Anaerobic Oxidation of Crude Oil Hydrocarbons by

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Environ. Sci. Technol. 2003, 37, 5213-5218

Anaerobic Oxidation of Crude Oil Hydrocarbons by the Resident Microorganisms of a Contaminated Anoxic Aquifer G . T O D D T O W N S E N D , †,‡ ROGER C. PRINCE,§ AND J O S E P H M . S U F L I T A * ,‡ Department of Botany and Microbiology and Institute for Energy and the Environment, University of Oklahoma, Norman, Oklahoma 73019, and ExxonMobil Research and Engineering Co., Annandale, New Jersey 08801

The biodegradation of two crude oils by microorganisms from an anoxic aquifer previously contaminated by natural gas condensate was examined under methanogenic and sulfate-reducing conditions. Artificially weathered Alaska North Slope crude oil greatly stimulated both methanogenesis and sulfate reduction. Gas chromatographic analysis revealed the entire n-alkane fraction of this oil (C13-C34) was consumed under both conditions. Naphthalene, 2-methylnaphthalene, and 2-ethylnaphthalene were also biodegraded but only in the presence of sulfate. Alba crude oil, which is naturally depleted in n-alkanes, resulted in a relatively modest stimulation of methanogenesis and sulfate reduction. Polycyclic aromatic hydrocarbon biodegradation was similar to that found for the Alaska North Slope crude oil, but a broader range of compounds was metabolized, including 2,6-dimethylnaphthalene and 2,7-dimethylnaphthalene in the presence of sulfate. These results indicate that n-alkanes are relatively labile, and their biodegradation in terrestrial environments is not necessarily limited by electron acceptor availability. Polycyclic aromatic hydrocarbons are relatively more recalcitrant, and the biodegradation of these substrates appeared to be sulfate-dependent and homologue-specific. This information should be useful for assessing the limits of in situ crude oil biodegradation in terrestrial environments and for making decisions regarding risk-based corrective actions.

Introduction The importance of crude oil to society can hardly be overstated. As the primary feedstock for the world’s liquid fuel and petrochemical industries, its extraction from the subsurface is the initial step in producing both the energy and the materials that industrialized societies demand. Currently, U.S. domestic crude oil production is over 950 million L per day, while worldwide crude oil production is more than 10 times that amount (1). The extraction and transport of crude oil has resulted in some of the most widely known incidents of environmental * Corresponding author phone: (405)325-5761; fax: (405)325-7541; e-mail: [email protected]. † Current address: AstraZeneca R&D Lund, S-221 87 Lund, Sweden. ‡ University of Oklahoma. § ExxonMobil Research and Engineering Co. 10.1021/es0264495 CCC: $25.00 Published on Web 10/22/2003

