Environ. Sci. Technol. 1997, 31, 590-596
Use of Stable Oxygen and Carbon Isotope Analyses for Monitoring the Pathways and Rates of Intrinsic and Enhanced in Situ Biodegradation PRADEEP K. AGGARWAL,* MARK E. FULLER, MICHELE M. GURGAS, JOHN F. MANNING, AND MICHAEL A. DILLON Environmental Research Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, Illinois 60439
Significant challenges remain in developing reliable techniques to monitor in situ biodegradation. Stable carbon and oxygen isotope analyses of the contaminants, products of degradation, and electron acceptor(s) may provide robust means for monitoring the occurrence, pathways, and rates of intrinsic or enhanced in situ biodegradation. Results of a laboratory study using diesel fuel and a mixed microbial culture show that combined stable carbon isotope analyses of carbon dioxide and stable oxygen isotope analyses of molecular oxygen allow monitoring of the occurrence and pathways of degradation. The first-order rate constants for contaminant degradation (about -0.04 day-1) obtained from oxygen and contaminant concentrations are in excellent agreement with those obtained from isotopic data for oxygen (-0.04 to -0.05 day-1), indicating that oxygen isotope analyses of molecular oxygen can be used for quantifying the rate of contaminant degradation. Based on our results and a review of the published literature on oxygen isotope systematics of molecular oxygen and other common electron acceptors (nitrate and sulfate), it is suggested that combined carbon and oxygen isotope analyses of carbon dioxide and the electron acceptors provide effective tools for monitoring intrinsic and enhanced in situ biodegradation of fuel or chlorinated hydrocarbons under aerobic and anaerobic conditions.
Introduction In situ bioremediation has emerged as one of the most important alternatives for the remediation of soils and groundwater contaminated with fuel and other hydrocarbons. Biomediation may be used as an intrinsic scheme, which relies on natural rates of biodegradation, or an active scheme, which seeks to enhance biotransformation rates by optimizing growth conditions for indigenous bacteria. Intrinsic bioremediation or natural attenuation has recently gained significant prominence as an attractive remedial alternative because of the large number of sites that require remediation (1, and references cited therein). Indeed, intrinsic bioremediation may be the only alternative for many sites where engineered remediation is not feasible due to site characteristics or where spatially large plumes exist with relatively low levels of contaminant concentrations. For intrinsic bioremediation to be a viable remedial action, however, it is imperative that the biotransformation processes be accurately * Corresponding author telephone: 630-252-7053; fax: 630-2525498; e-mail:
[email protected].
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understood and quantified. Quantification of intrinsic biodegradation may also reduce site remediation costs where engineered remediation is instituted. This is because engineered remediation may be carried out to achieve a target contaminant concentration that may be higher than the acceptable cleanup criterion but sufficiently low so that intrinsic biodegradation will further reduce contaminant concentrations to acceptable levels. In addition, methods for characterizing intrinsic biodegradation may be used for the optimal design and monitoring of engineered in situ biodegradation schemes. Occurrence and rates of intrinsic and enhanced biodegradation of chlorinated and fuel hydrocarbons have been characterized by using a variety of geochemical and microbiological methods based on field and laboratory studies. These methods include (1) mass balance-type approaches using changes in contaminant, metabolite, or electronacceptor concentrations and (2) comparison of laboratory and field microbiological assays. These approaches all have some merit but generally are found to be less than reliable in the field (2, 3), largely because of difficulties in (1) obtaining accurate mass balance due to natural inhomogeneities in the physical and hydraulic properties of soil and groundwater systems, (2) distinguishing between biotic and abiotic degradation processes, and (3) establishing a verifiable link between microbiological assays and microcosm studies and in