Methyl group oxidation, SN2-type hydrolysis, and SN1-type hydrolysis are suggested as natural transformation mechanisms of MTBE. This study reports for the first time MTBE isotopic fractionation during acid hydrolysis and for oxidation by permanganate. In acid hydrolysis, MTBE isotopic enrichment factors were epsilon(C) = -4.9 per thousand +/- 0.6 per thousand for carbon and epsilon(H) = -55 per thousand +/- 7 per thousand for hydrogen. Position-specific values were epsilon(C), reactive position = -24.3 per thousand +/- 2.3 oer thousand and epsilon(H,reactive position) = -73 per thousand +/- 9 per thousand, giving kinetic isotope effects KIE(C) = 1.025 +/- 0.003 and KIE(H) = 1.08 +/- 0.01 consistent with an SN1-type hydrolysis involving the tert-butyl group. The characteristic slope of deltadelta2H(bulk)/deltadelta13C(bulk) approximately epsilon(bulk,H)/ epsilon(bulk,C) = 11.1 +/- 1.3 suggests it may identify SN1-type hydrolysis also in settings where the pathway is not well constrained. Oxidation by permanganate was found to involve specifically the methyl group of MTBE, similar to aerobic biodegradation. Large hydrogen enrichment factors of epsilon(H) = -109 per thousand +/- 9 per thousand and epsilon(H,reactive position) = -342 per thousand +/- 16 per thousand indicate both large primary and large secondary hydrogen isotope effects. Significantly smaller values reported previously for aerobic biodegradation suggest that intrinsic fractionation is often masked by additional non-fractionating steps. For conservative estimates of biodegradation at field sites, the largest epsilon values reported should, therefore, be used.

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.

Carbon isotopic enrichment factors (epsilonC) measured during cometabolic biodegradation of methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) by Pseudonocardia tetrahydrofuranoxydans strain K1 were -2.3 +/- 0.2 per thousand, -1.7 +/- 0.2 per thousand, and -1.7 +/- 0.3 per thousand, respectively. The measured carbon apparent kinetic isotope effect was 1.01 for all compounds, consistent with the expected kinetic isotope effects for both oxidation of the methoxy (or ethoxy) group and enzymatic SN1 biodegradation mechanisms. Significantly, delta13C measurements of the tert-butyl alcohol and tert-amyl alcohol products indicated that the tert-butyl and tert-amyl groups do not participate in the reaction and confirmed that ether biodegradation by strain K1 involves oxidation of the methoxy (or ethoxy) group. Measured hydrogen isotopic enrichment factors (epsilonH) were -100 +/- 10 per thousand, -73 +/- 7 per thousand, and -72 +/- 20 per thousand for MTBE, ETBE, and TAME respectively. Previous results reported for aerobic biodegradation of MTBE by Methylibium petroleiphilum PM1 and Methylibium R8 showed smaller epsilonH values (-35 per thousand and -42 per thousand, respectively). Plots of Delta2H/Delta13C show different slopes for strain K1 compared with strains PM1 and R8, suggesting that different mechanisms are utilized by K1 and PM1/R8 during aerobic MTBE biodegradation.