 2003 American Chemical Society

contamination. The most infamous are marine accidents involving supertankers and offshore wells; however, terrestrial drilling and pipeline mishaps have created significant spills as well. Between 1994 and 2000, there were, on average, 79 crude oil spills reported in the United States each year due to pipeline accidents with an average yearly volume of over 11 million L (2). There are also an unknown number of smallscale releases related to the day-to-day exploration, extraction, and transportation of crude oil. Worldwide, the largest oil spill of any sort was the deliberate release of billions of liters onto the Kuwaiti desert (3). Understanding the processes that control the fate of spilled crude oil is important for determining the associated environmental risks and for designing appropriate remediation strategies. Crude oils are complex mixtures containing hundreds of individual compounds, the composition of which varies widely among oils, as do the resultant physical and toxicological characteristics. The hydrocarbon fraction includes alkanes, cycloalkanes, aromatic compounds, and naphthenaromatic hydrocarbons and represents 50-98% of the oil mass. Once released into the environment, the constituent hydrocarbons exhibit widely different susceptibilities to physical fate processes (i.e. volatilization, sorption, dissolution, photooxidation) and biodegradation. Aerobic hydrocarbon metabolism has been studied for many years, and the metabolic diversity and patterns of hydrocarbon biodegradation exhibited by aerobic microorganisms are wellknown (4). However, there is a relative lack of knowledge regarding the full potential for anaerobic hydrocarbon decay within the environment. Anaerobic hydrocarbon biodegradation was suggested over 70 years ago (5 and references therein), yet for decades the ability of microorganisms to biodegrade petroleum hydrocarbons in the absence of oxygen was not widely recognized and often discounted (6-8). Since the clear demonstration of benzene and toluene biodegradation in the absence of oxygen (9), much insight into anaerobic hydrocarbon biodegradation has been gained, and the phenomenon is now more widely accepted. This metabolism has been found to be surprisingly diverse with regards to the microorganisms that are involved, the hydrocarbons they consume, and the electron acceptors they utilize (see refs 10 and 11 for recent reviews). Due to initial laboratory successes and continued toxicological concerns, most studies have focused on the fate of aromatic compounds found in petroleum-based fuels, and consequently our understanding of the metabolism of benzene, toluene, ethylbenzene, and xylene isomers by anaerobic microorganisms is the most advanced (12). More recently, several studies have demonstrated that n-alkanes (13-16) and alicyclic compounds (17), saturated compounds that were previously held to be quite recalcitrant in the absence of oxygen, are anaerobically biodegraded. Still, much is not known about the anaerobic biodegradability of the enormous number of constituent hydrocarbons found in crude oil and petroleum fuels. Anaerobic hydrocarbon biodegradation studies have traditionally examined the fate of individual hydrocarbons and taken advantage of the resultant hydrocarbon-degrading enrichment cultures and isolates. Such studies are particularly well suited for examining the underlying physiology and metabolism of anaerobic hydrocarbon metabolism, but in many respects these experiments are ecologically unrealistic. The selective pressure exerted by a single substrate at high concentration bears little relation to the low concentration, multicomponent contamination associated with petroleum spills. Studies in which the actual contaminant is provided VOL. 37, NO. 22, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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as a substrate to unenriched microbial communities taken directly from the environment would provide a more realistic understanding of the behavior of these complex hydrocarbon mixtures. In this study, microorganisms from an anoxic aquifer that was chronically contaminated by natural gas condensate were challenged with two different crude oils. Organisms from this site exhibited broad anaerobic biodegradative activity toward the low molecular weight hydrocarbons that comprise natural gas condensate (18-20). The oils chosen for the study included an artificially weathered Alaska North Slope (ANS) crude oil, which is depleted in low molecular weight alkanes and monoaromatic compounds but retains its complement of mid-to-heavy range hydrocarbons (C15-C34), and an Alba crude oil, which is naturally depleted in n-alkanes of all sizes but retains most of its polycyclic aromatic hydrocarbons. By examining the change in crude oil composition over time, we documented the anaerobic biodegradation of the constituent hydrocarbons, determined the substrate preference of the indigenous microorganisms when presented with complex hydrocarbon mixtures, and examined the effect of electron acceptor availability on the biodegradation of individual hydrocarbon components. We found that aquifer sediments harbor microorganisms that are able to biodegrade a wide range of high molecular weight alkanes under both sulfate-reducing and methanogenic conditions. Selected naphthalenes were also biodegraded exclusively under the former conditions. The results also demonstrate the potential of microorganisms in anaerobic terrestrial environments to biodegrade hydrocarbons beyond those to which they are regularly exposed.

Methods Aquifer Samples. Samples were retrieved from a shallow aquifer that overlies a natural gas field in the Denver Basin near Ft. Lupton, CO (18, 21). This site has been impacted by natural gas condensate (96wt %/wt C5-C15 compounds) due to a leaking sump used to store liquids produced during natural gas recovery. Chronically contaminated sediments from within the saturated zone, located approximately 10 m downstream of this sump and 1.5 m below the surface, were obtained nonaseptically by hand boring. Sterile glass jars were filled to capacity with sediments and groundwater from the site, sealed, and stored on ice until delivery to the laboratory. Uncontaminated groundwater was similarly collected from a sampling well located approximately 10 m upgradient of the contamination and handled identically. Crude Oils. The ANS crude oil (initial API gravity ) 29°) was artificially weathered by distillation under reduced pressure as previously described (22, 23), resulting in an oil that is essentially depleted of compounds with less than 15 carbon atoms (Figure 1). It is thus similar to the residue that remains after the lighter components evaporate upon exposure in the environment (24). Alba crude oil originates from the UK sector of the North Sea, and its API gravity is 19.4° (Figure 1). It is characterized by a lack of n-alkanes and was used without any pretreatment. Aquifer Incubations. Aquifer slurries were constructed within an anaerobic glovebox. Sediment was initially sieved (1-cm2 openings) within the glovebox to remove large rocks and to thoroughly mix the samples. Groundwater was placed within the glovebox and amended with Na2S and cysteineHCl as reductants (both at 0.005%) and resazurin (0.0001%) as a redox indicator. Sediment (50 ( 0.5 g) and reduced groundwater (75 ( 0.5 mL) were added to 160 mL sterile serum bottles to construct aquifer incubations. The incubations were sealed with Teflon-lined rubber stoppers, crimpsealed, and removed from the glovebox. The headspaces of the bottles were then exchanged to N2/CO2 (80/20) at 15 kPa overpressure. Incubations used for sterile controls 5214