situ biodegradation. Methods based on changes in the concentrations of oxygen and carbon dioxide appear to be the most promising (4); however, the consumption of oxygen and the production of carbon dioxide may occur by the oxidation of natural soil organic matter or by abiotic processes (or both). In addition, carbon dioxide produced by biodegradation may be consumed by inorganic reactions (4). A number of studies have attempted to characterize the pathways and rates of intrinsic bioremediation of chlorinated and fuel hydrocarbons by using field and laboratory studies of geochemical and microbiological parameters (5). The fieldbased studies successfully used geochemical parameters (e.g., Eh, alkalinity, concentrations of oxygen, and other electron acceptors) as indicators of intrinsic biodegradation; however, quantification of intrinsic biodegradation on the basis of field studies or combined field and laboratory studies generally resulted in large uncertainties in the estimated rates of biodegradation (5-7). Similarly, the relationship between microbial respiration and contaminant degradation could not be reliably identified; for example, Odom et al. (7) observed significant sulfate reduction but no corresponding decrease in trichloroethylene (TCE) concentrations in microcosms using soils from contaminated sites. The natural abundance of stable isotopes of essential elements involved in the biodegradation processes (carbon and oxygen) may be used to monitor (1) the occurrence of in situ biodegradation, (2) the pathways of degradation, and (3) the rates and extent of biodegradation (8, 9). Monitoring of in situ biotransformation using stable isotopes may be achieved by the analysis of isotopic compositions of the products of degradation or the residual fractions of the contaminant or electron acceptors. Isotope composition are reported as per mil (‰) deviation from a standard using the δ notation:
δ)
(
)
Rx - 1 × 1000 Rstd
(1)
where R is the isotopic ratio (18O/16O or 13C/12C) in the sample (x) or standard (std). Carbon dioxide produced by various processes in the subsurface, such as plant root respiration,
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1997 American Chemical Society
TABLE 1. Composition of Mineral Medium Used for Biodegradation Experiments salt
concn (mg/L)
salt
concn (mg/L)
KH2PO4 K2HPO4 (NH4)2(SO4) MgSO4‚7H2O CaCl2‚2H2O FeSO4‚7H2O H3BO3
3470.0 4267.0 1234.0 460.0 17.6 1.0 0.3
CoCl2‚6H2O ZnSO4‚7H2O MnCl2‚4H2O Na2MoO4‚2H2O NiCl2‚6H2O CuCl2‚2H2O
0.2 0.1 0.03 0.03 0.02 0.01
soil organic matter oxidation, inorganic carbon dissolution, or contaminant hydrocarbon degradation, has characteristic δ13C values (8, 10). The δ13C of CO2 gas or dissolved inorganic carbon has been used as evidence for in situ biodegradation of hydrocarbons at contaminated sites (8, 11-14). Oxygen in the potential electron acceptors in aerobic or anerobic environments (molecular oxygen; nitrate; sulfate) also has characteristic δ18O values. The δ18O of atmospheric oxygen is about 23.5‰ on a global scale (15). Dissolution of oxygen in aqueous solutions induces only a small fractionation (0.7‰; 15), and the isotopic composition of dissolved oxygen in shallow groundwaters will be nearly the same as that of the atmospheric oxygen. The δ18O of sulfate and nitrate in groundwater systems generally is about 4-10 and 20‰, respectively (10, 16, 17). The oxygen isotopic compositions of molecular oxygen, nitrate, and sulfate in soil and groundwater systems are affected primarily by microbial processes, and isotopic fractionation during microbial respiration produces a significant change in the δ18O of the residual electron acceptors (10, 16-21). Aggarwal (9) used the isotopic compositions of soil gas CO2 and O2 to distinguish between aerobic and anaerobic production of CO2 and discussed the use of the isotopic compositions of soil gas O2 for quantifying microbial respiration rates. Except for that study, the combined use of stable oxygen and carbon isotopes to monitor and quantify in situ biodegradation of hydrocarbons does not appear to have been explored in the literature. In this paper, we present the results of a laboratory study designed to demonstrate the use of stable carbon and oxygen isotope analyses of carbon dioxide and molecular oxygen, respectively, for monitoring the occurrence, pathways, and rates of in situ biodegradation.
