Environ. Sci. Technol. 2000, 34, 2761-2763
Acetogenic Microbial Degradation of Vinyl Chloride PAUL M. BRADLEY* AND FRANCIS H. CHAPELLE U.S. Geological Survey, Stephenson Center, Suite 129, Columbia, South Carolina 29210
Under methanogenic conditions, microbial degradation of [1,2-14C]vinyl chloride (VC) resulted in significant (14 ( 3% maximum recovery) but transient recovery of radioactivity as 14C-acetate. Subsequently, 14C-acetate was degraded to 14CH4 and 14CO2 (18 ( 2% and 54 ( 3% final recoveries, respectively). In contrast, under 2-bromoethanesulfonic acid (BES) amended conditions, 14C-acetate recovery remained high (27 ( 1% maximum recovery) throughout the study, no 14CH4 was produced, and the final recovery of 14CO was only 35 ( 4%. These results demonstrate that 2 oxidative acetogenesis may be an important mechanism for anaerobic VC biodegradation. Moreover, these results (1) demonstrate that microbial degradation of VC to CH4 and CO2 may involve oxidative acetogenesis followed by acetotrophic methanogenesis and (2) suggest that oxidative acetogenesis may be the initial step in the net oxidation of VC to CO2 reported previously under Fe(III)-reducing, SO4reducing, and humic acids-reducing conditions.
Introduction Several studies have demonstrated that microorganisms indigenous to chloroethene contaminated groundwater and surface-water systems can mineralize vinyl chloride (VC) to CO2 (1-6) or CH4 and CO2 (7, 8) under anaerobic conditions. However, little is known about the mechanism(s) underlying these processes. For example, net anaerobic oxidation of [1,2-14C]VC to 14CO2 without detectable accumulation of intermediates has been demonstrated under Mn(IV)-reducing (6, Bradley unpublished results), Fe(III)-reducing (2-4), and SO4-reducing conditions (4). In all cases, the lack of a detectable lag between VC disappearance and 14CO2 production, the stoichiometric agreement between [1,2-14C]VC loss and 14CO2 recovery, and the lack of detectable intermediates suggested that any intermediate(s) formed during anaerobic VC oxidation were rapidly degraded to CO2. Nevertheless, the recently reported biodegradation of [1,2-14C]VC to 14CH4 under methanogenic conditions (7, 8) does suggest a possible mechanism for anaerobic VC oxidation. The observed production of equal amounts of 14CH4 and 14CO2 during [1,214C]VC degradation and the demonstrated lack of significant autotrophic methanogenesis under study conditions (7) led to the hypothesis that 14CH4 was produced via acetotrophic methanogenesis (8, 9). This hypothesis predicts that the significant intermediate of anaerobic VC mineralization is acetate which is readily metabolized to CH4 and CO2 via acetotrophic methanogenesis or to CO2 via anaerobic respiration. * Corresponding author phone: (803)750-6125; fax: (803)750-6181; e-mail:
[email protected]. 10.1021/es991371m Not subject to U.S. Copyright. Publ. 2000 Am. Chem. Soc. Published on Web 05/31/2000
FIGURE 1. Dissolved acetate concentrations in BES amended (20 mM) and unamended microcosms containing bed sediments from NAS Cecil Field. Microcosms were 20 mL serum vials containing 15 g of creek bed sediment, an atmosphere of helium, and approximately 0.5 µCi [1,2-14C]VC. The initial dissolved VC concentration was 8 µM (500 µg/L). Dissolved acetate concentrations were measured by ion exclusion high performance liquid chromatography. Data are means ( SD for triplicate unamended and BES-amended microcosms. Triplicate sterile controls were prepared as above and autoclaved for 1 h at 120 °C and 15 PSI. [1,2-14C]VC was obtained from NEN Dupont and had a radiochemical purity of 97%.