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FIGURE 1. Gas chromatograms of artificially weathered Alaska North Slope (ANS) and Alba crude oils extracted from sterile incubations. Several n-alkanes, pristane, and phytane are labeled. Contributions from the endogenous condensate in the inoculum are also indicated. The most prominent peak from this source is p-xylene. Note that pristane and phytane are present in both oils. were autoclaved for 20 min on 3 consecutive days. For methanogenic incubations, nonsterile incubations were kept for 1 month to allow endogenous sulfate levels to fall below 100 µM before the addition of the oils. Oil-amended incubations received 1.00 ((0.05) g of either the ANS or Alba crude oil. In addition, sulfate-reducing incubations and sulfate-amended sterile controls received an amendment of Na2SO4 from a sterile, anoxic stock solution to an initial concentration of approximately 20 mM. Autoclaved and oil-unamended incubations served as sterile and endogenous controls, respectively. All treatments were in triplicate, and incubations were kept at room temperature, in the dark, inverted, and without agitation. Quantitation of Microbial Respiration. The terminal electron-accepting processes in incubations were monitored over time. Methane production was monitored by gas chromatography (25), and sulfate depletion was determined by ion chromatography (26). When required, aqueous samples were removed by syringe, centrifuged to remove particulates, diluted 1:10 in Nanopure water, and analyzed immediately. All measurements were from triplicate incubations; data are presented as the mean ( the standard deviation. Hydrocarbon Analysis. At various times, entire incubation bottles were frozen and stored prior to hydrocarbon analysis. For oil analysis, bottles were defrosted, and the contents were extracted with two volumes of methylene chloride. The solvent was then filtered, dried over anhydrous sodium sulfate, and allowed to evaporate until the oil concentration was approximately 25 mg/mL. Care was taken to ensure that the samples did not evaporate to dryness. Gas chromatography/mass spectroscopy followed published procedures (27). Mass calibration and tuning of the instrument was accomplished with perfluorotributylamine followed USEPA method 8270C. Quantitation is based on the ratio of the individual hydrocarbon with hopane, a nonbiodegradable internal standard naturally present in crude oils (23). Hydrocarbon loss is reported as percentage lost relative to the amount of individual hydrocarbon present in the respective oils.

Results Terminal Electron-Accepting Processes. We monitored methanogenesis and sulfate reduction as quantitative indicators of anaerobic microbial metabolism in incubations amended with two different crude oils. The time course of

FIGURE 3. n-Alkane biodegradation in artificially weathered Alaska North Slope crude oil. (A) Chromatograms of residual oil after 13 months of incubation. (B) Time course of biodegradation of two n-alkanes. The incubations initially contained 3.62 mg of heptadecane and 1.13 mg of triacontane.

