Environ. Sci. Technol. 1997, 31, 2692-2696
Kinetics of DCE and VC Mineralization under Methanogenic and Fe(III)-Reducing Conditions PAUL M. BRADLEY* AND FRANCIS H. CHAPELLE U.S. Geological Survey, 720 Gracern Road, Suite 129, Columbia, South Carolina 29210
The kinetics of anaerobic mineralization of DCE and VC under methanogenic and Fe(III)-reducing conditions as a function of dissolved contaminant concentration were evaluated. Microorganisms indigenous to creek bed sediments, where groundwater contaminated with chlorinated ethenes continuously discharges, demonstrated significant mineralization of DCE and VC under methanogenic and Fe(III)-reducing conditions. Over 37 days, the recovery of [1,214C]VC radioactivity as 14CO ranged from 5% to 44% and 2 from 8% to 100% under methanogenic and Fe(III)-reducing conditions, respectively. The recovery of [1,2-14C]DCE radioactivity as 14CO2 ranged from 4% to 14% and did not vary significantly between methanogenic and Fe(III)reducing conditions. VC mineralization was described by Michaelis-Menten kinetics. Under methanogenic conditions, Vmax was 0.19 ( 0.01 µmol L-1 d-1 and the half-saturation constant, km, was 7.6 ( 1.7 µM. Under Fe(III)-reducing conditions, Vmax was 0.76 ( 0.07 µmol L-1 d-1 and km was 1.3 ( 0.5 µM. In contrast, DCE mineralization could be described by first-order kinetics. The first-order degradation rate constant for DCE mineralization was 0.6 ( 0.2% d-1 under methanogenic and Fe(III)-reducing conditions. The results indicate that the kinetics of chlorinated ethene mineralization can vary significantly with the specific contaminant and the predominant redox conditions under which mineralization occurs.
Introduction Dichloroethene (DCE) and vinyl chloride (VC) often contaminate shallow aquifer systems in the United States and, therefore, represent significant environmental hazards (13). Both DCE and VC are U.S. EPA priority pollutants (4), and their presence in groundwater frequently drives regulatory concerns for site remediation. For the majority of chlorinated ethene contaminated sites, the presence of DCE and VC is the result of microbial reductive dechlorination activity under anaerobic conditions (5-16). The frequently observed accumulation of DCE and VC under these conditions (5-16) and the lack of understanding of the factors controlling DCE and VC biodegradation in situ represent significant obstacles to the application of bioremediation as a remedial option in many systems. Two specific questions concerning the suitability of intrinsic bioremediation of the reduced chlorinated ethenes under anaerobic conditions are (1) the environmental significance of microbial mineralization of DCE to CO2 as a biodegradation mechanism and (2) the effect of DCE and VC concentrations on the rates of degradation of these con* Corresponding author telephone: 803-750-6125; fax: 803-7506181; e-mail:
[email protected].
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taminants. A number of investigators have observed complete oxidation of VC to CO2 under aerobic conditions (17-22). More relevant to the anaerobic conditions characteristic of most VC-contaminated aquifers, a recent laboratory investigation demonstrated rapid anaerobic mineralization of VC in aquifer material under Fe(III)-reducing conditions (18). Results of a continuous flow, fixed-film reactor study suggest that anaerobic mineralization may also represent a significant mechanism for DCE biodegradation in situ (23). The investigators reported recovery of 24% of [14C]PCE (tetrachloroethene) as 14CO2 under methanogenic conditions (23). However, a similar study conducted under methanogenic conditions indicated that microbial degradation of [14C]PCE stopped at ethene without significant conversion to 14CO2 (24). Although two recent studies have reported significant aerobic mineralization of DCE by soil (25), aquifer (25), and stream bed sediment (17) microbial communities, the potential for anaerobic mineralization of DCE by microorganisms indigenous to a chlorinated ethene contaminated site has not been investigated. A previous study also indicated that the rates of aerobic DCE and VC mineralization varied significantly with contaminant concentrations (17). In that study (17), aerobic mineralization of DCE and VC exhibited Michaelis-Menten kinetics, but the kinetics of anaerobic DCE and VC mineralization were not investigated. The purposes of the studies reported here were to (1) investigate the potential for anaerobic mineralization of DCE and VC in stream bed sediments under methanogenic and Fe(III)-reducing conditions and (2) evaluate the kinetics of anaerobic mineralization of DCE and VC in these sediments under methanogenic and Fe(III)-reducing conditions.