Experimental Section Experimental Design. The biodegradation of fuel hydrocarbons (no. 2 fuel oil or diesel fuel) was studied in batch experiments. A total of 50 mL of a mineral medium (Table 1) was dispensed into 250-mL bottles that were sealed with Mininert valves. The pH of the mineral medium was 6.9 and remained nearly the same throughout the experiment. Bottles containing the mineral medium were sterilized in an autoclave. A 50-µL aliquot of diesel fuel was aseptically added to each bottle. One group of bottles was designated as sterile controls; another group was inoculated with 0.5 mL of a mixed bacterial culture. The mixed bacterial culture was enriched from diesel-contaminated soil at a site near Joliet, IL. Although the culture was enriched under aerobic conditions, some anaerobic bacteria were also present in the culture; the presence of anaerobic bacteria was determined by observing microbial growth in anaerobic incubations. Inoculated and sterile control bottles were placed on a shaker at 175 rpm at room temperature (20-22 °C) for incubation. The experiments were run for up to 43 days. On designated days, duplicate sterile control and inoculated bottles were removed from the shaker for sampling and analyses of CO2 and O2 headspace concentrations, total petroleum hydrocarbon (TPH) concentrations, and 13C/12C and 18O/16O isotopic ratios. Carbon Dioxide and Oxygen Concentrations. An ∼10mL sample of the headspace was taken by using a gas-tight
locking syringe and was injected into an O2/CO2 analyzer equipped with an infrared detector (Illinois Instruments Model 3600). Ambient air (20.9% O2 and 0.0% CO2) and a synthetic gas mixture (9.9% CO2 and 91.1% nitrogen) were used as standards for calibration. Replicate analyses of samples and synthetic standards indicated a precision of (0.5% for measured concentrations greater than 10% and (1.4% for lower concentrations. Total Petroleum Hydrocarbons. After the CO2 and O2 measurements were made, 100 mL of methylene chloride was added to each bottle. The bottles were shaken by hand and degassed three times by loosening the cap and placed on a wrist-action shaker for 15 min. Approximately 2 mL of the methylene chloride extract was taken from the bottle for analysis using a gas chromatograph (GC) (Hewlett Packard Model 5890 T) equipped with a capillary column (Supelco PTE-5) and a flame ionization detector (FID). Helium was used as a carrier gas for the GC analysis. The TPH concentration was determined by using the total area counts in the chromatogram. The total area count for the sterile controls was used to normalize the diesel fuel concentration in the samples. The error of measurement was evaluated from the analyses of synthetic standards of variable diesel concentration and sterile controls and was estimated to be (6%. Stable Oxygen and Carbon Isotope Analysis. Samples for isotopic analyses were extracted and analyzed at the Coastal Science Laboratories, Austin, TX. A 40-mL aliquot of nitrogen gas (99.98%) was injected into the headspace through the septum in the Mininert valve and was mixed thoroughly by filling and emptying the syringe several times. The empty syringe was then removed, and up to three 10-mL aliquots were withdrawn for oxygen isotope analyses using different syringe assemblies. The bottle was then acidified by injecting 20 mL of laboratory-grade phosphoric acid to volatilize dissolved CO2. The bottles were shaken by hand, and two 10-mL aliquots of the headspace were withdrawn for isotopic analyses of CO2, which was cryogenically separated from water vapor and other gases by using dry ice-acetone and liquid nitrogen traps on a vacuum line. Aliquots for isotopic analyses of molecular oxygen were introduced into a vacuum line, and oxygen, nitrogen and other noncondensable gases were isolated by passing through dry ice and liquid nitrogen traps. Oxygen was converted to CO2 by circulating the noncondensable fraction over a graphite rod heated to about 800 °C and embedded with platinum wire to avoid the production of carbon monoxide (15). The CO2 produced was cryogenically separated from nitrogen and other gases. Bulk or fractions of diesel fuel separated using a GC were combusted to produce CO2, which was then separated cryogenically. Purified CO2 gas from the headspace and from oxygen or diesel fuel combustion was used for isotope ratio measurements using dual-inlet, isotope ratio mass spectrometers (VG/ SIRA Models 10 and 12). The isotopic ratios are reported with respect to international standards, Vienna Standard Mean Ocean Water (VSMOW) for oxygen and Vienna Pee Dee Belemnite (VPDB) for carbon (22). The δ18O of atmospheric oxygen extracted from ambient air samples was 23.1‰ (average of 14 measurements) with a standard deviation of 0.1‰. Synthetic air standards were prepared with CO2 concentrations ranging from 5% to 25% and O2 concentration ranging from 5% to 20%. The isotopic composition of oxygen in these standards was the same as that measured for ambient air samples. The measured δ18O of atmospheric oxygen in this study (23.1‰) is slightly lower than the 23.5% value reported in the literature (15). Consequently, all measured δ18O values here are reported after adjusting to an air value of 23.5‰. The precision of δ18O measurements for oxygen in the experimental samples was (0.3‰. The precision for δ13C and δ18O values of carbon dioxide was (0.2‰ for samples with CO2 concentrations