FIGURE 2. Cumulative methanogenesis and sulfate reduction in incubations amended with artificially weathered Alaska North Slope Crude (b) and Alba crude oil (2) compared to autoclaved (O) and unamended (9) control incubations. cumulative methanogenesis and sulfate reduction for these incubations is presented in Figure 2. In oil-unamended methanogenic and sulfate-reducing incubations, endogenous electron donors resulted in 314 (( 30) µmol of methane produced and 621 ((58) µmol of sulfate reduced, respectively, over the 475 d incubation. Autoclaved incubations did not produce methane, and only 203 ((35) µmol of sulfate loss was measured. Artificially weathered ANS and Alba crude oils stimulated both methanogenesis and sulfate reduction in oil-amended, nonsterile incubations, indicating that the indigenous anaerobic microflora could use constituents of these complex oils as electron donors. Amendments of the ANS crude oil resulted in a much greater stimulation of both methanogenesis and sulfate reduction than did amendments of the Alba crude oil (Figure 2). In both cases, crude oil-amended incubations held under sulfate-reducing conditions produced less than 70 µmol of methane. In ANS oil-amended incubations held under methanogenic conditions, there was a 60-80 day lag period, during which the rate of methanogenesis mirrored that observed in the oil-unamended samples. Following the lag, a substantial increase in the rate of methanogenesis was observed, and eventually 2610 ((503) µmol of methane was produced above the oil-unamended controls. A somewhat shorter lag period was observed under sulfate-reducing conditions, after which the consumption of this electron acceptor increased, resulting in a total of 2624 ((311) µmol of sulfate reduced above unamended incubations. Methanogenesis and sulfate reduction plateaued in ANS oil-amended incubations by the termination of the experiment (Figure 2). Alba crude oil resulted in a relatively modest stimulation and extended lag periods for both methanogenesis and sulfate-reduction. Nevertheless, by the termination of the experiment, incubations amended with Alba crude oil produced 595 ((98) µmol of methane and reduced 1128 ((46)

FIGURE 4. Gas chromatograms of oils extracted from Alba crude oil-amended incubations after 14 months. µmol of sulfate, both approximately double the amount measured in oil-unamended incubations (Figure 2). Hydrocarbon Biodegradation. We measured the loss of individual petroleum hydrocarbons from the two crude oils in incubations sacrificed over the course of the experiment. Neither of the oils underwent a significant change in sterile control incubations over the course of the experiment. The n-alkane fraction of the ANS crude oil, however, was completely removed under both sulfate-reducing and methanogenic conditions after 13 months of incubation (Figure 3A). The branched isoprenoid alkanes, pristane and phytane, were not altered within our limit of detection. The time course for degradation of two representative alkanes, heptadecane (C17H36) and triacontane (C30H62), is shown in Figure 3B. As illustrated by these examples, the entire range of n-alkanes was consumed simultaneously with only slightly reduced rates for the longer chain homologues. The n-alkanes were biodegraded approximately twice as fast in the presence of sulfate as they were in its absence. Nevertheless, by the end of the incubation period, all n-alkanes originating from the ANS crude oil were completely consumed under both incubation conditions. The chromatograms resulting from the extraction and analysis of the Alba oil-amended incubations are shown in Figure 4. Apart from loss of some small molecules from the condensate fraction, particularly p-xylene (see Figure 1), there are no differences between these chromatograms. Polycyclic aromatic hydrocarbons on the USEPA priority pollutant list (28) were similarly examined. The aquifer microorganisms in incubations amended with either oil and VOL. 37, NO. 22, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. Selective biodegradation of naphthalene, 2-methylnaphthalene, 2-ethylnaphthalene, and the coeluting 2,6- and 2,7dimethylnaphthalenes in Alba crude oil-amended incubations held under sulfate reducing conditions after 14 months. The m/z 128, 142, and 156 chromatograms are overlain.

FIGURE 6. Time course of naphthalene homologue biodegradation in Alba crude oil. held under sulfate-reducing conditions completely consumed naphthalene, 2-methylnaphthalene, and 2-ethylnaphthalene in order of preference (Figure 5). In incubations amended with Alba crude oil, the coeluting 2,6-and 2,7 dimethylnaphthalenes were also biodegraded, albeit somewhat more slowly than the other degradable naphthalene homologues (Figure 6). The 1-methylnaphthalene and 1-ethylnaphthalene homologues as well as the other dimethyl-substituted naphthalenes within these incubations resisted decomposition (Figure 6). No biodegradation of naphthalene or the alkyl homologues was seen in incubations held under methanogenic conditions.