Methods Chemicals. The ability of microorganisms to mineralize DCE and VC under anaerobic conditions was evaluated in sediment microcosms using [1,2-14C]DCE and [1,2-14C]VC. Neat [1,214C]DCE was obtained from Moravek Biochemicals, Inc. (Brea, CA). The [1,2-14C]DCE used in this study was a mixture of 29% trans and 71% cis isomers. The radiochemical purity of the DCE mixture was determined by radiometric detection gas chromatography to be greater than 99.9%. The chemical purity of the [1,2-14C]DCE was confirmed by GC/FID and GC/MS analysis. The [1,2-14C]VC was obtained from New England Nuclear Research Products, Du Pont (Boston, MA). The radiochemical purity of the VC was determined by radiometric/flame ionization detection GC to be greater than 97.6%. Study Site. Microcosm experiments were initiated using creek bed sediments from a former drum disposal area at the Naval Air Station, Cecil Field, located near Jacksonville, FL. The groundwater contamination at the site is a mixture of aliphatic and aromatic chlorinated solvents. The shallow groundwater flows eastward from the source area and discharges 330 m downgradient into a small creek. The site is characterized by a plume of cis-1,2-DCE-contaminated groundwater that extends from the source area to the creek. Dissolved concentrations of cis-1,2-DCE in shallow, groundwater monitoring wells range from 1900 µg/L (20 µM) at the source area to less than 10 µg/L (0.1 µM) in a well located 7 m upgradient of the creek. Concentrations of cis-1,2-DCE as high as 8.9 µg/L (0.1 µM) have been detected at a depth of 20 cm in the creek bed sediments, but no contamination of the upper 10 cm has been observed. Although this site has a history of VC contamination, at the time of this writing VC concentrations throughout the plume remain at or below the detection limit of 1 µg/L (0.02 µM). For the shallow groundwater and the creek water, the pH was 7.1 ( 0.3 and the temperature was 22 °C. S0013-936X(97)00110-7 This article not subject to U.S. copyright. Published 1997 by the American Chemical Society.
Microcosm Study. Microcosms consisted of 20-mL serum vials that were amended with 10 g of saturated bed sediment (water content ) 25% w/w) and sealed with Teflon-lined butyl rubber stopper/base trap assemblies (17, 18). Microcosms were created with a headspace of 100% helium and amended with 1.0 mL of anoxic, sterile distilled water (methanogenic treatments) or 1.0 mL of anoxic, sterile 10 mM Fe-EDTA (Fe(III)-reducing treatments). Anoxic conditions were confirmed in methanogenic and Fe-EDTA microcosms initially and at the end of the study by headspace analysis using thermal conductivity detection gas chromatography. Throughout the study, the lack of significant dissolved O2 ([O2] e 0.1 mg/L), NO3 ([NO3] e 0.2 µM), SO4 ([SO4] e 20 µM), and Fe(II) ([Fe(II)] e 0.01 mg/L) and the observed production of methane (up to 270 µmol/L headspace concentration) in microcosms amended only with distilled water indicate that methanogenesis was the predominant terminal electronaccepting process under these conditions. The lack of significant dissolved O2 ([O2] e 0.1 mg/L), NO3 ([NO3] e 0.2 µM), and SO4 ([SO4] e 20 µM) and the observed production of dissolved Fe (200 ( 96 µM Fe(II) produced in 37 days) in microcosms amended with Fe(III)-EDTA (data not shown) indicate that Fe(III) reduction was the predominant terminal electron-accepting process in these microcosms. Methane was detected in Fe(III)-reducing microcosms, but the fact that headspace methane concentrations did not change significantly during the incubation period indicates that significant methanogenesis did not occur under these conditions (data not shown). Killed controls were prepared in the same manner and autoclaved twice for 1 h at 15 psi and 121 °C. Duplicate experimental microcosms and a single killed control were prepared for each substrate at six dissolved substrate concentrations ranging from 0.2 to 57 µM for VC and from 1.4 to 80 µM for DCE. To sample, the base traps of individual microcosms were rinsed with 0.5 mL of sterile distilled water and filled with 0.3 mL of 3 M KOH. After a 12-h collection period, the KOH was removed, and the amount of trapped 14CO2 was quantified by scintillation counting. Sediment microcosms were incubated in the dark at room temperature for a period of 37 days. 14CO2 production was confirmed in select vials by the addition of barium chloride as described previously (18, 19, 23). The fact that no radioactivity was detected in the base traps of sterile serum vials, which contained radiolabeled substrate but no sediment, indicates that trapping of radiolabeled VC and DCE was not significant (less than 1%) in