Monitoring biodegradation of methyl tert-butyl ether (MTBE) using compound-specific carbon isotope analysis

Methyl tert-butyl ether (MTBE), the most common gasoline oxygenate, is frequently detected in surface water and groundwater. The aim of this study was to evaluate the potential of compound-specific isotope analysis to assess in situ biodegradation of MTBE in groundwater. For that purpose, the effect of relevant physical and biological processes on carbon isotope ratios of MTBE was evaluated in laboratory studies. Carbon isotope fractionation during organic phase/gas-phase partitioning (0.50 +/- 0.15@1000), aqueous phase/gas-phase partitioning (0.17 +/- 0.05@1000), and organic phase/aqueous-phase partitioning (0.18 +/- 0.24@1000) was small in comparison to carbon isotope fractionation measured during biodegradation of MTBE in microcosms based on aquifer sediments of the Borden site. In experiments with MTBE as the only substrate and a cometabolic experiment with 3-methypentane as primary substrate, MTBE became enriched in 13C by 5.1 to 6.9@1000 after 95 to 97% degradation. For both experiments, similar isotopic enrichment factors were obtained (-1.52 +/- 0.06 to -1.97 +/- 0.05@1000). Biodegradation of TBA, which accumulated transiently in the cometabolic microcosms, was also accompanied by carbon isotope fractionation, with an isotopic enrichment factor of -4.21 +/- 0.07@1000. This study suggests that carbon isotope analysis is a potential tool to trace in situ biodegradation of MTBE and TBA and thus to better understand the fate of these contaminants in the environment.     

Bioremediation of groundwater contaminated with gasoline hydrocarbons and oxygenates using a membrane-based reactor

The objective of this study was to operate a novel, field-scale, aerobic bioreactor and assess its performance in the ex situ treatment of groundwater contaminated with gasoline from a leaking underground storage tank in Pascoag, RI. The groundwater contained elevated concentrations of MTBE (methyl tert-butyl ether), TBA (tert-butyl alcohol), TBF (tert-butyl formate), BTEX (benzene, toluene, ethyl benzene, and xylene isomers), and other gasoline additives (tert-amyl methyl ether, di-isopropyl ether, tert-amyl alcohol, methanol, and acetone). The bioreactor was a gravity-flow membrane-based system called a Biomass Concentrator Reactor (BCR) designed to retain all biomass within the reactor. It was operated for six months at an influent flow rate that ultimately reached 5 gpm. The goal was to achieve a removal of all contaminants to <5 microg/L, which is the California Drinking Water advisory for MTBE. The concentration of TBA, an MTBE biodegradation byproduct, was consistently lower than that of MTBE. The other daughter compound detected in the influent, TBF, was degraded to concentrations below the detection limit of 0.02 microg/L. BTEX were consistently degraded to significantly lower levels in the effluent throughout the duration of the study (<1 microg/L). A similar high removal efficiency of the other gasoline oxygenates present in the groundwater (TAME, DIPE, and TAA) was also achieved. Dissolved organic carbon analysis demonstrated the ability of the bioreactor to produce high quality effluents with nonpurgeable organic carbon (NPOC) averaging approximately 50% lowerthan the NPOC concentrations in the influent contaminated groundwater.     

New evaluation scheme for two-dimensional isotope analysis to decipher biodegradation processes: application to groundwater contamination by MTBE

Compound-specific analysis of stable carbon and hydrogen isotopes was used to assess the fate of the gasoline additive methyl tert-butyl ether (MTBE) and its major degradation product tert-butyl alcohol (TBA) in a groundwater plume at an industrial disposal site. We present a novel approach to evaluate two-dimensional compound-specific isotope data with the potential to identify reaction mechanisms and to quantify the extent of biodegradation at complex field sites. Due to the widespread contaminant plume, multiple MTBE sources, the presence of numerous other organic pollutants, and the complex biogeochemical and hydrological regime atthe site, a traditional mass balance approach was not applicable. The isotopic composition of MTBE steadily changed from the source regions along the major contaminant plume (-26.4% to +40.0% (carbon); -73.1% to +60.3% (hydrogen)) indicating substantial biodegradation. Constant carbon isotopic signatures of TBA suggest the absence of TBA degradation at the site. Published carbon and hydrogen isotope fractionation data for biodegradation of MTBE under oxic and anoxic conditions, respectively, were examined and used to determine both the nature and the extent of in-situ biodegradation along the plume(s). The coupled evaluation of two-dimensional compound-specific isotope data explained both carbon and hydrogen fractionation data in a consistent way and indicate anaerobic biodegradation of MTBE along the entire plume. A novel scheme to reevaluate empiric isotopic enrichment factors (epsilon) in terms of theoretically based intrinsic carbon (12k/13k) and hydrogen (1k/2k) kinetic isotope effects (KIE) is presented. Carbon and hydrogen KIE values, calculated for different potential reaction mechanisms, imply that anaerobic biodegradation of MTBE follows a SN2-type reaction mechanism. Furthermore, our data suggest that additional removal process(es) such as evaporation contributed to the overall MTBE removal along the plume, a phenomenon that might be significant also for other field sites at tropic or subtropic climates with elevated groundwater temperatures (25 degrees C).     

