Environ. Sci. Technol. 2005, 39, 103-109
Carbon Isotopic Fractionation during Anaerobic Biotransformation of Methyl tert-Butyl Ether and tert-Amyl Methyl Ether PIYAPAWN SOMSAMAK,† HANS H. RICHNOW,‡ AND M A X M . H A¨ G G B L O M * , † Department of Biochemistry and Microbiology and Biotechnology Center for Agriculture and the Environment, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08901, and Department of Bioremediation, Center for Environmental Research (UFZ), Permoserstrasse 15, D-04318 Leipzig, Germany
The fuel oxygenate methyl tert-butyl ether (MTBE) has been frequently detected in groundwater and surface water. Since contaminated sites are often subsurface, anaerobic degradation of MTBE will likely be significant for remediation. As traditional approaches to evaluate biodegradation generally involve laboratory microcosm studies which require time and resources, innovative approaches are needed to demonstrate active in situ biodegradation of MTBE. This study was conducted to gather information at the laboratory level to evaluate the potential of applying carbon isotope fractionation as an indicator for in situ biodegradation of the fuel oxygenates MTBE and tert-amyl methyl ether (TAME). In this study, MTBE utilization was observed in a methanogenic sediment microcosm after a lengthy lag period of about 400 days. MTBE utilization was sustained upon refeeding and subculturing. tert-Butyl alcohol (TBA) was found to accumulate after propagation of cultures. The MTBE-grown cultures also utilized TAME and produced tert-amyl alcohol (TAA). The detection of TBA and TAA indicated that ether bond cleavage was the initial step in degradation for both compounds. Carbon isotope fractionation during anaerobic MTBE and TAME degradation was studied, and isotopic enrichment factors () with 95% confidence intervals of -15.6 ( 4.1‰ and -13.7 ( 4.5‰ were estimated for anaerobic MTBE and TAME degradation, respectively. Addition of 2-bromoethanesulfonic acid, an inhibitor of methanogenesis, substantially prolonged the lag period before transformation, but did not influence carbon isotope fractionation. Our experiment provided strong evidence of significant carbon isotope fractionation during anaerobic MTBE and TAME degradation, demonstrating that this technique can be used as an indicator for in situ MTBE and TAME degradation.
Introduction Methyl tert-butyl ether (MTBE) is a synthetic compound produced almost exclusively for use in gasoline as an octane * Corresponding author phone: (732)932-9763, ext 326; fax: (732)932-8965; e-mail:
[email protected]. † Rutgers, The State University of New Jersey. ‡ Center for Environmental Research. 10.1021/es049368c CCC: $30.25 Published on Web 11/19/2004
2005 American Chemical Society
enhancer and lately as a fuel oxygenate to reduce atmospheric concentrations of carbon monoxide and ozone in accordance with the United States Clean Air Act Amendments of 1990. Several other chemicals have also been used as fuel oxygenates, including ethyl tert-butyl ether (ETBE), tert-amyl methyl ether (TAME), diisopropyl ether (DIPE), tert-butyl alcohol (TBA), methanol, and ethanol (1). Because of its low cost, ease of production, and favorable transfer and blending characteristics, MTBE is the most commonly used fuel oxygenate (2-4). MTBE is currently the focus of public concern, particularly in the United States, as MTBE has been detected in groundwater and surface water across the United States (5, 6). MTBE has a very low taste and odor threshold, and thus even small quantities of MTBE will affect the quality of drinking water. There is also concern about its possible risk to human health, which is still inconclusive so far. Generally, contaminants can be naturally attenuated by various processes, including volatilization, adsorption, dispersion, hydrolysis, and biodegradation. Unlike other gasoline components, such as BTEX compounds (benzene, toluene, ethylbenzene, o-, m-, p-xylene), MTBE is very watersoluble, and it tends to partition from gasoline to the water phase. Once dissolved in water, the relatively low Henry’s law constant of MTBE does not lead to significant losses by partitioning into the gas phase. The relatively low Koc of MTBE implies that its movement is minimally retarded by soil particles, thus allowing MTBE plumes to travel at almost the same velocity as the groundwater stream. The reduction