1. Microbial solubilization and immobilization of toxic metals: key biogeochemical processes for treatment of contamination C. White, J.A. Sayer, G. M. Gadd Department of Biological Sciences, University of Dundee, Dundee, UK

2. Metal mobilization Metal immobilization Autotrophic and heterotrophic leaching Chelation by metabolites and siderospores Methylation Sorption to cell components Transport and intracellular sequestration Precipitation

3. Leaching by fungi Bioleaching : Dissolution of metals from their mineral source by organic acids produced by microorganisms BacteriaFungi Carried out by acidophilic bacteria Do not tolerate high pH values Yarrowia lipolytica (citric) Mucor spp.(fumaric, gluconic) Rhizopus spp (lactic, fumaric, gluconic) Aspergillus spp (citric, oxalic, malic, tartaric,α-ketoglutaric, itaconic) Penicillium spp (citric, tartaric, α-ketoglutaric, malic, gluconic)

4. Mechanisms : Metal cations may be directly displaced from the ore by hydrogen ions produced by the microorganisms resulting in a common acid leaching mechanism. Compounds produced by the microorganism can bind metals into soluble complexes by chelation.

5. Citrate  iron chelator, solubilizes Zn +2 from ZnO Oxalate  solubilizes phosphate, Al, Fe and Li, forms insoluble oxalates (mainly Ca oxalate)

6. Some examples: P.simplicissimum  leach Zn +2 in industrial filter dust by production of citric acid A.Niger  leach Cu from copper converter slag P.simplicissimum, A.Niger, P. notatum and Trichoderma viride  leach red mud

7. Microbial Metalloid Transformations Mechanisms Reduction of metalloid oxyanions to elemental forms Methylation of metalloids, metalloid oxyanions or organometalloids to methyl derivatives SeO 4 -2 and SeO 3 -2  Se o TeO 3 -2  Te o Se volatilization As volatilization Metalloids: B, Si, Ge, As, Sb, Te and Po selenate selenite

8. Metalloid transformations- Bioremediation Reduction to elemental forms: removal of Se in sediments, soil and water Methylation  volatilization: in situ bioremediation of soil and water at Kesterton Res. Simultaneous removal of NO 3 - and SeO 4 -2

9. Metal Precipitation by Sulfate- reducing Bacteria(SRB) Desulfovibrio desulfuricans www.sysbio.org/sysbio/ mechsensing/index.stm Low MW organic acids + SO 4 -2 HCO 3 - + H 2 S ATP

10. Immobilizing metals with SRB: Me +2 + S -2  MeS (s) Very stable under anaerobic conditions Optimum growth in pH 6-8 But sulfate reduction has been observed in pH of 3.25

11. Using SRB for bioremediation: Acid Mine Drainage(AMD)

12. What is AMD? 4Fe 2+ (aq) + 8SO 4 2- (aq) + 8H + (aq) When water discharges to surface: 4 Fe +2 + O 2 + 10 H 2 O  4 Fe(OH) 3(S) + 8 H + Yellow Boy

13. Remediation Options

15. (Clarified liquor) (sludge)

17. Sulfate-Reducing Permeable Reactive Zones (SR-PRZs) Aquifers Mine Tailings Contaminated Water Reactive Barrier Remediated Water 2 CH 2 O + SO 4 -2 → 2HCO 3 - + H 2 S Me +2 + S -2 → MeS

18. SR-PRZs Advantages Low cost In situ treatment Low maintenance Drawbacks Short lifetime Poor performance Long start-up time Understand role of microbial community Improve design of SR-PRZs

19. Carbon Flow Dynamics Polymers Oligomers Monomers Enzymatic Hydrolysis Organic Acids CO 2 Alcohols H2H2 Acetate CO 2 Fermentation Sulfate Reduction SO 4 H2SH2S Me 2+ MeS (s) CO 2 Hydrolytic processes Fermentative processes Sulfate-reduction processes Methanogenesis CH 4 + CO 2 Methanogenic processes

20. Objectives Compare microbial inocula performance in sulfate reducing barriers Column Experiments Identify and compare microbial communities PCR, Real Time PCR, DGGE, Cloning Develop microbial inocula to optimize SR- PRZs performance Batch, column and field experiments

21. Columns Set Up Simulated mine drainage water: 1.3204 g/L Na 2 SO 4 0.034 g/L FeSO 4 0.0268 g/L NH 4 Cl0.0088 g/L CdCl 2 0.0500 g/l ZnSO 4 pH 5.65 Substrate: Top layer:Bottom layer: Silica sand10% crushed pyrite 90% silica sand Middle layer: 22% wood chips45% silica sand 2% alfalfa11% pine bedding 5% limestone15% inoculum

24. Microbial Community Profiling Simulated mine drainage water effluent DNA extraction 16S rDNA PCR DGGE DNA sequencing Identification of microorganisms

25. DGGE Gel Acclimated Inoculum Column * Putative based on DNA sequence similarity Band 11: sulfate reducer* Std. 0 1 st wk 2 nd wk 3 rd wk 4 th wk Std. Band 9: Fermenter* Band 10: Fermenter* Band 5: Fermenter* Band 17: fermenter or cellulolytic bacteria* Band 21: sulfate reducer or fermenter* Band 7: sulfate reducer*

26. Batch Experiment Simulated mine drainage water Organic matter Inoculum Inocula: Dairy Manure Material from 2 SR- bioreactors Anaerobic Digester sludge Twice acclimated inoculum

27. Batch Experiment Will help identify inocula with key capabilities Inocula can be enriched or combined to enhance their performance Improved inocula can be tested in column and field experiments that better represent the SR-PRZs conditions

30. Conclusions Inocula play important role in column/batch reactors performance The wrong inoculum may not provide any advantage over no inoculum at all A suite of molecular methods has been developed which will allow for evaluation of microbial communities in the field and also to guide in construction of ideal inocula

31. What’s next? Complete DNA analysis Further test inocula in another batch experiment (this time inoculating with cells only to avoid matrix effects) Combine inocula that showed good performance to create improved inocula Test improved inocula in column experiments that better represent the conditions in the PRZs Do field experiments. Evaluate effect of pH, temperature and stresses on microbial community