Anaerobic Degradation of Methyl tert-Butyl Ether (MTBE) and tert-Butyl

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Environ. Sci. Technol. 2001, 35, 1785-1790

Anaerobic Degradation of Methyl tert-Butyl Ether (MTBE) and tert-Butyl Alcohol (TBA) KEVIN T. FINNERAN AND DEREK R. LOVLEY* Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003

The potential for anaerobic degradation of methyl tertbutyl 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 petroleumcontaminated subsurface environments.

Introduction The gasoline additive methyl tert-butyl ether (MTBE) is a ubiquitous groundwater contaminant (1). The U.S. Geological Survey National Water Quality Assessment Program identified MTBE in 27% of urban wells tested (2). A more recent survey indicated that between 5 and 10% of all community drinking water wells nationwide have detectable MTBE contamination (3). MTBE is mobile in the subsurface, in part because it is highly water soluble and does not significantly adsorb to aquifer sediments (4). Although the potential for significant aerobic degradation of MTBE has been observed in incubations of aquatic sediments (5), as well as a variety of pure cultures (6-10), there appears to be little intrinsic potential for MTBE degradation in aquifer sediments (11). Thus, plumes of MTBE can be much more extensive than the plumes of other petroleum co-contaminants (11, 12). * Corresponding author phone: (413)545-9651; fax: (413)5451578; e-mail: [email protected]. 10.1021/es001596t CCC: $20.00 Published on Web 03/30/2001

 2001 American Chemical Society

The persistence of MTBE in petroleum-contaminated aquifers has led to a search for methods for MTBE bioremediation. Two aerobic MTBE-degrading microorganisms have been isolated which can degrade MTBE when introduced into aquifer sediments in laboratory incubations (9, 10). Furthermore, there was a loss of MTBE from an oxygensupplemented portion of a petroleum-contaminated aquifer that was inoculated with high concentrations of a MTBEdegrading culture (12). It has been suggested that aerobic microorganisms that co-metabolize MTBE when growing on propane or butane might be used for either in situ remediation or to treat MTBE contamination in above-ground bioreactors (6, 7). Studies on aromatic hydrocarbon contaminants have suggested that anaerobic strategies for the in situ bioremediation of petroleum-contaminated subsurface environments may be as preferable as aerobic approaches (13-23). The source zone of petroleum-contaminated aquifers is invariably anaerobic (19). Introducing sufficient oxygen into these source zones can be technically difficult and expensive (14). Anaerobic electron acceptors can be easily added to the subsurface, and they do not react with other compounds present (20). These past studies serve as a basis from which to develop a potential strategy for anaerobic MTBE biodegradation. Fe(III) is frequently the most abundant potential electron acceptor for the anaerobic oxidation of organic contaminants in polluted subsurface environments (19). Previous studies have demonstrated that the anaerobic degradation of aromatic hydrocarbons, including benzene, can be stimulated in the Fe(III)-containing sediments of petroleum-contaminated aquifers with the addition of humic substances (HS). HS are considered to promote the activity of Fe(III)-reducing microorganisms by alleviating the need for direct contact between Fe(III) oxides and Fe(III)-reducing microorganisms (24). HS contain quinone moieties which Fe(III)-reducing microorganisms can use as electron acceptors for the oxidation of organic compounds (25-27). The hydroquinone moieties produced as the result of electron transfer to HS can abiotically reduce Fe(III) oxides, producing Fe(II) and regenerating oxidized HS that can again serve as an electron acceptor in the metabolism of Fe(III)-reducing microorganisms (27). Thus, even at very low concentrations, HS or other extracellular quinones can significantly accelerate the rate of Fe(III) oxide reduction in aquifer sediments via this electron shuttling mechanism (28). There appears to have been little investigation of the potential for anaerobic MTBE degradation. Thermodynamic calculations suggest that anaerobic oxidation of MTBE is possible with each of the most prevalent electron acceptors known to support anaerobic respiration in sedimentary environments (Table 1). A recent EPA study indicates that MTBE may be removed from groundwater under methanogenic conditions (29). However, the few studies that have been carried out under controlled anaerobic conditions suggest that MTBE is only slowly degraded, if at all, in the absence of oxygen. For example, in anaerobic MTBEcontaminated aquifer sediments, ca. 3% of the added [14C]MTBE was converted to 14CO2 in seven months (11). A similar study with aquatic sediments found no anaerobic oxidation of [14C]-MTBE (5). When freshwater sediments were amended with MTBE and incubated under anaerobic conditions, MTBE persisted in most of the sediments evaluated, with the exception of one bottle of sediment in which there was evidence for the conversion of some of the MTBE to tertbutyl alcohol (TBA), which was not further degraded (30, VOL. 35, NO. 9, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Calculated Free Energy Yields of Complete MTBE Oxidation Under a Variety of Terminal Electron Accepting Processesa acceptor

