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Bacterial demethylation of methylmercury in the South Florida Ecosystem

Project Proposal for 1998

Program: FRAGILE ENVIRONMENTS
Project Title: Bacterial demethylation of methylmercury in the South Florida Ecosystem.
Location of Study Area: Florida Everglades and Everglades Nutrient Removal Areas
Project Start Date: October 1, 1995
Project End Date: September 30, 1998
Project Number: 7-4387-40500
Project Chief: Ronald S. Oremland
Region/Division/Team/Section: WR, WRD, NRP, ACME
E-mail:roremlan@usgs.gov
Phone: (415) 329-4482
Fax: (415) 329-4463
Mailing Address:
USGS, ms 480, 345 Middlefield Road
Menlo Park, CA 94025
Program Element(s)/Task(s) TASK 3.3: Mercury cycling in the Everglades and the Everglades Nutrient Removal (ENR) Areas (renewal).
Panel:
Collaborators, Clients:
COLLABORATORS: D. Krabbenhoft (USGS) and C. Gilmour (Philadelphia Academy of Sciences).
COOPERATING AGENCY: Note that the US EPA funding has ended and therefore the salary of Dr. M. Marvin DiPasquale, technicians, travel costs, lab supplies, etc. are now requested entirely from the USGS.


BACKGROUND NARRATIVES

Project Summary: Bacterial methylation of mercury occurs widely in the anoxic sediments of the South Florida Ecosystem (SFE), however the methyl mercury (MeHg) formed is immediately subject to a bacterial degradative reaction (demethylation). The net difference between methylation and demethylation controls the amount of MeHg available for incorporation into food chains. This study will determine the rates of demethylation and the mechanisms by which it occurs.

Project Justification: The biota of the SFE contain high body burdens of MeHg. The in the SFE MeHg is formed from the methylation of Hg (11) carried out by certain types of anaerobic bacteria (e.g., sulfate-respirers). However, the MeHg formed is immediately subject to the demethylation reaction carried out by other types of bacteria, both aerobes and anaerobes. Hence, only by making simultaneous measures of methylation and demethylation rates can the net production of MeHg be gauged. This project has devised the techniques necessary to make measurements of demethylation at environmentally realistic concentrations, and has detected significant activity at all the sites assayed for methylation. Therefore, such information is critical for the accurate formulation of mathematical models of mercury dynamics in the SFE. Such models are needed in order to achieve and implement effective managerial controls on the extent of the MeHg contamination of the biota in the SFE.

Project Objectives: We have employed 14C-MeHg and confirmed that oxidative demethylation (OD) is far more significant than the alternative organomercurial lyase pathway as the mechanism for MeHg degradation in sediments, periphyton, and waters of the SFE. Applied levels of 14C-MeHg that we now use are 4 orders of magnitude lower than used previously, and are in the range of ~20 - 40 ng MeHg/g dry sediment, which approach the in situ levels of MeHg detected. Furthermore, we found that rate constants for OD are linear over several orders of magnitude of applied MeHg concentrations, and that this extrapolates down to in situ levels of MeHg. We will continue these investigations over the next 12 months in order to determine if there are spatial and seasonal trends with regard to the rates of OD, the products of MeHg, and the mechanisms by which OD occurs. We will carry out further mechanistic investigations on periphyton, a "hot spot" for both methylation and demethyaltion. We will combine our efforts on demethylation with diel microelectrode investigations of vertical chemical gradients (pH, sulfide, oxygen) within the periphyton/microbial mats. Naturally, this effort will be closely linked to studies of Hg methylation and Hg speciation made by other teams (Gilmour and Krabbenhoft). This will allow us to determine: 1) the fate of the Hg in OD; 2) the diel cycle of methylation/demethylation and Hg speciation in periphyton and mats; and 3) the microorganisms responsible for OD.