Anaerobic degradation of methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA)

The potential for anaerobic degradation of methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA) was investigated in laboratory incubations of sediments from a petroleum-contaminated aquifer and in aquatic sediments. The addition of humic substances (HS) stimulated the anaerobic degradation of MTBE in aquifer sediments in which Fe(III) was available as an electron acceptor. This is attributed to the fact that HS and other extracellular quinones can stimulate the activity of Fe(III)-reducing microorganisms by acting as an electron shuttle between Fe(III)-reducing microorganisms and insoluble Fe(III) oxides. MTBE was not degraded in aquifer sediments without Fe(III) and HS. [14C]-MTBE added to aquatic sediments adapted for anaerobic MTBE degradation was converted to 14CO2 in the presence or absence of HS or the HS analog, anthraquione-2,6-disulfonate. Unamended aquatic sediments produced 14CH4 as well as 14CO2 from [14C]-MTBE. The aquatic sediments also rapidly consumed TBA under anaerobic conditions and converted [14C]-TBA to 14CH4 and 14CO2. An adaptation period of ca. 250-300 days was required prior to the most rapid anaerobic MTBE degradation in both sediment types, whereas TBA was metabolized in the aquatic sediments without a lag. These results demonstrate that, under the appropriate conditions, MTBE and TBA can be degraded in the absence of oxygen. This suggests that it may be possible to design strategies for the anaerobic remediation of MTBE in petroleum-contaminated subsurface environments.     

Variations in 13C/12C and D/H enrichment factors of aerobic bacterial fuel oxygenate degradation

Reliable compound-specific isotope enrichment factors are needed for a quantitative assessment of in situ biodegradation in contaminated groundwater. To obtain information on the variability on carbon and hydrogen enrichment factors (epsilonC, epsilonH) the isotope fractionation of methyl tertiary (tert-) butyl ether (MTBE) and ethyl tert-butyl ether (ETBE) upon aerobic degradation was studied with different bacterial isolates. Methylibium sp. R8 showed a carbon and hydrogen isotope enrichment upon MTBE degradation of -2.4 +/- 0.1 and -42 +/- 4 per thousand, respectively, which is in the range of previous studies with pure cultures (Methylibium petroleiphilum PM1) as well as mixed consortia. In contrast, epsilonC of the beta/-proteobacterium L108 (-0.48 +/- 0.05 per thousand) and Rhodococcus ruber IFP 2001 (-0.28 +/- 0.06 per thousand) was much lower and hydrogen isotope fractionation was negligible (epsilonH < or = -0.2 per thousand). The varying isotope fractionation pattern indicates that MTBE is degraded by different mechanisms by the strains R8 and PM1 compared to L108 and IFP 2001. The carbon and hydrogen isotope fractionation of ETBE by L108 (epsilonC = -0.68 +/- 0.06 per thousand and epsilonH = -14 +/- 2 per thousand) and IFP 2001 (epsilonC = -0.8 +/- 0.1 per thousand and epsilonH = -11 +/- 4 per thousand) was very similar and seemed slightly higher than the fractionation observed upon MTBE degradation by the same strains. The low carbon and hydrogen enrichment factors observed during MTBE and ETBE degradation by L108 and IFP 2001 suggest a hydrolysis-like reaction type of the ether bond cleavage compared to oxidation of the alkyl group as suggested for the strains PM1 and R8. The variability of carbon and hydrogen enrichment factors should be taken into account when interpreting isotope pattern of fuel oxygenates with respect to biodegradation in contamination plumes.     

