenase (NAH), toluene monooxygenase (TOL), toluene dioxygenase (TOD), and biphenyl dioxygenase (BPH4) genes were routinely detected in BTEX-impacted wells. Aromatic oxygenase genes were not detected in sentinel wells outside the plume indicating that elevated levels of oxygenase genes corresponded to petroleum hydrocarbon contamination. Total aromatic oxygenase gene copy numbers detected in impacted wells were on the order of 106–109 copies L−1. PHE, RMO, NAH, TOD, and BPH4 gene copies positively correlated to total BTEX concentration. Mann–Kendall analysis of benzene concentrations was used to evaluate the status of the dissolved BTEX plume. The combination of trend analysis of contaminant concentrations with quantification of aromatic oxygenase genes was used to assess the feasibility of MNA as corrective measures at both sites.