Environ. Sci. Technol. 2000, 34, 892-896
Carbon Isotope Fractionation during Anaerobic Biodegradation of Toluene: Implications for Intrinsic Bioremediation JASON M. E. AHAD,† B A R B A R A S H E R W O O D L O L L A R , * ,† ELIZABETH A. EDWARDS,‡ GREG F. SLATER,† AND BRENT E. SLEEP§ Stable Isotope Laboratory, Department of Geology, University of Toronto, Toronto, Ontario, Canada, M5S 3B1, Department of Chemical Engineering & Applied Chemistry, University of Toronto, Toronto, Ontario, Canada, M5S 3E5, and Department of Civil Engineering, University of Toronto, Toronto, Ontario, Canada, M5S 1A4
Carbon isotope fractionation produced by anaerobic biodegradation of toluene was evaluated in laboratory experiments under both methanogenic and sulfate-reducing conditions. A small (∼2‰) but highly reproducible 13Cenrichment in the residual toluene at advanced stages of microbial transformation was observed in both cultures. The maximum isotopic enrichment observed in the residual toluene was +2.0‰ and +2.4‰ for the methanogenic and sulfate-reducing cultures, respectively, corresponding to isotopic enrichment factors () of -0.5 and -0.8. Because the accuracy and reproducibility associated with gas chromatograph-combustion-isotope ratio mass spectrometry (GC/C/IRMS) is (0.5‰, delineating which of these two terminal electron-accepting processes (TEAP) is responsible for the biodegradation of toluene at field sites will not be possible. However, the potential does exist to use compound-specific isotope analysis (CSIA), in conjunction with other methodologies, as a means of validating advanced stages of intrinsic bioremediation in anaerobic systems. Caution is urged that relating this small (∼2‰) fractionation to biodegradation at complex field sites will prove a challenge.
Introduction Due to widespread use, petroleum hydrocarbons are ubiquitous groundwater contaminants. They enter the subsurface environment via leakage from underground storage tanks, industrial discharge, improper disposal techniques, and accidental spills. Approximately 15% of regular gasoline is comprised of benzene, toluene, ethylbenzene, and m-, p-, and o-xylene (BTEX) (1), relatively water-soluble monoaromatic hydrocarbons which are toxic and confirmed or suspected carcinogens (2). Many cleanup efforts have focused on bioremediation and in particular on in situ or intrinsic biodegradationstaking advantage of the ability of indigenous microbial populations to degrade hydrocarbons (3). * Corresponding author phone: (416)978-0770; fax: (416)978-3938; e-mail:
[email protected]. † Department of Geology. ‡ Department of Chemical Engineering & Applied Chemistry. § Department of Civil Engineering. 892
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The ability of microorganisms to biodegrade BTEX compounds under aerobic conditions is well documented in the literature (4, 5). While less is known about anaerobic BTEX biodegradation, laboratory results have shown that certain BTEX compounds can be degraded under denitrifying (6, 7), iron(III)-reducing (8, 9), sulfate-reducing (10-12), and methanogenic (13, 14) conditions. These results have also been verified in the field (15, 16), where many groundwater systems are anaerobic (17, 18). Although the rate of microbial transformation of BTEX slows down in the absence of molecular oxygen, anaerobic biodegradation nonetheless provides significant remediation potential at many sites (18). Techniques traditionally used to verify the occurrence of intrinsic bioremediation at contaminated field sites include monitoring indirect indicators of biological activity such as depletion of contaminant and electron acceptors and production of dissolved inorganic carbon (DIC) (15) or methane (CH4) (19), and the enumeration of BTEX-degrading microorganisms (20). Although determining the geochemical and microbiological characteristics at a specific location is essential to any remediation protocol, this approach alone will not provide irrefutable proof of intrinsic bioremediation. This stems from the difficulty in obtaining accurate mass balances of contaminant, electron acceptors and end products in heterogeneous soil and groundwater systems, the inability to distinguish between biodegradation and contaminant concentration decreases due to physical processes such as sorption, dissolution, and volatilization, and the inability to extrapolate laboratory-based microbiological assays and microcosm studies to intrinsic biodegradation in the field (21, 22). Stable carbon isotopes provide a promising new method of validating intrinsic bioremediation. Carbon has two stable isotopes, with 12C comprising 98.89% and 13C comprising 1.11% of the total abundance (23). Because of the size of this abundance gap, the ratios of 13C to 12C in carbon-bearing compounds are expressed as per mil (‰) differences relative to a standard, or δ13C, where
