Objective: Determine factors controlling bacterially-mediated Hg methylation and demethylation with an emphasis on redo

1. Mark E. Hines Isaac Adatto Soujanya L. Rallabandi Department of Biological Sciences UMass Lowell Objective: Determine factors controlling bacterially-mediated Hg methylation and demethylation with an emphasis on redox changes

2. CO2 + Hg2+ Oxidative Process CH3Hg+ CH4 + CO2 + Hg2+ Hg0 Hg2+ CH4 + Hg0 Reductive Process merB merA CO2 + Hg2+ CH3Hg+ Oxidative Process Oxic Water Anoxic Sediments SRB Hg2+ (Hg(HS)2)

4. Rates Bog Floating Mat SOIL Sedge SURF Grass & Sedge SURN

5. Approach • Potential Hg methylation • 203Hg2+ - toluene extraction and scintillation counting • Potential methylmercury demethylation • 14C-MeHg chloride - separation of gases (CO2 and CH4) • Reductive demethylation 14CH4; oxidative 14CO2 • Microbial metabolism • Incubation and gas and solute changes • Methanogenesis, sulfate reduction, nitrate reduction, acetogenesis, carbon dioxide

6. 0.30 Rates 0.25 0.20 Bog Floating Mat Percent per day-1 Percent day-1 SOIL 0.15 Sedge SURF Grass & Sedge SURN 0.10 0.05 0 Soil Bog SURF SURN Sedge Floating Mat Mixed Grass/Sedge Hg Methylation Potential June (Dry) • Methylation rates are very low • Riparian soil near creek is most active, whereas sedge and upland soil least active ‘Demethylation not detected’

7. Rates Bog Sedge Bog Upper Floating Mat Bog Lower SURF Lower SURF Soil SURF Upper SURN Lower SURN Upper Floating Mat Mixed Grass/Sedge Percent day-1 SOIL Sedge SURF Grass & Sedge SURN Hg Methylation Potential Fall (wet) 2.5 Methylation 2.0 1.5 Percent per day-1 1.0 0.5 0 -0.5 • Methylation most active in riparian and bog • Sedge and soil least active

8. Rates Bog Floating Mat Percent day-1 SOIL Sedge SURF Grass & Sedge SURN MeHg Demethylation Potential 3.5 Demethylation 3.0 CO2 CH4 2.5 2.0 Percent per day-1 1.5 1.0 0.5 0 • Demethylation generally low and highly variable (some replicates were undetectable), but active in boggy areas not riparian regions • Anoxic regions usually support oxidative path • Despite similar rate constants, methylation probably greatly outweighs demethylation Sedge Bog Upper Bog Lower SURF Soil SURF Lower Floating Mat SURF Upper SURN Lower SURN Upper Mixed Grass/Sedge

9. 2,500 2,000 1,500 Total C degraded* (nmol ml-1 day-1) 1,000 500 0 F. MAT BOG S MV SURF SURN Microbial ‘Respiration/Fermentation’ *total C degraded = sum of CH4, CO2 and acetate (x2) accumulation

10. Importance of Alternate Electron Acceptors and Fermentation Polymers  Monomers  CO2 • Hg methylation, demethylation and mobility should be greatly affected by redox status including changes in the use of various electron acceptors during bacterial respiration • Fermentation becomes an increasingly important metabolic path as the system becomes more reducing Polymers  Monomers  Fermentation products  CO2

11. Polymers LMW acids/ alcohols H2 + CO2 CO2, CH4 Acetate Acetate Methanogenic pathways Intermediate Monomers Fermentation H2 + CO2 Acetate Acetogenesis 2o Fermentation Acetogenesis Terminal step Terminal step Terminal step

12. 800 700 CO2 NO3 SO4 600 acetate propionate 500 nmol ml-1 day-1 400 300 200 100 0 F. MAT BOG S MV SURF SURN -100 Microbial ‘Respiration/Fermentation’ • Methanogenesis was practically non-existent except at the floating mat where it was a few nmol ml-1 day-1. • Sulfate reduction occurred only in the floating mat • Nitrate reduction was noted only in the sedge communities • Acetogenesis was an important feature everywhere except the sedge communities

13. Microbial ‘Fermentation’ 1600 1400 • Acetate was a dominant end product and by far the dominant organic product • Propionate accumulation was high at the same sites as acetate 1200 Acetate-C Production Propionate-C Production (x10) 1000 VFA Production (nmol ml-1 day-1) 800 600 400 200 0 F. MAT BOG S MV SURF SURN

