Environ. Sci. Technol. 1998, 32, 2556-2563
Bacterial Methylmercury Degradation in Florida Everglades Peat Sediment MARK C. MARVIN-DIPASQUALE* AND RONALD S. OREMLAND U.S. Geological Survey, Water Resources Division/MS 480, 345 Middlefield Road, Menlo Park, California 94025
Methylmercury (MeHg) degradation was investigated along an eutrophication gradient in the Florida Everglades by quantifying 14CH4 and 14CO2 production after incubation of anaerobic sediments with [14C]MeHg. Degradation rate constants (k) were consistently e0.1 d-1 and decreased with sediment depth. Higher k values were observed when shorter incubation times and lower MeHg amendment levels were used, and k increased 2-fold as in-situ MeHg concentrations were approached. The average floc layer k was 0.046 ( 0.023 d-1 (n ) 17) for 1-2 day incubations. In-situ degradation rates were estimated to be 0.02-0.5 ng of MeHg (g of dry sediment)-1 d-1, increasing from eutrophied to pristine areas. Nitrate-respiring bacteria did not demethylate MeHg, and NO3- addition partially inhibited degradation in some cases. MeHg degradation rates were not affected by PO43- addition. 14CO production in all samples indicated that oxidative 2 demethylation (OD) was an important degradation mechanism. OD occurred over 5 orders of magnitude of applied MeHg concentration, with lowest limits [1-18 ng of MeHg (g of dry sediment)-1] in the range of in-situ MeHg levels. Sulfate reducers and methanogens were the primary agents of anaerobic OD, although it is suggested that methanogens dominate degradation at in-situ MeHg concentrations. Specific pathways of OD by these two microbial groups are proposed.
Introduction High levels of the neurotoxin monomethylmercury (MeHg) have been reported for the biota of the Florida Everglades (1-5). The major source of mercury (Hg) to this aquatic system is atmospheric deposition of inorganic Hg(II) (6), much of which is initially deposited to the sediment as it readily adsorbs to settling particles (7). Inorganic mercury methylation (MeHg production) is subsequently carried out by sulfate-reducing bacteria (SRB) within the anoxic sediment (8, 9). However, not all of the MeHg produced is translocated out of the sediment and into local food chains. Some fraction is microbially degraded within the sediment or is buried. Thus, whether particular sediments are a net source or sink of MeHg depends on the relative rates of sediment accumulation, mercury methylation, and MeHg degradation (10, 11). While many studies of environmental Hg cycling have assessed mercury methylation, far fewer have included MeHg degradation. As a result, less is known about the latter process in natural systems. * Corresponding author phone: 650-329-4442; fax: 650-329-4463; e-mail:
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There are two known pathways for microbial MeHg demethylation, namely, via organomercurial-lyase (OML) (12) and oxidative demethylation (OD) (13). The mer-B operon encodes for the OML enzyme that forms CH4 and Hg2+ from MeHg. Both methanogens and sulfate reducers have been shown to be involved in OD, the defining characteristic of which is the oxidation of the MeHg methyl group to CO2, either with or without concurrent CH4 production (13, 14). However, the specific pathway invoked by each bacterial group and their relative contribution to observed end product gas ratios is still unclear. It is also unknown under which environmental conditions either OML or OD dominates at in-situ MeHg concentrations. The current research is part of the larger multi-investigator ACME (Aquatic Cycling of Mercury in the Everglades) program being conducted in south Florida. In this paper we report on MeHg degradation within Everglades anoxic sediments, including the detection of OD at in-situ MeHg concentrations, a linear response of degradation rates as a function of applied MeHg concentration, and low (95%. Similarly, the recovery (as 14CO2) of >95% of radiolabled [H]14CO2 added to autoclaved sediment samples verified that the HCl addition was sufficient to