Environ. Sci. Technol. 2003, 37, 3859-3863
Chlorine Isotope Fractionation during Microbial Reduction of Perchlorate N E I L C . S T U R C H I O , * ,† PAUL B. HATZINGER,‡ MARTHA D. ARKINS,‡ CHRISTY SUH,† AND LINNEA J. HERATY† Department of Earth & Environmental Sciences, University of Illinois at Chicago, Chicago, Illinois 60607, and Shaw Environmental and Infrastructure, Princeton Research Center, Lawrenceville, New Jersey 08648
Perchlorate contamination of surface water and groundwater is an emerging public health problem that has adversely affected the drinking water supplies of millions of people in the western United States. Microbial reduction has shown promise as a cost-effective means for in situ bioremediation of perchlorate-contaminated water. Measurements of stable isotope ratios of light elements (H, C, N, O, S, Cl) can often be used to distinguish biodegradation of organic and inorganic molecules from abiotic loss mechanisms such as adsorption, dispersion, or volatilization because of the relatively large kinetic isotope effects accompanying biodegradation. We quantified chlorine isotope fractionation during perchlorate biodegradation by a common perchlorate-reducing bacterium, Dechlorosoma suillum, initially isolated from a perchloratecontaminated groundwater source in southern California. The values of the chlorine isotopic fractionation factor R derived from two microcosm experiments were R ) 0.9834 ( 0.0001 (R 2 ) 0.9999) and R ) 0.9871 ( 0.0008 (R 2 ) 0.9832). These R values indicate that the rate of the 35ClO reduction is ∼1.3-1.7% faster than that of the 4 37ClO reduction. This relatively large kinetic isotope effect 4 indicates that chlorine isotope analysis provides a sensitive technique by which to document in situ bioremediation of perchlorate in groundwater.
Introduction Ammonium perchlorate has been used since the 1940s in the United States as the primary oxidizer in the solid propellant for many types of rockets and missiles. Perchlorate salts are also commonly used in fireworks, munitions, air bags, highway flares, and matches (1). Past disposal practices in the military and aerospace industry, which include burning perchlorate-containing materials and discharging wastewater produced during the replacement of outdated propellant in missiles and rockets, has led to perchlorate contamination in groundwater in numerous states, including California, Maryland, Texas, Massachusetts, New Mexico, Utah, and Nevada (2-4). In addition, both Lake Mead and the Colorado River contain measurable levels of perchlorate (2). Although * Corresponding author phone: (312)355-1182; fax: (312)4132279; e-mail:
[email protected]. † University of Illinois at Chicago. ‡ Princeton Research Center. 10.1021/es034066g CCC: $25.00 Published on Web 07/29/2003
2003 American Chemical Society
the total scope of perchlorate contamination in the United States remains unclear, recent estimates suggest that the drinking water of more than 15 million people may be affected (5). According to data compiled by the California Department of Health Services (CDHS), since 1997, perchlorate has been detected in 80 of 912 public water supplies tested in the state and 292 of 5205 private drinking water sources sampled contained traces of the oxidant (6). Based on current toxicological data, California has established a provisional action level of 4 µg/L for perchlorate in drinking water. Other states, including Maryland, Nevada, and Texas, have also instituted advisory levels for perchlorate, and it is expected that the U.S. EPA will establish a reference dose (RfD) for the compound in 2003. The perchlorate anion is nonvolatile, its common salts are highly soluble in water (e.g., the solubility of ammonium perchlorate is 200 g/L), and it is chemically stable in aqueous solution. As a result, conventional water treatment technologies such as air-stripping, carbon adsorption, ultrafiltration, and advanced oxidation are not effective for removing the oxidant from water supplies (2, 7). However, research conducted during the past several years has revealed that there are a variety of bacteria that can degrade perchlorate under anoxic conditions by using the molecule as a terminal electron acceptor (8-11). In addition, recent data suggests that these perchlorate-reducing organisms are widely distributed in many environments including soils, groundwater, and surface water (5, 9, 12, 13). During biological reduction of perchlorate, there appears to be an initial two-step reduction of perchlorate (ClO4-) to chlorate (ClO3-) and then chlorite (ClO2-), which is catalyzed by a perchlorate reductase enzyme (14, 