Role for Acetotrophic Methanogens in Methanogenic Biodegradation

Introduction. Although microbial degradation of vinyl chloride (VC) under methanogenic conditions is well-known (1-9), the extent to which methanogens...
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Environ. Sci. Technol. 1999, 33, 3473-3476

Role for Acetotrophic Methanogens in Methanogenic Biodegradation 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, stream-bed sediment microorganisms rapidly degraded [1,2-14C]vinyl chloride to 14CH and 14CO . Amendment with 2-bromoethanesulfonic 4 2 acid eliminated 14CH4 production and decreased 14CO2 recovery by an equal molar amount. Results obtained with [14C]ethene, [14C]acetate, or 14CO2 as substrates indicated that acetotrophic methanogens were responsible for the production of 14CH4 during biodegradation of [1,2-14C]VC.

Introduction Although microbial degradation of vinyl chloride (VC) under methanogenic conditions is well-known (1-9), the extent to which methanogens contribute to this process remains uncertain (6, 8, 10-12). To date, two VC biodegradation mechanisms have been documented under methanogenic conditions, but recent evidence suggests that neither process requires the presence of methanogens (6, 8, 10-12). The elevated H2 concentrations that characterize methanogenic conditions favor reduction of VC to ethene (3, 8, 13); however, reduction of VC has been demonstrated in the absence of methanogenesis and subsequently attributed to reductive dechlorinating bacteria (10-12, 14). Likewise, microbial oxidation of VC to CO2 has been demonstrated under methanogenic conditions (4-6), but subsequent investigation indicated that the observed oxidation of VC was coupled to humic acids reduction and not dependent on methanogenesis (6). However, recent observations of 14CH4 accumulation during biodegradation of [1,2-14C]VC under methanogenic conditions suggested a possible role for methanogens in VC degradation (15). The research presented in this paper clarifies the role of methanogens in the observed degradation of VC to CH4.

Experimental Results Methanogenic VC degradation was investigated using bed sediments collected from a black-water stream at Naval Air Station (NAS) Cecil Field, FL (Figure 1). The study site and the general methods for microcosm preparation and monitoring have been described in detail previously (4-6, 15, 16). In contrast to previous investigations conducted at this site (4-6, 16), the bed sediment samples used in Figure 1 were collected under springtime, high-flow conditions, and consequently, the leaf litter accumulation and organic content (less than 1% of dry weight) of the sediments were relatively low. Anaerobic microcosms that were prepared with a helium headspace and amended with [1,2-14C]VC demonstrated extensive methane production [33.2 ( 0.2 µmol (L of * Corresponding author telephone: (803)750-6125; fax: (803)7506181; e-mail: [email protected]. 10.1021/es990395q Not subject to U.S. Copyright. Publ. 1999 Am. Chem. Soc. Published on Web 08/27/1999

FIGURE 1. Percentage recovery of [1,2-14C]VC radioactivity as VC and its nonchlorinated products in microcosms containing bed sediments from NAS Cecil Field. Microcosms were 20-mL serum vials containing 15 g of fresh, creek bed sediment, an atmosphere of helium, and approximately 0.5 µCi of [1,2-14C]VC. The initial dissolved VC concentration was 500 µg/L. Analytical methods were described in detail previously (15). Data are means ( SD for triplicate 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%. headspace)-1 day-1] and complete removal of [1,2-14C]VC within 72 days (Figure 1). Periodic examination of relevant geochemical indicators verified that O2, NO3, Fe(III), and SO4 reduction were insignificant under these culture conditions. The removal of [1,2-14C]VC was attributable to biological activity because the decrease in [1,2-14C]VC in control microcosms was only 11% (Figure 1). Degradation of [1,214C]VC was accompanied by immediate and rapid production of equal amounts of 14CH4 and 14CO2. The final recovery of [1,2-14C]VC radioactivity as 14CH4 and 14CO2 was about 21% each. In contrast to previous investigations at this site (4-6, 16), significant production of [14C]ethene was observed, but not until day 49. [14C]Ethene concentrations subsequently declined as [14C]ethene was further reduced to [14C]ethane. By the end of the study, [14C]ethene and [14C]ethane represented about 50% of the recovered radioactivity. These results demonstrate that CH4 can be a significant product of VC biodegradation in methanogenic sediments and suggest that methanogens may be directly involved in this process. To test the hypothesis that methanogens contribute to the degradation of VC to CH4, the effects of 20 mM BES (2bromoethanesulfonic acid, an inhibitor of methanogenesis) on overall methanogenesis and [1,2-14C]VC mineralization were investigated in microcosms containing sediment material collected from the same location at NAS Cecil Field. In contrast to the previous experiment (Figure 1), sediments for this and all subsequent experiments were collected under late summer, low-flow conditions, and consequently, the leaf litter accumulation and organic content (2-5% of dry weight) of the sediments were relatively high. These conditions were similar to those under which samples were collected for previous investigations conducted at this site (4-6, 16). Anaerobic microcosms amended with [1,2-14C]VC demonstrated rapid VC degradation (data not shown) and methane production (Figure 2A). [1,2-14C]VC degradation was VOL. 33, NO. 19, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Percentage Recovery of 14C Radioactivity as 14CO2 and 14CH4 in Bed Sediment Microcosms after 24 Daysa 14C-labeled