release essentially all 14CO2 formed from [14C]MeHg. This CH4 combustion-CO2 trapping (C-T) method had a 14C detection limit of 0.05 nCi sample-1, which was 2 orders of magnitude lower than the GC-GPC method. A direct comparison of the two methods was conducted with site 2B sediment using two replicate (n ) 3) sample sets that were amended with 62 nCi of [14C]MeHg (mL of wet sediment)-1 and incubated for 11 days. ANOVA was used to determine if the measured amount of 14CH4 and 14CO2 produced during the incubation was the same for the two methods. VOL. 32, NO. 17, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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July and December 1995 samples were assayed using low specific activity [14C]MeHgI (3.7 mCi mmol-1; ICN Radiochemicals). Higher specific activity [14C]MeHgI (54 mCi mmol-1; Amersham) was used during December 1996 and April 1997. The combined use of the C-T method and the higher specific activity radiotracer allowed us to incubate samples for much shorter time intervals (1-2 days) than via GC-GPC and to decrease the quantity of applied MeHg 3 orders of magnitude to 2 ng of MeHg (mL wet sediment)-1. This amendment level is about 4-100 times greater than in-situ concentrations considering the range of 0.2-5 ng of MeHg (g of dry sediment)-1 reported by Gilmour et al. (9) for the same floc sediments and assuming an average sediment bulk density of 0.1 g of dry sediment (mL of wet sediment)-1. Concentration Dependence. MeHg degradation in floc layer sediment (sites F1 and U3) was studied over a range of applied [14C]MeHg concentrations [2, 10, 31, and 155 ng of MeHg (mL of wet sediment)-1] during December 1996. Sample setup, incubation, replication, and analysis (via the C-T method) was conducted as previously described. Concentration dependence was again examined in April 1997 using site HS floc sediment and lower amendments [0.1, 0.4, 0.9, and 1.7 MeHg (mL of wet sediment)-1] (1-18 MeHg (g of dry sediment)-1), which were achieved by increasing the sediment volume to 80 cm3 per sample. Duplicate samples per treatment were run, and the flushing times used during the postincubation extraction of 14CH4 and 14CO2 were increased to 30 min per fraction. Values of k were calculated from the linear regression slopes of degradation rates versus applied MeHg concentrations.
Results Spatial and Temporal MeHg Degradation. Potential rates of MeHg degradation generally decreased with sediment depth, and surface floc sediment rates increased from nutrient enriched to less eutrophied sites along the north to south (ENR f 2Bs) sampling transect (Figure 2). Rates measured during December 1996 were 3 orders of magnitude lower than those measured during July and December 1995 due to the decrease in applied [14C]MeHg concentrations. Spatial trends in the corresponding 14CO2/14CH4 ratios typically paralleled degradation rates and were consistently 1 at all sites sampled. Both BES and MoO42- decreased total MeHg degradation as compared with controls. MoO42partially or totally inhibited 14CO2 production, and thus decreased 14CO2/14CH4 ratios at all sites.
Discussion Degradation Rates and Rate Constants. The observed increase in floc layer MeHg degradation rates along the ENR f 2Bs eutrophication transect (Figure 2) was primarily due to a decrease in sediment bulk density and a corresponding increase in applied MeHg concentration (Figure 3), as values of k in floc sediment exhibit no distinct spatial trend along this transect (Figures 3 and 4). Increases along this eutrophication gradient have been reported for in-situ MeHg sediment concentrations and mercury methylation rates (9), as well as total Hg concentrations in sediment (24), water (7), and biota (4). The low sediment Hg concentrations in cattail-dominated areas were shown to be a consequence of the higher sedimentation rates, which dilutes aeolian-derived Hg, in eutrophied relative to pristine areas (24). In a similar way, our standard MeHg additions were diluted with increased sediment particulate matter at eutrophied sites.
