Final Report: Importance of Reductive Dechlorination in Chesapeake Bay Sediments Role of Sulfate RespirationEPA Grant Number: R822444
Title: Importance of Reductive Dechlorination in Chesapeake Bay Sediments Role of Sulfate Respiration
Investigators: Capone, Douglas G. , Baker, Joel E. , Gilmour, Cynthia C.
Institution: University of Maryland
EPA Project Officer: Hiscock, Michael
Project Period: October 1, 1995 through September 1, 1998
Project Amount: $286,703
RFA: Exploratory Research - Chemistry and Physics of Water (1995) RFA Text | Recipients Lists
Research Category: Water , Land and Waste Management , Engineering and Environmental Chemistry
Objective:The primary objectives of the research project were to: (1) assess whether the capacity for microbial reductive dechlorination of aryl chlorides was widely distributed in estuarine sediments of the Chesapeake Bay; and (2) determine the role that sulfate reducing bacteria (SRB) had in this process. Using the model aryl chloride compound, 2,4-dichlorophenol (DCP), the effects of sulfate concentration, electron donor availability, and metabolic inhibitors on the reductive dechlorination process and its acclimation period were characterized. Several SRB isolates from the Chesapeake Bay were screened for their ability to dechlorinate DCP. In addition, these observations of aryl chloride transformations and reductive dechlorination were related to the more extensive suite of parameters collected in studies of benthic biogeochemical processes and aryl chloride distributions along the salinity gradient of the Chesapeake Bay. Three sites chosen were the Upper Bay (UB), Mid Bay (MB), and Lower Bay (LB), representing low-, mid-, and high-salinity locations, respectively, along the Bay's main stem. Sulfate reduction (SR) dominates anaerobic carbon flow at all three sites. Manipulative experiments were conducted on sediment slurries as well as on determinations of reductive dechlorination on intact sediment cores.
Summary/Accomplishments (Outputs/Outcomes):Field Studies. The capacity for the short-term onset of reductive dechlorination of DCP added to spatially distinct depth horizons in sediment cores from the three primary sites in the Chesapeake Bay was evaluated. The onset of DCP dechlorination was compared to depth specific rates of SR and levels of sulfate, sulfide, methane, acetate, hydrogen, redox potential, and sediment organic matter.
The rapid onset of reductive dechlorination activity in relatively short-term assays (6?12 days) along the salinity and sulfate gradient of the Bay revealed that dechlorinating bacteria were widely distributed among locations, and with depth in the sediments. Surprisingly, short-term reductive dechlorination activity was not correlated to any number of environmental variables such as sulfate concentration, salinity, redox potential, or total organic matter, except at the MB site during summer. In MB surface sediment, very rapid reductive dechlorination rates were observed upon depletion of sulfate, which coincided with rapid rates of net H2 production. Similar rapid reductive dechlorination rates were not observed in lower depths, where sulfate was limiting, but net H2 production rates were lower. It was concluded that the turnover rate of electron donors, rather than their pool sizes, ultimately determined reductive dechlorination rates. The relative rates of reductive dechlorination with depth among sites mirrored relative rates of SR that, in turn, follow the distribution of labile carbon. At the non-bioturbated MB site, the greatest rates of both electron accepting processes were observed in surface sediments and decreased with depth. At the UB and LB sites, where the activities of infauna distribute carbon more evenly with depth, profiles of SR and reductive dechlorination rates were more vertically homogeneous.
Hydrogen concentrations in Chesapeake Bay sediments were determined for the first time during this research. These measurements may reveal information on the dominant terminal electron-accepting process and whether a system is in steady state. Relative H2 concentrations in sediments among sites supported suppositions by other investigators that electron acceptors that are more energetic than sulfate could contribute to anaerobic carbon mineralization, particularly at the UB site and in the surface sediments at site MB during certain seasons. Sulfate accumulated in the presence of molybdate in anaerobic sediments from both the MB and LB sites, indicating that mechanisms for anaerobic sulfide oxidation were present at both locations. The presence of oxidants for reduced sulfur at all three sites suggests that anaerobic sulfur cycling may be more important and play a greater role in carbon cycling than previously recognized. Partially oxidized sulfur species (such as sulfite and thiosulfate) at all three sites would provide substrates for bacteria that make a living by disproportionating or reducing these compounds. Because their activities would be tightly coupled to and dependent on SRB activity, it is suggested that this group could be involved in ortho reductive dechlorination of DCP. The finding that the majority of chlorophenol dechlorinating bacteria isolated thus far belong to this physiological group of unusual sulfidogens supports this supposition.