Experimental Results To test the hypothesis that acetate is a significant intermediate formed during anaerobic VC mineralization, the production and accumulation of acetate and 14C-acetate were evaluated in methanogenic microcosms amended with [1,2-14C]VC (Figures 1 and 2). Microcosms were prepared using bed sediments collected from a black-water stream at the Naval Air Station (NAS) Cecil Field, FL. The study site and the general methods for microcosm preparation and monitoring have been described in detail previously (7, 8). The bed sediment samples used in this study were collected under low streamflow conditions and consequently the leaf litter accumulation, organic content (g4% dry weight), and dissolved acetate concentration (130 ( 30 µM) of the sediments were relatively high. Previous investigations conducted under these organic-rich conditions have demonstrated that methanogenesis and microbial humic acids reduction occur concomitantly (5, 8). Anaerobic microcosms, that were prepared with a helium headspace and amended with [1,2-14C]VC, demonstrated extensive methane production (33 ( 3 µmol per L headspace per day), complete removal of [1,2-14C]VC (data not shown), and a 90% decrease in dissolved acetate concentrations within 37 days (Figure 1). Each of these changes was attributable to biological activity because no significant changes in these parameters were observed in sterile control microcosms. Periodic examination of relevant geochemical indicators verified that O2-, NO3-, Fe(III)-, and SO4-reduction were insignificant under these culture conditions. Degradation of [1,2-14C]VC resulted in immediate but transient recovery of 14C-acetate which reached a maximum of 14 ( 3% at day one but decreased to 2 ( 1% by day seven (Figure 2). No 14CH4 or 14CO2 was recovered in experimental microcosms on day one. The decrease in 14C-acetate recovery after day one, was accompanied by rapid accumulation of 14CH4 and 14CO2 (18 ( 2% and 54 ( 3% final recoveries, respectively). The decrease in 14C-acetate recovery and the simultaneous accumulation VOL. 34, NO. 13, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Percentage recovery of 14C-radiolabel as 14CH4, 14CO2, 14C-acetate, and 14C-ethene in unamended and in BES-amended (20 mM) microcosms. Microcosms were prepared as for Figure 1. 14CH4, 14CO , and 14C-ethene were quantified by radiometric detection gas 2 chromatography as described previously (7, 8). The 14C-acetate fraction was separated as described in Figure 1, and the radioactivity was quantified by liquid scintillation counting. Data are means ( SD for triplicate experimental, BES amended, and sterile control microcosms. Recovery data are not corrected for losses due to sampling. No [1,2-14C]VC degradation was observed in control microcosms (82 ( 3% final recovery). of 14CH4 and 14CO2 observed in this study are consistent with acetotrophic methanogenesis as hypothesized previously (8, 9). The excess recovery of 14CO2 relative to 14CH4 was approximately 36% and is consistent with VC mineralization coupled to humic acids reduction under these conditions (5, 8). These results demonstrate that acetate can be a significant intermediate formed during anaerobic mineralization of VC and suggest that the acetate produced by this process is subsequently consumed by acetotrophic methanogens and humic acids reducers. To further evaluate the significance of acetate as a product of VC degradation and the importance of acetotrophic methanogenesis as a sink for acetate in this system, the effects of 20 mM BES (2-bromoethanesulfonic acid, an inhibitor of methanogenesis) on the recovery of 14CH4, 14CO2, and 14Cacetate were investigated (Figure 2). Addition of 20 mM BES completely inhibited 14CH4 production, and decreased the final 14CO2 recovery by a similar amount (about 19% of theoretical, Figure 2). Under BES amended conditions, 14Cacetate accumulation (27 ( 1% maximum accumulation) was approximately double that observed under unamended conditions and remained high throughout the study (20 ( 1% final recovery, Figure 2). The dissolved acetate concentration also increased under BES amended conditions from 130 ( 25 µM to 790 ( 50 µM (Figure 1). The lack of 14CH , the associated decrease in 14CO recovery, and the 4 2 marked increase in the accumulation and persistence of 2762
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acetate and 14C-acetate under BES amended conditions further demonstrate the importance of acetotrophic methanogensis as the final step in the degradation of [1,2-14C]VC to 14CH4 and as a major sink for acetate under these conditions. The results of the BES amendment suggest that acetate also may be a significant intermediate for the nonmethanogenic oxidation of [1,2-14C]VC to 14CO2 reported in previous studies (5, 8). The fact that the recovery of 14CO2 remained high (35 ( 4%) even with BES amendment demonstrated that a nonmethanogenic mechanism also contributed to the oxidation of [1,2-14C]VC to 14CO2 (Figure 2). This observation is consistent with the previous finding that, under methanogenic conditions, a significant fraction of the microbial oxidation of [1,2-14C]VC to 14CO2 in these bed sediments