reactants

products

∆G°′ in kJ/reaction

nitrate nitrate iron sulfate methanogenesis

C5H12O + 6NO3- + H+ C5H12O + 3.75NO3- + 2.5H+ + 2.75H2O C5H12O + 30Fe(OH)3 + 55H+ C5H12O + 3.75SO42- + 2.5H+ C5H12O + 2.75H2O

5HCO3- + 3N2 + 4H2O 5HCO3- + 3.75NH4+ 5HCO3- + 30Fe2+ + 76H2O 5HCO3- + 3.75H2S +H2O 3.75CH4 + 1.25HCO3- + 1.25H+

-3054.8 -1951.2 -347.4 -275.2 -238.7

a Free energy calculated from the standard free energies of formation of the products and reactants (44, 45) and by assuming standard conditions except for pH 7.

31). In another study, MTBE persisted in most water-saturated soils, but was slowly degraded in one soil, amended with starch and nutrients, that was assumed to be methanogenic (32), but it was not clear that the loss of MTBE that was observed could be attributed to anaerobic processes because the soils were not incubated under strict anaerobic conditions. In order to learn more about the potential for anaerobic degradation of MTBE, MTBE degradation was investigated in anaerobic sediments from a MTBE-contaminated aquifer in which rates of MTBE degradation were previously reported to be slow (11), as well as in aquatic sediments which previous studies have demonstrated can anaerobically degrade relatively recalcitrant hydrocarbon constituents such as benzene (33, 34).

Materials and Methods Sediment Incubations. Aquifer sediments were collected from a previously described (11) petroleum-contaminated aquifer in Beaufort, SC. This is a military installation on Port Royal Island in coastal South Carolina which formerly had a leaking underground storage tank (with reformulated gasoline) that has since been excavated. The site has been well characterized by the U.S. Geological Survey (USGS) as part of the Toxic Substances Hydrology Program. The source area is still highly contaminated with aromatic hydrocarbons and MTBE and is anaerobic. Sediments from the Fe(III)reducing zone were collected with a hand auger. Freshwater aquatic sediments were collected from the Potomac River at the previously described location (35) by using an Eckman dredge. These sediments had previously been found to have the potential for the anaerobic degradation of other hydrocarbon contaminants, including benzene (33, 34) All sediments were placed in sealed, anaerobic containers and shipped overnight to be processed. The sediments were homogenized in a N2-filled glovebag, and dispensed into the appropriate serum bottles or anaerobic pressure tubes, as described below. The vessels were sealed with thick butyl rubber stoppers. Upon removal from the glovebag the headspace of all the vessels was flushed with N2/CO2 (93:7) that had been passed through heated, reduced copper filings to remove any traces of oxygen. All sediments were incubated at 20 °C in the dark. The terminal electron accepting process (TEAP) in the sediment was determined by monitoring production of 14CO and 14CH from [2-14C]-acetate as previously described 2 4 (14). Approximately 12 g of sediment was dispensed into two sets of triplicate anaerobic pressure tubes. One series of sediment incubations was amended with Na2MoO4 (1 mM) in order to inhibit sulfate reduction. For studies on degradation of MTBE and TBA, sediments (30 g) were incubated in 60-mL serum bottles, as previously described (22). Killed controls were heat-sterilized at 121 °C for one hour per day, for three consecutive days. All amendments were added anaerobically from anaerobic, sterile stock solutions. No more than 1 mL of liquid was added to any of the sediments. Headspace volume in these 1786