of MTBE mass by physical processes in groundwater is probably insignificant, because volatilization in aquifers is not very efficient and the hydrolysis of MTBE at almost neutral pH values is very slow (7). Therefore, bioremediation may play a significant role in mass reduction of MTBE at contaminated sites. Although early reports indicated that MTBE was resistant to biodegradation, aerobic MTBE biodegradation has been clearly demonstrated (see refs 8-10 for reviews) along with the biodegradation of other structurally related fuel oxygenates such as TAME, ETBE, and TBA. Recently, MTBE has also been shown to be biodegradable anaerobically under methanogenic (11, 12), denitrifying (13), iron(III)-reducing (14), and sulfate-reducing (15) conditions. TBA is often detected as an intermediate of MTBE biodegradation, suggesting that cleavage of the ether bond is the initial step in the degradation pathway. Under both aerobic and anaerobic conditions, the slow degradation of TBA indicated by an enrichment of these components suggests that the degradation of the metabolite is a crucial step in MTBE mineralization. Anaerobic MTBE degradation is extremely important for natural attenuation as a remediation option, since MTBEcontaminated sites are often subsurface with limited oxygen available for biodegradation. Moreover, cocontamination with a mixture of gasoline hydrocarbons leads to a rapid consumption of oxygen in aquifers. Although MTBE degradation has been conclusively demonstrated both in laboratory studies and at field sites, evaluation of in situ biodegradation is a unique challenge, in particular when natural attenuation is taken into account as a remediation option. The general strategy to confirm in situ biodegradation should include three types of evidence: (i) documented loss of contaminants from the site, (ii) laboratory assays showing that microorganisms in site samples have the potential to transform the contaminants under the expected site conditions, and (iii) one or more lines of evidence showing that the biodegradation potential is actually realized in the field (16). Considering the time and resources required, innovative VOL. 39, NO. 1, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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approaches are needed to demonstrate active ongoing biodegradation of MTBE as well as to evaluate the effectiveness of various cleanup technologies applied in the field. Isotope fractionation has been reported for biodegradation of environmental contaminants, such as petroleum hydrocarbons (17-22) and chlorinated compounds (23-26), and may serve as an indicator for in situ biodegradation. Compound-specific stable carbon isotope analysis has been proposed as a method for verifying in situ MTBE biodegradation (27-29). Hunkeler et al. (27) demonstrated that partitioning between environmental media revealed a very low carbon isotope fractionation in comparison to carbon isotope fractionation measured during biodegradation of MTBE in aquifer sediment microcosms. Isotope enrichment factors of -1.5‰ to -2.4‰ were estimated for aerobic MTBE degradation (27, 28). In contrast, much larger isotope enrichment factors were obtained for anaerobic MTBE degradation; however, the specific degradation conditions such as electron acceptors were not documented (29). To quantify in situ biodegradation, isotope fractionation factors representative for the biogeochemical conditions governing in situ biodegradation are essential. Therefore, the extent of isotope fractionation in particular degradation pathways should be investigated before quantification of in situ biodegradation. To date, only one study has reported carbon isotopic fractionation during anaerobic MTBE degradation (29), and it remains to be determined whether different microbial communities will produce similar degrees of carbon isotope fractionation. Since anaerobic biodegradation is significant for remediation of MTBE-contaminated sites, this study was conducted to gather information at the laboratory level to evaluate the potential of applying carbon isotope fractionation as an indicator for in situ biodegradation of MTBE and TAME. The objective of this study was to examine carbon isotope fractionation during MTBE and TAME degradation by anaerobic enrichment cultures and to estimate the isotope enrichment factor for these processes.