15). The chlorite is then further degraded by chlorite dismutase to chloride (Cl-) and oxygen (O2) (9). A molecular probe, which is based on the highly conserved chlorite dismutase gene, has recently been developed for the detection of perchlorate-reducing strains in natural environments (16). Full-scale bioreactor systems have been successfully applied at several sites for removing perchlorate from groundwater (13, 17). In addition, in situ treatment of perchlorate-contaminated aquifers using electron donor addition to stimulate naturally occurring bacteria also appears to be a promising remedial approach, and several field trials are underway to demonstrate this technology (13, 18, 19). However, one of the difficulties in evaluating the performance of in situ bioremediation is distinguishing chemical loss due to biodegradation from other loss mechanisms, such as dilution, dispersion, adsorption, or unexpected groundwater flow patterns during system operation (i.e, bringing uncontaminated groundwater into the treatment zone). In addition, it is often difficult to document slow biodegradation processes that are occurring naturally over time (i.e, intrinsic biodegradation). There has been very little research or consideration of intrinsic perchlorate biodegradation, although this process may be a significant natural sink for the molecule in anoxic environments where there is cocontamination with an organic electron donor or where a naturally occurring electron donor is present. Stable isotope analysis is one technique whereby biodegradation of organic and inorganic molecules can be successfully distinguished from other nonbiological loss mechanisms. In this paper, we evaluate the potential use of this procedure to document perchlorate biodegradation by measuring the Cl isotopic fractionation accompanying anaerobic growth of a common perchlorate-degrading bacterium, Dechlorosoma suillum. VOL. 37, NO. 17, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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The use of stable isotope ratio measurements for understanding the geochemical behavior of natural and anthropogenic compounds in the environment is a major field of research within the earth, atmospheric, oceanic, and environmental sciences. Recent developments in the measurement and application of the stable isotope ratios of carbon and chlorine in chlorinated aliphatic hydrocarbons (20-22) led to important advances in characterizing the behavior of these compounds in contaminated groundwater aquifers (23-26). Particularly important is the discovery that microbial degradation of chlorinated aliphatic hydrocarbons under aerobic (27) and anaerobic (28, 29) conditions is accompanied by significant kinetic isotope effects for both carbon and chlorine, allowing evaluation of the extent of biodegradation through measurement of isotope ratios. Stable isotope ratio measurements have not yet (to our knowledge) been reported for perchlorate from field settings or microbial culture experiments. Chlorine has two stable isotopes, 35Cl and37Cl, in the ratio 35 Cl/37Cl ) 3.087. Stable isotope ratios are normally reported in δ notation. For chlorine, this is defined as δ37Cl ) [(Rsample/ Rstandard)-1] × 1000, where R ) 37Cl/35Cl, and the standard is Standard Mean Ocean Chloride (30). δ37Cl values are expressed in units of ‰ (per mil or parts per thousand). Because chlorine in the environment occurs mainly as the chloride ion, there are no redox reactions that induce significant changes in bonding energy and concomitant large isotopic variations among chloride-bearing compounds. Thus, natural variations in δ37Cl values are relatively small, e.g., δ37Cl values for chloride in surface waters have a relatively narrow range from -2 to +1‰ (31). Reported δ37Cl values for perchlorate are limited to data for three samples of reagent NaClO4 ranging from +0.2 to +2.3‰ (32). The variability in chlorine isotope ratios of manufactured and naturally occurring perchlorate has not yet been adequately characterized. Likewise, the chlorine isotope effect accompanying microbial reduction has not been measured and cannot be accurately predicted; therefore, it must be measured experimentally in a variety of anaerobic microcosms before field studies of chlorine isotopes in perchlorate can be meaningfully interpreted. Accurate knowledge of the chlorine kinetic isotope effect for perchlorate reduction by the indigenous (or representative) microbial population and the initial δ37Cl values of the perchlorate will enable reasonably precise estimates of the extent of microbial reduction of perchlorate in field settings.