FIGURE 2. Sediment depth profiles of MeHg degradation rates and the corresponding 14CO2/14CH4 ratios for sites along the major northsouth eutrophication gradient (ENR f 2Bs). Error bars for potential rates represent (1 standard error (n ) 3). The Y-coordinate represents the interval midpoint depth. The horizontal dashed line represents the transition zone between surface floc sediment and underlying peat sediment. Since calculated potential rates depend on the MeHg amendment concentration and vary as a function of sediment bulk density, it may be more useful to consider how k values alone varied among sites and sampling dates. The increase in k values between the 1995 samplings and the subsequent dates (Figure 4) was presumably due to the switch from the GC-GPC to the C-T method. The long incubation times
needed for the former approach likely resulted in a number of bottle effects, including the buildup of metabolites, sulfate and nutrient depletion, and shifts in microbial populations. In addition, high amendment levels may also have resulted in enrichment of MeHg degrading bacteria in 1995 samples, or conversely, some degree of microbial Hg toxicity as suggested by the consistent temporal trend in floc k VOL. 32, NO. 17, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Sediment depth profiles of MeHg degradation rate constants (k), sediment bulk density, and applied MeHg concentrations along the ENR f 2Bs eutrophication transect during December 1996. The Y-coordinate represents the interval midpoint depth. The horizontal dashed line represents the transition zone between surface floc sediment and underlying peat sediment. Error bars indicate (1 standard deviation for both k values (n ) 3) and bulk density measurements (n ) 2).
FIGURE 4. MeHg degradation rate constants (k) measured in variable depth floc layer sediment for all sites and sampling periods. Sites are arranged from north to south (left to right) on the X-axis. Error bars indicate (1 standard deviation (n ) 3). (December 1995 < July 1995 < December 1996) (Figure 4), which corresponded to standard MeHg additions of 4480, 1280, and 2 ng of MeHg (mL of wet sediment)-1, respectively. The lower k values for 1995 may also reflect the longer incubation if the degradation rate was not constant with time but actually decreased or became zero after the initial pool of bioavailable [14C]MeHg was consumed. The existence of multiple MeHg fractions, with differing microbial availability, is likely since MeHg has been shown to bind and/or complex with dissolved and particulate organic matter (25), metal oxides, and clays (26) to varying degrees. Our results illustrate the importance of using both incubation times and MeHg amendment concentrations as small as practical in making these types of measurements. Thus, the floc layer average k (0.046 ( 0.023 d-1), calculated from C-T data only, 2560
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may be more accurate than the lower estimate (0.037 ( 0.025 d-1), which includes 1995 (GC-GPC) data for both the reasons above and because of the noted underestimated 14CO2 concentrations measured via by GC-GPC. Values of k were consistently e0.1 d-1 (Figures 4-6) for all sites, depths, and sampling periods over a very large range of applied concentrations [1-200 000 ng of MeHg (g of dry sediment)-1]. However, in controlled amendment experiments, k values increased 2-fold as in-situ MeHg concentrations were approached (Figure 4). Thus, the k values derived from our nontracer level standard MeHg additions may underestimate actual k by a factor of at least 2. Consequently, the above estimate of the floc layer average k would be closer to 0.1 d-1. Gilmour et al. (9) reported a range of floc sediment in-situ MeHg concentrations of roughly 0.2-5 ng of MeHg
FIGURE 6. Individual treatment degradation rate constants (k) as a function of applied MeHg concentration for December 1996 and April 1997 floc sediment concentration amendment experiments.
