Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
BACTERIAL METHYLATION OF MINE-DERIVED INORGANIC MERCURY IN LAKE AND ESTUARINE SEDIMENTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0201896
Grant No.
(N/A)
Project No.
CA-D-MMG-7352-H
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2009
Project End Date
Sep 30, 2014
Grant Year
(N/A)
Project Director
Nelson, D.
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Microbiology & Molecular Genetics
Non Technical Summary
Californias legacy of inorganic mercury pollution from abandoned mines is of concern due to its potential conversion to methylmercury. Bacteria living in oxygen-depleted sediments produce this especially toxic form of mercury, which is readily biomagnified in predatory fish and birds near the apex of aquatic food webs. We have recently shown that a group called "iron-reducing bacteria" are as active at producing methylmercury as other bacteria, called "sulfate-reducers", which were previously believed to perform the bulk of these transformations in marine and freshwater sediments. The current proposal will continue to refine experiments based on natural sediments to determine the general importance of iron-reducers as mercury methylators throughout the sediments of a lake and an estuary impacted by typical mine-derived mercury. Pure cultures of abundant iron-reducing bacteria will also be isolated from mine-impacted marine sediments and assayed for their ability to produce methylmercury from the divalent inorganic form. A variety of stakeholder groups have been interested in our basic research findings on these and related topics to date. The PI will continue to keep these groups informed of our new findings and any possible implications for remediation actions.
Animal Health Component
0%
Research Effort Categories
Basic
25%
Applied
60%
Developmental
15%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1330210110020%
1124010119020%
1120210119020%
1334010110040%
Knowledge Area
133 - Pollution Prevention and Mitigation; 112 - Watershed Protection and Management;

Subject Of Investigation
0210 - Water resources; 4010 - Bacteria;

Field Of Science
1100 - Bacteriology; 1190 - Limnology;
Goals / Objectives
The research objectives for this project are as follows: (1) For mine-impacted sediments of Clear Lake, determine the relative contribution of sulfate-reducing bacteria to methylation of mercury while altering native sediment properties and inorganic mercury levels as little as possible. (2) For mine-impacted sediments of Clear Lake that are first manipulated to biologically deplete sulfate and oxidized iron, determine the relative rates of mercury methylation upon supplementation with each biological oxidant separately and both together. (3) For mine-impacted sediments of Walker Creek Estuary and a control site, determine the proportional contribution of sulfate-reducing bacteria to methylation of mercury while altering native sediment properties and inorganic mercury levels as little as possible. (4) For a spectrum of sediment types from Walker Creek Estuary, isolate pure cultures of marine iron-oxidizing bacteria and test the per-cell rates of production of methylmercury for representative cultures. (5) Use bioaccumulation of methylmercury in the muscle tissue of the lined shore crab, PACHYGRAPSUS CRASSIPES, to determine the extent and magnitude of the impact of mercury from Walker Creek on biota around Tomales Bay; a site showing minimal impact will be selected as control sediment for the third objective. . Under the earlier version of this project the PI presented new basic research findings that have implications for mercury management policy to the following stakeholder groups: Delta Tributaries Mercury Council, San Francisco Estuary Institute, San Francisco Bay Water Board. These presentations, made in person or via dissemination of unpublished research findings, were in response to requests from these groups, and we will continue to disseminate our findings in this manner as they become available. Additionally, our report on our Walker Creek Estuary studies, which has been posted on the UC Office of the President Coastal Environmental Quality Initiative website (http://repositories.cdlib.org/ucmarine/ceqi/040), had 742 full-text downloads in the first 30 months of posting (2006-12-13) and continues to be downloaded at a steady pace. We will continue to present our findings at scientific meetings and in research journal articles. A recent peer-review of an earlier version of our pending manuscript on the Walker Creek Estuary studies characterized our 2006 publication (Fleming et al., 2006, Mercury methylation from unexpected sources: molybdate-inhibited freshwater sediments and an iron-reducing bacterium. Applied and Environmental Microbiology 72:457-464) as follows: "In this reviewers opinion, that finding was one of the most significant advances in Hg biogeochemistry in recent years, because for over 20 years prior to the 2006 paper, SRB [sulfate-reducing bacteria] were the focus of all research on Hg methylation." Thus, we believe that our current basic research emphasis on establishing the generality of those earlier findings continues to have strong implications for environmental policy and remediation of contaminated sites.
Project Methods
The current proposal aims to refine sediment incubation methods used to study microbial methylation of inorganic mercury to a new level. For our previous groundbreaking studies of methylation in Clear Lake sediments, one volume of anoxic sediment was mixed with two volumes of anoxic lake water and supplemented to a final Hg++ concentration of roughly 100 ppm (as HgCl2). This approach produced the uniform replicate incubation mixtures required for our experimental design, but also obviously significantly diluted all pore water constituents except Hg++, which was greatly enhanced. The current experiments will employ "minimal-dilution" mixing techniques and anaerobic incubations in gas-tight, multi-layer, plastic bags. For sediment studies in general this approach was introduced by other researchers, and our recent experiments on estuarine sediments from Walker Creek are the only published reports on the extension of these methods to natural sediment studies of microbial production of methylmercury. The extremely good statistical agreement we achieved for replicate incubations of the same treatment is indeed promising. We also determined that diminishing the Hg++ addition by about 80-fold (to 1.2 ppm) still gave an extremely strong signal of methylmercury production; however, even this much lower level of Hg++ (a bactericide at high concentrations) a transient inhibitory effect on bacterial processes in general, if one can judge from the impact of this Hg++ level on sulfate reduction. Thus, the studies envisioned in the current proposal will be performed with an addition of only 0.1 ppm Hg++, "minimal dilution" mixing and gas-tight anaerobic bag incubations. All other procedures will be similar to those previously employed in our published studies or the published works of others. Determinations of total methylmercury will be accomplished by sending samples to Batelle Marine Sciences Laboratory (Sequim, WA); determinations of total mercury in crab muscle tissue (well established as an excellent surrogate for methylmercury in that matrix) will be determined on a direct mercury analyzer (DMA-80; Milestone, Inc.) in the laboratory of the PI. Dilution-to-extinction culturing of marine iron-reducing bacteria will follow published procedures previously employed in our laboratory, except that the media will be modified to make them suitable for marine strains. Pure cultures of marine iron-reducing bacteria will be assayed for per-cell rates of production of methylmercury as performed previously.