substrate [1-14C]acetate [2-14C]acetate H14CO3 14CH 4

[1,2-14C]etheneb

treatment

14CO 2 recovery (%)

14CH 4 recovery (%)

experimental sterile control experimental sterile control experimental sterile control experimental sterile control experimental sterile control

75 ( 2 0(0 55 ( 7 0(0 98 ( 4 100 ( 4 0(0 0(0 0(0 0(0

0(0 0(0 19 ( 5 0(0 2(2 0(0 100 ( 6 99 ( 4 0(0 0(0

a Recoveries are expressed as a percentage of the radioactivity initially added as [1-14C]acetate (carboxyl labeled), [2-14C]acetate (methyl labeled), H14CO3, 14CH4, or [1,2-14C] ethene. Data are means ( SD for triplicate experimental and triplicate autoclaved control microcosms. Approximately 0.1 µCi of 14C-labeled substrate was added to each treatment. [14C]Acetate and H14CO3 (g98% radiochemical purity) were obtained from Sigma Chemicals (St. Louis, MO). [14C]Ethene (g98% radiochemical purity) was obtained from Moravek Biochemicals (Brea, CA). 14CH4 (g99% radiochemical purity) was obtained from NEN Dupont (Boston, MA). b After a 24-day incubation, 19 ( 15% of the radioactivity was recovered as [14C]ethene, and 45 ( 11% was recovered as [14C]ethane in the experimental microcosms. No other radiolabeled products were detected in these microcosms. In the autoclaved control microcosms, 84 ( 2% of the radioactivity was recovered as [14C]ethene. No other radiolabeled products were detected in the autoclaved controls.

FIGURE 2. Effect of 20 mM BES amendment on methanogenesis (A), percentage mineralization of [1,2-14C]VC to 14CH4 (B), and percentage mineralization of [1,2-14C]VC to 14CO2 (C). Microcosms were prepared with approximately 0.1 µCi of [1,2-14C]VC. The initial dissolved VC concentration was 100 µg/L. Methane production was measured by GC-TCD and quantified as nmol/0.5 mL of headspace sample. 14CH and 14CO were quantified as described previously (15). Data 4 2 are means ( SD for triplicate experimental, BES-amended, and sterile control microcosms. accompanied by a concomitant production of 14CH4 and 14CO (Figure 2B,C). After 24 days, the recovery of [1,2-14C]VC 2 radioactivity as 14CH4 was 14 ( 2% (Figure 2B), and the recovery as 14CO2 was 63 ( 3% (Figure 2C). Addition of 20 mM BES completely inhibited 14CH4 production and decreased 14CO2 accumulation by an equal amount (13 ( 2%). The fact that the recovery of 14CO2 remained high (51 ( 2%) even with BES amendment demonstrated that a nonmethanogenic mechanism was primarily responsible for oxidation of [1,2-14C]VC to 14CO2 (Figure 2C). This observation is consistent with a previous investigation which demonstrated that, under methanogenic conditions, microbial oxidation of [1,2-14C]VC to 14CO2 in these bed sediments was coupled primarily to humic acids reduction and not dependent on methanogenesis (6). No [14C]ethene or [14C]ethane accumulation was observed in this experiment (data not shown). The complete inhibition of 14CH4 accumulation and the stoichiometric decrease in 14CO2 recovery in this experiment indicated that methanogens were responsible for the observed production of 14CH4 from [1,2-14C]VC and confirmed that the mechanism involved in this process yielded equimolar amounts of 14CH4 and 14CO2. The fact that 14CH4 production in the first experiment (Figure 1) was immediate and preceded [14C]ethene accumulation by at least 24 days indicated that the observed 14CH was not the result of [1,2-14C]VC degradation to [14C]4 ethene followed by reduction to 14CH4. The immediate accumulation of 14CH4 and the absence of detectable [14C]ethene or [14C]ethane accumulation in the second experiment (Figure 2) were consistent with this conclusion. To test the hypothesis that [14C]ethene was not the precursor of 14CH4, 3474