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bottles was approximately 28 mL. Poorly crystalline Fe(III) oxide was synthesized as previously described (35) and added to provide 40-100 mmol/kg of sediment. Fe(III) chelated with nitrilotriacetic acid (NTA) and Fe(III) chelated with ethylene diamine tetraacetic acid (EDTA) were added to a final concentration of 10 mM. Anthraquinone-2,6-disulfonate (AQDS) and humic acids (Aldrich, St. Louis, MO) were added to final concentrations of ca. 100 µM and 0.15 g/kg of sediment, respectively. MTBE and TBA were added to provide a final concentration of ca. 50 mg/L and the concentrations of MTBE and TBA were monitored over time. Additional studies were conducted with the aquatic sediments in which [14C]-MTBE or [14C]-TBA was added to monitor the degradation products of MTBE and TBA. [U-14C]-MTBE (5.0 mCi/mmol; New England Nuclear, Boston, MA) or [U-14C]-TBA (5.0 mCi/mmol, Moravek Biochemicals, Inc., Brea, CA) were added to provide 0.5 µCi. The radiochemical purity of the tracers was 99%. Production of 14CO2 and 14CH4 were monitored over time. Analytical Techniques. 14CO2 and 14CH4 were measured with a gas chromatograph coupled to a gas proportional radiochromatography detector as previously described (14). In order to monitor degradation of MTBE and TBA, headspace samples were collected with a syringe and needle and were separated by gas chromatography on a HP-1 fuel oxygenates capillary column (Hewlett Packard, Wilmington, DE; 60 m length × 0.25 mm i.d.) connected to a flame ionization detector. Concentrations of HCl-extractable Fe(III) and Fe(II) in the sediments were determined with ferrozine as previously described (36).

Results The potential for anaerobic MTBE degradation was first evaluated in the aquifer sediments. No [14C]-MTBE was available for these studies so MTBE degradation was monitored by measuring the loss of MTBE over time. There was no loss of MTBE over 275 days in sediments that received no amendments or in sediments amended with only with poorly crystalline Fe(III) oxide, NTA, Fe(III)-NTA, or Fe(III)-EDTA (data not shown). However, in one of the three bottles amended with HS and Fe(III), the MTBE was depleted below the detection limit (1 mg/L) during this time. When MTBE (ca. 50 mg/L) was added back to the sediments, it was consumed without a lag (Figure 1). MTBE was added back with repeated consumption of the MTBE until after 180 days when MTBE consumption stopped. By this time the sediments were very dark compared to the reddish-brown color when the Fe(III) was first added, suggesting that most of the Fe(III) had been reduced. When more Fe(III) oxide and MTBE were added to the sediment degradation eventually resumed (Figure 1). In the other two replicates of the HS-amended sediments, the concentration of MTBE was ca. 17 mg/L after the first 275 days of incubation. Additional MTBE was added to these sediments and the loss of MTBE was monitored over time. MTBE was degraded without a lag in both of these sediment samples (Figure 2), but at a slower rate than the other

FIGURE 1. Anaerobic loss of MTBE in one of three replicate samples of an aquifer sediment enrichment after being adapted for 275 days in the presence of MTBE and amended with Fe(III) oxide and humic substances. The initial concentration of MTBE added was ca. 50 mg/L. Short arrows indicate re-addition of MTBE. The long arrow indicates re-addition of MTBE and Fe(III) oxide.

FIGURE 2. Anaerobic loss of MTBE in the remaining two replicates of the aquifer sediment enrichments that had been adapted to MTBE for 275 days and comparable killed controls. The initial concentration of MTBE added was ca. 50 mg/L. Killed control data are the means of triplicate analyses. sediment sample of the triplicate series (Figure 1). Sediments amended with the humics analog, AQDS, and Fe(III) degraded approximately 60% of the MTBE, but none of the replicates went below detectable levels (data not shown). The potential for anaerobic degradation of MTBE, as well as TBA, was evaluated in freshwater aquatic sediments from the Potomac River. When freshly collected sediments were amended with [2-14C]-acetate, both 14CH4 and 14CO2 were produced over time. When molybdate was added to inhibit sulfate reduction in the sediments, the rate of 14CO2 production was diminished with an increase in the production of 14CH , but there was still significant 14CO production. The 4 2 sediments contained ca. 10.8 µmol of HCl-extractable Fe-

FIGURE 3. Anaerobic loss of TBA in sediment from the Potomac River. The initial concentration of TBA added was ca. 50 mg/L. Arrows indicate re-addition of TBA. Data are the means of triplicate analyses.

