APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1999, p. 327–329 0099-2240/99/$04.0010 Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Vol. 65, No. 1
2-Bromoethanesulfonate, Sulfate, Molybdate, and Ethanesulfonate Inhibit Anaerobic Dechlorination of Polychlorobiphenyls by Pasteurized Microorganisms DINGYI YE,1,2* JOHN F. QUENSEN III,1 JAMES M. TIEDJE,1,3
AND
STEPHEN A. BOYD1
Department of Crop and Soil Sciences1 and Center for Microbial Ecology,3 Michigan State University, East Lansing, Michigan 48824, and Department of Biology, Hong Kong Baptist University, Hong Kong, People’s Republic of China2 Received 17 June 1998/Accepted 15 October 1998
Dechlorination of Aroclor 1242 by pasteurized microorganisms was inhibited by 2-bromoethanesulfonate (BES), sulfate, molybdate, and ethanesulfonate. Consumption of these anions and production of sulfide from BES were detected. The inhibition could not be relieved by hydrogen. Taken together these results suggest that pattern M dechlorination is mediated by spore-forming sulfidogenic bacteria. These results also suggest that BES may inhibit anaerobic dechlorination by nonmethanogens by more than one mechanism. extracted, and analyzed by capillary gas chromatography with an electron capture detector as previously described (14). No methane production was detected in any pasteurized slurries as previously reported (22), indicating no growth of methanogens (5). Elimination of methanogens was also established by the following: (i) we previously reported that the Hudson River pasteurized microorganisms survived not only pasteurization but also ethanol treatment, which should eliminate thermophilic methanogens (22), and (ii) no methane was detected in triplicate pasteurized slurries containing no PCBs after 4 months of incubation (data not shown), ruling out the unlikely possibility that some thermophilic methanogens happened to survive the pasteurization and that methane was not detected due to a shift of electron flow to dechlorination. In this preliminary inhibition experiment (Fig. 1), the initial concentrations of BES and molybdate were 50 and 5 mM, respectively. To replenish the inhibitors, the same amounts of molybdate and half as much BES were refed at 2, 4, 6, and 8 weeks. The initial concentration of sulfate was 20 mM, and the same amount was added at 4 and 8 weeks. The dechlorination pattern observed was the typical metapreferential dechlorination pattern M (2). Dechlorination was completely inhibited in bottles with all three anions throughout 12 weeks of incubation (Fig. 1). The slurries amended with either sulfate or BES turned dark black after 2 weeks of incubation. Additionally, these slurries emitted a strong sulfide smell when they were sampled while being flushed with N2CO2. In contrast, slurries amended with molybdate were yellowish and did not emit the sulfide smell when sampled. Inhibitor fate and concentration effect. Since BES, sulfate, and molybdate inhibited the dechlorination in the preliminary experiment, further experiments were done to evaluate the dose-response relationship and to determine whether the inhibitors were metabolized. Two types of experiments were conducted; both were prepared in the same way as the preliminary inhibition experiment except as follows: the inocula were taken from a culture previously pasteurized at 90°C for 15 min and retained the pattern M dechlorination activity, and then they were repasteurized at 90°C for 10 min before inoculation; the experimental vessels were 28-ml serum tubes (Bellco Glass Inc., Vineland, N.J.); each tube received 1 g of sediment, 4.5 ml of RAMM, and 0.5 ml of inoculum; the concentration of Aroclor 1242 was 500 mg/g of dry sediment; and the tubes were
Effects of 2-bromoethanesulfonate (BES), sulfate, and molybdate on dechlorination of polychlorinated biphenyls (PCBs) have been reported (for a review, see reference 2) and were also investigated in our previous studies with nonpasteurized microorganisms (21). However, in all these studies, the microbial communities contained methanogens. Due to the complicated relationships between methanogens and sulfidogens (9, 10, 17, 20), it is usually difficult to interpret the results. In this study pasteurization eliminated methanogens and still retained a partial dechlorination activity (pattern M [2]), thus simplifying the dechlorinating community. Therefore, we investigated the effects of the same anions on PCB dechlorination by microorganisms that withstood repeated pasteurization. Information from such inhibition study should provide some information about the composition of the dechlorinating community and consequently facilitate isolation of the PCB-dechlorinating microorganisms. Preliminary inhibition experiment. The inoculum was collected from site H7 sediments, upper Hudson River, N.Y. (3). Inoculum preparation and pasteurization were as described elsewhere (22). Each 60-ml serum bottle contained 10 g of PCB-free Hudson River sediments and was prepared as previously described (22). The final volumes of the revised anaerobic mineral medium (RAMM) (16) and inoculum in each bottle were 20 and 10 ml, respectively, and the final concentration of PCBs (Aroclor 1242; Monsanto Co., St. Louis, Mo.) was 800 mg/g of dry sediment. Stock solutions of BES, sulfate, and molybdate (all were sodium salts) were bubbled with N2, autoclaved, and then introduced. The controls were autoclaved twice, 1 h each time with an interval of incubation at 37°C for 5 h before PCBs were added. After addition of PCBs the samples were shaken for 1 h and then incubated at 25°C in the dark. Methane production was determined by gas chromatography with a flame ionization detector (23). The headspace gas was analyzed to measure methane production after a culture was shaken and before the slurry was sampled for PCB analysis (23). To analyze PCBs, 2 ml of the sediment slurry was shaken,
* Corresponding author. Mailing address: Department of Biology, Hong Kong Baptist University, 224, Waterloo Rd., Kowloon Tong, Hong Kong, People’s Republic of China. Phone: (852) 2339 7062. Fax: (852) 2336 1400. E-mail:
[email protected]. 327
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FIG. 1. Inhibition by BES, sulfate, and molybdate of anaerobic dechlorination of Aroclor 1242 by pasteurized microorganisms. The error bars indicate standard deviations of triplicate samples. The concentrations of the anions are given in the text.
