1. Biogeochemical Framework to Evaluate Mercury Methylation Potential During in-situ Remediation of Contaminated Sediments NIEHS R01ES024344 2014-2018 Heileen Hsu-Kim, Marc Deshusses Duke University Dwayne Elias Oak Ridge National Lab

2. Project team Duke University Helen Hsu-Kim (PI) – Aquatic Geochemistry Marc Deshusses (Co-PI) – Bioremediation Oak Ridge National Lab Dwayne Elias (Co-PI) – Microbial Ecology Other Collaborators Steven Brown,Ph.D., Dow Chemical, Berry’s Creek Study Group

3. Challenges of the mercury problem: Selin, 2009, Annu. Rev. Environ. Resour. Objective: To establish biogeochemical indicators for methylmercury production potential Many sources to biosphere Long range transport Food web accumulation Mechanisms of MeHg production

4. Factors contributing towards mercury methylation potential Framework to predict methylation potential Productivity of methylating microorganisms Bioavailability of Hg ? ?

5. High methylation potential Low methylation potential HgH x S 2 x-2 Hg-thiol Hg-DOM Amorphous or crystalline HgS nanoparticles Aggregated or micro-crystalline HgS (s) DOM-capped polynuclear HgS clusters Dissolved Hg(II) complexes Geochemical Forms of Mercury in Sediments

6. Microorganisms that Methylate Mercury Deltaproteobacteria Gilmour et al., ES&T, 2013 Firmicutes, Clostridia ARCHAEA Euryarchaeota Obligate anaerobes Phylogenically diverse hgcAB: two gene cluster

7. Freshwater Saline water Ambient Hg: 2 nmol g -1 Spike Hg: 2 nmol g -1 [SO 4 2- ] 0 < 0.07 mM [SO 4 2- ] 0 = 15 mM Biomethylation in Sediment Slurry Microcosms Bioavailability-limited methylation Productivity-limited methylation Zhang et al., ES&T, 2014 dissolved Hg+sulfide nano-HgS microcrystalline HgS dissolved Hg+sulfide nano-HgS microcrystalline HgS

8. Bioavailability vs. Productivity Threshold? Kucharzyk et al., ICMGP, 2013 Mixed microbe community enriched from sediments Dissolved Hg added Nano HgS added C-substrate for growth

9. Project Objectives: Sediment-water microcosms with samples from Superfund site Aim 1: Activity of methylating microbes hgcAB gene abundance/expression Microbial diversity Aim 2: Hg bioavailability Size fractionation, solid-water partitioning of Hg Thiol-extraction potential Passive sampler with thiolated resin Aim 3: Delineation of the controls on methylation potential Limited by microbial activity Limited by bioavailability Aim 4: Effects of sediment amendments Activated carbon Clay mineral particles Ferrous iron (FeCl 2 ) Measurements of Methylation Potential Site Characterization and Remediation To establish biogeochemical indicators for methylmercury production potential To test the effectiveness of in-situ remediation

10. Design schematic for PCR, qPCR and RT- qPCR primers. Activity of Methylating Microbes Parks et al., Science, 2013 Gilmour et al., ES&T, 2013 Amino acid sequence alignments for hgcAB for all predicted Hg-methylating organisms

11. Quantification of Hg bioavailability Thiol-based selective extraction microbial culture: D. priopionicus 1pr3 bulk-scale HgS dissolved Hg+sulfide nano-HgS Zhang et al., ES&T, 2012

12. Evaluation of Hg methylation potential in sediments Anaerobic sediment- water microcosms Major experimental variables: Sediment origin Type of Hg added (dissolved, nanoHgS, Hg-FeS) differentiated by Hg isotope Measurements of methylation potential: MeHg concentration or net production rate Gene abundance & expression Hg bioavailability (thiol selective extraction) MeHg production rate hgcAB abundance or expression bioavailable [Hg] Data Analysis: Calibration of measurement methods Assess the effectiveness of remediation Field samples of benthic sediments (Oak Ridge, TN; Berry’s Creek Study Area; other sites?) Range of characteristics: Salinity Organic Carbon MeHg concentration (relative to total Hg)