Experimental Section Culture, Growth Conditions, and Sample Collection. The perchlorate-degrading culture used for isotope enrichment studies, Dechlorosoma suillum JPLRND, was initially isolated from a groundwater sample collected in southern California. The culture was purified using traditional enrichment and plating methods and identified by 16S rRNA gene sequencing (Acculabs, Newark, DE). A similar bacterium was recently described (8). During our initial experiment on Cl isotopic fractionation during perchlorate biodegradation (microcosm #1), the culture was grown aerobically on agar plates (R2A Agar; Becton Dickinson and Co., Sparks, MD), and colonies were transferred into a 4 L bottle containing 3 L of a basal salts medium (BSM), ∼10 mM perchlorate, and ∼17 mM acetate as the electron donor. For the second experiment, microcosm #2, growth conditions were the same, except that a 35-mL aliquot of active culture from microcosm #1 was used to inoculate the 3 L flask. The lag time in the second study was significantly reduced because the culture had been actively degrading perchlorate, rather than growing aerobically, prior to inoculation. As a control, we reanalyzed perchlorate concentrations in some of the filtered microcosm samples used for Cl isotope analysis, after more than a year 3860
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in storage, and found no significant change in perchlorate concentration compared to measurements made at the time of the microcosm experiment. To verify that the concentrations of transient intermediates chlorate and chlorite were negligible with respect to perchlorate and that all perchlorate was converted stoichiometrically to chloride (14, 15), we performed a third experiment (microcosm #3) using a low chloride version of the BSM medium. This experiment was inoculated from an agar plate of the pure culture. The basal salts medium (BSM), which was modified from (33), contained 62.5 mg/L of NaH2PO4(H2O), 265 mg/L of K2HPO4(3 H2O), 125 mg/L of NH4Cl, 200 mg/L of MgSO4(7 H2O), 12 mg/L of FeSO4(7 H2O), 3.0 mg/L of MnSO4(H2O), 3.0 mg/L of ZnSO4(7 H2O), 1.0 mg/L of CoCl2(6 H2O), 1.0 mg/L of CaCl2(2 H2O), 2.4 mg/L of Na2MoO4(2 H2O), 1.4 mg/L of Al2(SO4)3(H2O), 1.0 mg/L of CuSO4(5 H2O), and 1.0 mg/L of NiSO4(6 H2O). For the low chloride version of BSM used in microcosm experiment #3, NH4Cl was replaced by (NH4)2SO4. The BSM was prepared using distilled water, boiled under nitrogen gas for 15 min, and then autoclaved. The solution was cooled, placed in a Coy Environmental Chamber (N2 headspace), amended with sterile solutions of sodium acetate and sodium perchlorate to achieve the desired concentrations, and then inoculated with D. suillum JPLRND. The bottle was sealed with a sterile rubber stopper and incubated at room temperature. Subsamples were taken periodically in the Coy chamber, and these samples were immediately filtered through a 0.2 µm pore-size cellulose acetate filter units (Corning Inc., Corning, NY) and placed at 4 °C. The samples for Cl isotopic analysis of perchlorate were shipped to the University of Illinois at Chicago overnight on ice and stored at 4 °C until the samples were processed for isotopic analysis. Determination of Anion Concentrations. Perchlorate concentrations in filtered microcosm solutions used for Cl isotopic analysis of perchlorate were determined by using a Dionex 100 ion chromatograph with an IonPac AS16 analytical column (4 mm) and an IonPac AG16 guard column (4 mm). The injection volume was 1 mL, the eluent was 30 mM NaOH, and the flow rate was 1.6 mL/min. Calibrations were performed with mass dilutions of a 999 µg/mL perchlorate reference solution obtained from Alltech Associates, Inc. Each sample was injected in triplicate. The relative standard deviation of these triplicate perchlorate determinations was better than (2%, and the accuracy was better than (6%. For microcosm #3, perchlorate concentration was determined using EPA method 314.0, and chlorate, chlorite, and chloride