δ13C )
[
]
(13C/12C)sample - (13C/12C)standard (13C/12C)standard
× 1000 (1)
Isotopically distinct molecules will participate in reactions at slightly dissimilar rates. This is known as the kinetic isotopic effect and occurs as the result of differences in activation energies of the isotopic forms caused by differences in mass (24). In particular, biologically mediated reactions tend to favor the lighter isotope. For stable carbon isotopes (13C and 12C), this typically results in the residual substrate becoming more enriched in 13C (i.e., a less negative δ13C value) as the reaction proceeds. This phenomenon has been observed in a variety of microbial processes; for example, large isotopic shifts have been recorded during the bacterial oxidation of methane (25, 26) and during the biodegradation of chlorinated hydrocarbons (27-30). In the past few years, the development of continuous flow compound-specific isotope analysis (CSIA) has made it possible to perform rapid isotopic analyses of organic contaminants present as dissolved constituents in groundwater at very low concentrations (31-34), providing the potential to use CSIA as a means of validating bioremediation at BTEX-contaminated sites. Within the accuracy and reproducibility typically associated with CSIA ((0.5‰), recent studies have demonstrated that dissolution (34), sorption (35), and volatilization (34, 35) do not significantly alter the 10.1021/es990797y CCC: $19.00
2000 American Chemical Society Published on Web 01/22/2000
TABLE 1. Stoichiometry and Energetic Equations of Toluene (C7H8) Oxidation under Methanogenic and Sulfate-Reducing Conditionsb type of equation stoichiometrica
condition
energetic stoichiometrica
methanogenic methanogenic sulfate-reducing
energetic
sulfate-reducing
a
equation +
C7H8 + 0.072NH4 + 7.102H2O f 2.318HCO3- + 0.072C5H7O2N + 4.32CH4 + 2.39H+ C7H8 + 7.5H2O f 2.5HCO3- + 4.5CH4 + 2.5H+ C7H8 + 0.144NH4+ + 2.568H2O + 4.14SO42- f 6.28HCO3- + 0.144C5H7O2N + 2.07H2S + 2.07HS- + 0.214 H+ C7H8 + 3H2O + 4.5SO42- f 7HCO3- + 2.25H2S + 2.25HS- + 0.25H+
Includes carbon incorporated into microbial cell (C5H7O2N) formation.
b
Adapted from Edwards et al. (10) and Edwards and Grbic´ -Galic´ (14).
isotopic signature of aromatic hydrocarbons. Until recently, however, less was known about carbon isotope fractionation produced during biodegradation. Sherwood Lollar et al. (27) found no significant change in the isotopic composition of the residual toluene during aerobic biodegradation of toluene carried out in laboratory experiments using two mixed consortia cultured from different field sites. Only a small isotopic fractionation effect (∼2‰ isotopic enrichment in contaminant residual) was observed during aerobic biodegradation of benzene by a mixed microbial culture (36). In contrast, Meckenstock et al. (37) reported larger isotopic enrichments in residual toluene, 3-6‰ and up to 10‰ during anaerobic and aerobic biodegradation experiments, respectively. What these results indicate is that isotopic fractionation effects may be different for different compounds, terminal electron-accepting processes (TEAP), degradative metabolic pathways, or microbial populations. Significantly, Hall et al. (38) found that two different species of bacteria capable of aerobically degrading phenol produced distinctive fractionation signals in the respired CO2. More detailed characterization of the magnitude of carbon isotope fractionation associated with these different parameters must be carried out before the potential of using CSIA as a tool for monitoring biodegradation of aromatic hydrocarbons can be fully assessed. The goal of this experiment was to characterize carbon isotope fractionation associated with anaerobic biodegradation of toluene under two different TEAP.