Experimental Section Establishment of Methanogenic MTBE-Degrading Cultures. The methanogenic cultures investigated in this study were established from anaerobic microcosms using a sediment inoculum from the Arthur Kill inlet, an intertidal strait between New Jersey and Staten Island, NY. The microcosms were set up to examine the potential of indigenous microorganisms to biodegrade MTBE. The microcosms were established in anaerobic medium as previously described (15), without the addition of terminal electron acceptors (except for carbonate). Sediment slurries (1:10 v/v sediment) were divided into aliquots in serum vials capped with Tefloncoated rubber stoppers and crimped with aluminum seals. Each vial contained 50 mL of slurry and had a 10 mL headspace of N2/CO2 (70:30 v/v) passed over hot reduced copper filings to remove traces of O2. Sterile controls were autoclaved three times on consecutive days before the experiment was initiated. MTBE (Aldrich, Milwaukee, WI) was added to all enrichments and autoclaved controls to a final concentration of 100 mg L-1. Relatively high substrate levels were used to enrich for anaerobic organisms that could utilize the fuel oxygenate as a source of carbon and energy. Strict anaerobic microbial techniques were used throughout all experimental manipulations. Microcosms were set up in triplicate and incubated under static conditions in the dark at 28 °C. Substrate concentrations were monitored periodically. Active cultures were refed and monitored for the depletion of substrate. Finally, active cultures were repeatedly transferred (1:5 v/v) to fresh medium to establish stable cultures utilizing MTBE as the sole carbon source. 104
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Experimental Setup. For the isotope fractionation experiments, the sixth-generation cultures (approximately 10-5 dilution of the original microcosm) were used. The transferred cultures were fed once with 10 mg L-1 MTBE to establish activity, after which the supernatant liquid was discarded and 75 mL of fresh methanogenic medium was added. This enrichment slurry was used as the inoculum. A total of 15 serum vials were prepared for each live treatment. Each vial received 1 mL of enrichment slurry and 9 mL of methanogenic medium. The serum vials were capped with gray Teflonlined butyl rubber septa and crimped with aluminum seals. Anaerobic MTBE and TAME solutions were prepared by adding MTBE or TAME (Aldrich) neat to anaerobic medium to a final concentration of 120 mg L-1. A 2 mL sample of anaerobic MTBE or TAME solution was added to yield a final substrate concentration of 20 mg L-1. Abiotic controls were also prepared the same way, except that 1 mL of anaerobic medium was used instead of enrichment slurry. After addition of substrate, all live cultures and sterile control vials were shaken for 30 min at 100 rpm and allowed to settle for 12 h, after which a 1 mL liquid sample was taken. Two vials of each treatment were sacrificed immediately by addition of 1 mL of saturated NaCl solution and adjusted to pH 1 by addition of 3 M HCl. Substrate concentrations in all remaining live cultures and abiotic controls were measured by headspace analysis. Thereafter, the remaining live cultures and sterile control vials were incubated at 28 °C without shaking. The headspace concentration of each vial was monitored periodically, and the biodegradation of MTBE and TAME was initially evaluated by comparing the headspace concentration to the initial concentration of that respective vial. At different stages of substrate utilization, cultures were sacrificed. Before the addition of NaCl solution and HCl, a 1 mL liquid sample was taken and analyzed immediately by GC injection for the concentration of substrate