Progress 10/01/09 to 09/30/14

Outputs
Target Audience: A broad range of researchers are continuing to access our findings. As a measure of the impact of this research, a technical report "Contribution of Iron-Reducing Bacteria to Mercury Methylation in Marine Sediments", which was supported by the Coastal Environmental Quality Initiative (UCOP), has been viewed or downloaded more than 1300 times to date. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project was accomplished with a graduate student. The student was trained in a variety of laboratory techniques and assays, and taught to analyze data. How have the results been disseminated to communities of interest? A technical report "Contribution of Iron-Reducing Bacteria to Mercury Methylation in Marine Sediments", which was supported by the Coastal Environmental Quality Initiative (UCOP), has been viewed or downloaded more than 1300 times to date. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The continued overall focus of this project is the identification and cultivation of the Bacteria that are major contributors to the pathways of mercury methylation in organic-rich sediments driven by the impact of two Coast Range mine sites from which mercury ore was previously mined. One mine impacts freshwater lake sediments and the other estuarine sediments; thus, the receiving bodies represent the two prominent niches where fish and birds are impacted by the environmental pollutant, mono-methylmercury, which is bio-accumulated at higher trophic levels. It has been known for some time that methylmercury is formed in sediments mainly via the activity of anaerobic Bacteria and perhaps Archaea interacting with various forms of inorganic mercury. The literature paradigm accepted for more than 20 years was that almost all anaerobic mercury-methylation can be ascribed to the activity of sulfate-reducing bacteria. We used the molybdate anion, a specific inhibitor of this bacterial process, to test this assertion. Molybdate acts to inhibit sulfate reducing-bacteria by competing with sulfate in the first step in the dissimilatory sulfate-reduction pathway, which is an ATP-consuming reaction. The resultant molybdenum compound is unstable and decomposes with a resulting futile consumption of ATP. Previous years' research showed that sulfate reduction in mercury-amended sediments from Walker Marsh and Clear Lake was completely inhibited by molybdate at concentrations that were roughly 10% of ambient sulfate values. By contrast, at these same molybdate levels, 50% or more of ambient mercury methylation persisted. If sulfate-reducing bacteria were the only microbes responsible for methylation, full inhibition of methylation would have been the expected result. Furthermore, our previous pure culture studies showed that certain freshwater strains of iron-reducing bacteria were as prolific at mercury methylation as sulfate-reducing bacteria. Recently, we adapted the use of gas-tight sediment incubation bags to confirm our earlier findings under conditions that more closely mimic natural sediments. Under anaerobic conditions we homogenized a sample of the top 10 cm of Walker Marsh sediments, which included an upper suboxic but oxidized zone where oxidized iron and oxidized manganese are the dominant electron acceptors and a deeper more reduced zone where sulfate-reducing bacteria will predominate. This homogenized sediment was then distributed under anoxic conditions into replicate gas-tight bags that were subsequently amended uniformly in various combinations with molybdate and divalent mercury in a novel approach that altered ambient volumes of pore water by less than 1%. Most-probable-number determinations indicated that there were approximately 106 to 108 iron-reducing bacteria per gram of Walker Marsh sediment. We are continuing attempts to isolate marine iron-reducing bacteria from Walker Marsh in pure culture as a prerequisite to assaying their ability to produce mono-methyl mercury from divalent mercury. These experiments generated more support for the view that bacteria other than sulfate-reducers are responsible for a substantial fraction of mercury methylation in marine and freshwater sediments.