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TABLE 2. TPH, Carbon Dioxide, and Oxygen Concentrations and Isotopic Compositions of Carbon Dioxide and Oxygen in Biodegradation Experimentsa TPH time (day) run TPH-11 0 1 8 35 run TPH-13 0 2 8 10 15 22 24 31 36 43
CO2
O2
concn (vol %)c
δ13C
δ18O
(‰, VPDB)
(‰, VSMOW)
0.101 NA NA 0.030
0.0 0.3 7.3 9.7
-10.0 -26.5 -30.3 -28.3
0.101 0.085 0.061 0.064 0.057 0.053 0.039 0.013 0.028 0.035
0.0 0.9 11.0 11.8 11.4 18.5 17.9 17.8 20.5 20.2
-10.0 -31.8 -30.7 -30.6 -30.2 -28.5 -28.4 -29.4 -28.6 -28.3
concn (vol %)b
concn (vol %)c
δ18O (‰, VSMOW)d
NA NA NA NA
20.7 20.3 10.6 6.2
23.5 23.6 35.3 16.4
NA 32.2 35.5 34.7 35.0 34.6 35.0 34.9 34.7 35.2
20.7 20.2 11.4 9.8 10.2 9.4 9.4 7.1 7.1 5.9
23.5 26.1 33.4 33.8 37.8 42.1 45.7 49.1 26.1 26.0
a All data are averages of duplicate experiments. The errors of measurement are as discussed in the text. NA, not analyzed. b TPH dissolved in mineral medium; calculated by normalizing the total area count for a sample to that of a sterile control that had a diesel concentration of 1 µL/mL. c Headspace concentration. d Adjusted to an atmospheric air value of 23.5‰.
greater than 0.3% and (2‰ for samples with lower CO2 concentrations.
Results and Discussion The degradation of diesel with a mixed bacterial culture was studied with two experimental runs, TPH-11 and TPH-13. Run TPH-11 was conducted with limited sampling, primarily to determine the duration and sampling frequency for a more detailed run. The results of the gas concentration, TPH, and isotopic analyses are listed in Table 2. As noted earlier, all experiments were conducted in duplicate. Therefore, each data point in the two runs represents the average of duplicate analyses. In addition, isotopic analyses were obtained on at least two aliquots of a sample, and each isotopic analysis is an average of at least four measurements. Concentrations of TPH, CO2, and O2 in the sterile controls remained within analytical precision of the initial values (0.1%, 0.0%, and 20.9%, respectively) throughout the experiment. The δ18O of molecular oxygen in the sterile controls also was within the analytical precision of the initial value. The discussion below is based primarily on the results of run TPH-13. Occurrence and Pathways of Degradation. Figure 1 shows the TPH, CO2, and O2 concentrations for run TPH-13. The diesel concentration decreased from 0.101% to 0.085% (15% degradation) in the first two days of incubation, which resulted in an increase in the CO2 concentration (to ∼1%) and a decrease in the O2 concentration (to ∼20%). The CO2 and O2 concentrations on day 8 were 11.0 and 11.4%, respectively, while the diesel concentration decreased to 0.061% (about 40% degradation), indicating rapid microbial growth and diesel degradation during this period. Between days 8 and 43, the concentration of diesel decreased at a much lower rate, changing from 0.061% to about 0.03%. During the same period, the change in O2 concentration (from 11.4% to 5.9%) also was relatively small, indicating a period of lower microbial growth following rapid growth observed through day 8. The CO2 concentration increased from 11.0% to 20.2% from day 8 to day 43. Most of this increase occurred rapidly between days 15 and 22 when CO2 concentration increased to 18.5%, probably as a result of the mineralization of a nonvolatile, intermediate product formed during the early degradation of diesel or of some of the microbial biomass. The lack of a correspondingly sharp decrease in diesel concentration is consistent with this hypothesis. The mineralization process occurred in a period of lowered microbial
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FIGURE 1. Concentrations of diesel fuel (TPH), CO2, and O2 during the degradation of diesel by a mixed bacterial culture (run TPH-13). Also shown are the diesel and O2 concentrations in the sterile controls. The sharp increase in CO2 concentration on day 22 is considered to reflect the mineralization of a nonvolatile, intermediate product of diesel degradation or biomass.