remaining and intermediates formed. The sacrificed vials were then stored at -20 °C for later carbon isotope analysis. Inhibition of Anaerobic MTBE Degradation by BES. Live cultures and abiotic controls were prepared as described above. Anaerobic 2-bromomethanesulfonic acid (BES) as a selective inhibitor of methanogenesis was added from an anaerobic stock solution to each vial to a final concentration of 20 mM (30). Analytical Methods. The concentration of MTBE and TAME was determined with a static headspace method. A 100 µL headspace sample was analyzed for MTBE and TAME with a Hewlett-Packard 5890 gas chromatograph equipped with a 0.53 mm × 30 m DB1 column (J&W Scientific, Folsom, CA) and a flame ionization detector. The GC column temperature was first held at 35 °C for 3 min, increased to 120 °C at a rate of 5 °C min-1, and then held for 1 min. In addition, the concentrations of MTBE and TAME were confirmed by direct injection of a 1 µL aqueous sample using the same instrument and temperature program. Intermediates were identified by comparison of their retention times to authentic standards. Standard curves for each compound were generated with aqueous standards of 1, 5, 10, and 25 mg L-1. Detection limits were 0.5 mg L-1 for MTBE and TAME, and 1.0 mg L-1 for TBA and tert-amyl alcohol (TAA). Methane was detected by headspace analysis using the same instrument described above at an oven temperature held constant at 35 °C. Because the volume of methane produced was too low to be accurately measured (no overpressure), methane concentrations during MTBE degradation were estimated on the basis of the peak area from headspace injection. Stable Carbon Isotope Analysis. Stable isotope analyses were conducted at the Stable Isotope Laboratory of the Center for Environmental Research (UFZ), Leipzig-Halle. The system consisted of a gas chromatograph (6890 series, Agilent Technology) coupled with a combustion interface (Thermo
FIGURE 1. Anaerobic MTBE loss in three replicates (solid tilted squares, replicate 1; solid triangles, replicate 2; solid circles, replicate 3) of intertidal sediment microcosms without amendment of the external electron acceptor. The initial concentration of MTBE added was ca. 100 mg L-1. Autoclaved control data are the means of triplicate analyses (open circles). Finnigan GC-combustion III, Bremen, Germany) and a Finnigan MAT 252 isotope ratio mass spectrometer (ThermoFinnigan, Bremen). The organic substances in the CG effluent were oxidized to CO2 on a CuO/Ni/Pt catalyst held at 960 °C. A Poraplot Q column (0.32 mm × 25 m, Chrompack, The Netherlands) was used for separation. Helium at a flow rate of 1.5 mL min-1 was used as the carrier gas. The GC temperature program was held at 150 °C for 15 min, then increased to 220 °C at a rate of 3 °C min-1, and then held for 10 min isothermally. Samples were injected in split mode with a split ratio 1:1 into a hot injector held at 220 °C. Headspace injection volumes ranged from 0.2 to 1 mL based on the concentration of MTBE determined previously. Each sample was analyzed at least in triplicate. The direct headspace method had a detection limit of approximately 4 mg L-1 for MTBE and 6 mg L-1 for TAME. The carbon isotopic compositions (R) are reported as δ notation in parts per thousand (denoted as ‰) enrichments or depletions relative to a standard (V-PDB; Vienna Pee Dee Belemnite standard) of known composition. δ values of carbon were calculated as follows:
δ(13C) (‰) ) (Rsample/Rstandard - 1) × 1000 where Rsample and Rstandard represent 13C/12C ratios of the sample and the international standard. The direct headspace analysis of standard MTBE and TAME had a mean isotope composition of -30.5 ( 0.4 ‰ (n ) 8) and -22.3 ( 1.7 ‰ (n ) 8), respectively.