incubated at 30°C instead of 25°C. Inhibitor concentrations were as plotted in Fig. 2 and 3. In the experiment shown in Fig. 2, sulfate and molybdate amended at 4 mM were analyzed by high-pressure ion chromatography using an HPIC AS4A column (20 cm) and a conductivity detector. The mobile phase was 1.7 mM NaHCO3–1.8 mM Na2CO3, and the flow rate was 2.3 ml/min. Before the cultures were sampled, the tubes were vortexed for 10 min. After the sediments had settled, the headspace gas was analyzed for methane production, and then 1 ml of the liquid portion was withdrawn while the vessel was flushed with N2. The liquid samples were acidified with 1 N HCl while being bubbled with N2 for 5 min to drive out H2S. The samples were then centrifuged, filtered (0.22-mm-pore-size filters; Millipore Co., Bedford, Mass.), and analyzed for sulfate and molybdate. In the experiment shown in Fig. 3, BES and ethanesulfonate were analyzed by high-pressure liquid chromatography according to the method described by Lo ¨ffler et al (8). Both BES and sulfate completely inhibited the dechlorination at concentrations of $1 mM (Fig. 2 and 3). Statistical analysis (analysis of variance; data not shown) showed that there was no significant difference in molybdate inhibition at 2, 4, and 8 mM. Ion chromatography results showed that after 4 weeks of incubation, sulfate and molybdate in the 4 mMamended group decreased 23.1 and 27.7%, respectively, com-
FIG. 2. Effects of BES, sulfate, and molybdate at different concentrations ($1 mM) on anaerobic dechlorination of Aroclor 1242 by the pasteurized microorganisms. The error bars indicate standard deviations of triplicate samples.
APPL. ENVIRON. MICROBIOL.
FIG. 3. Effects of BES, sulfate, molybdate, and ethanesulfonate at different concentrations (#1 mM) on anaerobic dechlorination of Aroclor 1242 by the pasteurized microorganisms. The error bars indicate standard deviations of triplicate samples.