Publications


    Progress 01/01/13 to 09/30/13

    Outputs
    Target Audience: A broad range of researchers are continuing to access our findings. As a measure of the impact of this research, a technical report "Contribution of Iron-Reducing Bacteria to Mercury Methylation in Marine Sediments", which was supported by the Coastal Environmental Quality Initiative (UCOP), has been viewed or downloaded more than 1300 times to date. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project was accomplished with a graduate student. The student was trained in a variety of laboratory techniques and assays, and taught to analyze data. How have the results been disseminated to communities of interest? A technical report "Contribution of Iron-Reducing Bacteria to Mercury Methylation in Marine Sediments", which was supported by the Coastal Environmental Quality Initiative (UCOP), has been viewed or downloaded more than 1300 times to date. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? The continued overall focus of this project is the identification and cultivation of the Bacteria that are major contributors to the pathways of mercury methylation in organic-rich sediments driven by the impact of two Coast Range mine sites from which mercury ore was previously mined. One mine impacts freshwater lake sediments and the other estuarine sediments; thus, the receiving bodies represent the two prominent niches where fish and birds are impacted by the environmental pollutant, mono-methylmercury, which is bio-accumulated at higher trophic levels. It has been known for some time that methylmercury is formed in sediments mainly via the activity of anaerobic Bacteria and perhaps Archaea interacting with various forms of inorganic mercury. The literature paradigm accepted for more than 20 years was that almost all anaerobic mercury-methylation can be ascribed to the activity of sulfate-reducing bacteria. We used the molybdate anion, a specific inhibitor of this bacterial process, to test this assertion. Molybdate acts to inhibit sulfate reducing-bacteria by competing with sulfate in the first step in the dissimilatory sulfate-reduction pathway, which is an ATP-consuming reaction. The resultant molybdenum compound is unstable and decomposes with a resulting futile consumption of ATP. Previous years' research showed that sulfate reduction in mercury-amended sediments from Walker Marsh and Clear Lake was completely inhibited by molybdate at concentrations that were roughly 10% of ambient sulfate values. By contrast, at these same molybdate levels, 50% or more of ambient mercury methylation persisted. If sulfate-reducing bacteria were the only microbes responsible for methylation, full inhibition of methylation would have been the expected result. Furthermore, our previous pure culture studies showed that certain freshwater strains of iron-reducing bacteria were as prolific at mercury methylation as sulfate-reducing bacteria. Recently, we adapted the use of gas-tight sediment incubation bags to confirm our earlier findings under conditions that more closely mimic natural sediments. Under anaerobic conditions we homogenized a sample of the top 10 cm of Walker Marsh sediments, which included an upper suboxic but oxidized zone where oxidized iron and oxidized manganese are the dominant electron acceptors and a deeper more reduced zone where sulfate-reducing bacteria will predominate. This homogenized sediment was then distributed under anoxic conditions into replicate gas-tight bags that were subsequently amended uniformly in various combinations with molybdate and divalent mercury in a novel approach that altered ambient volumes of pore water by less than 1%. Most-probable-number determinations indicated that there were approximately 106 to 108 iron-reducing bacteria per gram of Walker Marsh sediment. We are continuing attempts to isolate marine iron-reducing bacteria from Walker Marsh in pure culture as a prerequisite to assaying their ability to produce mono-methyl mercury from divalent mercury. These experiments generated more support for the view that bacteria other than sulfate-reducers are responsible for a substantial fraction of mercury methylation in marine and freshwater sediments.