Experimental Section Anaerobic biodegradation of toluene was carried out under both methanogenic and sulfate-reducing conditions. The methanogenic culture used in this study was a well-defined mixed consortium enriched from gasoline-contaminated aquifer sediments from Pensacola, FL (14). This consortium, which has been maintained on toluene as the sole carbon and energy source for over 10 years, was found to be composed primarily of two eubacterial and two archaeal (methanogenic) species, with each species carrying out a defined role (39). The initial steps of toluene degradation in this culture are believed to proceed via methyl hydroxylation to benzyl alcohol, followed by further oxidation steps to benzaldehyde and benzoate (14, 39). The major end products of methanogenic degradation are HCO3- (CO2) and CH4 (Table 1). The sulfate-reducing culture used in this study was a mixed consortium originally collected from an oil refinery site in Oklahoma chronically exposed to hydrocarbon contamination. Although this consortium has not been microbially characterized, sulfate and methane concentrations were analyzed during this study to confirm that toluene degradation did indeed occur via sulfate-reduction. The major end products of toluene degradation using sulfate as an electron acceptor are HCO3- (CO2), HS-, and H2S (Table 1). For both types of electron acceptors, each biodegradation experiment was run twice. In addition, for each experiment, cultures were prepared and run in duplicate, giving a total of four replicates of the degradation (Figure 1). The cultures
FIGURE 1. Toluene concentrations (mg/L) in controls (b) and methanogenic and sulfate-reducing cultures over time (hours) during anaerobic biodegradation experiments. Squares (9) and triangles (2) represent culture bottles from the two repeat experiments. Each experiment was run in duplicate. Error bars represent ( 7% on concentrations. were prepared in 250 mL autoclaved glass bottles filled with 80 mL of a liquid medium described by Edwards and Grbic´Galic´ (14) and were inoculated with either the methanogenic or sulfate-reducing culture in suspension. The pH of this medium was maintained between 6.8 and 7.3. Sulfate (ca. 14 mM Na2SO4) was added only to the sulfate-reducing consortia to provide a sufficient quantity of the necessary electron acceptor. The cultures were incubated at 25 ( 2 °C in an anaerobic glovebox (Coy Laboratory Products) with an atmosphere consisting of the following gas mixture: 10% H2, 10% CO2, and 80% N2. All glassware was preincubated in the glovebox for at least 1 day to remove all traces of oxygen. The methanogenic cultures were amended with 0.075 mmol (8 µL) of neat toluene (Fisher Scientific, 99.9779% purity), yielding an aqueous concentration (Co) of 55 mg/L. Due to a concern regarding concentration toxicity, the sulfatereducing consortium was amended with only 0.028 mmol (3 µL) of neat toluene (21 mg/L). All four culture bottles were sealed with screw-cap Mininert valves (Precision Sampling VOL. 34, NO. 5, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Corp.) and manually shaken for approximately 1 min to ensure partitioning between the air and liquid phases. Two controls, also prepared in duplicate, were used for these experiments. Controls consisted of 250 mL autoclaved and acid-washed glass bottles filled with 80 mL of distilled water and two different concentrations of toluene (55 and 21 mg/L). In abiotic control experiments run at the University of Toronto it has been shown that there are no changes in toluene controls due to reaction with substrates in the sterile medium. Hence distilled water is a good substitute for sterile (autoclaved) medium in this set of experiments. The purpose of these controls was to ascertain whether any isotope effects were associated with the sampling technique and also to provide initial toluene concentrations and isotopic compositions (Co and δ13Co, respectively) for the samples. Like the cultures, all four control bottles were sealed with Mininert valves, manually shaken for 1 min, and stored inside the anaerobic glovebox. Headspace samples from all cultures and controls were analyzed for both toluene concentration and toluene isotopic composition as biodegradation proceeded. All toluene concentrations (controls and cultures) and methane concentrations (cultures only) were determined by removing 200 µL of headspace using a 500 µL Pressure-Lok