were determined using EPA method 300.0. Determination of Chlorine Isotope Ratio in Perchlorate. Amounts of perchlorate extracted for the isotope ratio measurements ranged from 15 to 52 micromoles. Perchlorate was extracted from experimental solutions using Empore Anion-SR (#2252) solid-phase extraction disks. Each disk was pretreated by rinsing sequentially with 10-mL each isopropyl alcohol, deionized water, 1 M NaOH solution, and deionized water. Each disk was then eluted with three 10-mL aliquots of 0.1 M NaNO3 and rinsed with deionized water to remove Cl prior to application for perchlorate extraction. Perchlorate was extracted from the experimental solutions at a flow rate of about 1 mL/s. Perchlorate was coeluted from the extraction disk along with chloride (and other extracted anions) using three 10-mL aliquots of 0.1 M NaI, followed by 10 mL of deionized water. The perchlorate-containing solution was acidified with 1 mL of 8 M HNO3, and 4.5 mL of 1 M AgNO3 (or more, to ensure excess) was added, to precipitate Cl and I (as AgCl and AgI). While adding the excess AgNO3, the solution was stirred and then heated to near boiling before placing in a dark cabinet overnight. The solution was then passed through a new, deionized water-rinsed 0.45 µm polypropylene syringe filter to remove AgCl and AgI pre-
cipitates. Precipitates retained on the filter were washed twice with 0.01 N HNO3 and once with deionized water, and these washings were added to the perchlorate-containing solution. A drop of 1 M AgNO3 was added to verify removal of Cl and I from the solution. The chloride- and iodide-free, perchlorate-bearing solution with AgNO3 was then evaporated to near dryness, transferred to a Pyrex combustion tube, and evaporated to dryness under filtered air at 105 °C. The tube was then evacuated while being cooled to liquid nitrogen temperature, warmed to room temperature under vacuum, sealed, and then combusted for 1 h at 550 °C to produce AgCl as a product of the quantitative perchlorate decomposition. The resulting AgCl was then purified in the tube by flushing out nitrogen oxide gases and then leaching excess soluble nitrates with 2 mL of 0.05 M HNO3 with the tube agitated in an ultrasonic bath at 80 °C. The purified AgCl was then dried at 115 °C, the tube was evacuated, and excess CH3I was added. The tube was then resealed and combusted for 2 h at 300 °C to produce CH3Cl. Subsequent cryogenic purification of CH3Cl for mass spectrometric analysis of the Cl isotope ratio followed the method of Holt et al. (20). The method as described above gave perchlorate yields of 82-106% when tested with up to 8 L of a synthetic solution having concentrations of 20 µM perchlorate and 1000 µM chloride. Replicate Cl isotope ratio measurements of a perchlorate solution made by dissolving anhydrous NaClO4 reagent (Mallinckrodt) yielded a δ37Cl value of +2.72 ( 0.07‰ (n ) 3), using a CH3Cl reference gas calibrated against seawater chloride. The mean precision of replicate δ37Cl measurements for perchlorate from microcosm experiments was (0.3‰, and perchlorate yields determined from capacitance manometer and ion gauge readings ranged from 73 to 109%. Estimation of Isotopic Fractionation Factors. The isotopic fractionation factor, R, is defined as
R ) RA/RB
(1)
(2)
where R and R0 are the Cl isotope ratios (37Cl/35Cl) of the residual perchlorate and the initial, unreacted perchlorate, respectively, and F is the fraction of perchlorate remaining. In terms of the δ37Cl value of the perchlorate
(δ37Cl + 1000)/(δ37Cl0 + 1000) ) FR-1
(3)
where δ37Cl is the Cl isotopic composition at any value F, and δ37Cl0 is the Cl isotopic composition at F ) 1. The value of R can thus be derived simply by taking the natural log of eq 3 and rearranging:
R-1 ) ln(δ37Cl + 1000)/(δ37Cl0 + 1000) - ln F
(4)