Discussion Despite the relatively recent acceptance of anaerobic hydrocarbon metabolism as an important environmental fate process, relatively little is known about the extent of this process. We examined the fate of two crude oils in incubations containing anoxic aquifer sediments that were previously exposed to natural gas condensate. To obtain results that might best reflect in situ biodegradative activities, no effort was made to optimize culture conditions by the addition of media components or by further enrichment of the indigenous microbiota. Nevertheless, the resident microflora exhibited a broad range of biodegradative activities toward crude oil hydrocarbons under both sulfate-reducing and methanogenic conditions. This was true despite the lack of any previous chronic exposure of the microflora to high molecular weight hydrocarbons. Quantification of hydrocarbon loss was measured relative to an internal hopane 5216

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biomarker (23). Recently, Wang et al. (29) reported the apparent biodegradation of hopanes in oil remaining from the 1974 Metula spill, and Bost et al. (30) reported degradation of hopanes and norhopanes by an aerobic microbial consortium enriched from a creosote-contaminated site. Although we have no evidence that biodegradation of 17R(H)21β(H)hopane occurred in any of our samples, if it did, our estimates of the extent of biodegradation of other compounds would be underestimates. Oil-unamended incubations displayed background rates of sulfate reduction and methanogenesis, likely driven by the natural gas condensate hydrocarbons present as endogenous contaminants in the samples. A dramatic increase in both terminal electron accepting processes in ANS oilamended incubations reflected robust biodegradation of the crude oil hydrocarbons. The artificial weathering of the ANS crude oil prior to its use as substrate removed the low molecular weight volatile hydrocarbons to which the indigenous microbial populations were previously exposed. Nevertheless, hydrocarbon analysis revealed that the resident microorganisms were able to metabolize a wide range of medium to long chain alkanes (up to C34) under both conditions as well as selected naphthalenes when sulfate was available. The Alba crude oil lacks the readily degradable n-alkane fraction, which likely accounts for the relatively modest stimulation in methanogenesis and sulfate reduction observed in incubations amended with this oil, and other components of Alba crude oil must have been metabolized to account for the stimulation in methanogenesis and sulfate reduction that was noted. The stimulation of the latter process can be at least partially attributed to the anaerobic biodegradation of the naphthalene fraction, and Alba oil-amended incubations did demonstrate a broader range of naphthalene biodegradation than was seen in incubations containing the ANS crude oil. While naphthalene, 2-methylnaphthalene, and 2-ethylnaphthalene were biodegraded in both crude oils, 2,6and 2,7-dimethylnaphthalene were only consumed in the Alba oil-amended incubations. It seems likely that the n-alkane fraction in the ANS oil represented preferred substrates to the resident microflora relative to the more recalcitrant naphthalenes. The preferred anaerobic metabolism of 2-substituted methylnaphthalenes over 1-substituted isomers has been reported in both a naphthalene-degrading enrichment culture (31) and primary enrichments (10). Our results extend this observation to the ethyl- and dimethyl-substituted homologues. We believe this is the first report of 2-ethylnaphthalene and of 2,6- and 2,7-dimethylynaphthalene biodegradation under anaerobic conditions. A similar preference for naphthalene, 2-methylnaphthalene, and 2-ethylnaphthalene over 1-methylnaphthalene and 1-ethylnaphthalene has been reported for aerobic marine isolates (32). Although the enzymatic mechanisms of attack must differ, the similar homologue preferences exhibited by anaerobic and aerobic microorganisms suggest that the selective removal of naphthalenes in situ will also be similar, regardless of the prevailing redox condition. We did not observe biodegradation of larger molecular weight polycyclic aromatic hydrocarbons, though their degradation has been reported in anaerobic marine ecosystems under sulfate-reducing conditions (33, 34). The complex composition of crude oil makes precise determination of the expected stoichiometry of terminal electron-accepting processes difficult. Although analysis of the dominant hydrocarbons found in crude oil is relatively straightforward, these oils also contain large amounts of high molecular weight resins, asphaltenes, and other hydrocarbons that are not as amenable to quantitative molecular analysis. The artificially weathered ANS crude oil