Air-water transfer of MTBE, its degradation products, and alternative fuel oxygenates: the role of temperature

The gasoline oxygenate methyl tert-butyl ether (MTBE) has become one of the world's mostwidespread groundwater and surface water contaminants. As a result, there has been increasing interest in the environmental behavior of MTBE and its degradation products, mainly tert-butyl formate (TBF) and tert-butyl alcohol (TBA). In contrast, the environmental behavior of the proposed alternatives to MTBE, namely ethyl tert-butyl ether (ETBE), tert-amyl methyl ether (TAME), and diisopropyl ether (DIPE) has hardly been studied yet, although some of them are already in substantial use in various countries. A key parameter for the assessment of the fate, transport, and possible remediation of these contaminants is the air-water partitioning constant (KiH). The KiH is highly temperature dependent, and it is therefore necessary to obtain reliable experimental values at relevant temperatures. Hence, the KiH of MTBE, ETBE, TAME, and DIPE, along with the degradation products, TBF and methyl acetate, were determined from 5 degrees C-40 degrees C. The alternatives to MTBE generally had a higher KiH, which implies that, upon emission into the environment, the alternatives partition more readily into the air phase than MTBE. This may favor their use, as it is in the air phase where dilution and degradation are the most effective. The degradation products of MTBE, with the exception of TBF, have much lower KiH values at all temperatures. Hence, the degradation products will have a stronger affinity for the water phase. The temperature dependency of the kinetics of air-watertransfer is discussed using a boundary layer model. Only for TBA but not for the ethers a significant effect of temperature was found.     

Avoiding hydrolysis of fuel ether oxygenates during static headspace analysis

The determination of fuel ether oxygenates in groundwater was found to be problematic when samples are preserved at pH < 2 and then analyzed using heated headspace sampling. Acid catalyzed the hydrolysis of tert-amyl methyl ether, ethyl tert-butyl ether, and methyl tert-butyl ether during headspace sampling when aqueous samples were heated at 80 degrees C, a typical temperature used for heated headspace sampling. Hydrochloric acid at pH 2 did not cause hydrolysis of oxygenate ethers in samples stored for 28 d at 4 degrees C. When trisodium phosphate was used to preserve the sample or to adjust the pH of samples preserved with acid before headspace sampling, the recovery of spiked ethers was excellent. The heated headspace method was also applicable for the determination of other fuel oxygenates including ethanol, tert-butyl alcohol (TBA), tert-amyl alcohol (TAA), isopropyl alcohol (IPA), acetone, and monoaromatic compounds found in gasoline including benzene, toluene, ethylbenzene, xylenes, and trimethylbenzenes. The method detection limits range from 0.1 to 0.2 microg/L for the ethers and aromatics. For alcohols and acetone, the method detection limits were 0.8 microg/L for TBA, 18 microg/L for ethanol, 1.2 microg/L for TAA, 5.5 microg/L for IPA, and 3.3 microg/L for acetone. The heated headspace method yielded accurate results for ether oxygenates in samples containing a wide range of gasoline concentrations (2500-100000 microg/L).     

Carbon and hydrogen isotopic fractionation during biodegradation of methyl tert-butyl ether

Carbon and hydrogen isotopic fractionation during aerobic biodegradation of MTBE by a bacterial pure culture (PM1) and a mixed consortia from Vandenberg Air Force Base (VAFB) were studied in order to assess the relative merits of stable carbon versus hydrogen isotopic analysis as an indicator of biodegradation. Carbon isotopic enrichment in residual MTBE of up to 8.1/1000 was observed at 99.7% biodegradation. Carbon fractionation was reproducible in the PM1 and VAFB experiments, yielding similar enrichment factors (epsilon) of -2.0/1000 +/- 0.1/1000 to -2.4/1000 +/- 0.3/1000 for replicates in the PM1 experiment and -1.5/1000 +/- 0.1/1000 to -1.8/1000 +/- 0.1/1000 for replicates in the VAFB experiment. Hydrogen isotopic fractionation was highly reproducible for the PM1 pure cultures, with epsilon values of -33/1000 +/- 5/1000 to -37/1000 +/- 4/1000 for replicate samples. In the VAFB microcosms, there was considerably more variability in epsilon values, with values of -29/1000 +/- 4/1000 and -66/1000 +/- 3/1000 measured for duplicate sample bottles. Despite this variability, hydrogen isotopic fractionation always resulted in 2H enrichment of the residual MTBE of >80/1000 at 90% biodegradation. The reproducible carbon fractionation suggests that compound-specific carbon isotope analysis may be used to estimate the extent of biodegradation at contaminated sites. Conversely, the large hydrogen isotopic fractionation documented during biodegradation of MTBE suggests that compound-specific hydrogen isotope analysis offers the most conclusive means of identifying in-situ biodegradation at contaminated sites.     

