Environ. Sci. Technol. 1993, 27,976-970
COMMUNI CAT IONS Anaerobic Biodegradation of Known and Potential Gasoline Oxygenates in the Terrestrial Subsurface Joseph M. Sufllta' and Melanie R. Mormlle Department of Botany and Microbiology, The University of Oklahoma, Norman, Oklahoma 73019
at the expense of the more labile hydrocarbons and consume oxygen (13). This process continues until anaerobic conditions develop. The continued metabolism of some gasoline components can then be linked with the consumption of alternate terminal electron acceptorswhen anaerobic conditions prevail (13). The microbial destruction of oxygenates could substantially diminish the health and environmental risks associated with the widespread use of these materials. While the microbial metabolism of some gasoline oxygenates has been established (141,we are unaware of any reports of the biodegradation of MTBE. In fact, aerobic experimentation attests to the recalcitrance of MTBE relative to other gasoline components (15-17). Since unanticipated environmental consequences are often associated with the use and release of persistent chemicals, prudence would dictate a search for substitute oxygenates that fulfill the desired role and biodegrade once released into the environment. We sought to (i) examine known and potential gasoline oxygenates for their susceptibility to anaerobic decay, (ii) identify structural features that tended to favor or retard the anaerobic destruction of these materials, and (iii) identify potential gasoline oxygenates which may be more environmentally acceptable than the additives currently employed.
The octane enhancer tetraethyllead has been largely phased out of automobile fuels due to environmental and health concerns, and recent US. legislation mandates the reformulation of gasoline to increase its oxygen content (1, 2 ) . Oxygenates such as methyl tert-butyl ether (MTBE),ethanol, methanol, and tert-butyl alcohol provide both octane enhancement and oxygen content to gasolines. The use of such additives is believed to be environmentally acceptable since oxygenated fuels reduce the impact of hydrocarbon combustion on the atmosphere ( 3 ) . MTBE is the most popular additive because it can be produced within refineries, it blends easily without phase separation in gasoline, and transfer of the reformulated mix can be accomplished through existing pipelines (1). From 1980 to 1986, the commercial production of MTBE increased a t a rate of about 40% per year ( 4 ) . In 1991, domestic production of MTBE was 4.35 billion kg, seventh in rank of organic compounds produced in the United States (5). MTBE can represent up to 15% of the volume of gasoline, and projections are that the production of this chemical will increase by 25 92 per year over the next several years (6). It is our contention that the environmental and health risks associated with the use of gasoline oxygenates has not been fully evaluated since the fate of gasoline has not been taken into account. Gasoline is often spilled or leaks from storage facilities and contaminates surrounding environments, including aquifers (7). In the United States alone, tens of millions of gallons of gasolinemay be released from storage tanks to the ground each year (8). The more water-soluble and more mobile components of gasoline have the highest potential of impacting humans and recipient environments. Oxygenates are far more watersoluble than gasoline hydrocarbons and have poor adsorption characteristics ( 4 , 9 ) . Incidents of groundwater contamination with MTBE are well known (10, 11, 4 ) . The toxicity characteristics associated with MTBE appear to be mild relative to other gasoline constituents, but a recent assessment of the health risks associated with unleaded gasoline identified the additive as a major component of concern for humans (7).MTBE is a known irritant that can cause odor problems in water supplies ( 4 ) and induce an anesthetic response in rats that were orally challanged (12). Microorganisms play a prominent role in governing the fate of fuel components spilled in aquifers. When such spills occur, aerobic heterotrophic microorganisms respire
A series of alcohols, ketones, esters, and ethers (Table I) were tested for the ability of the compounds to be completely biodegraded to methane in aquifer slurries. Sediment and groundwater were collected from a methanogenic portion of a shallow anoxic aquifer polluted by municipal landfill leachate (18). Slurries were prepared by placing 50 g of sediment and 75 mL of groundwater in sterile 160-mL serum bottles. The bottles were sealed with Teflon-lined stoppers and incubated in the dark a t room temperature. The biodegradability assay was essentially similar to several previous studies (e.g., ref 19). Each compound was added to the incubation mixtures to reach an initial substrate concentration of 50 ppm C (18, 19). An oxygenate concentration of 50 ppm C was used since initial experiments indicated that these levels did not inhibit methanogenesis and yet allowed for the easy detection of methane above background controls. Pressure increases resulting from biogas formation (CHI and COz) were monitored with an automated pressure transducer system (20). The acclimation period was estimated as the amount of time where no significant pressure differences
Environ. Sci. Technol., Vol. 27, No. 5, 1993
0 1993 American Chemical Society
Table I. Anaerobic Biodegradation of a Series of Oxygenate Chemicals in Aquifer Slurriesa rate
oxygenate methanol ethanol 2-propanol tert-butanol
research octane rating 133* 129* 118h 103h
acclimation period (days)
Alcohols 5 25-30 15-20 >252
methyl acetate ethyl acetate methyl propionate methyl butyrate methyl isobutyrate
Ketones 15-20 25 21-28 Esters llOd 0-15 117' 0-7 113' 5 110' 0-7 llOd 15
methyl tert-butyl ether methyl tert-amyl ether ethyl tert-butyl ether isopropyl ether diethyl ether propyl ether butyl ether butyl methyl ether butyl ethyl ether
Ethers 118' >249 1111 >182 118' 2182 110 >252 UNKNf >182 UNKN >182 UNKN >182 UNKN 84 UNKN >182
116 methyl ethyl ketone 115 acetone methyl isobutyl ketone 10Sd
methane recovery ( % theo(PPm Caday-]) retical)
7.4 f 0.7 17.9 f 0.6 7.6 k 0.3 0
103 91 112
9.4 f 1.9 7.3 k 0.6 0.9 f 0.9
90 89 46
16.6 f 5.4 13.7 k 2.4 7.3 f 3.6 5.3 f 1.3 4.1 rt 1.0
101 94 109 93 84
0 0 0 0 0 0
0 0 0
0 0.5 k 0.1 0
T h e rates reported reflect t h e average measured in triplicate incubations. b Reference 22. Reference 23. Reference 24. e Reference 25. f U N K N = unknown.