Experimental Studies?Mid Bay Summary. The mesohaline MB site was the primary site and the focus of repeated observations of reductive dechlorination activity during different seasons and years. In surficial MB sediments, two rates of ortho reductive dechlorination of DCP (approximately 100 mM) could be described: (1) a slow, steady rate in the presence of sulfate; and (2) a rapid rate upon depletion of sulfate, with concomitant increases in electron donor levels (e.g., labile organic carbon and H2). In sulfate depleted sediment, the ortho dechlorinated metabolite 4-chlorophenol (4-CP) was produced in near stoichiometric balance with the amount of DCP degraded, and persisted. Degradation of 4-CP apparently was dependent on sulfate concentration and a slow rate of SR. Molybdate, a "specific" inhibitor of SRB, severely inhibited reductive dechlorination, implicating SRB involvement in dechlorination. However, it also was observed that molybdate may inhibit non-target physiological groups. Furthermore, three of the MB SRB isolates (two Desulfovibrio and one Desulfotomaculum) did not dechlorinate DCP under sulfate-reducing conditions.
Sulfate inhibited reductive dechlorination by various degrees in different experiments with sediments from MB. Seven experiments with sulfate-replete surficial sediment were undertaken during four different years and three seasons. Sulfate had the greatest inhibitory effect on reductive dechlorination when SR was limited by electron donor availability, such as organic carbon or H2. Sediments collected closest to the time of the spring phytoplankton bloom appeared to support greater rates of reductive dechlorination activity in both the presence and absence of sulfate. During the time that sediments or slurries were incubated?prior to the start of any experiment?labile substrates were being consumed. Under initial sulfate-replete conditions, there was a positive linear relationship between the length of the pre-incubation time and length of the subsequent lag period. Based on these results, it is proposed that, while sulfate partially inhibits ortho reductive dechlorination of DCP, the greatest controlling variable on reductive dechlorination rates at this site is electron donor availability, rather than sulfate status.
The above relationships supported the hypothesis that the length of the lag period before reductive dechlorination activity is observed in sulfidogenic sediments is a function of the availability of suitable electron donors. The fact that reductive dechlorination was evident shortly after dosing DCP in many of the experiments indicates that bacteria responsible for this activity were present in sufficient numbers and did not require the lengthy lag periods for induction or derepression of dehalogenating enzymes observed in many other freshwater systems. Because reductive dechlorination activity could be enriched, growth of dechlorinating bacteria from initially small numbers, during certain seasons, also may be a factor contributing to lag periods in this system.
Although the Chesapeake Bay system is very productive, most of the primary production is consumed in the MB water column, except during brief periods in the spring when phytoplankton bloom material is deposited to the MB site. The intensity of this deposition depends on the magnitude of the spring Susquehanna freshet. The timing and magnitude of spring bloom deposition determine subsequent SR rates in warmer months, and this labile carbon pool is turned over rapidly. The relationship between carbon availability and lag periods in sulfate-replete sediment reveals that in very fresh sediment, there are enough labile substrates to initiate reductive dechlorination in very short time periods concurrent with SR. The surprising finding was that for samples from this productive site during certain times of the year, labile carbon could be consumed rapidly (2?3 weeks of incubation), thereby constraining reductive dechlorination or SR. By late summer in certain years, it is likely that the majority of labile electron donor substrates already have been metabolized and would limit both reductive dechlorination and SR under long-term incubation in a closed system.