Enrichment of stable carbon and hydrogen isotopes during anaerobic biodegradation of MTBE: microcosm and field evidence

The conventional approach to evaluate biodegradation of organic contaminants in groundwater is to demonstrate an increase in the concentration of transformation products. This approach is problematic for MTBE from gasoline spills because the primary transformation product (TBA) can also be a component of gasoline. Compound-specific stable isotope analysis may provide a useful alternative to conventional practice. Changes in the delta13C and deltaD of MTBE during biodegradation of MTBE in an anaerobic enrichment culture were compared to the delta13C and deltaD of MTBE in groundwater at nine gasoline spill-sites. The stable isotopes of hydrogen and carbon were extensively fractionated during anaerobic biodegradation of MTBE. The stable isotope enrichment factor for carbon (epsilonC) in the enrichment cultures was -13 (-14.1 to -11.9 at 95% confidence level), and the hydrogen enrichment factor (epsilonH) was -16 (-21 to -11 at 95% confidence level). The isotope enrichment factors for carbon and hydrogen during anaerobic biodegradation indicate that the first reaction is enzymatic hydrolysis of the O-Cmethyl bond. The ratio of epsilonH to epsilonC was consistent between the enrichment culture and the field site that provided the inoculum, and with the other eight sites, suggesting a common degradation pathway. Compound-specific isotope evidence is discussed in terms of its utility for monitoring in situ biodegradation, in particular, for measuring how much MTBE was degraded. For the studied field sites, significant biodegradation of the original mass of MTBE is suggested, in some cases exceeding 90%.     

Use of compound-specific stable carbon isotope analyses to demonstrate anaerobic biodegradation of MTBE in groundwater at a gasoline release site

Currently it is unclear if natural attenuation is an appropriate remedial approach for groundwater impacted by methyl tertiary butyl ether (MTBE). Site-characterization data at most gasoline release sites are adequate to evaluate attenuation in MTBE concentrations over time or distance. But, demonstrating natural biodegradation of MTBE requires laboratory microcosm studies, which could be expensive and time-consuming. Recently, compound-specific carbon isotope ratio analyses (13C/12C expressed in delta13C notation) have been used to demonstrate aerobic biodegradation of MTBE in laboratory incubations. This study explored the potential of this approach to distinguish MTBE biodegradation from other abiotic processes in an anaerobic groundwater plume that showed extensive decrease in MTBE concentrations. To our knowledge, this is the first study to use delta13C of MTBE data in groundwater and laboratory microcosms to demonstrate anaerobic biodegradation of MTBE. The delta13C of MTBE in monitoring wells increased by up to 31 per thousand (-25.5 per thousand to +5.5 per thousand) along with a 40-fold decrease in MTBE concentrations. Anaerobic incubations in laboratory microcosms indicated up to 20-fold reduction in MTBE concentrations with a corresponding increase in delta13C of MTBE of up to 33.4 per thousand (-28.7 per thousand to +4.7 per thousand) in live microcosms. Little enrichment was observed in autoclaved controls. These results demonstrate that anaerobic biodegradation was the dominant natural attenuation mechanism for MTBE at this site. The estimated isotopic enrichment factors (epsilon(field) = -8.10 per thousand and epsilon(lab) = -9.16 per thousand) were considerably larger than the range (-1.4 per thousand to -2.4 per thousand) previously reported for aerobic biodegradation of MTBE in laboratory incubations. These observations strongly suggest that delta13C of MTBE could be potentially useful as an "indicator" of in-situ MTBE biodegradation.     