Overall Strategy, Study Design, and Planned Major Products: We will conduct a series of seasonal measurements of demethylation in sediments, periphyton/mat materials, and waters along an expanded north to south gradient of stations including the ENR, WCA-2A, WCA-2B, and WCA-3A regions, We will apply 14C-MeHg at ~20-40 ng/g dry wt. sediment and incubate samples for short duration's (e.g., <24 h). Such a protocol of an expanded number of stations, seasonal samplings, and experimental replication will allow us to test with statistical reliability as to whether there are seasonal and spatial trends in the demethylation data. Analysis of the endproduct gases (14CO2 and/or 14CH4) will allow us to determine the relative contributions of the oxidative demethylation (OD) pathway vs. the organomercurial lyase pathway to the destruction of MeHg. To date our data suggest that the OD pathway predominates, and that the fate of the Hg is this reaction is its recycling as Hg (ll) rather than as escape as volatile Hg (0). We will conduct further experiments to confirm our initial observations on the fate of Hg, but these results are important since it implies that a "mercury loop" exists whereby the regeneration of inorganic Hg associated with OD of MeHg makes it reavailable for methylation.

DETAILS ON OVERALL FY 1998 PROJECT JUSTIFICATION, OBJECTIVES, PLANS, AND STRATEGY: Bacterial methylation of inorganic mercury occurs in anoxic sediments and is recognized as the basis for the mercury contamination of food chains such as the SFE. Quantification of the extent to which methylation occurs for incorporation into mathematical models must be balanced by processes which destroy MeHg. This later process, termed demethylation, must also be quantified in order to arrive at values for net methylation (or demethylation) of mercury which are most suited for incorporation into mathematical models. Hence, simultaneous assays of methylation and demethylation produce M/D ratios which are commonly used in ecosystem-based surveys, such as SFE (Vaithiyanathan et al., 1996).

The most-studied mechanism for demethylation is that of the bacterial mer operon which encodes for enzymes which cleave the carbon-mercury bond (organomercurial lyase) and reduce Hg (11) to Hg (0) (mercuric reductase). The first reaction results in the formation of methane. However, another type of bacterial demethylation was discovered in which carbon dioxide, in addition to methane, is produced from cleavage of the carbon-mercury bond. This process is termed oxidative demethylation (Oremland et al., 1991; 1995). The distinction between these two demethylation processes is significant because they are carried out by vastly different organisms and enzyme systems. Hence, while the mer operon holds the potential for removal of mercury from ecosystem by its volatilization as Hg (0) (Barkay et al., 1991), the fate Hg after undergoing oxidative demethylation is not known. It is possible that oxidative demethylation merely regenerates the Hg (11) ion, thereby retaining mercury in the system and either making it available again for methylation or for reaction with sulfide and removal by precipitation as HgS. Therefore, not only are estimates of the in situ rate of demethylation needed to determine M/D ratios (i.e., rates of net methylation), but process-oriented studies are needed to determine which type of demethylation reactions are occurring and what is the fate of the mercury. This is obviously required for any attempts to understand mercury mass balances in the SFE. Since this is a process-oriented study, we anticipate publication of the results in peer-reviewed journals.

WORK PLAN

Overall: Four field trips will be conducted corresponding to the seasonal samplings of the ACME group. Stations sampled and assayed will be closely coordinated with the Gilmour and Krabbenhoft teams as has been accomplished in the past. This will allow the ACME team to attain a complete temporal/spatial data base with respect to methylation, demethylation, and mercury speciation in the SFE. Ancillary measures of other significant biogeochemical (e.g., sulfate-reduction, methanogenesis, denitrification) or chemical characterizations (nutrients, pH, DO, inorganic ions) will be divided between the groups as has been done previously. We will also conduct experimental work with selected materials (e.g., mats and periphyton) to determine volatilization rates of Hg (0) from MeHg, and the influence of photosynthetic oxygen production in microbial mats on the rates and nature of demethylation. This work will be coupled with the use of microelectrode technology to measure gradients of sulfide, oxygen, and pH within microbial mats and periphyton communities. Analysis of endproduct gases from 14C-MeHg demethylation will be done by an oxidation/trapping procedure in conjunction with liquid scintillation spectrometry. Hg (0) formed during demthylation of MeHg will be analyzed by cold vapor fluorescence atomic absorption spectrometry.

Timeline: A new fact sheet will be submitted by March, 1998. Research findings will also be submitted for inclusion in the SFE web page as requested. The major products of this research will be a series of publications in peer-reviewed scientific journals. It is not possible at this time to state the specific number, topics, or publication dates of such products because they will have to undergo the necessary data reduction, interpretation, writing, editing, and review for submission. We anticipate that several papers will be derived from this work, some of which will be written by the Oremland/Di Pasquale team and other will be written in collaboration with the larger ACME group.