    Publications


      Progress 01/01/12 to 12/31/12

      Outputs
      OUTPUTS: The continued overall focus of this project is the identification and cultivation of the Bacteria that are major contributors to the pathways of mercury methylation in organic-rich sediments driven by the impact of two Coast Range mine sites from which mercury ore was previously mined. One mine impacts freshwater lake sediments and the other estuarine sediments; thus, the receiving bodies represent the two prominent niches where fish and birds are impacted by the environmental pollutant, mono-methylmercury, which is bio-accumulated at higher trophic levels. It has been known for some time that methylmercury is formed in sediments mainly via the activity of anaerobic Bacteria and perhaps Archaea interacting with various forms of inorganic mercury. The literature paradigm accepted for more than 20 years was that almost all anaerobic mercury-methylation can be ascribed to the activity of sulfate-reducing bacteria. We used the molybdate anion to test this assertion. Molybdate acts to inhibit sulfate reducing-bacteria by competing with sulfate in the first step in the dissimilatory sulfate-reduction pathway, which is an ATP-consuming reaction. The resultant molybdenum compound is unstable and decomposes with a resulting futile consumption of ATP. Previous years' research showed that sulfate reduction in mercury-amended sediments from Walker Marsh and Clear Lake was completely inhibited by molybdate at concentrations that were roughly 10% of ambient sulfate values. By contrast, at these same molybdate levels, 50% or more of ambient mercury methylation persisted. If sulfate-reducing bacteria were the only microbes responsible for methylation, full inhibition of methylation would have been the expected result. Furthermore, our previous pure culture studies showed that certain freshwater strains of iron-reducing bacteria were as prolific at mercury methylation as sulfate-reducing bacteria. Recently, we adapted the use of gas-tight sediment incubation bags to confirm our earlier findings under conditions that more closely mimic natural sediments. Under anaerobic conditions we homogenized a sample of the top 10 cm of Walker Marsh sediments, which included an upper suboxic but oxidized zone where oxidized iron and oxidized manganese are the dominant electron acceptors and a deeper more reduced zone where sulfate-reducing bacteria will predominate. This homogenized sediment was then distributed under anoxic conditions into replicate gas-tight bags that were subsequently amended uniformly in various combinations with molybdate and divalent mercury in a novel approach that altered ambient volumes of pore water by less than 1%. Most-probable-number determinations indicated that there were approximately 10^6 to 10^8 iron-reducing bacteria per gram of Walker Marsh sediment. We are continuing attempts to isolate marine iron-reducing bacteria from Walker Marsh in pure culture as to assay their ability to produce mono-methyl mercury from divalent mercury. These experiments generated more support for the view that bacteria other than sulfate-reducers are responsible for a substantial fraction of mercury methylation in marine and freshwater sediments. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: A broad range of researchers are continuing to access our findings. As a measure of the impact of this research, a technical report "Contribution of Iron-Reducing Bacteria to Mercury Methylation in Marine Sediments", which was supported by the Coastal Environmental Quality Initiative (UCOP), has been viewed or downloaded almost 1300 times to date. PROJECT MODIFICATIONS: Not relevant to this project.

      Impacts
      The vast majority of mercury mined in the U.S. was extracted from the Coast Range of California, and this mining legacy continues to impact our environment. Concerns about mercury derive from the ability of highly toxic methyl mercury to biomagnify in animals, including humans, near the top of food webs. Therefore, an understanding all significant microbial sources of methyl mercury in impacted environments is essential for coherent design of remedial actions.

      Publications

      • No publications reported this period


      Progress 01/01/11 to 12/31/11

      Outputs
      OUTPUTS: The ongoing focus of this project continues to be the identification and cultivation of the prokaryotes that are major contributors to the pathways of mercury methylation in organic-rich sediments driven by the impact of two Coast Range mine sites from which mercury ore was previously mined. One mine impacts freshwater lake sediments and the other estuarine sediments; thus, the receiving bodies represent the two prominent niches where fish and birds are impacted by the environmental pollutant, mono-methylmercury, which is bio-accumulated at higher trophic levels. It has been known for some time that methylmercury is formed in sediments mainly via the activity of anaerobic Bacteria and perhaps Archaea interacting with various forms of inorganic mercury. The literature paradigm accepted for more than 20 years has been that almost all anaerobic mercury-methylation can be ascribed to the activity of sulfate-reducing bacteria. We used the molybdate anion, a specific inhibitor of this bacterial process, to test this assertion. Molybdate acts to inhibit sulfate reducing-bacteria by competing with sulfate in the first step in the dissimilatory sulfate-reduction pathway, which is an ATP-consuming reaction. Previous years' research showed that sulfate reduction in mercury-amended sediments from Walker Marsh and Clear Lake was completely inhibited by molybdate at concentrations that were roughly 10% of ambient sulfate values. By contrast, at these same molybdate levels, 50% or more of ambient mercury methylation persisted. If sulfate-reducing bacteria were the only microbes responsible for methylation, full inhibition of methylation would have been the expected result. Furthermore, our previous pure culture studies showed that certain freshwater strains of iron-reducing bacteria are as prolific at mercury methylation as sulfate-reducing bacteria. Recently, we adapted the use of gas-tight sediment incubation bags to confirm our earlier findings under conditions that more closely mimic natural sediments. Under anaerobic conditions we homogenized a sample of the top 10 cm of Walker Marsh sediments, which included an upper suboxic but oxidized zone where oxidized iron and oxidized manganese are the dominant electron acceptors and a deeper more reduced zone where sulfate-reducing bacteria will predominate. This homogenized sediment was then distributed under anoxic conditions into replicate gas-tight bags that were subsequently amended uniformly in various combinations with molybdate and divalent mercury in a novel approach that altered ambient volumes of pore water by less than 1%. Most-probable-number determinations indicated that there were approximately 10^6 to 10^8 iron-reducing bacteria per gram of Walker Marsh sediment. We are continuing attempts to isolate marine iron-reducing bacteria from Walker Marsh in pure culture as a prerequisite to assaying their ability to produce mono-methyl mercury from divalent mercury. These experiments generated more support for the view that bacteria other than sulfate-reducers are responsible for a substantial fraction of mercury methylation in marine and freshwater sediments. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: The PI is currently sharing unpublished data with various stakeholders including the San Francisco Estuary Institute and State Water Board personnel formulating policy for daily loads of mercury to Tomales Bay. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