Evaluation of the impact of fuel hydrocarbons and oxygenates on groundwater resources

The environmental behavior of fuel oxygenates (other than methyl tert-butyl ether [MTBE]) is poorly understood because few data have been systematically collected and analyzed. This study evaluated the potential for groundwater resource contamination by fuel hydrocarbons (FHCs) and oxygenates (e.g., tert-butyl alcohol [TBA], tertamyl methyl ether [TAME], diisopropyl ether [DIPE], ethyl tert-butyl ether [ETBE], and MTBE) by examining their occurrence, distribution, and spatial extent in groundwater beneath leaking underground fuel tank (LUFT) facilities, focusing on data collected from over 7200 monitoring wells in 868 LUFT sites from the greater Los Angeles, CA, region. Excluding the composite measure total petroleum hydrocarbons as gasoline (TPHG), TBA has the greatestsite maximum (geometric mean) groundwater concentration among the study analytes; therefore, its presence needs to be confirmed at LUFT sites so that specific cleanup strategies can be developed. The alternative ether oxygenates (DIPE, TAME, and ETBE) are less likely to be detected in groundwater beneath LUFT facilities in the area of California studied and when detected are present at lower dissolved concentrations than MTBE, benzene, or TBA. Groundwater plume length was used as an initial indicator of the threat of contamination to drinking water resources. Approximately 500 LUFT sites were randomly selected and analyzed. The results demonstrate MTBE to pose the greatest problem, followed by TBA and benzene. The alternative ether oxygenates were relatively localized and indicated lesser potential for groundwater resource contamination. However, all indications suggest the alternative ether oxygenates would pose groundwater contamination threats similar to MTBE if their scale of usage is expanded. Plume length data suggest that in the absence of a completely new design and construction of the underground storage tank (UST) system, an effective management strategy may involve placing greater emphasis on UST program for ensuring adequate enforcement and compliance with existing UST regulations.     

Pathway-dependent isotope fractionation during aerobic and anaerobic degradation of monochlorobenzene and 1,2,4-trichlorobenzene

Stable carbon isotope fractionation is a valuable tool for monitoring natural attenuation and to establish the fate of groundwater contaminants. In this study, we measured carbon isotope fractionation during aerobic and anaerobic degradation of two chlorinated benzenes: monochlorobenzene (MCB) and 1,2,4-trichlorobenzene (1,2,4-TCB). MCB isotope fractionation was measured in anaerobic methanogenic microcosms, while 1,2,4-TCB isotope experiments were carried out in both aerobic and anaerobic microcosms. Large isotope fractionation was observed in both the anaerobic microcosm experiments. Enrichment factors (ε) for anaerobic reductive dechlorination of MCB and 1,2,4-TCB were -5.0‰ ± 0.2‰ and -3.0‰ ± 0.4‰, respectively. In contrast, no significant isotope fractionation was found during aerobic microbial degradation of 1,2,4-TCB. The cleavage of a C-Cl σ bond occurs during anaerobic reductive dechlorination of MCB and 1,2,4-TCB, while no σ bond cleavage is involved during aerobic degradation via dioxygenase. The difference in isotope fractionation for aerobic versus anaerobic biodegradation of MCB and 1,2,4-TCB can be explained by the difference in the initial step of aerobic versus anaerobic biodegradation pathways.     

Impact of ethanol on the natural attenuation of MTBE in a normally sulfate-reducing aquifer

Side-by-side experiments were conducted in an aquifer contaminated with methyl-tert-butyl ether (MTBE) at a former fuel station to evaluate the effect of ethanol release on the fate of pre-existing MTBE contamination. On one side, for approximately 9 months we injected groundwater amended with 1-3 mg/L benzene, toluene, and o-xylene (BToX). On the other side, we injected the same, adding approximately 500 mg/L ethanol. The fates of BToX in both sides ("lanes") were addressed in a prior publication. No MTBE transformation was observed in the "No Ethanol Lane." In the "With Ethanol Lane", MTBE was transformed to tert-butyl alcohol (TBA) underthe methanogenic and/or acetogenic conditions induced by the in situ biodegradation of the ethanol downgradient of the injection wells. The lag time before onset of this transformation was less than 2 months and the pseudo-first-order reaction rate estimated after 7-8 months was 0.046 d(-1). Our results imply that rapid subsurface transformation of MTBE to TBA may be expected in situations where strongly anaerobic conditions are sustained and fluxes of requisite nutrients and electron donors allow development of an active acetogenic/methanogenic zone beyond the reach of inhibitory effects such as those caused by high concentrations of ethanol.     