A
B
FIGURE 2. Isotopic compositions during the degradation of diesel by a mixed bacterial culture (in TPH-13): (A) δ18O of molecular O2; (B) δ13C of CO2. The δ13C of diesel was -28.5‰. Note the slight increase (∼2‰) in δ13C after day 15, which is considered to result from the mineralization of an intermediate product or biomass. The sharp decrease in δ18O after day 31 is believed to have occurred by isotopic exchange during bacterial reduction of sulfate. growth rate (Figure 1) so that the overall consumption of oxygen appears to have remained the same before and during the sharp increase in CO2 concentration. The isotopic compositions of CO2 and O2 are shown in Figure 2. Isotopic compositions changed as a result of both the extent and the pathways of degradation. The δ13C of CO2 shifted from near -10‰ (atmospheric CO2) at the start to about -31‰ within two days of incubation and remained nearly the same through day 15. These δ13C values were slightly lower as compared to that of the diesel (δ13C ) -28.5‰). After day 15, the δ13C of CO2 increased to about -28.5‰ (Figure 2). The increase of about 2‰ in the δ13C values of CO2 after day 15 coincides with the sharp increase in the concentration of CO2 during the same period. The small increase in the δ13C of CO2 and nearly identical δ13C values for CO2 and diesel after day 15 are consistent with the hypothesis that mineralization of an intermediate product or microbial biomass formed in the early degradation of diesel may have caused the sharp increase in CO2 concentration between days 15 and 22. The effects of the formation and mineralization of intermediate products or biomass after day 22, if any, may not be apparent because the incremental amount of CO2 produced after this time is negligible as compared with the total CO2 in the headspace. Changes in the δ13C of CO2 produced by hydrocarbon degradation may also result from the selective degradation of fractions of diesel fuel that may have different isotopic compositions. Gas chromatograms of residual hydrocarbons on days 2, 15, 22, and 43 (Figure 3) do not indicate selective degradation of a particular fraction of diesel between days 15 and 22. To further verify whether isotopic changes may have occurred due to isotopic variability in diesel fuel, three fractions of the diesel were isolated and analyzed for carbon isotopic composition. The saturate, aromatic, and NSO (nitrogen-sulfur-oxygen-containing compounds) fractions were obtained by using a GC and different solvents (hexane,
toluene, and methanol, respectively) in a silica gel column. The δ13C of these three fractions (-27.9 to -28.5‰) were nearly the same as that of the composite sample, indicating that variability in the isotopic composition of source hydrocarbons did not cause the shift in the δ13C of CO2 after day 15. Except for the first sample (day 2), the δ18O values of CO2 were the same (∼35‰) throughout the experiment (Table 2). Oxygen isotopic exchange between CO2 and water at normal temperatures and pressures occurs rapidly (10), and therefore, the δ18O of CO2 gas is governed by isotopic equilibrium with water. At 25 °C, the δ18O of CO2 is about 41.1‰ higher than that of water (10). The δ18O of water used in our experiment was -6.2‰, and the expected δ18O of CO2 will be 34.9‰, similar to the measured values. The slightly lower δ18O value of CO2 in the day 2 sample is due to a lack of complete isotopic equilibration across the gas-water interface in the presence of high diesel concentrations. The δ18O of O2 increased from 23.5‰ (atmospheric air) to 26.1‰ on day 2 of the experiment (Figure 2), consistent with the increase expected from aerobic respiration. The change in the isotopic composition of residual oxygen from day 2 to day 31 was quite substantial (from 26.1 to 49.1‰). This large change in isotopic composition contrasts with the relatively small change in oxygen concentration during the same period (Figure 1). After day 8, as the degradation of diesel progressed beyond about 30%, the relative change in oxygen concentration was small as compared to the error of measurement ((1.4%). However, the corresponding change in δ18O was much greater than the error of measurement ((0.3‰). This indicates