were measured between substrate-amended and unamended controls. At the end of the incubation period, the depletion of the parent substrate (or lack thereof) and the formation of methane over background controls were confirmedwith a Varian 3300 gas chromatograph equipped with a flame ionization detector. A 1.8 m X 0.32 cm 801 100 porapak Q column or a 0.2% Carbowax 1500 on Carbopack C column were used for headspace methane analyses and oxygenate determinations, respectively. Nitrogen (30 mL/min) served as the carrier gas for both analyses. Autoclaved controls were similarly assayed and were uniformly unable to exhibit methane formation or oxygenate disappearance. The rate of substrate depletion was determined in incubations receiving a subsequent addition of the oxygenate. The amount of methane formed in aquifer incubations was compared to that theoretically expected based on the Buswell equation (21). Results and Discussion Alcohols such as methanol, ethanol, and 2-propanolhave relatively high octane values and were anaerobically degraded in our assay as evidenced by substrate removal and the production of close to the theoretically expected amount of methane (Table I). Previous studies have documented the methanogenic fermentation of methanol, ethanol, and 2-propanol (ref 26 and references therein), and such findings are confirmed in our experiments. Methanol had the shortest acclimation time, while the other alcohols showed a variable period of 15-30 days prior to the mineralization of the parent substrate. However, no evidence for the anaerobic destruction of the structurally more complex tert-butyl alcohol could be obtained. tert-Butyl alcohol is a known gasoline additive, and the anaerobic destruction of this chemical in aquifers is slow and may be concentration dependent (14).
To our knowledge, methyl ethyl ketone is not a fuel additive but is a common pollutant in the terrestrial subsurface (27). Simple ketones like methyl ethyl ketone and acetone possess reasonable octane ratings, and these compounds were mineralized following a 2-4-week acclimation period to close to the theoretically expected amounts of methane (Table I). Methanogenie enrichment cultures that convert acetone to CH4 and COz have been reported (28). However, the highly branched methyl isobutyl ketone was much more persistent. Following a long acclimation period of 112 days, this compound slowly degraded relative to the other ketones, and less than half of the theoretically expected amount of methane was eventually recovered. Like the other potential oxygenates,the esters possessed comparable octane ratings, and all were degraded at similar rates after relatively short acclimation periods (Table I). As a group, the esters were the most easily degraded class of potential oxygenatesrelative to the other test chemicals. Presumably, the esters were hydrolytically cleaved prior to their ultimate mineralization under anaerobic conditions. In contrast, the ethers were relatively persistent molecules that generally resisted anaerobic destruction (Table I). After at least 182 days, no evidence for the anaerobic destruction of known gasoline oxygenates such as MTBE, ethyl tert-butyl ether, or tert-amyl ether could be obtained. Only butyl methyl ether, the straight chain analog of MTBE, was capable of being converted to 99% of the theoretically expected amount of methane. However, a 84-day acclimation period was measured prior to gas formation with this compound, and the rate of substrate degradation was relatively slow. Our findings illustrate the importance of chemical structure on the susceptibility of fuel additives to anaerobic decay. Oxygenates containing a tertiary or quaternary carbon atom proved much more recalcitrant than their unbranched or moderately branched chemical analogs. Other studies attest to the resistance of MTBE to aerobic microbialdestruction (15-1 7). The importance of chemical structure on the biodegradation of pollutant chemicals has been appreciated for decades (e.g.. refs 29-31). Consequently, we believe the initial results presented here will prove to be generalizing. We are currently examining the influence of other electron acceptors on the biodegradation of gasoline oxygenates using inocula obtained from a variety of pristine and hydrocarbon-polluted habitats. Future reports will detail the latter experiments. Because of the general recalcitrance associated with most of the gasoline additives currently employed, our findings suggest that such materials may not be as environmentally compatible as would be desirable. That is, efforts taken to ameliorate the problems of CO emissions and urban ozone levels have led to the introduction of relatively recalcitrant chemicals to the environment. Since it is unreasonable to predict every environmental consequence associated with the use of persistent chemicals, we suggest the investigation of alternate fuel additives that (i) function adequately as octane boosters and still reduce air emissions, (ii) possess the necessary handling and performance characteristics to be used efficiently by the petroleum infrastructure, (iii) possess negligible or minimal toxicity characteristics, and (iv) still biodegrade in the likely event they are released into the environment. In this respect, the biodegradable alcohols, ketones, esters, and butyl Envlron. Scl. Technol., Vol. 27, No. 5, 1993 077
methyl ether should be considered as candidate molecules. Considering that all but the latter chemical can be derived from renewable biomass feedstocks, their use may also help mitigate potential effects on global warming trends. However, such deliberations should be made relative to the net environmental and health concerns.
Acknowledgments This research was funded by the American Petroleum Institute. The opinions, findings, and conclusions expressed in the paper are those of the authors and not necessarily those of the American Petroleum Institute. We thank J. Robertson and D. P. Nagle, Jr., for technical assistance and constructive comments, respectively.
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Received for review November 18, 1992. Revised manuscript received February 1, 1993. Accepted February 2, 1993.