Comparative Assessments of Benzene, Toluene, and Xylene Natural Attenuation by Quantitative Polymerase Chain Reaction Analysis of a Catabolic Gene, Signature Metabolites, and Compound-Specific Isotope Analysis

A controlled-release study conducted at Vandenberg Air Force Base involved the injection of anaerobic groundwater amended with benzene, toluene, and o-xylene (BToX; 1-3 mg/L each) in two parallel lanes: lane A injectate contained no ethanol, whereas lane B injectate contained approximately 500 mg/L ethanol. As reported previously by Mackay and co-workers, ethanol led to slower BToX disappearance in lane B. Here, we report on assessments of BToX natural attenuation by three independent and specific monitoring approaches: signature metabolites diagnostic of anaerobic TX metabolism (benzysuccinates), compound-specific isotope analysis (CSIA), and quantitative polymerase chain reaction (qPCR) analysis of a catabolic gene involved in anaerobic TX degradation (bssA). In combination, the three monitoring methods provided strong evidence of in situ TX biodegradation in both lanes A and B; however, no single method provided strong evidence for TX biodegradation in both lanes. Benzylsuccinates were detected almost exclusively in lane B, where slower TX degradation and higher residual TX concentrations led to higher metabolite concentrations. In contrast, CSIA provided evidence of TX biodegradation almost exclusively in lane A, as greater degradation rates led to more pronounced isotopic enrichment. qPCR analyses of bssA were more complex. Evidence of increases in bssA copy number (up to 200-fold) after the release started was stronger in lane A, but higher absolute bssA copy number (and bacterial abundance, based on 16S rRNA genes) was observed in lane B, where bacteria genetically capable of anaerobic TX degradation may have been growing primarily on ethanol or its metabolites rather than TX.