contains approximately 45.8 mg of alkanes per g (22). Accordingly, 1335 µmol of methanogenesis or sulfate reduction would be expected if these hydrocarbons were completely oxidized in 1 g of oil. Our sulfate-reducing and methanogenic incubations consumed over 2600 µmol of sulfate and produced a similar amount of methane. Therefore, there was more than adequate sulfate reduction and methanogenesis to account for the complete oxidation of the ANS crude oil’s alkane fraction. This contention is also supported by the obvious changes in the gas chromatographic profiles in this oil as a function of biodegradation (Figure 3). The similar amounts of sulfate reduction and methanogenesis observed in oil-amended incubations indicates that, though some differences in biodegradation rates and substrate specificities were seen, comparable amounts of oil can be biodegraded under both of these electron-accepting conditions. The anaerobic biodegradation of crude oil n-alkanes by marine sediment microorganisms under sulfate reducing conditions has been previously reported (35). Hydrocarbonoclastic bacteria have been isolated from a saline, sulfidogenic oil-water separator (13) as well as marine (36, 37) and estuarine (38) environments. Our study extends the observation of n-alkane biodegradation in complex hydrocarbon mixtures with sulfate as electron acceptor to terrestrial environments. The small amount of methane produced in sulfate-amended incubations demonstrates that this electron acceptor successfully shifted the overall metabolism toward sulfate reduction and away from methane formation. Although early reports strongly suggested the occurrence of methanogenic n-alkane biodegradation (5), recent studies, with improved anaerobic and analytical techniques, conclusively demonstrated this phenomenon. Microorganisms from ditch mud incubated with hexadecane produced methane over a period of 800 days (15). The role of sulfatereducing bacteria in this process has been questioned as a small amount of sulfate was reported to enhance methanogenesis, and the culture contained a 16S rRNA sequence that was consistent with a cell phylogenetically related to Desulfovibrio. Anderson and Lovley demonstrated the conversion of [14C]hexadecane to radiolabeled carbon dioxide and methane by microorganisms from a crude oil-contaminated aquifer (39). Our study demonstrates that methanogenic n-alkane biodegradation is far broader than previously recognized and that methanogenesis can be relied upon as a terminal electron-accepting process for the biodegradation of these and other hydrocarbon fractions. In the absence of sulfate, we can envision at least two modes of methanogenic alkane metabolism by the organisms from the Ft. Lupton sediments. A fermentative population of hydrocarbon-oxidizing microorganisms that depend on hydrogen- and/or acetate-consuming microorganisms may form a syntrophic association with methanogens when sulfate is limiting. Alternatively, hydrocarbon-oxidizing sulfate reducing bacteria may, in the absence of sulfate, syntrophically metabolize these compounds, relying upon hydrogenand/or acetate-reducing methanogens to dispose reducing equivalents. Meckenstock succeeded in creating a hydrocarbon-degrading syntrophic coculture by growing a toluenedegrading sulfate reducer with a hydrogen-consuming nitrate reducer with nitrate present as the sole electron acceptor (40). The sulfate dependency for naphthalene biodegradation is consistent with other studies that suggest that this compound is largely recalcitrant under methanogenic conditions (34, 41, 42). While future research may indicate otherwise, the fate of the naphthalenes may largely be a function of electron acceptor availability. A characteristic of alkane-degrading isolates, whether aerobic or anaerobic, is their relatively restricted substrate range (10, 43). The microorganisms in our incubations were

previously exposed in situ to natural gas condensate that is rich in short- to medium-chain alkanes (C5-C15); however, they were able to oxidize alkanes up to 34 carbons in length. It may be that these sediments harbored microorganisms with a much broader substrate range than previously recognized or that the resident bacteria were able to rapidly adapt to higher molecular weight alkanes. Regardless, these results demonstrate that n-alkane biodegradative activities can transcend the range of previous hydrocarbon exposure within a particular environmental compartment. Previous studies examining the anaerobic biodegradation of crude oil have often relied on enrichment cultures or isolates (36, 44, 45). Though these studies confirm the ability of anaerobic bacteria to metabolize individual hydrocarbons within crude oil, they do not illustrate the wider range of biodegradative activities that may be manifested in situ. Our study, using microorganisms from contaminated subsurface sediments, revealed previously unrecognized potential for anaerobic hydrocarbon metabolism. The biodegradation of the complete range of n-alkanes, regardless of the availability of sulfate, may be an important in situ metabolic process affecting both terrestrial crude oil spills and oil reservoirs. The electron acceptor dependent and homologue specific biodegradation of naphthalenes may serve as a diagnostic indicator of the state of in situ biodegradative processes. That is, alkane-depleted but naphthalene-rich oils, such as the Alba crude oil used in this study, may have been biodegraded in the reservoir under methanogenic, not sulfate-reducing, conditions. In any case, knowledge of the metabolic fate of crude oil and other complex hydrocarbon mixtures will shed light on the extent to which in situ biodegradative processes are acting upon residual contamination and influence decisions regarding the need for remedial action.

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Received for review December 19, 2002. Revised manuscript received August 27, 2003. Accepted August 29, 2003. ES0264495