Results Enrichment of Methanogenic MTBE-Degrading Consortia. MTBE degradation profiles of the original methanogenic microcosms established with intertidal sediment are shown in Figure 1. With an initial concentration of 108.4 ( 3.9 mg L-1, there was no evidence of any substrate utilization observed during the first 300 days of incubation. However, on day 390, the MTBE concentration in one live microcosm was decreased by 20%. The MTBE concentration in that particular enrichment culture gradually decreased over time, and complete depletion was observed after 550 days of incubation. TBA, the common MTBE degradation intermediate, was not detected by GC analysis of aqueous samples. Even though comparable levels of methane were detected in all three replicates (data not shown), no depletion of MTBE in comparison to sterile controls was observed in the other two microcosms. About 20% depletion of the MTBE concentration due to abiotic processes or losses was observed in autoclaved controls over 550 days.
FIGURE 2. Transformation of MTBE (solid squares) to TBA (open squares) in the active microcosm upon respiking with MTBE. The arrow indicates addition of MTBE. After depletion of MTBE in the one active microcosm it was respiked with 20 mg L-1 MTBE to confirm utilization. Depletion of MTBE was faster compared to the initial spike, and MTBE was removed below the detection limit of 0.5 mg L-1 within 44 days (Figure 2). A TBA concentration of 6.6 mg L-1 was measured on day 44. The microcosm was fed with 20 mg L-1 MTBE two more times. During the experiment TBA accumulated to a concentration of 32.7 mg L-1 after the third addition of MTBE. The detection of TBA confirmed anaerobic MTBE biotransformation. After the third spike, 10 mL of the microcosm slurry was transferred into 40 mL of fresh methanogenic medium and spiked with MTBE. After establishment of activity, these culture transfers (1:5) were sequentially repeated, and MTBE was fed as the sole substrate throughout the course of the subsequent incubation. The enrichments maintained their capability to transform MTBE. The headspace methane concentration increased as MTBE utilization proceeded, and TBA was accumulated in stoichiometric amounts to MTBE utilized in more diluted enrichments (data not shown). MTBE/TAME Degradation Profiles. The sixth generation of MTBE-grown cultures (approximately 10-5 dilution of the original microcosm) were tested for their capability to biodegrade TAME, as well as for the role of methanogenesis in MTBE and TAME biodegradation. Individual 12 mL cultures were established and spiked with MTBE or TAME with or without BES to inhibit methanogenesis. Headspace substrate concentrations were monitored over time, and cultures were sacrificed at different stages of biodegradation for analysis. With an average initial MTBE concentration of 20.5 ( 0.8 mg L-1, MTBE degradation in live cultures without BES proceeded after a lag period of 30 days (Figure 3a). In the experiments with BES, minimal MTBE degradation was observed during the first 120 days of incubation (Figure 3a). From an average initial MTBE concentration of 20.6 ( 0.9 mg L-1, between 10% and 90% MTBE transformation was observed at day 160. The remaining of live cultures with BES were sacrificed on day 185 with an MTBE concentration range from 0.9 to 14.3 mg L-1. MTBE depletion in abiotic controls was less than 10% at the end of the experiments on day 185. As MTBE was utilized, with and without BES, stoichiometric amounts of TBA accumulated (Figure 4a). Without BES, headspace methane concentrations increased corresponding to MTBE utilized, while methane was not detectable in cultures with BES, indicating that methanogenesis was inhibited (Figure 4a). Figure 3b shows TAME degradation profiles by MTBEgrown methanogenic cultures with and without BES. Under methanogenic conditions, TAME utilization began after a lag period of about 7-15 days (Figure 3b). TAME concentrations in all live cultures decreased over time from an average initial concentration of 20.4 ( 1.0 mg L-1. In the presence of BES, TAME utilization began after 30 days of incubation. TAME concentrations in these cultures were not monitored VOL. 39, NO. 1, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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and in the absence of BES, with concentrations accounting for 50-80% of TAME utilized (Figure 4b). Without BES, headspace methane concentrations increased corresponding to the amount of TAME utilized, while methane was detected at relatively low concentrations in a few cultures with BES, indicating that methanogenesis was almost completely inhibited (Figure 4b). Isotope Fractionation during Anaerobic Biotransformation of MTBE and TAME. Individual cultures from the previous experiment were sacrificed at different stages of substrate utilization. All live samples with MTBE concentrations greater than 4 mg/L-1 were analyzed for carbon isotope composition. The δ(13C) of the MTBE standard was -30.5 ( 0.4‰, and the mean δ(13C) of two sample vials sacrificed on day 0 was -29.0 ( 0.3‰. In live cultures, δ(13C) of MTBE increased as biodegradation proceeded. A total shift of δ(13C) of 24.4‰ was observed at 77.8% MTBE degradation, indicating a strong enrichment of 13C in the residual MTBE fraction. The mean δ(13C) of MTBE of two abiotic control vials collected on day 125 (-29.0 ( 0.1‰) was comparable to the initial values of both live cultures (-29.0 ( 0.3‰) and abiotic controls (-29.1 ( 0.1‰). Because the differences of δ(13C) of MTBE in abiotic controls at the beginning and the end of the experiment were minimal, the isotope ratios were not analyzed for other abiotic controls collected during the course of the experiment. FIGURE 3. (a) MTBE concentrations in live cultures with MTBE only (solid squares), MTBE + BES (solid circles), and abiotic controls (open squares). (b) TAME concentrations in live cultures with TAME only (solid triangles), TAME + BES (solid tilted squares), and abiotic controls (open circles). Error bars represent the standard deviation, n ) 5.
The mean δ(13C) values of TAME in two live cultures and two abiotic vials sacrificed on day 0 were -19.2 ( 0.5‰ and -20.1 ( 0.4‰, respectively. The values were slightly enriched in 13C compared to the carbon isotope composition of the TAME standard (-22.3 ( 1.7‰). In the live cultures an enrichment of 13C in the residual fraction during TAME biodegradation was observed. The isotope composition of TAME was enriched in 13C to -8.1‰ and -5.1‰ after 50.3% and 64.0% TAME utilization, respectively. A reliable determination of the isotope composition of residual TAME at a more advanced stage of transformation was not possible. The mean isotope composition of TAME of two abiotic control vials collected at the end of the experiment was -18.4 ( 0.3‰. With the addition of BES to inhibit methanogenesis, the transformations of MTBE and TAME were also accompanied by an enrichment of 13C in the residual parent substrates. At 72.1% MTBE degradation, a δ(13C) value of -8.1‰ was measured in the residual MTBE, which gives a total shift of 21.5‰ compared to the initial isotope composition. Analysis of δ(13C) of TAME in live enrichments also demonstrated strong enrichment of 13C in the residual TAME fraction. A total shift of 15.1‰ was observed in live enrichments at 72.6% TAME utilization.