gastight syringe (Precision Sampling Corp.). The headspace samples were injected onto a gas chromatograph (Hewlett-Packard 5890 Series II) equipped with a Supel-Q plot column (0.53 mm × 30 m, Supelco Co.) and flame ionization detector (FID). The column was held isothermally at 160 °C, with the injector set at 200 °C and detector at 250 °C. A three point calibration was done using external standards, and aqueous concentrations were calculated using Henry’s law. Methane concentrations were calibrated using gas cylinder standards containing 0.01 and 1% methane in a N2 matrix. Reproducibility on concentration measurements was (7% for toluene and (5% for methane. All toluene isotopic compositions (controls and cultures) were determined by removing 50-1000 µL of headspace using a 1000 µL Pressure-Lok gastight syringe (Vici Precision Sampling Inc.). Larger injection volumes for the cultures were required during the latter stages of biodegradation in order to obtain a sufficiently large toluene peak on the gas chromatograph-combustion-isotope ratio mass spectrometer (GC/C/IRMS). The headspace samples were injected onto a GC/C/IRMS system consisting of a Varian 3400 gas chromatograph equipped with a 30 m × 0.25 mm i.d. DB624 column and Finnigan MAT 252 gas source isotope ratio mass spectrometer. The column was held isothermally at 80 °C, and the injector was set at 200 °C. Samples were run against external CO2 isotopic standards, and all δ13C values are reported relative to the V-PDB standard (40). While internal reproducibility based on duplicate injections of a given sample is generally e0.3‰, differences between samples (error bars) are assigned a value of 0.5‰ to incorporate both reproducibility and the accuracy associated with GC/C/IRMS analysis after Dempster et al. (33) and Slater et al. (34). Aqueous samples (1 mL) were taken from each culture bottle for sulfate analysis. Sulfate was analyzed on a Dionex Series 4000i ion chromatograph (IC) with an electrochemical conductivity detector. The eluant for the IC was 0.75 mM sodium bicarbonate-2.2 mM sodium carbonate (2 mL/min), and the regenerant was 0.025 N sulfuric acid. The IC was calibrated with external standards.
Results and Discussion In Figure 1, toluene concentrations in methanogenic culture bottles and controls are plotted in duplicate for both experiments. Over approximately 170 h, the methanogenic 894
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cultures degraded toluene from an initial concentration (Co) of 55 mg/L to 90% (f ) 0.1) and >70% (f ) 0.3) degradation in the methanogenic and sulfatereducing consortia, respectively. This observation does not change even if a smaller assessment of error incorporating reproducibility alone (0.3‰) is used. Enrichment factors () for each culture were calculated by a least squares linear regression of the data on a plot of ln(f) versus ln[(δ13Ctol/1000 + 1)/(δ13Co/1000 + 1)] after Mariotti et al. (41). Enrichment factors of -0.5 (r 2 ) 0.82) and -0.8 (r 2 ) 0.82) were obtained for the methanogenic and sulfate-reducing cultures, respectively. If a straight-line relationship results from such a plot it indicates that degradation is controlled by a single-order one-step reaction. While the r2 for these regressions (0.82) is not so good as to
FIGURE 2. δ13C values of residual toluene versus the fraction of toluene remaining undegraded in the methanogenic and sulfatereducing cultures from both experiments. Initial δ13C of toluene before biodegradation is -28.4‰ at f ) 1.0. Vertical error bars represent an accuracy and reproducibility of (0.5‰ on δ13C values. Horizontal error bars represent (10% on the fraction of toluene remaining, which was calculated based on a Co of 55 and 21 mg/L for the methanogenic and sulfate-reducing cultures, respectively. The area between the dotted lines depicts the range of uncertainty incorporating both accuracy and reproducibility ((0.5‰) associated with the initial isotopic signature of the toluene. be entirely conclusive, it may indicate that fractionation during degradation is controlled by a single reaction step. Implications for Intrinsic Bioremediation. Anaerobic biodegradation of toluene, carried out under both methanogenic and sulfate-reducing conditions, using two distinct microbial communities, produced a small (∼2‰) 13Cenrichment in the residual toluene due to extensive microbial transformation. This isotopic fractionation was