This function is commonly used to describe the familiar Rayleigh-type isotopic fractionation that accompanies a variety of natural processes (34). The isotopic fractionation factor being estimated here does not represent isotope exchange between two substances at equilibrium but rather the kinetic isotope effect (KIE) of the unidirectional enzymecatalyzed perchlorate reduction reaction, and the effective definition of R can be thought of as
R ) 1/KIE
time
(5)
[ClO4-], mM
δ37Cl of ClO4-
Microcosm #1 (d) 10.1
0 10
8.52
16
1.99
17
+2.34 +2.73 +4.65 +4.80 +5.55 +30.63 +29.86 +29.23 +40.80
1.05
0.00 0.83
Microcosm #2 (h) 9.01 7.77
1.50
6.00
3.00
2.89
4.25
0.49
5.00
0.15
+2.93 +6.42 +6.40 +9.32 +11.86 +21.06 +20.53 +37.32 +37.13 +61.36
TABLE 2. Stoichiometry of Cl-Bearing Reactant and Products during Microbial Perchlorate Reduction time (h)
[ClO4-] (mM)
[ClO3-] (mM)
[ClO2-] (mM)
[Cl-] total (mM)
Cl- producta (mM)
0 145 167 174 191 216
12.04 11.31 1.683 0.000 0.000 0.000
0.000 0.144 0.011 0.000 0.000 0.000
0.000 0.000 0.000 0.000 0.000 0.000
0.620 1.324 10.17 12.65 12.73 12.62
0.000 0.704 9.549 12.03 12.11 12.00
a
where R is an isotope ratio, and A and B are two substances (or, as in the present case, product and reactant, respectively). For Cl isotope ratios, R represents 37Cl/35Cl. Values of R are derived from the experimental results by assuming the exponential function
R/R0 ) FR-1
TABLE 1. Chlorine Isotopic Fractionation during Microbial Perchlorate Reduction in Microcosms
Product [Cl-] ) total [Cl-] - 0.620 mM.
where KIE is the ratio of the reaction rates (k) of the light and heavy Cl isotopes, k35/k.37
Results and Discussion Two experiments (microcosms #1 and #2) were performed to measure the chlorine kinetic isotope effect during the microbial reduction of perchlorate. Experimental results for perchlorate concentrations and δ37Cl values from these two experiments are given in Table 1. A third experiment (microcosm #3) was performed to evaluate the levels of the potential intermediates chlorate and chlorite and to quantify the conversion of perchlorate to chloride. Results for this experiment are given in Table 2. Complete reduction of perchlorate was achieved after about 18 days in microcosm #1, after about 5.5 h in microcosm #2, and after about 7 days in microcosm #3. Figures 1 and 2 show the perchlorate concentration in each microcosm as a function of time. There was a long initial lag period in microcosm #1, probably because the culture was previously grown under aerobic conditions rather than by using perchlorate as an electron acceptor. In microcosm #2, the culture was transferred during active perchlorate degradation, and there was no lag period before perchlorate degradation was observed. Microcosm #3 was prepared in the same way as microcosm #1; therefore, there was a lag period before perchlorate degradation began to occur. Figure 2 shows the concentrations of chlorate, chlorite, and secondary chloride in microcosm #3. There was no detectable chlorite at any time, and chlorate was detectable only at about 1.3% of perchlorate concentration during the active phase of perchlorate degradation. Conversion of perchlorate to secondary chloride was stoichiometric. VOL. 37, NO. 17, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 3. Comparison of Various Chlorine Kinetic Isotope Effects r
reaction
reference
0.9974-0.9991 0.9962 0.9955 0.994-0.995 0.9934 0.992 0.991 0.989 0.988 0.987-0.991 0.983-0.987
volatilization of pure chlorinated aliphatic hydrocarbons microbial dechlorination of dichloromethane enzyme-catalyzed dechlorination of 1,2-dichloroethane microbial dechlorination of trichloroethene enzyme-catalyzed dechlorination of 1-chlorobutane abiotic dechlorination of benzyl chloride by Na-borohydride abiotic dechlorination of DDT enzyme-catalyzed chlorination of 3,5-dimethylphenol enzyme-catalyzed chlorination of 1,3,5-trimethoxybenzene microbial dechlorination of perchloroethene microbial reduction of perchlorate
(24, 38) (27) (39) (28) (39) (40) (41) (42) (42) (28) this work
FIGURE 3. Variation of δ37Cl value with perchlorate concentration (log mg/L) in the two microcosm experiments. Analytical error is smaller than data points.