FIGURE 4. Anaerobic production of 14CO2 and 14CH4 from [14C]-TBA in sediment from the Potomac River. Data are the means of triplicate analyses. Bars designate one standard deviation. (III), and ca. 85 µmol of Fe(II) per gram (dry weight) of sediment. These results, coupled with there being no detectable nitrate, indicated that methane production, sulfate reduction, and Fe(III) reduction were taking place simultaneously in the sediments (19). There was an immediate loss of the TBA added to Potomac River sediments that had received no other amendments (Figure 3). When TBA was added back to the sediments it continued to be degraded over time. When [14C]-TBA was added to the sediments it was converted to 14CO2 and 14CH4 (Figure 4). The concentration of MTBE in MTBE-amended Potomac River sediments appeared to decline slowly over a 300 day incubation period when no amendments were made or when HS and Fe(III) or AQDS and Fe(III) were added (data not shown). After this preincubation in the presence of MTBE, VOL. 35, NO. 9, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. Anaerobic production of 14CO2 and 14CH4 from [14C]-MTBE in sediment from the Potomac River. Sediments were unamended (A), amended with poorly crystalline Fe(III) oxide and humic substances (B), or amended with poorly crystalline Fe(III) oxide and anthraquinone2,6-disulfonate (C). Open symbols represent 14CO2. Closed symbols represent 14CH4. The data from each of the triplicate incubations for each treatment are shown.

Discussion

FIGURE 6. Anaerobic production of 14CO2 from [14C]-MTBE in sediment from the Potomac River. Sediments were amended with poorly crystalline Fe(III) oxide. The data from each of the triplicate incubations are shown. the potential for the sediments to metabolize [14C]-MTBE was evaluated in order to provide greater sensitivity in the ability to detect MTBE degradation. The concentration of MTBE in the sediments at the time of [14C]-MTBE addition was ca. 6 mg/L. After a short lag period, one of the triplicate unamended sediment samples converted ca. 30% of the added [14C]-MTBE to 14CO2 within 130 days, whereas the other two unamended sediment samples converted approximately 19-23% of the added [14C]-MTBE to 14CO2 in the same period of time (Figure 5). Small amounts of 14CH4 were also produced in all of these sediments. In two of the triplicate sediments treated either with HS or AQDS and Fe(III), [14C]-MTBE was immediately oxidized to 14CO2 (Figure 5). No 14CH4 was produced in these sediments. All Fe(III) oxide-only amended bottles converted ca. 23% of the [14C]-MTBE to 14CO2 at comparable rates (Figure 6). None of the other amendments which included Fe(III) ) NTA and Fe(III) ) EDTA converted more than 3% of the added [14C]MTBE to 14CO2 (data not shown). 1788

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The results demonstrate that MTBE and TBA can be degraded in anaerobic sediments. After an adaptation period, MTBE was actively consumed in aquifer sediments in which the activity of Fe(III)-reducing microorganisms was stimulated with the addition of HS. The aquatic sediments had an intrinsic capacity to degrade MTBE without the need for amendments. The initial concentration of MTBE that was used in this study (50 mg/L) is comparable to concentrations likely to be found in the anaerobic source zones of petroleumcontaminated aquifers (11). These results suggest that it may be possible to develop anaerobic strategies for MTBE bioremediation within the source zone of petroleum-contaminated aquifers. Anaerobic Degradation of MTBE. Previous reports of MTBE degradation in aquifer sediment (11, 30) or soils (32), or aquatic sediments (5, 31) have suggested that MTBE is not readily degraded under in situ, anaerobic conditions. In a similar manner, the aquifer sediments evaluated in this study also had little intrinsic potential to degrade MTBE. MTBE was only rapidly degraded in the aquifer sediments when HS were added and when Fe(III) concentrations were maintained at high levels. Based on previous studies of the oxidation of organic matter coupled to Fe(III) oxide reduction in the presence of HS (25, 27, 28, 36), the degradation of MTBE in the presence of added HS and Fe(III) is assumed to result from Fe(III)reducing microorganisms oxidizing MTBE with the transfer of electrons to HS. Once reduced, the HS can abiotically transfer electrons to the Fe(III). This regenerates the HS to the oxidized form. Thus, HS promote electron shuttling to Fe(III) in a catalytic manner and small amounts of HS can continue to promote Fe(III) reduction through multiple reduction and oxidation cycles. Studies with laboratory cultures have demonstrated the potential for HS to serve as an electron shuttle to Fe(III) oxides in this manner (25, 27, 37). It has also previously been demonstrated that the addition of HS or the HS analog, AQDS, can stimulate the reduction of Fe(III) in sediments (28, 38), accompanied by the growth of Fe(III)-reducing microorganisms (39). This strategy has been used to promote degradation of other recalcitrant compounds (21, 36, 40). The studies with the aquatic sediments provided preliminary evidence that anaerobic MTBE degradation coupled to the reduction of a variety of electron acceptors may be