      Impacts
      The vast majority of mercury mined in the U.S. was extracted from the Coast Range of California, and this mining legacy continues to impact our environment. Concerns about mercury derive from the ability of highly toxic methyl mercury to biomagnify in animals, including humans, near the top of food webs. Therefore, an understanding all significant microbial sources of methyl mercury in impacted environments is essential for coherent design of remedial actions.

      Publications

      • No publications reported this period


      Progress 01/01/10 to 12/31/10

      Outputs
      OUTPUTS: This project continues to focus on identifying the types of microbes that are major contributors to the pathways of mercury methylation in organic-rich sediments driven by the impact of two Coast Range mine sites from which mercury ore was previously mined. One mine impacts freshwater lake sediments and the other estuarine sediments; thus, the receiving bodies represent the two prominent niches where fish and birds are impacted by the environmental pollutant, mono-methylmercury, which is bio-accumulated at higher trophic levels. It has been known for some time that methylmercury is formed in sediments mainly via the activity of anaerobic Bacteria and perhaps Archaea interacting with various forms of inorganic mercury. The literature paradigm accepted for more than 20 years has been that almost all anaerobic mercury-methylation can be ascribed to the activity of sulfate-reducing bacteria. We used the molybdate anion, a specific inhibitor of this bacterial process, to test this assertion. Molybdate acts to inhibit sulfate reducing-bacteria by competing with sulfate in the first step in the dissimilatory sulfate-reduction pathway, which is an ATP-consuming reaction. The resultant molybdenum compound is unstable and decomposes with a resulting futile consumption of ATP. Previous years' research showed that sulfate reduction in mercury-amended sediments from Walker Marsh and Clear Lake was completely inhibited by molybdate at concentrations that were roughly 10% of ambient sulfate values. By contrast, at these same molybdate levels, 50% or more of ambient mercury methylation persisted. If sulfate-reducing bacteria were the only microbes responsible for methylation, full inhibition of methylation would have been the expected result. Furthermore, our previous pure culture studies showed that certain strains of iron-reducing bacteria are as prolific at mercury methylation as sulfate-reducing bacteria. In our most recent studies we adapted the use of gas-tight sediment incubation bags to confirm our earlier findings under conditions that more closely mimic natural sediments. Under anaerobic conditions we homogenized a sample of the top 10 cm of Walker Marsh sediments, which included an upper suboxic but oxidized zone where oxidized iron and oxidized manganese are the dominant electron acceptors and a deeper more reduced zone where sulfate-reducing bacteria will predominate. This homogenized sediment was then distributed under anoxic conditions into replicate gas-tight bags that were subsequently amended in various combinations with molybdate and divalent mercury. Most-probable-number determinations indicated that there were approximately 10^6 to 10^8 iron-reducing bacteria per gram of Walker Marsh sediment. We are continuing attempts to isolate marine iron-reducing bacteria from Walker Marsh in pure culture as a prerequisite to assaying their ability to produce mono-methyl mercury from divalent mercury. These experiments generated more support for the view that bacteria other than sulfate-reducers are responsible for a substantial fraction of mercury methylation in marine and freshwater sediments. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: The PI is currently sharing unpublished data with various stakeholders including the San Francisco Estuary Institute and State Water Board personnel formulating policy for daily loads of mercury to Tomales Bay. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

      Impacts
      The vast majority of mercury mined in the U.S. was extracted from the Coast Range of California, and this mining legacy continues to impact our environment. Concerns about mercury derive from the ability of highly toxic methyl mercury to biomagnify in animals, including humans, near the top of food webs. Therefore, an understanding all significant microbial sources of methyl mercury in impacted environments is essential for coherent design of remedial actions.