Excess H2 that was added to sulfate-replete sediments did little to stimulate reductive dechlorination. Instead, exogenous H2 appeared to select for fast-growing SRB, which effectively competed with dechlorinating bacteria for this electron donor. This competitive exclusion of dechlorination may explain the negative dechlorination results (above) with fast-growing SRB isolates. Although the levels of H2 added were much greater than the apparent in situ concentration, they were not sufficiently high to reduce the entire sulfate pool. When non-limiting levels of electrons from the acetone solvent used to deliver DCP eventually became available, competition for electron donors was relieved, and members of all electron-accepting groups, (i.e., SRB, methanogens, and dechlorinators) benefitted. Likewise, when sulfate became depleted, the production of H2 temporarily outpaced consumption, allowing for rapid utilization in reductive dechlorination. Thus, when production of electron donors outpaces consumption by the dominant electron-accepting process, rapid rates of reductive dechlorination were observed, regardless of sulfate levels. It was at these times of rapid electron donor production and reductive dechlorination rates that 4-CP consumption coupled to SR was inhibited even when sulfate was present, perhaps by selecting against slower-growing SRB utilizing 4-CP. MB sediments displayed depth-specific differences in microbial responses to DCP concentration. Surficial sediments always displayed faster rates of reductive dechlorination, presumably due to the greater availability of labile substrates. SRB at the surface also were not nearly as sensitive to high concentrations of DCP (> 1 mol/g) as they were below the surface, where SR was inhibited. The inhibition of SR by DCP observed in lower depths was temporary, as SR resumed once DCP was dechlorinated. During the inhibition of SR by DCP in the lower depths, reductive dechlorination was eventually observed. The potential for DCP to disrupt SR, if released in large enough quantities to result in DCP concentrations > 0.6 µmol/g, could have large impacts on benthic metabolism. Because a substantial fraction (32 percent) of annual Bay primary production is mineralized via SR at this site, this restriction of SR could result in larger ecosystem-level effects.
At low, more environmentally relevant concentrations, the pathway of DCP degradation changed. Low concentrations of DCP (10 nmol/g) were degraded without a lag in sulfate-reducing sediments from all depths examined and para dechlorination became more important. The production of 2-chlorophenol, the para metabolite, could be the result of some exoenzymatic or abiotic reduction. Para dechlorination is the favored pathway thermodynamically, although bacteria favor the less energetic ortho dechlorination in most systems. These results stress the importance of testing a range of target compound concentrations when evaluating their fate in the environment.
Lower and Mid Bay Comparisons. DCP was dechlorinated without a lag when added to either sulfate replete slurries or whole sediment from the high salinity LB site. Reductive dechlorination rates in the presence of sulfate were not biphasic, as observed at the MB site. Sulfate did not appear to inhibit reductive dechlorination at LB, as was frequently observed at the MB site. Short-term reductive dechlorination rates were similar with depth and during different seasons, in contrast to activities observed at MB. At both sites, molybdate inhibited ortho reductive dechlorination of DCP, implicating SRB in the reaction. The 4-CP degradation was coupled to SR at both sites. SRB were evidently involved in phenol degradation at the LB site as well. Phenol was detected as an intermediate metabolite in DCP degradation in LB sediments when either sulfate was limiting or molybdate was present, but not at MB.
This research demonstrates that reductive dechlorination is an important fate for the model compound, DCP, in relatively unpolluted anoxic sediments of the main stem of the Chesapeake Bay. The presence of sulfate does not exclude reductive dechlorination, although it slows rates of reductive dechlorination in the mesohaline portion, likely through more successful competition for electron donors with sulfate. In sulfate-reducing MB sediments, the same conditions that limit SR also limit ortho reductive dechlorination of DCP. SR does not completely consume electron donors when the supply of labile substrates is non-limiting. The presence of sulfate promotes more complete degradation of DCP, as it is required for metabolite 4-CP degradation at both the MB and LB sites, and for phenol degradation at LB.
These links between SR and dechlorination allow speculation on the fate of chlorinated compounds, such as DCP, in Chesapeake Bay sediments and factors controlling dechlorination. Previous basic research on benthic metabolism and biogeochemistry at the selected sites provide predictable relationships between seasonal temperature effects and dechlorination rates. It is known that flow dynamics of the Susquehanna River, the largest tributary of the Chesapeake Bay, is important in controlling benthic metabolism and, by extension, dechlorination in the main stem of the Bay. The pulse of labile phytoplankton biomass in the spring would likely fuel moderate rates of dechlorination in the presence of sulfate until this labile carbon is consumed. As temperatures warm in later months, increased rates of benthic metabolism often exceed rates of sulfate diffusion in MB, resulting in sulfate depletion. This non-steady state situation results in increased rates of dechlorination when sufficient electron donors are available to fuel the reaction. With lower concentrations of DCP, there appears to be a sufficient rate of supply of electron donors to support dechlorination without a lag at all sites and sediment depths examined, regardless of season or sulfate concentration. Finally, SRB are implicated in dechlorination in both the LB and MB, and collective evidence suggests that they may be the dechlorinating bacteria, particularly at MB. The finding that some SRB are active at very low sulfate levels also may explain why molybdate inhibits reductive dechlorination when sulfate is at or below levels generally considered limiting to SR in this region of the Bay. If SRB are not the dechlorinators, then bacteria closely associated with or participating in sulfur cycling are strongly implicated.