Groundwater microbial analysis to assess enhanced BTEX biodegradation by nitrate injection at a gasohol-contaminated site

Abstract

Groundwater samples from a gasohol contaminated aquifer were analyzed to investigate the effects of nitrate injection on microbial communities associated with benzene, toluene, ethylbenzene and xylenes (BTEX) biodegradation. Real-time quantitative PCR was used to quantify total bacteria (16S rDNA), nitrate-, iron-, sulfate-reducing bacteria and methanogens. Anaerobic BTEX degradation potential was assessed by targeting the bssA gene which encodes for benzylsuccinate synthase (BSS), an enzyme that initiates the biodegradation of toluene and xylenes. Aerobic BTEX biodegradation potential was assessed by targeting the catabolic genes: toluene dioxygenase (TOD), naphthalene dioxygenase (NAH), ring hydroxylating monooxygenase (RMO), phenol hydroxylase (PHE), and/or biphenyl dioxygenase (BPH). 16S rDNA gene copies were higher (∼4 × 10 5 cells ml −1) at the plume centerline coinciding with the highest concentrations of BTEX (∼26 mg-total l −1) and ethanol (∼3 mg l −1). Regions with high nitrate consumption coincided with the increased nitrate-reducing bacteria population. The establishment of Fe(III)-reducing zones were unlikely associated with bacteria belonging to Geobacter genus. Sulfate-reducing bacteria and methanogens were not detected corroborating with the geochemical footprints. Nitrate did not stimulate the fortuitous growth of anaerobic BTEX degraders as indicated by the absence of bssA amplification. Nitrate alleviated the high biological oxygen demand (BOD) associated with BTEX and ethanol biodegradation thus maintaining microaerophilic niche that supported the growth of aerobic BTEX degraders as indicated by the presence of PHE gene copy numbers (∼2 × 10 5 PHE gene copies ml −1). Among the genes tested, the non-functional 16S rDNA showed significant correlation ( r 2 = 0.94; p < 0.05) with BTEX (but not ethanol) first-order ( k′) biodegradation rates. Further investigations are required, however, to validate qPCR accuracy and reliability to estimate contaminants removal rates for a wide range of contaminated sites.