highly reproducible and greater than the analytical uncertainty incorporating both accuracy and reproducibility. Hence the potential exists to use this fractionation as a means of identifying advanced stages of intrinsic biodegradation at contaminated field sites. Since the magnitude of fractionation observed is the same for both TEAP within uncertainty however, carbon isotopic fractionation effects will not provide a means to distinguish between which of these two types of microbial populations is active at a given site. All experimental data to date indicate that carbon isotopic fractionation due to anaerobic biodegradation of aromatic hydrocarbons is small. Enrichment factors reported by Meckenstock et al. (37) for biodegradation of toluene via nitrate-, ferric-iron, and sulfate-reducing bacterial strains are slightly larger but the same order of magnitude as those reported in this study. Both studies confirm that even at advanced stages of biodegradation, isotopic enrichment in the residual contaminant will be on the order of a few ‰
rather than several tens of ‰ reported for chlorinated hydrocarbons (27, 30). In contrast, Meckenstock et al.’s (37) results for isotopic fractionation during aerobic biodegradation of toluene are significantly different than those previously reported for aromatic hydrocarbons (27, 36). Sherwood Lollar et al. (27) reported that at even 90% biodegradation of toluene by two separate mixed aerobic consortia, no significant isotopic fractionation was observed. Meckenstock et al.’s (37) study of aerobic biodegradation of toluene by the pure culture Pseudomonas putida mt-2 shows more than 10‰ enrichment in residual toluene at >90% conversion. These contrasting results for aerobic biodegradation underscore the necessity for more detailed experiments to be carried out to determine the extent to which different microbial strains and pure versus mixed microbial cultures control isotopic fractionation effects. The key implication of this study is that applying stable carbon isotope signatures in the field as a means of identifying anaerobic biodegradation will pose a significant challenge. Since values are small, measured changes in the δ13C of toluene at contaminated sites are not likely unless degradation is nearly complete. Furthermore, if samples are only taken from wells where biodegradation is occurring but has not yet reached advanced stages, one may make the false assumption that intrinsic bioremediation is not occurring in the field. In addition, both analytical uncertainty and possible isotopic variability at the site due to the presence of different sources of aromatic hydrocarbons with different initial δ13C values (31, 33, 35) have the potential to dampen the signal produced by biodegradation. Reliable application of this method will require a large enough number of sampling points both in the degraded perimeter of the plume and undegraded core of the plume to ensure that any observed isotopic variation accurately reflects spatial variation in biodegradation rather than the influence of other complicating factors. Nonetheless, if samples taken from the perimeter of a contaminant plume (where the greatest percentage of degradation is expected to occur) contain δ13C values which are consistently isotopically enriched, then CSIA, used carefully in conjunction with other methodologies, may be used as a means of validating intrinsic bioremediation. In fact, significant changes in δ13C values might suggest that intrinsic bioremediation is nearly complete.
Acknowledgments The authors wish to thank Neil Arner and Hong Li (Department of Geology, University of Toronto) for providing technical support in the Stable Isotope Laboratory and Kirsten Krastel (Department of Chemical Engineering and Applied Chemistry, University of Toronto) for assisting with the maintenance of the microbial cultures. Funding for this project was made available through the Natural Sciences and Engineering Research Council of Canada Strategic Research Grant Program. Additional support was generously provided by the University Consortium Solvents-in-Groundwater Research Program, General Electric Research and Development Centre, United Technologies Corporation, and Water and Earth Science Associates Ltd.
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Received for review July 16, 1999. Revised manuscript received November 12, 1999. Accepted November 18, 1999. ES990797Y