FIGURE 5. Floc layer sediment MeHg degradation rates versus applied MeHg concentration. The linear least squares regression line (dashed) and the corresponding 14CO2/14CH4 ratios are shown. Error bars represent (1 standard error (n ) 3 for December 1996, n ) 2 for April 1997). When absent, error bars were smaller than the symbol. The degradation rate constant (k) (i.e., regression line slope) and regression r 2 value are inset for each case. (g of dry sediment)-1, which increased along the ENR f 3A15 transect. Multiplying this range by an assumed constant average k of 0.1 d-1, we estimate that MeHg degradation rate increased from 0.02 to 0.5 ng of MeHg (g of dry sediment)-1 d-1 along the same transect. Comparing these values to MeHg production rates [1-10 ng of MeHg (g of dry sediment)-1 d-1] (9), we conclude that MeHg production exceeds MeHg degradation in Everglades floc sediments. However, the degree to which these processes vary seasonally is unclear since only one field sampling (December 1996) presented here used freshly collected sediment and low MeHg amendments. Nutrients. While PO43- is the nutrient limiting macrophyte primary production in the Everglades (17, 20), its addition had no affect on MeHg degradation rates at any site (Figure 7), indicating that the bacteria involved in MeHg degradation were not PO42- limited. Similarly, NO3- addition did not stimulate degradation, indicating that nitraterespiring bacteria were not directly involved in MeHg degradation. However, the decrease in rates at 2Bs and an increase in 14CO2/14CH4 ratios at F1 and U3 suggest that NO3may play a role in controlling the magnitude and pathway of MeHg degradation. Drake et al. (27) reported an inhibitory affect of NO3- on methanogenesis in Everglades sediment,
and this phenomenon is common in benthic systems (28). Thus, in environments where concentrations are high, NO3may exert a secondary influence on MeHg degradation by inhibiting methanogens and possibly sulfate reducers. An inhibition of mercury methylation due to NO3- has been previously noted in experiments with lake sediment (29), although the NO3- concentrations that resulted in inhibition greatly exceeded in-situ levels. Oxidative Demethylation. [14C]MeHg degradation via OD was observed during each sampling period, at all sites, and over the full range of applied MeHg concentrations. Although a linear response of demethylation rates to MeHg concentrations has been previously reported (11, 23), the current results represent the first evidence of this relationship for OD (Figure 5). Furthermore, the April 1997 amendments [1-18 ng of MeHg (g of dry sediment)-1] represent the lowest concentrations, reported to date, for which OD has been shown to occur and fall within the range of in-situ MeHg concentrations reported for these same sites (9). Pak and Bartha (30) recently confirmed the involvement of both SRB and methanogens in MeHg degradation using pure cultures and suggested that the production of 14CO2 from [14C]MeHg may represent the enzymatically catalyzed mercury methylation reaction (31) running in reverse. While 14CO production provides evidence that some degree of OD 2 had occurred, the use of 14CO2/14CH4 ratios in determining the specific pathways of MeHg degradation is complicated by a number of unknowns, namely, What fraction of 14CH4 was attributable OML degradation as opposed to methanogen-linked OD? What was the relative contribution of SRB and methanogens to MeHg degradation in the natural environment? Do these two bacterial groups degrade MeHg in the same fashion and with the same stoichiometric end product ratios? The CH4 production profiles indicated that the most active zone of methanogenesis was within or just below the organicrich floc layer (Marvin-DiPasquale, unpublished). The floc layer also had the highest rates of sulfate reduction (9). Hence, both methanogens and SRB were active in the zone of most rapid mercury methylation and highest in-situ MeHg concentration (9). While SRB will outcompete methanogens for acetate and H2 (32, 33), the coexistence of both groups within VOL. 32, NO. 17, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 7. Nutrient and inhibitor amendment experiments (December 1995). Bars represent the average 0-8 cm depth integrated potential MeHg degradation rate for each treatment, and lines represent the corresponding 14CO2/14CH4 ratios. Error bars indicate (1 standard error (n ) 3) in both cases. the same sediment interval suggests the use of noncompetitive substrates by methanogens (34) or to a surfeit of electron donor (35). The importance of both SRB and methanogens in OD has been previously demonstrated (13, 14). We hypothesize that the cometabolism of MeHg by methanogens proceeds by an OD pathway analogous to monomethylamine degradation (36): +
4CH3NH3 + 2H2O f 3CH4 + CO2 + 4NH4
+
(1)
such that