      Publications

      • No publications reported this period


      Progress 01/01/09 to 12/31/09

      Outputs
      OUTPUTS: This project focuses on identifying the types of microbes that are major contributors to the pathways of mercury methylation in organic-rich sediments driven by the impact of two Coast Range mine sites from which mercury ore was previously mined. One mine impacts freshwater lake sediments and the other estuarine sediments; thus, the receiving bodies represent the two prominent niches where fish and birds are impacted by the environmental pollutant, mono-methylmercury, which is bio-accumulated at higher trophic levels. It has been known for some time that methylmercury is formed in sediments mainly via the activity of anaerobic Bacteria and perhaps Archaea interacting with various forms of inorganic mercury. The literature paradigm accepted for more than 20 years has been that almost all anaerobic mercury-methylation can be ascribed to the activity of sulfate-reducing bacteria. We used the molybdate anion, a specific inhibitor of this bacterial process, to test this assertion. Molybdate acts to inhibit sulfate reducing-bacteria by competing with sulfate in the first step in the dissimilatory sulfate-reduction pathway, which is an ATP-consuming reaction. The resultant molybdenum compound is unstable and decomposes with a resulting futile consumption of ATP. Previous years' research showed that sulfate reduction in mercury-amended sediments from Walker Marsh and Clear Lake was completely inhibited by molybdate at concentrations that were roughly 10% of ambient sulfate values. By contrast, at these same molybdate levels, 50% or more of ambient mercury methylation persisted. If sulfate-reducing bacteria were the only microbes responsible for methylation, full inhibition of methylation would have been the expected result. Furthermore, our previous pure culture studies showed that certain strains of iron-reducing bacteria are as prolific at mercury methylation as sulfate-reducing bacteria. In our most recent studies we adapted the use of gas-tight sediment incubation bags to confirm our earlier findings under conditions that more closely mimic natural sediments. Under anaerobic conditions we homogenized a sample of the top 10 cm of Walker Marsh sediments, which included an upper suboxic but oxidized zone where oxidized iron and oxidized manganese are the dominant electron acceptors and a deeper more reduced zone where sulfate-reducing bacteria will predominate. This homogenized sediment was then distributed under anoxic conditions into replicate gas-tight bags that were subsequently amended in various combinations with molybdate and divalent mercury. Most-probable-number determinations indicated that there were approximately 10^6 to 10^8 iron-reducing bacteria per gram of Walker Marsh sediment. We are continuing attempts to isolate marine iron-reducing bacteria from Walker Marsh in pure culture as a prerequisite to assaying their ability to produce mono-methyl mercury from divalent mercury. These experiments generated more support for the view that bacteria other than sulfate-reducers are responsible for a substantial fraction of mercury methylation in marine and freshwater sediments. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: The PI is currently sharing unpublished data with various stakeholders including the San Francisco Estuary Institute and State Water Board personnel formulating policy for daily loads of mercury to Tomales Bay. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

      Impacts
      The vast majority of mercury mined in the U.S. was extracted from the Coast Range of California, and this mining legacy continues to impact our environment. Concerns about mercury derive from the ability of highly toxic methyl mercury to biomagnify in animals, including humans, near the top of food webs. Therefore, an understanding all significant microbial sources of methyl mercury in impacted environments is essential for coherent design of remedial actions.

      Publications

      • No publications reported this period


      Progress 01/01/08 to 12/31/08

      Outputs
      OUTPUTS: This project continues to focus on identifying the types of microbes that are major contributors to the pathways of mercury methylation in organic-rich sediments driven by the impact of two Coast Range sites where mercury ore was previously mined. One mine impacts freshwater lake sediments and the other estuarine sediments; thus, the receiving bodies represent the two prominent niches where fish and birds are impacted by this environmental pollutant that is bio-accumulated at higher trophic levels. It has been known for some time that methylmercury is formed in sediments mainly via the activity of anaerobic Bacteria and perhaps Archaea interacting with various forms of inorganic mercury. The literature paradigm accepted for more than 20 years has been that almost all anaerobic mercury-methylation can be ascribed to the activity of sulfate-reducing bacteria. We used the molybdate anion, a specific inhibitor of this bacterial process, to test this assertion. Molybdate acts to inhibit sulfate reducing-bacteria by competing with sulfate in the first step in the dissimilatory sulfate-reduction pathway, which is an ATP-consuming reaction. Previous years' research showed that sulfate reduction in mercury-amended sediments from Walker Marsh and Clear Lake was completely inhibited by molybdate at concentrations that were roughly 10% of ambient sulfate values. By contrast, at these same molybdate levels, 50% or more of ambient mercury methylation persisted. If sulfate-reducing bacteria were the only microbes responsible for methylation, full inhibition of methylation would have been the expected result. Furthermore, our previous pure culture studies showed that certain strains of iron-reducing bacteria are as prolific at mercury methylation as sulfate-reducing bacteria. In our most recent studies we adapted the use of gas-tight sediment incubation bags to confirm our earlier findings under conditions that more closely mimic natural sediments. Under anaerobic conditions we homogenized a sample of the top 10 cm of Walker Marsh sediments, which included an upper suboxic but oxidized zone where oxidized iron and oxidized manganese are the dominant electron acceptors and a deeper more reduced zone where sulfate-reducing bacteria will predominate. This homogenized sediment was then distributed under anoxic conditions into replicate gas-tight bags that were subsequently amended in various combinations with molybdate and divalent mercury. Excellent statistical agreement between samples from replicate bag incubations demonstrated that this approach has great potential for producing more environmentally realistic studies of mercury methylation. Most-probable-number determinations for iron- reducing bacteria indicated 10^6 to 10^8 of these bacteria per gram of Walker Marsh sediment. Furthermore, 16S rRNA analysis of randomly selected bacterial clones from positive enrichments showed that approximately half of these were from known iron-reducing bacteria. These experiments generated more support for the view that bacteria other than sulfate-reducers are responsible for a substantial fraction of mercury methylation in marine and freshwater sediments. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: The PI summarized these findings both for marine and freshwater sediments in an invited presentation to a stakeholders group (Delta Tributaries Mercury Council) in Sacramento in May 2008. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