experimental microcosms. The amount of radioactivity initially present in the experimental microcosms as 14CO2 was estimated based on the percentage recovery observed in killed and sediment-free control microcosms to be less than 1% of the total radioactivity added as VC or DCE. The 14CO2 production data were corrected for the recovery efficiency of H14CO3 and the amount of radioactivity present initially in sediment microcosms. The recovery efficiency was quite consistent but relatively low (52 ( 3%) due to the circumneutral sediment pH and the 12 h nonsacrificial (no acidification) recovery method employed in this study. Initial rates (first 8 days) of DCE and VC mineralization observed in bed sediment microcosms at different substrate concentrations were estimated by simple linear regression of the 14CO2 evolved as a function of time. The microcosm headspace gas was analyzed at the completion of the study for the presence of the daughter products of reductive dechlorination of VC and DCE. The presence of cis-DCE, trans-DCE, and VC in bed sediment microcosms was determined after the final base collection by vigorously shaking the microcosms and analyzing the headspace using flame ionization detection gas chromatography. The presence of ethene, ethane, and methane in the microcosm headspace was determined in the same manner using thermal conductivity detection gas chromatography. Because the adsorbed and final dissolved phases were not
FIGURE 1. Percentage recovery of [1,2-14C]VC radioactivity as 14CO2 under Fe(III)-reducing (a) and methanogenic (b) conditions as a function of time (days) and dissolved VC concentrations. Data are means ( SD for duplicate experimental microcosms. No significant mineralization of [1,2-14C]VC was observed in killed control microcosms. quantified, a mass balance analysis of the original substrates and the reduced daughter products is not appropriate. Thus, the results of the final gas analyses are presented here as presence/absence data solely to provide a qualitative indication of the intermediates that had accumulated in the headspace to detectable levels by the termination of the mineralization experiment. The amount of dissolved Fe(II) produced in the bed sediment microcosms under methanogenic and Fe-EDTAamended conditions was also measured as an indication of active microbial Fe(III) reduction. A 0.5-mL aliquot of microcosm slurry was removed using a filtered syringe (0.2 µm pore diameter), diluted as needed with distilled water, and analyzed for dissolved Fe using a ferrozine colorimetric method (Hach Company, Loveland, CO).
Results and Discussion The microorganisms indigenous to the creek bed sediments at NAS Cecil Field demonstrated rapid mineralization of VC under methanogenic and Fe(III)-reducing conditions (Figure 1). After 37 days, the recovery of [1,2-14C]VC radioactivity as 14CO ranged from 5% to 44% in methanogenic microcosms 2 and from 8% to 100% in Fe(III)-reducing microcosms (Figure 1). For all treatments, VC mineralization was attributable to biological activity because the recovery of 14CO2 in killed control microcosms was approximately 3%, the purity level for the [1,2-14C]VC (data not shown). In general, the recovery of 14CO2 in Fe(III)-reducing microcosms was approximately twice that observed in methanogenic microcosms (Figure 1). The rapid mineralization of VC by stream bed sediment microorganisms observed in the present study under Fe(III)reducing conditions is consistent with the rapid VC mineralization observed previously in Fe(III)-reducing aquifer sediments (18). The mineralization of VC observed in the present study under methanogenic conditions is consistent with a report of significant PCE mineralization in a methanogenic, continuous-flow, fixed-film reactor (23) and indicates that significant mineralization of VC can occur under methanogenic conditions. Differences between methanogenic and Fe(III)-reducing microcosms were also reflected in the final microcosm
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TABLE 1. Presence (+) or Absence (-) of DCE Isomers, VC, Ethene, and Ethane in Headspace of Bed Sediment Microcosms Incubated under Methanogenic and Fe(III)-Reducing Conditionsa treatment
substrate VC
Fe-reducing methanogenic
DCE
Fe-reducing methanogenic
experimental control experimental control experimental control experimental control
t-DCE
c-DCE
VC
ethene
ethane
NAb
NA NA NA NA + + + +
+ + + + + + -
+ + + -
+ + + -
NA NA NA + + + +
a Presence (+) is defined as having a headspace concentration greater than the analytical detection limits of 20 nmol/L for cis- and trans-DCE, 8 nmol/L for VC, and 50 nmol/L for ethene and ethane. b Not applicable.