Predicting methyl tert-butyl ether, tert-butyl formate, and tert-butyl alcohol levels in the environment using the fugacity approach

Through its extensive use as a fuel oxygenate, methyl tert-butyl ether (MTBE) is found nearly ubiquitouslythroughout the environment. To better understand the environmental fate of MTBE, fugacity models are commonly used. However, models developed by the scientific community and by governmental bodies differ in their predictions of relative MTBE concentrations for relevant environmental compartments and of seasonal concentration variations; further, to date they have not considered the formation of transformation products. In this study, the sensitivity of predicted environmental concentrations of MTBE and its two major degradation products, tert-butyl formate (TBF) and tert-butyl alcohol (TBA), to all types of model input parameters is analyzed in a probabilistic sensitivity analysis. This analysis allowed for an assessment of the most influential parameters for predicting soil, water, and air concentrations and thereby provided insight into why previous modeling studies on MTBE differed. Further, the information from the sensitivity analysis was used to parametrize a multispecies transformation model for predicting European concentration levels of MTBE and, for the first time, TBF and TBA. Water and air concentrations of MTBE predicted with the transformation model were in good agreement with measurements of environmental samples. No studies are available on environmental TBF and TBA levels to compare with model predictions; however, the modeling results indicate that, in the water phase, TBA concentrations may reach appreciable levels. One major uncertainty identified regarding the prediction of TBA levels was the fraction of TBA formed from atmospheric MTBE and TBF.     

Insight into methyl tert-butyl ether (MTBE) stable isotope fractionation from abiotic reference experiments

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.     

Simultaneous determination of fuel oxygenates and BTEX using direct aqueous injection gas chromatography mass spectrometry (DAI-GC/MS)

A direct aqueous injection-gas chromatography/mass spectrometry (DAI-GC/MS) method for trace analysis of gasoline components in water is presented. The method allows for the simultaneous quantification of the following solutes: methyl tert-butyl ether (MTBE), its major degradation products (tert-butyl formate, tert-butyl alcohol (TBA), methyl acetate, and acetone), and possible substitutes of MTBE as an octane enhancer in gasoline (tert-amyl methyl ether, ethyl tert-butyl ether) as well as benzene, toluene, ethylbenzene, p-xylene, m-xylene, and o-xylene (BTEX). No enrichment or pretreatment steps are required, and sample volumes of only 50 microL are needed for analysis. The detection limits in two different matrixes (spiked lake water and contaminated groundwater) are < or = 2 microg/L for most analytes and < 0.2 microg/L for MTBE, benzene, and toluene. The accuracy of the DAI-GC/MS method was excellent as determined from comparison with headspace-GC/MS and purge-and-trap-GC/MS. The DAI-GC/MS method has been applied to various environmental studies, which demonstrated its versatility. The applications comprised both laboratory (MTBE degradation in water treatment, quantification of polar gasoline components) and field (MTBE degradation ata gasoline spill site) investigations.     

Extending the Rayleigh equation to allow competing isotope fractionating pathways to improve quantification of biodegradation

The Rayleigh equation relates the change in isotope ratio of an element in a substrate to the extent of substrate consumption via a single kinetic isotopic fractionation factor (alpha). Substrate consumption is, however, commonly distributed over several metabolic pathways each potentially having a different alpha. Therefore, extended Rayleigh-type equations were derived to account for multiple competing degradation pathways. The value of alpha as expressed in the environment appears a function of the alpha values and rate constants of the various involved degradation pathways. Remarkably, the environmental or apparent alpha value changes and shows non-Rayleigh behavior over a large and relevant concentration interval if Monod kinetics applies and the half-saturation constants of the competing pathways differ. Derived equations were applied to previously published data and enabled (i) quantification of the share that two competing degradation pathways had on aerobic 1,2-dichloroethane (1,2-DCA) biodegradation in laboratory batch experiments and (ii) calculation of the extent of methyl tert-butyl ether (MTBE) biodegradation shared over aerobic and anaerobic degradation at a field site by means of an improved solution to two-dimensional (carbon and hydrogen) compound-specific isotope analysis (CSIA).     