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an anaerobic microcosm study was conducted using [1,214C] ethene as a substrate. [14C]Ethene was rapidly degraded to [14C]ethane under methanogenic conditions with 19 ( 15% of the radioactivity recovered as [14C]ethene and 45 ( 11% recovered as [14C]ethane after 24 days (Table 1). No 14CH or 14CO accumulation was observed in this study (Table 4 2 1). These results indicate that [14C]ethene was not the precursor for the 14CH4 observed in Figures 1 and 2. Methanogens have been shown to metabolize only a limited number of carbon substrates and have been separated into two nutritional groups based upon their characteristic utilization of inorganic versus organic carbon substrates (17). Autotrophic methanogens utilize the inorganic substrates, CO2 and H2, according to the general equation:

CO2(aq) + 4H2(aq) f CH4(aq) + 2H2O(l)

(1)

∆G°′ ) -192.9 kJ/mol CH4 In contrast, heterotrophic methanogens produce methane by cleaving the methyl group from organic substrates (methanol, methylamines, and acetate). In surface water sediments, acetate is the primary substrate for methanogenesis and is estimated to account for 70% of the CH4 produced in these systems (18, 19). Acetotrophic methanogenesis can be described as

CH3COO-(aq) + H+(aq) f CH4 (aq) + CO2(aq) (2) ∆G°′ ) -51.1 kJ/mol CH4 An assessment of the potential contribution of these two processes to microbial degradation of [1,2-14C]VC to 14CH4 was made by examining the ability of Cecil Field, bed sediment microorganisms to produce 14CH4 via autotrophic methanogenesis of 14CO2 or acetotrophic methanogenesis of [14C]acetate. The fact that 14CH4 and 14CO2 were formed concomitantly and without an apparent lag (Figures 1 and 2) suggested that the observed 14CH4 accumulation was not the result of microbial oxidation of [1,2-14C]VC to 14CO2 followed by autotrophic methanogenesis. To test this hypothesis, metha-

nogenic microcosms were prepared with 14CO2 as the substrate. In contrast to the [1,2-14C]VC degradation experiments (Figures 1 and 2B), no 14CH4 accumulation was observed in 14CO2-amended microcosms during the first 10 days of incubation (data not shown). By day 24, trace amounts of 14CH4 were recovered in one experimental microcosm, but the mean recovery for the triplicate experimental microcosms was not significant (2 ( 2%; Table 1). In contrast, the accumulations of 14CH4 in the two [1,2-14C]VC degradation experiments were 19 ( 2% (Figure 1) and 14 ( 2% (Figure 2B) by 24 days. The lack of significant 14CH4 accumulation in 14CO2-amended microcosms and the immediate recovery of 14CH4 in [1,2-14C]VC-amended microcosms over the same incubation period demonstrated that autotrophic methanogenesis was not responsible for the rapid production of 14CH4 observed in this study during [1,2-14C]VC biodegradation. A number of observations suggest that the mechanism by which 14CH4 is produced during [1,2-14C]VC biodegradation may involve acetotrophic methanogenesis. Analysis of water samples from Cecil Field during late summer indicated that the creek water and bed sediment interstitial water contained dissolved acetate at concentrations of 200 ( 10 and 470 ( 20 µM, respectively. This observation indicates that an active community of acetogens is present in the Cecil Field bed sediments and suggests that the bed sediment environment may support significant acetotrophic methanogenesis. In addition, acetate is the only two-carbon (carbon linked) compound known to serve as a substrate for methanogenesis (17-19). Finally and most importantly, acetate is the only known methanogenic substrate that characteristically yields equal amounts CH4 and CO2 (17, 19). Likewise, biodegradation of [1,2-14C]VC under methanogenic conditions produced equal amounts of 14CH4 and 14CO2 in the first experiment of this study (Figure 1). Production of equal amounts of 14CH4 and 14CO2 were also attributable to methanogen-mediated (BES-inhibited) [1,2-14C]VC degradation to 14CH4 in the second experiment (Figure 2B,C). To test the hypothesis that microorganisms capable of acetotrophic methanogenesis are present in the bed sediments at NAS Cecil Field, anaerobic microcosm studies were initiated using [1-14C]acetate (carboxyl labeled) and [2-14C]acetate (methyl labeled). In microcosms amended with [1-14C]acetate, 75 ( 2% of the added radioactivity was recovered as 14CO2 within 24 days (Table 1). The total mineralization observed in microcosms amended with [2-14C]acetate was the same (74 ( 7%) with 55 ( 7% and 19 ( 5% of the initial radioactivity recovered as 14CO2 and 14CH4, respectively (Table 1). During acetotrophic methanogenesis, the methyl group is the precursor of CH4, while the carboxyl group is oxidized to CO2 (19). Thus, the difference in 14CO2 recovery between [1-14C]acetate- and [2-14C]acetate-amended microcosms reflects the contribution of methanogens (about 20%) to the recovery of 14CO2 in the [1-14C]acetate microcosms. The 55 ( 7% recovery of [2-14C]acetate radioactivity as 14CO2 indicates that nonmethanogenic mechanisms were primarily responsible for acetate oxidation in these sediments. This observation is consistent with a previous demonstration that oxidation of organic compounds coupled to humic acids reduction is significant in these sediments under methanogenic conditions (6). Oxidation of acetate coupled to humic acids reduction has been demonstrated previously (20). The immediate mineralization of [14C]acetate to 14CH4 and 14CO2 observed in this study confirmed that acetotrophic methanogens are active in these sediments. The existence of acetotrophic methanogens in these sediments is consistent with the hypothesis that the mechanism of [1,2-14C]VC degradation to 14CH4 involves acetotrophic methanogenesis. Although these results demonstrate that microbial degradation of VC to CH4 and CO2 can occur and indicate that