14. 2.5 2.0 1.5 Acetate-C/CO2 1.0 0.5 0.0 F. MAT BOG S MV SURF SURN Importance of Acetate as an End Product • Acetate-C is produced as readily as CO2, especially in boggy sites and riparian areas

15. Polymers LMW acids/ alcohols H2 + CO2 CO2, CH4 Acetate Acetate Decoupling of Terminal Processes Monomers 1° Fermentation H2 + CO2 Acetate Acetogenesis X 2o Fermentation Acetogenesis ? X Methanogenesis X X Methanogenesis Methanogenesis

16. H2S MeSH Pectin Hg Methanol DMS MeHg CO2 CO2, CH4 Who Cares? C-1 Compounds * O2, NO3-, Fe3+, SO42- Methane Producers

17. NH AK 4 500 400 3 1.5 300 Acetate (mM) CH4 (µM) 1.3 DMS (µM) 2 200 1.0 1 100 0.8 0.5 0 0 0 2 4 6 8 0 2 4 6 8 0.3 Days 0.0 Methane, Acetate and C1 Compounds are Produced Simultaneously In no cases do acetate or C1 compounds accumulate independently

18. 1600 Control 1200 + Fe(III) Acetate (µM) 800 Acetate (and C1) is consumed by all other processes except methanogenesis + NO3- 400 + O2 0 160 450 700 650 300 120 Nitrate (uM) Reduced Fe (uM) 600 150 CH4 (µM) 550 0 500 0 1 2 3 4 80 Time (day) 40 0 6 0 2 4 Days

19. Methylation is an Anaerobic Process Riverbanks Sediments 203Hg2+ CH3203Hg+ 20 Wet (Anoxic) 16 12 Methylation (% /day) 8 Wet (Oxic) Dry (Anoxic) 4 0 0 1 2 3 4 5 6 7 8 9 Days Oxygen exposure rapidly inhibits methylation. However, even deposits that have been dry and oxic for months retain the ability to methylate when anoxia is restored.

20. CH4 CO2 Demethylation changes pathway • Oxidative is replaced rapidly by reductive during drying. • Upon reintroduction of anoxic conditions, the reductive path persists • After several weeks of anoxia, path switches back to oxidative. • Samples maintained wet and anoxic eventually support methanogenesis, which increases importance of methane as an end product, but does not indicate a reductive path. 12 Dry (Anoxic) 10 8 6 4 2 Demethylation (% perday) 0 1 4 20 34 2 56 85 100 10 Wet (Anoxic) 8 6 4 2 0 85 1 2 4 20 34 56 100 Days

21. Conclusions, So Far • Methylation occurs primarily during wet periods. • Methylation is highest in riparian soils near the stream, but is also active in “boggy” areas. • Methylation is insignificant in “sedge” soils, which may be due to easy drainage. • Demethylation appears to be quite low, but is significant in “boggy” areas. • The lack of active demethylation in riparian soils, in conjunction with active methylation, may help explain high methylmercury levels there. • As noted in other sites, methylation tends to occur via the oxidative path in anoxic sites, but this is not universal.

22. Conclusions, So Far (con’t) • Bog and riparian areas support the highest decomposition rates, i.e., highest microbial activity. • Methanogenesis is very slow, but highest in wet bog sites. • Acetate is an important end product of anaerobic metabolism in bog and riparian areas, which indicates a decoupling of primary (and secondary) fermentation from terminal decomposition. • Since acetate is a “carboxylated” C1 compound that behaves biochemically like real C1 compounds, it is possible that the demethylation of MeHg is inhibited to some degree. • Methylation is very sensitive to oxygen, but extended oxidation does not preclude methylation in the future

23. Conclusions So Far (con’t) • Oxygen presence also affects the path (and probably the magnitude) of demethylation. • Drying and oxygenation leads to a rapid shift to reductive demethylation (mer), whereas a shift back to anoxia does not lead to oxidative demethylation for several weeks to months.

24. Future • Concentrate on factors affecting methylation and demethylation (or the lack thereof) in riparian regions, but other wetlands deserve attention • Effects of wetting and drying • Effects of alternate electron acceptors • Potential for the inhibition of demethylation including more thorough investigation of ability to demethylate • Tie microbial diversity to Hg transformations • More detailed field study of temporal changes in microbial activities including respiration/fermentation and Hg transformations (including Hg speciation)