4CH3Hg+ + 2H2O + 4H+ f 3CH4 + CO2 + 4Hg2+ + 4H2 (2) Because sulfate reduction rates were rapid (e800 nmol cm-3 d-1) and pore water SO42- concentrations were e400 uM along the sampling transect (9), we expect that in-situ SO42- pools were depleted within 1-2 days in bottle incubations. Thus, methanogenesis likely dominated MeHg degradation for a majority of the long-term incubations conducted during July and December 1995. Ratios of 14CO2/ 14CH were often near or below 0.3 during these two periods 4 (Figure 2), which is the ratio predicted when degradation proceeds via reaction (2). We would also expect SO42- limited or depleted conditions during the 2.5 day April 1997 standard incubations, as these were not initiated until 3 days after sediment collection. In this case, 14CO2/14CH4 ratios ranged from 0.1 to 1.4 with an average (( standard error) of 0.40 ( 0.17. For these three incubation periods, ratios 0.3 for 1 day incubations conducted during December 1996 (Figure 2), and a positive correlation (Pearson coefficient r ) 0.70) was found between porewater SO42- concentrations and the percent of MeHg degraded to 14CO2 in floc layer sediment during this period (data not shown). Further, ratios exceeded 2 in all SO42amended samples, while molybdate virtually eliminated 14CO2 production (Figure 7). These results indicate that CO2 production from MeHg degradation is enhanced under sulfate-reducing conditions and suggest that SRB oxidize the methyl group of MeHg entirely to CO2. Such a come2562
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tabolism of MeHg might be analogous to the oxidation of acetate by SRB:
SO42- + CH3COO- + 3H+ f H2S + 2CO2 + 2H2O (3) such that
SO42- + CH3Hg+ + 3H+ f H2S + CO2 + Hg2+ + 2H2O (4) The increase of 14CO2/14CH4 ratios with MeHg concentration (Figure 5a,b) may thus reflect a shift from methanogendominated demethylation at the low concentrations to SRBdominated demethylation at higher concentrations. Above ≈800 ng of MeHg (g of dry sediment)-1], the 14CO2/14CH4 ratio remained constant, suggesting that the individual contributions of both groups to total MeHg degradation also remained constant. At in-situ level MeHg amendments (Figure 5c) the consistently low ratio (≈0.1) suggested that methanogens dominated degradation at in-situ concentrations. It is reasonable to assume that both methanogens and SRB can simultaneously degrade MeHg via OD, since it is proposed that MeHg is simply cometabolized along with primary substrates and does not represent an energy-yielding substrate for either group. In both eqs 2 and 4, it is proposed that for OD the fate of Hg is to remain in the +2 valence. Indeed, there is no a priori reason to assume that a Hg(II) reductase is associated with OD, as has been demonstrated for the mer-mediated Hg detoxification pathway (12). If OD is not associated with a subsequent Hg(II) reduction, this has major implications for natural systems where OD dominates. The production of Hg0 from Hg(II) has been cited as a major sink for Hg from aquatic systems (37-39). In lieu of significant Hg0 production in sediments, liberated Hg(II) may be readily remethylated or react with reduced S species, particularly in reducing environments such as Everglades sediment. A tightly coupled methylation/demethylation cycle may effectively maintain a MeHg pool that is continually available for bioaccumulation. This may partially explain why MeHg is found to build up in the Everglades food chain. Determining if Hg(II) reduction to Hg0 occurs under conditions favoring OD represents an important focus area for future research. Accurate estimates of rate constants are essential for modeling Hg dynamics in any system. Here we provide the first estimates of k for benthic MeHg degradation in the Everglades. These results will be used in the Hg model currently being developed for this system and may be
applicable to other benthic systems. Methodological advances that allow for true tracer level stable or radioisotope MeHg amendments will help determine if the k values observed in this study are representative of in-situ degradation rates in anoxic sediments or if even higher rates can be expected as suggested by our concentration dependence experiment. Further investigations aimed at identifying the fraction of readily bioavailable MeHg are needed as ecosystem level Hg models become more refined. Finally, the occurrence of OD at in-situ MeHg concentrations indicates that this pathway, as well as the mer pathway, should be included in ecosystem Hg models. This will be particularly important if it is found that the fate of Hg differs for the two pathways.
Acknowledgments The authors thank D. Krabbenhoft, C. Gilmour, and J. Benoit for initial reviews of this manuscript and P. Dowdle, L. Miller, T. Hancock, K. Goodwin, C. Sasson, J. Agee, and A. Burns for field and laboratory assistance. This work was supported by the U.S. Environmental Protection Agency (Grant DW14936802-01-0) and the U.S. Geological Survey (Fragile Ecosystems Program) both awarded to R.S.O. and was carried out as part of the postdoctoral work of M.C.M. as a National Research Council Associate.
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Received for review December 18, 1997. Revised manuscript received June 10, 1998. Accepted June 12, 1998. ES971099L
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