that under field conditions, where a low rate of degradation may be reached relatively quickly, the isotopic data would provide a more accurate monitoring of in situ biodegradation than may be achieved by using O2 concentrations. The δ18O of O2 decreased dramatically between days 31 and 36 (Figure 2). The oxygen concentration, however, remained low (∼7%) and did not change during this period (Figure 1). It should be noted that the data in Table 2 represent averages of duplicate experiments. The observed change in δ18O in both experimental runs, therefore, reflect a process that occurred reproducibly in six different incubations. In addition, the reproducibility of the observed isotopic shifts in the two runs eliminates the possibility of the leakage of atmospheric oxygen (δ18O ) 23.5‰) or analytical error as causes of lower δ18O. The δ18O of oxygen in a closed system may decrease by isotopic exchange or mixing with oxygen from a source with low δ18O value. Potential sources of oxygen in our experiments with low δ18O values were water, phosphate, and sulfate. Isotopic exchange between molecular oxygen and other liquid or gaseous phases is negligible at normal temperatures and pressures (10). However, isotopic exchange between sulfate and water or other phases may occur during bacterial reduction of sulfate. The dissimilatory reduction of sulfate by bacteria is a complex process (23) and may proceed by the intermediate formation of sulfite or enzyme-sulfate complexes (24, 25). These intermediate products are known to facilitate isotopic exchange between water and sulfate oxygen (24-26). Similarly, isotopic exchange between molecular oxygen and sulfate may also occur during the bacterial reduction of sulfate. The dissolved oxygen concentration in our experiments between days 31 and 36, when the observed change in isotopic composition of O2 occurred, is estimated to be about 2.8 mg/L by using a Henry’s law constant of 0.0013 mol atm-1 (27). Thermodynamically, sulfate reduction is not favored in the presence of oxygen. The postulated occurrence of sulfate reduction may result from redox disequilibrium due to differences in the kinetics of microbially mediated redox reactions at low concentrations of dissolved oxygen (28). Although the bottles in our experiment were continuously
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FIGURE 3. Chromatograms of residual diesel fuel at days 2, 15, 22, and 43 in run TPH-13. Selective degradation of a particular fraction of diesel fuel is not apparent throughout the experiment. shaken, anaerobic microenvironments are known to form around particles as small as 100 µM (29). Formation of microbial biomass in the experiments, therefore, could have facilitated redox disequilibrium. Alternatively, the mixed bacterial culture used in our experiments may contain sulfatereducing bacteria that become active at low oxygen concentrations. Although many of the sulfate-reducing bacteria are obligate anaerobes, recent studies have shown that some sulfate reducers are oxygen-tolerant and may also reduce O2 to H2O (23, 29, 30). Dilling and Cypionka (31) have shown aerobic respiration by sulfate-reducing bacteria. Other examples of sulfate-reducing activity in a variety of aerobic or microaerobic environments have been reviewed by Smith (29). Thus, sulfate reduction according to the reaction
SO42- + 2H+ ) H2S + 2O2
(2)
and the consumption of an equivalent amount of O2 either by reduction to H2O or by aerobic respiration would result in shifting the isotopic composition of molecular oxygen in the headspace (by isotopic exchange with low δ18O oxygen in sulfate) without a change in its concentration, as observed in our experiments. Isotopic exchange during sulfate reduction appears to be the only plausible mechanism for the observed change in the isotopic composition of oxygen. Sulfate reduction was indicated by the presence of the characteristic odor of H2S gas in the culture headspace. To further confirm the presence of H2S, we conducted an additional experiment for headspace
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analysis. Semi-quantitative analysis by using a GC-mass spectrometer indicated trace amounts of headspace H2S (