FIGURE 2. Percentage recovery of [1,2-14C]DCE radioactivity as 14CO2 under Fe(III)-reducing (a) and methanogenic (b) conditions as a function of time (days) and dissolved DCE concentrations. Data are means ( SD for duplicate experimental microcosms. No significant mineralization of [1,2-14C]DCE was observed in killed control microcosms. headspace compositions (Table 1). Under methanogenic conditions, ethene and ethane were detected in experimental but not control microcosms. Accumulation of ethene and ethane during degradation of chlorinated ethenes under methanogenic conditions has been reported previously (2630). The lack of ethene and ethane accumulation in Fe(III)reducing microcosms is similar to the results of aerobic VC mineralization studies (17-22) and is consistent with direct oxidation of VC to CO2 under Fe(III)-reducing conditions (18). The microorganisms indigenous to the creek bed sediments at NAS Cecil Field also demonstrated significant mineralization of [1,2-14C]DCE under methanogenic and Fe(III)-reducing conditions (Figure 2). In contrast to the VC mineralization results, no significant difference in DCE mineralization was observed between methanogenic and Fe(III)-reducing microcosms. The mean recoveries ranged from 4% to 14% for Fe(III)-reducing and methanogenic treatments (Figure 2). For all treatments, [1,2-14C]DCE mineralization was primarily attributable to biological activity, because the mean recovery of 14CO2 in killed control microcosms was 1 ( 1% after 37 days (data not shown). The lack of difference in isomeric ratios between final microcosm headspace analyses and the headspace analyses of the [1,214C]DCE stock solution (data not shown) indicated that both cis and trans isomers of DCE were degraded by the bed
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FIGURE 3. Change in the rate (µmol L-1 d-1) of microbial mineralization of VC as a function of dissolved VC concentration (µM) under Fe(III)-reducing (a) and methanogenic (b) conditions. For each substrate concentration, data are means ( SD for duplicate experimental microcosms. Michaellis-Menten parameter estimates were obtained by nonlinear regression analysis. No significant mineralization of [1,2-14C]VC was observed in killed control microcosms. sediment microorganisms in approximately equimolar amounts (17). VC, ethene, and ethane were detected in experimental microcosms but not in killed control microcosms under both methanogenic and Fe(III)-reducing conditions (Table 1). Rates of VC mineralization varied significantly with the initial dissolved concentration of VC (Figure 3). Under methanogenic and Fe(III)-reducing conditions, VC mineralization increased linearly over the first 8 days of incubation (Figure 1), and these data were used to calculate initial rates of VC mineralization for each dissolved VC concentration. A plot of the rate (µmol L-1 d-1) of VC mineralization by bed sediment microorganisms over this range of substrate concentrations indicates that mineralization under methanogenic and Fe(III)-reducing conditions follows MichaelisMenten kinetics (Figure 3). A first-order rate expression did not adequately describe the kinetics of VC mineralization in this study, because the increase in the rate of VC mineralization was not linear over the range of substrate concentrations investigated (31). Similarly, a zero-order rate expression did not adequately describe the kinetics of VC mineralization in this study, because the rate was not constant over the range of substrate concentrations investigated (31). Thus, a non-
FIGURE 4. Relationship between the rate of mineralization of DCE (% d-1) and dissolved DCE concentration (µM) under Fe(III)-reducing (a) and methanogenic (b) conditions. For each dissolved DCE concentration, data are means ( SD for duplicate experimental microcosms (b) and single killed control microcosms (O). linear regression package (Sigmaplot 3.0, Jandel Scientific, San Rafael, CA) was utilized to estimate the Michaelis-Menten kinetic parameters, Vmax and km, for VC mineralization under methanogenic and Fe(III)-reducing conditions. Under methanogenic conditions, Vmax was 0.19 ( 0.01 µmol L-1 d-1 ((standard error of the parameter estimate), and the halfsaturation constant, km, was 7.6 ( 1.7 µM. Under Fe(III)reducing conditions, Vmax was 0.76 ( 0.07 