FIGURE 1. Perchlorate concentration (expressed as C/C0) vs time (days) in (A) microcosm experiment #1 and (B) microcosm experiment #2.
FIGURE 2. Concentrations of perchlorate, chlorate, chlorite, and chloride (mM) vs time (days) in microcosm experiment #3. The δ37Cl value of perchlorate was measured as a function of perchlorate concentration in each of the first two culture experiments. Data from these two experiments are compared in Figure 3, which shows δ37Cl vs log [ClO4-]. Each experimental data set yields an approximately linear array of data points. Isotopic fractionation factors (R) were calculated by linear regression of eq 4. The results of the linear regressions 3862
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FIGURE 4. Variation of Cl isotope ratio (ln R/R0) vs fraction of perchlorate remaining (ln F) in (A) microcosm experiment #1 and (B) microcosm experiment #2. Values of the chlorine isotope fractionation factor, r, calculated by linear regression are 0.9834 ( 0.0001 (R2 ) 0.9999) and 0.9871 ( 0.0008 (R2 ) 0.9832), respectively, for experiments #1 and #2. are shown in Figure 4; the R value derived from microcosm #1 is 0.9834 ( 0.0001 (R2 ) 0.9999) and that for microcosm #2 is 0.9871 ( 0.0008 (R2 ) 0.9832). The higher R value measured in microcosm #2 may reflect a diffusive transport limitation such that the microbial demand for electron acceptors overwhelmed the rate at which perchlorate diffusion could maintain the characteristic kinetic isotope effect expressed in microcosm #1. This interpretation is consistent with the fact that the isotopic fractionation factor in
microcosm #2 appears to diverge from that of microcosm #1 only during the last two-thirds of the reaction, when the microbial activity was presumably at a maximum. The measured R values of both experiments, however, indicate that the rate of the 35ClO4 reduction reaction is 1.3-1.7% faster than that of the 37ClO4 reduction reaction. The magnitude of this chlorine kinetic isotope is large compared to other reported chlorine kinetic isotope effects (Table 3). It is comparable, however, to other isotopic fractionations occurring during microbial reduction of oxyanions of nitrogen, sulfur, and selenium (35-37). Assuming that the measured chlorine isotope effect of microbial perchlorate reduction is representative of that which may be observed during in situ bioremediation in field settings, it is possible to estimate the sensitivity of chlorine isotope ratio measurements to the extent of microbial perchlorate degradation. For example, if an R value of 0.985 ( 0.002 (the mean of the two measured values) is assumed, and the typical precision of a δ37Cl measurement of perchlorate extracted from groundwater is (0.3‰, then as little as 2% biodegradation of perchlorate can be detected. Significant perchlorate biodegradation could occur through natural attenuation processes without being detected by perchlorate concentration measurements by ion chromatography. The enhanced sensitivity of isotopic measurements enables more precise evaluation of the extent and rate of natural perchlorate attenuation. In addition, chlorine isotope ratio analysis will be a useful technique to monitor and verify the progress of in situ perchlorate bioremediation in applications where electron donors are supplied to the subsurface for this purpose. When characteristic chlorine (and oxygen) isotope ratios of perchlorates of different origin are defined, perchlorate source tracing may also become feasible. These new capabilities using isotope analysis are likely to find widespread application for assessing perchlorate bioremediation and improving the management of impacted public water supplies.
Acknowledgments The Strategic Environmental Research and Development Program provided partial support for this research. Ben Holt and Kent Orlandini (Argonne National Laboratory) provided invaluable assistance in developing methods for perchlorate extraction and isotopic analysis.
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Received for review January 24, 2003. Revised manuscript received May 6, 2003. Accepted June 20, 2003. ES034066G
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