possible. In the absence of other amendments, [14C]-MTBE was converted to 14CO2 and 14CH4. The addition of Fe(III) with or without HS or AQDS inhibited the production of 14CH , presumably the result of Fe(III)-reducing microorgan4 isms outcompeting methanogenic microorganisms for electron donors (41). Although the highest rates of MTBE oxidation were observed in some of the sediment samples amended with HS or AQDS, the high variability in the rate of MTBE oxidation within the different treatments made it impossible to determine whether the addition of HS significantly stimulated MTBE oxidation. The least variability was seen in Fe(III)-only amended bottles, but more research is needed to determine if Fe(III) has a stimulatory effect on MTBE degradation. High variability in the rate of MTBE degradation in replicate samples of sediments from within the same site has been observed previously (30, 32). A similar phenomenon was reported in studies in which benzene degradation was stimulated in aquifer sediments with Fe(III) chelators (23). The reasons for this variability have yet to be investigated in detail. However, a likely explanation is that the populations involved in the degradation of the contaminants are initially very small. In this case, heterogeneities in the sediments might result in much lower numbers of the appropriate organisms in one sample rather than another. Thus, the time necessary to build up a sufficiently large population of organisms that can degrade the contaminants may vary between sediments, resulting in a significant difference in the lag period prior to detectable degradation of the contaminant. Anaerobic Degradation of TBA. An additional concern resulting from contamination of groundwater with gasoline amended with oxygenates is whether TBA will be degraded. TBA may be a product of MTBE degradation (6, 30), and/or an original component of the gasoline (42). Several studies have concluded that TBA is only slowly degraded or persists under anaerobic conditions (5, 35, 30, 32). To our knowledge the results presented here provide the first indication that TBA can be rapidly degraded under strict anaerobic conditions. The rates of TBA degradation reported here under anaerobic conditions are comparable to those previously reported for aerobic degradation in TBA-adapted sediments (5). [14C]-TBA was converted to a combination of 14CH4 and 14CO . If TBA was converted to methane and carbon dioxide 2 solely according to the reaction

(CH3)3COH + H2O f 3CH4 + CO2 then it would be expected that 14CH4 and 14CO2 would be produced in a ratio of 3:1. However, the results with [2-14C]acetate suggested that methanogenesis was not the only terminal electron-accepting process taking place in these sediments. The ratio of 14CH4 to 14CO2 from [2-14C]-acetate was 1:2, whereas this ratio is typically 4:1 in sediments in which methane production is the predominant terminal electron-accepting process. The actual ratio of 14CO2 to 14CH was 3:1 for [14C]-TBA oxidation. Therefore, it appears 4 that, as with acetate, TBA was being converted to methane and carbon dioxide, by a variety of terminal electronaccepting processes. Implications for MTBE Remediation. There is increasing reliance on natural degradation processes to remove many petroleum contaminants from impacted subsurface environments (43). The results suggest that in some instances, anaerobic microbial communities may eventually develop which can degrade MTBE, but there are many sites at which this has not happened in a timely manner that would prevent significant spread of MTBE. Thus, methods for stimulating MTBE degradation in sediments are needed. This study

suggests that, in some instances, it may be possible to stimulate the rate of anaerobic MTBE degradation. However, a much better understanding of the mechanisms for anaerobic MTBE degradation will be required before it will be possible to design reliable anaerobic bioremediation strategies for MTBE.

Acknowledgments We thank R.T. Anderson for his help in setting up the sediment incubations and radiotracer studies. We also thank F.H. Chapelle and J.E. Landmeyer of the USGS, Columbia, SC, for the Laurel Bay sediment samples and their technical assistance. This research was supported by the American Petroleum Institute.

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Received for review August 16, 2000. Revised manuscript received February 5, 2001. Accepted February 5, 2001. ES001596T