Critical evaluation of the 2D-CSIA scheme for distinguishing fuel oxygenate degradation reaction mechanisms

Although the uniform initial hydroxylation of methyl tert-butyl ether (MTBE) and other oxygenates during aerobic biodegradation has already been proven by molecular tools, variations in carbon and hydrogen enrichment factors (ε(C) and ε(H)) have still been associated with different reaction mechanisms (McKelvie et al. Environ. Sci. Technol. 2009, 43, 2793-2799). Here, we present new laboratory-derived ε(C) and ε(H) data on the initial degradation mechanisms of MTBE, ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) by chemical oxidation (permanganate, Fenton reagents), acid hydrolysis, and aerobic bacteria cultures (species of Aquincola, Methylibium, Gordonia, Mycobacterium, Pseudomonas, and Rhodococcus). Plotting of Δδ(2)H/ Δδ(13)C data from chemical oxidation and hydrolysis of ethers resulted in slopes (Λ values) of 22 ± 4 and between 6 and 12, respectively. With A. tertiaricarbonis L108, R. zopfii IFP 2005, and Gordonia sp. IFP 2009, ε(C) was low (<|-1|‰) and ε(H) was insignificant. Fractionation obtained with P. putida GPo1 was similar to acid hydrolysis and M. austroafricanum JOB5 and R. ruber DSM 7511 displayed Λ values previously only ascribed to anaerobic attack. The fractionation patterns rather correlate with the employment of different P450, AlkB, and other monooxygenases, likely catalyzing ether hydroxylation via different transition states. Our data questions the value of 2D-CSIA for a simple distinguishing of oxygenate biotransformation mechanisms, therefore caution and complementary tools are needed for proper interpretation of groundwater plumes at field sites.     

Hydrolysis of tert-butyl methyl ether (MTBE) in dilute aqueous acid

tert-Butyl methyl ether (MTBE) is generally considered to be resistant to chemical transformation in aqueous solution. This lack of reactivity has led to concerns of the long-term impacts of MTBE in groundwater. Although hydrolysis in the presence of strong acids has been recognized as a mechanism for MTBE transformation, it has been discounted as a significant reaction under environmental conditions. In this study, we have examined the fate of MTBE and other ether oxygenates under moderately acidic conditions (> or=pH 1). The results demonstrate that MTBE is sensitive to acid-catalyzed hydrolysis reaction that generates tert-butyl alcohol (TBA) and methanol as products. The reaction is first-order with respect to the concentration of MTBE and hydronium ion with a second-order rate constant of about 0.9 x 10(-2) M(-1) h(-1) at 26 degrees C. Commercially available acidic ion-exchange resins were also shown to catalyze the hydrolysis of MTBE at near neutral pH. Pseudo-first-order rate constants were observed to be as high as 0.03 h(-1) at 25 degrees C and 0.12 h(-1) at 35 degrees C. These findings are discussed in terms of their possible implications for the treatment and environmental fate of MTBE and other gasoline oxygenates.     

Combined carbon and hydrogen isotope fractionation investigations for elucidating benzene biodegradation pathways

Recently, combined carbon and hydrogen isotope fractionation investigations have emerged as a powerful tool for the characterization of reaction mechanisms relevant for the removal of organic pollutants. Here, we applied this approach in order to differentiate benzene biodegradation pathways under oxic and anoxic conditions in laboratory experiments. Carbon and hydrogen isotope fractionation of benzene was studied with four different aerobic strains using a monooxygenase or a dioxygenase for the initial benzene attack, a facultative anaerobic chlorate-reducing strain as well as a sulfate-reducing mixed culture. Carbon and hydrogen enrichment factors (epsilon(C), epsilon(H)) varied for the specific pathways and degradation conditions, respectively, so that from the individual enrichment factors only limited information could be obtained for the identification of benzene biodegradation pathways. However, using the slope derived from hydrogen vs carbon isotope discriminations or the ratio of hydrogen to carbon enrichment factors (lambda = deltaH/ deltaC approximately epsilon(H)/epsilon(C)), benzene degradation mechanisms could be distinguished. Although experimentally determined lambda values partially overlapped, ranges could be determined for different benzene biodegradation pathways. Specific lambda values were < 2 for dihydroxylation, between 7 and 9 for monohydroxylation, and > 17 for anaerobic degradation. Moreover, variations in lambda values suggest that more than one reaction mechanism exists for monohydroxylation as well as for anaerobic benzene degradation under nitrate-reducing, sulfate-reducing, or methanogenic conditions. Our results show that the combined carbon and hydrogen isotope fractionation approach has potential to elucidate biodegradation pathways of pollutants in field and laboratory microcosm studies.