methanogens play a pivotal role, additional investigation is required to identify the responsible organism(s), specific mechanism, and important intermediates of the process. Taken in their entirety, however, the results of this study do suggest a general mechanism. The accumulation of acetate in the bed sediments at Cecil Field, the presence of acetotrophic methanogens in these sediments, the concomitant production of equal amounts of 14CH4 and 14CO2 during [1,2-14C]VC degradation, the demonstrated lack of significant autotrophic methanogenesis during the study period, and the inhibition of methanogenesis and [1,2-14C]VC degradation to 14CH4 in BES-amended treatments indicate that acetotrophic methanogenesis is the final step in the biodegradation of VC to CH4. This hypothesis in turn suggests coupling the following general reactions to yield acetate as an intermediate product and CH4 and CO2 as the final products of VC biodegradation:

CH2CHCl(g) + 2H2O(l) f CH3COO-(aq) + H2(aq) +

2H+(aq) + Cl- (aq) (3)

∆G°′ ) -62.4 kJ/mol CH3COO-(aq) + H+(aq) f CH4(aq) + CO2(aq) (2) ∆G°′ ) -51.1 kJ/mol These reactions lead to an overall equation that is consistent with the observed microbial degradation of VC to equal amounts CH4 and CO2:

CH2CHCl(g) + 2H2O(l) f CH4(aq) + CO2(aq) + H2

(aq) + H+(aq) + Cl-(aq) (4)

∆G°′ ) -113.4 kJ/mol CH4 It is noteworthy that both steps in this hypothetical mechanism are exergonic and have the potential for supporting microbial energy production and growth. A preliminary assessment of acetate as an intermediate in the biodegradation of VC to CH4 was made by analyzing bed sediment interstitial water from [1,2-14C]VC-amended microcosms for the presence of radiolabeled acetate. Samples collected following the conclusion of the [1,2-14C]VC biodegradation study shown in Figure 2 were immediately stored at 4 °C and subsequently analyzed by ion exclusion HPLC. Eluant fractions corresponding to the acetate elution window were collected and analyzed for radioactivity using liquid scintillation counting. As much as 2.8% of the radioactivity initially added as [1,2-14C]VC was recovered as [14C]acetate in experimental microcosms. No significant difference was observed between BES-amended and unamended treatments. No [14C]acetate was detected in autoclaved control microcosms. While the presence of [14C]acetate in experimental microcosms and the absence of [14C]acetate in control microcosms are consistent with the hypothesis that acetate is an intermediate in VC biodegradation to CH4, the recovery of [14C]acetate in this study was less than the impurity level of the [1,2-14C]VC and must be considered inconclusive. These results suggest a previously unrecognized pathway for VC biodegradationsVC transformation to acetate followed by acetotrophic methanogenesissthat may contribute to VC bioremediation in a variety of hydrologic sysems. Further investigation of this process and its significance at other VCcontaminated sites is warranted.

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Received for review April 7, 1999. Revised manuscript received July 12, 1999. Accepted July 30, 1999. ES990395Q