      Impacts
      The vast majority of mercury mined in the U.S. was extracted from the Coast Range of California, and this mining legacy continues to impact our environment. Concerns about mercury derive from the ability of highly toxic methyl mercury to biomagnify in animals, including humans, near the top of food webs. Therefore, an understanding all significant microbial sources of methyl mercury in impacted environments is essential for coherent design of remedial actions.

      Publications

      • No publications reported this period


      Progress 01/01/07 to 12/31/07

      Outputs
      This project focuses on the microbial portion of the biotic pathways of mercury methylation driven by the impact of two Coast Range sites where mercury ore was previously mined. One mine impacts freshwater lake sediments and the other estuarine sediments; thus, the receiving bodies represent the two prominent niches where fish and birds are impacted by this environmental pollutant. It has been known for some time that methylmercury is formed mainly in sediments via anaerobic bacteria interacting with various forms of inorganic mercury. The accepted literature paradigm is that mercury-methylation can be ascribed to the activity of sulfate-reducing bacteria. We used the molybdate anion, a specific inhibitor of this bacterial process, to test this assertion. Molybdate acts to inhibit sulfate reducing-bacteria by competing with sulfate in the first step in the dissimilatory sulfate-reduction pathway, which is an ATP-consuming reaction. The resultant molybdenum compound is unstable and decomposes with a resulting futile consumption of ATP. Previous years' research showed that sulfate reduction in mercury-amended sediments from Walker Marsh and Clear Lake was completely inhibited by molybdate at concentrations that were roughly 10% of ambient sulfate values. By contrast, at these same molybdate levels, 50% or more of ambient mercury methylation persisted. If sulfate-reducing bacteria were the only microbes responsible for methylation, full inhibition of methylation would have been the expected result. An iron-reducing bacterium cultured from Clear Lake was reported previously (2005) to convert inorganic mercury to methyl mercury at per cell rates comparable to active sulfate reducing bacteria. In the current year repeated molecular and culturing studies of Walker Marsh sediments demonstrated that high populations of iron-reducing bacteria were present as sites that yielded high rates of molybdate-insensitive methylation. Furthermore, laboratory based studies were undertaken to assess an alternative explanation for this methylation. It had been established previously by others that sulfate-reducing bacterial species such as DESULFOBULBUS PROPIONICUS could also grow in the absence of sulfate (via fermentation) and would produce methyl mercury just as actively as if they were reducing sulfate. Thus, this mode of growth had to be considered as a possible explanation for molybdate-insensitive methylation observed in our experiments. Pure culture studies with DESULFOBULBUS PROPIONICUS strain 1pr3 showed that 2mM molybdate caused equally active cell death and lysis under either set of conditions. That is, even though growing by fermentation, these bacteria appear to be constitutive for the first step in sulfate reduction and, therefore, still inhibited by molybdate. These experiments generate more support for the view that bacteria other than sulfate-reducers are responsible for a substantial fraction of mercury methylation in marine and freshwater sediments.

      Impacts
      The vast majority of mercury mined in the U.S. was extracted from the Coast Range of California, and this mining legacy continues to impact our environment. Concerns about mercury derive from the ability of highly toxic methyl mercury to biomagnify in animals, including humans, near the top of food webs. Therefore, an understanding all significant microbial sources of methyl mercury in impacted environments is essential for coherent design of remedial actions.

      Publications

      • Mercury methylation by sulfate-reducing bacteria and iron-reducing bacteria in situ and in culture. 2007. Emily J. Fleming, PhD dissertation, UC Davis, 130 pp.