Planned Deliverables/Products:
Marvin-DiPasquale, M., R. Oremland, and P. Dowdle. 1996. Demethylation of methyl-mercury by methanogenic bacteria in Florida Everglades peat sediments. Abs. Ann. Meeting, Amer. Soc. Microbiology, # Q 210.

Marvin DiPasquale, M.C., and R.S. Oremland. 1997. Methyl-mercury degradation in the Florida Everglades sediment, water, and microbial mats. Amer. Soc. Limnol. Oceanogr. Ann. Meet., Santa Fe, NM, p. 233.

Marvin-DiPasquale, M.C., and R.S. Oremland. Bacterial methyl-mercury degradation potentials in Florida Everglades peat sediment. in preparation for Biogeochemistry.

Planned Outreach Activities: N/A

Prior Accomplishments in Proposed Area of Work: N/A

New Directions, Expansion of Continuing Project (if applicable): N/A

ACCOMPLISHMENTS, OUTCOMES, PRODUCTS, OUTREACH

Accomplishments and Outcomes, Including Outreach: The work undertaken in 1998 will build on our results of the previous years, and our interactions/collaborations with other teams involved, including those led by Dr. C. Gilmour and Dr. D. Krabbenhoft. One of the findings of these two investigators was that periphyton and certain bacterial mats appear to be hot spots of methylation activity, and we have determined that they are also hot spots for demthylation. Because these sites appear to sustain sulfate-reducing bacteria, and that these microbes have been implicated as principal methylators, it is likely that there are strong chemical gradients of oxygen, sulfide, and pH that these gradients fluctuate vertically (scale = millimeters) over a diel cycle as is typical of microbial mats. We propose to undertake field studies employing microelectrodes for measurement of these parameters, and will build our own systems to achieve this end. Joint incubation experiments were performed whereby rates of methylation and demethylation were both assayed using 203Hg (Gilmour group) and 14C_ tracers (Oremland group). Preliminary results indicate that the rate constants for both processes are similar, however because the Hg (11) pool is much higher than the MeHg pool, the rate of methylation exceeds that of demethylation, thereby achieving very high rates of net methylation. However, the concentrations of MeHg in these systems is too low to justify the extrapolated rates of net methylation. This suggests that the methylation rates are overestimated and we must conduct further joint experiments of this type to ascertain whether this is the case, or whether there are some physical constraints, such as adsorption of Hg (11), which cause the methylation rate to be overestimated. Oxidative demethylation has been shown to be the main mechanism for MeHg destruction, even at very low applied levels of the MeHg radiotracer. A change in Hg speciation from Hg (11) to Hg (0) has thus far not been observed, indicating that a mercuric reductaser is not involved with OD. This work has been conducted in collaboration with the Krabbenhoft group.

In addition to the above investigations, we will continue to sample and assay our extended field stations in the SFE in order to acquire a 2-year final data set of rates to determine if seasonal trends are evident. In all of these demethylation studies we will perform work with a new high specific activity 14C-MeHg which allows us to work in the range of <40 ng MeHg/g dry sediment. We will also conduct investigations, employing cold vapor atomic fluorescence spectroscopy to determine the fate of the mercury (as opposed to the carbon) in sediments having demethylation activity.

References:
Barkay, T., R.R. Turner, A. VanderBrook, and C. Liebert. 1991. The relationships of Hg (0) volatilization from a freshwater pond to the abundance of mer genes in the gene pool of the indigenous microbial community. Microbial Ecology 21 ~ 151 - 161.

Oremland, R.S., C.W. Culbertson, and M.R. Winfrey. 1991. Methylmercury decomposition in sediments and bacterial cultures: involvement of methanogens and sulfate reducers in oxidative demethylation. Appl. Environ. Microbiol. 57: 130 - 137.

Oremland, R.S., L.G. Miller, P. Dowdle, T. Connell, and T. Barkay. 1995. Methylmercury oxidative degradation potentials in contaminated and pristine sediments of the Carson River, Nevada. Appl. Environ. Microbiol. 61.- 2745 - 2753.

Vaithiyanathan, P., C,J, Richardson, R.G. Kavanaugh, C.B. Craft, and T. Barkay. 1996. Relationships of eutrophication to the distribution of mercury and to the potential for methylmercury production in the peat soils of the Everglades. Environ. Sci. Technology (in press).


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