was coupled to humic acids reduction and not dependent on methanogenesis (5, 8). The observed increase in 14CO2 recovery and the associated decrease in 14C-acetate recovery after 14 days argue for 14CO2 being produced at the expense of 14C-acetate (Figure 2). Furthermore, the fact that production of 14CO2 did not begin until after 14C-acetate accumulation (Figure 2) is consistent with the hypothesis that 14C-acetate, rather than [1,2-14C]VC, is the immediate substrate for 14CO2 production. Acetate oxidation coupled to humic acids reduction has been demonstrated previously (10). In addition to acetogenic oxidation of VC (this study) followed by acetate consumption via acetotrophic methanogenesis (8, 9, this study) and humic acids reduction (5, 10, this study), reductive dechlorination to ethene was a significant mechanism for anaerobic VC degradation in this study (Figure 2). Consistent with previous investigations conducted with these sediments under methanogenic conditions (7, 8), significant production of 14C-ethene was observed (9 ( 1% recovery) but not until well after the onset of 14C-acetate, 14CH4, and 14CO2 production (day 37, Figure 2). The fact that 14C-ethene production followed 14C-acetate accumulation raises the possibility that 14C-ethene was a product of 14C-acetate transformation. However, based on a demonstrated lack of 14C-ethene production in these sediments under 14C-acetate amended, methanogenic conditions (8), the 14C-ethene observed in this study is attributable to reductive dechlorination of [1,2-14C]VC rather than acetate transformation. Moreover, the low rate of 14C-ethene production observed in this study is entirely consistent with numerous reports that microbial reductive dechlorination of VC to ethene is characteristically slow (11-15). The complete inhibition of 14C-ethene production observed in this study under BES amended conditions also is consistent with reductive dechlorination of [1,2-14C]VC to 14C-ethene. BES has been shown to be an effective inhibitor of microbial reductive dechlorination (16). Because acetate can serve as an electron donor for reductive dechlorination of chloroethenes (13, 17, 18), however, the possibility exists that some of the 14C-acetate formed in this study was oxidized during reductive dechlorination of VC. This study has important implications for the current understanding of the microbial ecology of anaerobic VC biodegradation. The fact that acetate is an intermediate of anaerobic VC mineralization indicates that acetogens are responsible for the initial anaerobic degradation of VC to a nontoxic product. This conclusion is consistent with the consensus that acetogens are a metabolically diverse group of microorganisms capable of degrading a wide variety of substrates (19, 20) including chlorinated compounds (2124). Moreover, this conclusion suggests that the subsequent processes of acetotrophic methanogenesis and anaerobic respiration contribute only indirectly to VC degradation by consuming the acetate produced by acetogens and thereby promoting further acetogenesis. Thus the oxidation of VC to
Literature Cited
FIGURE 3. Proposed model for anaerobic degradation of VC to non-chlorinated products. Pathways depicted by open arrows have not been established. non-chlorinated products was significantly lower in the absence of acetotrophic methanogenesis (55 ( 3% final recovery, Figure 2) than in uninhibited experimental microcosms (74 ( 2% final recovery not including 14C-ethene, Figure 2). Likewise, the final recovery of radioactivity as [1,2-14C]VC was 29 ( 4% in the BES amended microcosms, but no [1,2-14C]VC remained in experimental microcosms after 37 days. Based on the cumulative evidence from this and previous studies, a conceptual model for anaerobic microbial degradation of VC to non-chlorinated products can be proposed (Figure 3). According to this model, VC can be reduced to ethene via the ubiquitous but characteristically inefficient process of anaerobic cometabolic reductive dechlorination (25) or via the relatively efficient process of chlororespiration (15). Alternatively, VC can be degraded via oxidative acetogenesis as shown in this study. The acetate formed during oxidative acetogenesis of VC can then be mineralized to CO2 and CH4 via acetotrophic methanogenesis or to CO2 via microbial humic acids reduction or microbial reduction of various inorganic electron acceptors. In addition, it has been suggested that, in the presence of an appropriate terminal electron acceptor, individual microorganisms may be able to directly catalyze the anaerobic oxidation of VC to CO2 (2). In light of the current results, however, further investigation is needed to determine if anaerobic oxidation of VC to CO2 is possible within individual microorganisms or if the process requires the cooperation of multiple metabolic groups as observed in this study.
Acknowledgments We thank Ron Oremland and James Landmeyer for their critical reviews. This investigation 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 December 13, 1999. Revised manuscript received April 3, 2000. Accepted April 17, 2000. ES991371M
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