      Progress 01/01/06 to 12/31/06

      Outputs
      This study focuses on the microbial portion of the biotic pathways of mercury methylation driven by the impact of two Coast Range sites where mercury ore was previously mined. One mine impacts freshwater lake sediments and the other estuarine sediments; thus, the receiving bodies represent the two prominent niches where fish and birds are impacted by this environmental pollutant. It has been known for some time that methylmercury is formed in anoxic sediments via certain anaerobic bacteria interacting with various forms of inorganic mercury. The accepted literature paradigm is that mercury-methylation is generally attributed to the activity of sulfate-reducing bacteria, and we used the molybdate anion, a specific inhibitor of this bacterial process, to test this assertion. Molybdate acts to inhibit sulfate reducing-bacteria by competing with sulfate in the first step in the dissimilatory sulfate-reduction pathway, which is an ATP-consuming reaction. The resultant molybdenum compound is unstable and decomposes with a resulting futile consumption of ATP. For mercury-amended sediments from Walker Marsh (and in parallel with rates observed with sediments incubated in the absence of mercury), 3mM molybdate was completely inhibitory and 0.3mM molybdate decreased sulfate reduction rates by 74-91% vs. no-molybdate controls. Mercury methylation in uninhibited sediments occurred at rates seen in other systems. At final concentrations of 0.3mM and 3mM added molybdate, mercury methylation rates decreased by 22 and 47%, respectively. If sulfate-reducing bacteria were the only microbes responsible for methylation one would expect full inhibition at 3mM molybdate. This observed incomplete inhibition of mercury methylation has not been reported for other marine systems, but the results parallel our earlier findings for Clear Lake, CA. The recently described ability of iron-reducing bacteria to methylate mercury in these freshwater sediments (2005 Progress Report, this project) expands the kinds of anaerobic bacteria and terminal electron acceptors known to be important in the production of methylmercury. Concentrations of reducible-iron measured in Walker Marsh sediments were equivalent to those in systems where iron-reduction was determined to be the dominant pathway for anaerobic carbon oxidation. Inoculated with sediments from Walker Marsh, enrichments using media rich in reducible-iron yielded several genera of bacteria that were present in high numbers and were identified through molecular sequencing to be members of the genus DESULFUROMONAS or PELOBACTER. Members of these genera are known from the literature to be active in iron reduction and to play a significant role in carbon cycling in marine sediments. Based on the possibly widespread ability of these organisms to methylate mercury and their presence in these sediments, marine mercury methylation that is not attributable to sulfate-reducing bacteria may well be due to the activity of these or related iron-reducing bacteria.

      Impacts
      The vast majority of mercury mined in the U.S. was extracted from the Coast Range of California, and this mining legacy continues to impact our environment. Concerns about mercury derive from the ability of highly toxic methylmercury to biomagnify in animals, including humans, near the top of food webs. Therefore, an understanding all significant microbial sources of methylmercury in impacted environments is essential for coherent design of remedial actions.

      Publications

      • Fleming, E.J., Mack, E.E., Green, P.G. and Nelson, D.C. 2006. Mercury methylation from unexpected sources: molybdate-inhibited freshwater sediments and an iron-reducing bacterium. Appl. Environ. Microbiol. 72:457-464.


      Progress 01/01/05 to 12/31/05

      Outputs
      This study focuses on the microbial portion of the biotic pathways of mercury methylation driven by the impact of two Coast Range mine sites. One mine impacts freshwater lake sediments and the other estuarine sediments; thus, the receiving water bodies represent the two prominent niches where fish and birds are impacted by this environmental pollutant. It has been known for some time that methylmercury is formed in anoxic sediments by the activity of certain anaerobic bacteria interacting with various forms of divalent inorganic mercury. During the reporting period we published evidence, based on circum-neutral pH portions of Clear Lake sediments, that the accepted literature paradigm, namely that sulfate-reducing bacteria are the principal methylators of inorganic mercury in anoxic sediments, is too simplistic. These experiments, which were based on use of the molybdate anion as a specific inhibitor of sulfate-reducing bacteria, showed that molybdate concentrations sufficient to inhibit an average of 87% of all sulfate reduction activity inhibited mercury methylation by an average of only 28%. These results emphasize that additional types of bacteria are performing the majority of mercury methylation in these sediments. One additional candidate identified by our research is a group called "iron-reducing bacteria", which use soluble or solid forms of oxidized iron, Fe(III), as anaerobic electron acceptors. We demonstrated that these bacteria are present at a minimum of one million cells per gram of Clear Lake sediments. Additionally, the first pure culture we obtained from this sediment source (a GEOBACTER species, designated strain CLFeRB) was able to methylate inorganic mercury at a per cell rate comparable to the most active sulfate reducing bacteria previously tested by other researchers. This strain converted divalent mercury into mono-methylmercury regardless of whether it was in exponential or stationary growth phase and regardless of whether the oxidized iron was supplied in a solid or chelated form.

      Impacts
      The vast majority of mercury mined in the United States was extracted from the Coast Range of California, and its legacy continues to impact our environment. Concerns about mercury derive from the ability of highly toxic forms of this element to biomagnify in animals, including humans, near the top of aquatic food webs. Understanding major microbial sources of methylmercury in impacted environments is essential for coherent design of remedial actions.

      Publications

      • No publications reported this period