pared to their killed-cell controls. Differences in the color of the slurries amended with sulfate, BES, and molybdate were also observed. Unlike the nonamended group, the 8 and 16 mM sulfate-amended slurries and the 16 mM BES-amended slurry turned black, while the molybdate-amended slurry was yellowish. This observation was consistent with that in the preliminary inhibition experiment and has also been observed in similar experiments with nonpasteurized microorganisms (21). Molybdate partially inhibited the dechlorination at a lower concentration (1 mM) and did not inhibit the dechlorination at concentrations of #0.5 mM (Fig. 3). The inhibitory effects of sulfate and BES also decreased with a decrease in concentrations, and no inhibitory effects were observed when their concentrations were #0.1 mM. In this experiment, ethanesulfonate, a structural analog of BES, also inhibited the dechlorination and no significant difference in the inhibitory effect between ethanesulfonate and BES was observed. Concentrations of BES and ethanesulfonate used for amendment at 1 mM were assayed with high-pressure liquid chromatography and were found to have decreased by 47.1 and 42.3%, respectively, compared to their killed-cell controls. In the BESamended samples, no ethanesulfonate, the potential debromination product of BES, was detected. To examine whether the inhibition could be relieved by hydrogen, an additional eight samples were prepared with 60-ml serum bottles, instead of 28-ml tubes, to increase the capacity for headspace gas, and were amended with (in duplicate) either 1 mM sulfate, 1 mM BES, 1 mM ethanesulfonate, or 16 mM molybdate. These samples were flushed with H2-CO2 (80:20) twice a week to completely displace the headspace gas. Inhibition of the dechlorination could not be relieved by replenishment of hydrogen. Organosulfonates have been reported to be used as electron acceptors (6, 7, 15), and reduction of organosulfonate to sulfide has also been documented (6). To determine whether the sulfonic moiety of BES may be reduced to sulfide, an experiment similar to that described by Ha¨ggblom and Young (4) was performed with the following modifications: the experimental vessels were 28-ml serum tubes containing 4.5 ml of medium (the freshwater medium described in reference 4) and 0.5 ml of inoculum; sulfide was quantified by the methylene blue method (19); H2S was driven off by nitrogen; and the incubation time was 3 weeks. The freshwater growth medium was modified as follows: Na2SO4 was replaced by 2 mM BES; 1.5 mM Na2S was
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BES also inhibits anaerobic aromatic dechlorination by nonmethanogens. In our case, however, production of sulfide from BES was detected; this is the first report confirming reduction of the sulfonic moiety of BES to sulfide. It appears that BES may inhibit anaerobic dechlorination by nonmethanogens by more than one mechanism.
FIG. 4. Amount of sulfide S recovered from different amendment cultures. The error bars indicate standard deviations of triplicate samples.
This work was supported in part by the General Electric Co., Michigan State University Institute for Environmental Toxicology, the Great Lakes and Mid-Atlantic Hazardous Substance Research Center of EPA, the SERDP Bioconsortium, and Hong Kong Baptist University. We thank Linda Schimmelpfennig for technical assistance. Investigations of ethanesulfonate and hydrogen were suggested by reviewers, and we appreciate this suggestion.
reduced to 0.5 mM; ascorbic acid was added at concentration of 0.1 g/liter; a mixture of lactate, pyruvate, and acetate (1 g of each per liter; all were sodium salts) was chosen as the substrate; the headspace gas was H2-CO2 (80:20); and the trace elements and vitamins were replaced by those used in RAMM (16). A molybdate (20 mM)-inhibited BES-containing culture and a culture without BES served as controls. The amount of sulfide S recovered from the BES-amended culture was more than doubled the 72 mg of sulfide S from Na2S (reductant in the medium) (Fig. 4). This result proved production of sulfide from BES. Results of the inhibitor concentration experiments suggest that the inhibition of the dechlorination by BES, sulfate, molybdate, and ethanesulfonate was probably not due to general toxic effects of these anions. Effects of BES on a wide variety of microorganisms, including the spore-formers genera Clostridium and Bacillus and different types of anaerobes, were investigated, and 25 mM BES had no significant side effect (12, 18). Similarly, it has also been documented that ;2 mM molybdate is not toxic (10). In our experiment, 1 mM BES completely inhibited dechlorination and 1 mM molybdate partially inhibited the dechlorination. The effective concentration of molybdate in the slurries should have been even lower because some molybdate should have adsorbed onto the clay surfaces (13) present in the sediment slurries and become nonbioavailable. The dechlorination was also completely suppressed by 1 mM sulfate, and general toxicity of sulfate at this concentration has never been reported. Both the bromide moiety and the sulfonic moiety of BES are potential electron acceptors (7, 11, 15) and may compete with PCBs for electrons (11). In our experiment, (i) no debromination product (ethanesulfonate) was detected in the BESamended samples, (ii) reduction of the sulfonic moiety to sulfide was detected, and (iii) ethanesulfonate also inhibited dechlorination. Based on these observations, we suggest that the inhibition is mainly due to the sulfonic moiety. Apparently the tested anions inhibited dechlorination by selectively affecting certain target microorganisms. Molybdate, a specific inhibitor of sulfate-reducing bacteria (SRB), is able to cause the death of SRB by rapidly depleting their ATP pools (12). Sulfate is a electron acceptor of SRB, while both BES and ethanesulfonate are potential electron acceptors of SRB, and production of sulfide from BES was detected in this experiment. Together, these data suggest that pattern M dechlorination is mediated by spore-forming sulfidogens. BES has long been regarded as a specific inhibitor of methanogens (1, 12). Recently, Lo ¨ffler et al. reported that BES inhibited dechlorination of chloroethanes in the absence of methanogens, but no change in BES was observed under their experimental conditions (8). Our results provide evidence that
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