FIGURE 4. Concentrations of headspace methane (solid symbols) and tertiary alcohol (open symbols) at different stages of (a) MTBE and (b) TAME degradation in live cultures with MTBE only (squares), MTBE + BES (circles), TAME only (triangles), and TAME + BES (tilted squares). between day 59 and day 165. On day 165, all of the cultures were sacrificed. TAME concentrations in most vials were below 2 mg L-1 except for three cultures with TAME concentrations of 5.8, 7.8, and 8.0 mg L-1. TAA was detected as the TAME transformation product both in the presence 106
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Estimation of the Isotopic Fractionation Factor (R) and Isotopic Enrichment Factor (). Calculations were based on the Rayleigh equation Rt/R0 ) (ct/c0)(1/R-1) for a closed system (31, 32), where R is the isotope ratio, C is the concentration, and the index (0, t) describes the incubation time at the beginning (0) and during the reaction time (t) of the experiment. When ln(Rt/R0) versus ln(ct/c0) is plotted (Figure 5), the kinetic isotope fractionation factor (R) and isotopic enrichment factor () can be determined from the slope of the curve (b), with b ) 1/R - 1 and ) 1000b. Isotope ratios (Rt/R0) were determined from the equation Rt/R0 ) (δt/1000 + 1)/(δ0/1000 + 1). Linear regression was used to estimate the slope of each data set. The enrichment factors of the individual anaerobic MTBE and TAME degradation were -15.6‰ and -13.7‰, respectively. The addition of BES to inhibit methanogenesis in parallel batch experiments resulted in enrichment factors of -14.6‰ and -11.2‰, which are slightly lower compared to those of the fractionation experiments without BES addition.
FIGURE 5. Double logarithmic plot according to the Rayleigh equation of the isotopic composition versus the residual concentration of substrates: (a) MTBE, (b) MTBE + BES, (c) TAME, (d) TAME + BES. Solid symbols represent live cultures. Open symbols represent abiotic controls.
TABLE 1. Carbon Fractionation Factors (r),a Carbon Enrichment Factors (E) with a 95% Confidence Interval,b and Intrinsic Carbon Enrichment Factors (EIntrinsic) for MTBE and TAME Biotransformation by Methanogenic Cultures r MTBE, methanogenic MTBE + BES TAME, methanogenic TAME + BES
E (‰)
Eintrinsic
R2
1.0159 ( 0.0015 -15.6 ( 4.1 -78.4 0.9662 1.0148 ( 0.0023 -14.6 ( 5.2 -73.0 0.8634 1.0139 ( 0.0011 -13.7 ( 4.5 -82.2 0.9885 1.0113 ( 0.0010 -11.2 ( 3.2 -67.2 0.9759
With an uncertainty of (1σ. b 95% confidence interval obtained from regression analysis of the Rayleigh equation. a
The intrinsic isotope fractionation factor Rintrinsic and isotope enrichment factor intrinsic were also calculated from the equation Rintrinsic ) 1/[(n/R) - (n - 1)] and the related intrinsic ) (1/Rintrinsic - 1) × 1000 (33). The equations take into account the number of carbon atoms (n) which do not undergo isotope fractionation. This approach allows the comparison of isotope fractionation of organic compounds with different numbers of carbon atoms. The isotopic enrichment factor () and the intrinsic enrichment factor (intrinsic) estimated for each treatment are summarized in Table 1.
Discussion The prospect of using stable isotope fractionation as a tool to demonstrate in situ MTBE degradation has received increased attention. Only a few studies, however, have been reported so far. Hunkeler et al. (27) demonstrated that carbon isotope fractionation during partitioning between environmental compartments was small in comparison to carbon isotope fractionation measured during aerobic biodegradation. Aerobic degradation experiments with MTBE as the only substrate and a cometabolic transformation experiment with 3-methylpentane yielded similar isotope enrichment factors (-1.52 ( 0.06‰ to -1.97 ( 0.05‰). Gray et al. (28) reported carbon isotope enrichment factors of -2.0 ( 0.1‰ to -2.4 ( 0.3‰ and -1.5 ( 0.1‰ to -1.8 ( 0.1‰ during aerobic biodegradation of MTBE by a bacterial pure culture
and a mixed consortium, respectively. The carbon enrichment factors obtained in those two studies show small variability associated with the cleavage of the methyl group under aerobic conditions. In contrast, much stronger enrichment factors were observed during anaerobic MTBE and TAME degradation. Our study presents evidence of significant 13C enrichment in residual MTBE and TAME during anaerobic biodegradation due to the faster reaction rates of lighter compared to heavier isotopomers. Isotopic enrichment factors () with a 95% confidence interval of -15.6 ( 4.1‰ and -13.7 ( 4.5‰ were estimated for anaerobic MTBE and TAME degradation, respectively. Kolhatkar et al. (29) found an enrichment factor for MTBE degradation in an undefined anaerobic microcosm of -9.16 ( 5.0 ‰, which is slightly lower but associated with a high uncertainty. Considering the upper limit of the uncertainty, it is similar to the factors found in our experiments. Nevertheless, our observation and previous results strongly indicate that carbon isotope analysis has the potential to demonstrate active anaerobic MTBE and TAME biodegradation, especially in subsurface environments where oxygen is limited. A small carbon isotope enrichment (