µmol L-1 d-1 and km was 1.3 ( 0.5 µM. These results are similar to a previous investigation demonstrating that aerobic VC mineralization follows Michaelis-Menten kinetics (17). The results of the current study indicate that the maximum rate of VC mineralization by the bed sediment microbial community under Fe(III)-reducing conditions was four times faster than that observed under methanogenic conditions (Figure 3), but 16 times slower than the maximum rate (12.4 µmol L-1 d-1) observed under aerobic conditions (17). Moreover, the lower km observed under Fe(III)-reducing conditions indicates that the overall microbial process involved in VC mineralization under these conditions has a greater affinity for VC than that found under methanogenic (this study) or aerobic (km ) 12.1 µM) (17) conditions. In contrast to VC mineralization, the percentage rates (% d-1) of anaerobic DCE mineralization observed in the bed sediment microcosms did not vary significantly with substrate concentration under Fe(III)-reducing or methanogenic conditions (Figure 4). Under both methanogenic and Fe(III)reducing conditions, the mean rate of DCE mineralization was 0.6 ( 0.2% d-1. These results demonstrate that over the range of dissolved DCE concentrations examined in this study, the kinetics of DCE mineralization in these sediments is firstorder. The differences observed in the apparent kinetics of DCE and VC mineralization under different redox conditions may reflect fundamental differences in the mechanisms and microbial populations involved under differing redox conditions. The lack of enhancement in the rate of DCE mineralization under Fe(III)-reducing conditions in spite of the facts that VC appears to be a significant intermediate formed during DCE mineralization (Table 1) and VC mineralization is significantly enhanced under Fe(III)-reducing conditions (Figures 1 and 3) is consistent with the hypothesis that the controls on anaerobic DCE and VC mineralization are not
the same. Based on these results, it is proposed that in these stream bed sediments the initial step in DCE mineralization is a reductive dechlorination, which is rate-limiting and exhibits first-order kinetics over the dissolved DCE concentration range examined in this study (1.4-80 µM). The fundamental differences in the kinetics of DCE mineralization under aerobic (17) and anaerobic (this study) conditions and the lack of enhancement in the rate of DCE mineralization in the presence of a strong oxidant (Fe(III)-EDTA, this study) are consistent with the hypothesis that anaerobic DCE mineralization involves an initial, rate-limiting reductive step. Selection of an appropriate model for describing DCE and VC biodegradation is a matter of determining the km for degradation relative to the environmentally significant ranges of dissolved DCE and VC concentrations. In situ, dissolved VC concentrations ranging from non-detectable up to 16, 25, and 71 µM have been reported for Dover AFB, DE (9), Plattsburg AFB, NY (11), and St Joseph, MI (13), respectively. Over this range of concentrations, the results of the current study indicate that the Michaelis-Menten model adequately describes the rates of VC mineralization under methanogenic or Fe(III)-reducing conditions. In situ, dissolved DCE concentrations ranging from non-detectable up to 20, 100, 530, and 1300 µM have been reported for NAS Cecil Field (this study), Dover AFB (9), Plattsburg AFB (11), and St Joseph, MI (13), respectively. Based on the results of the present study, the first-order degradation model appears appropriate for the NAS Cecil Field site. The extent to which these results have relevance to other sites or to dissolved concentrations outside the range examined here remains to be determined.
Acknowledgments We thank Cliff Casey and Mike Maughon of the Naval Facilities Engineering Command for assistance in collecting sediments and providing background information for the site. This research was supported by the U.S. Geological Survey Toxic Substances Hydrology Program and the Southern Division Naval Facilities Engineering Command.
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Received for review February 10, 1997. Revised manuscript received May 22, 1997. Accepted May 29, 1997.X ES970110E X
Abstract published in Advance ACS Abstracts, July 15, 1997.
