1. MICROBIAL INTERACTIONS IN MINE TAILINGS GEOL 7740 Topic in Environmental Geosciences Stanislaw Lozecznik Department of Civil Engineering University of Manitoba

2. Bacterial Activity Singer and Stumm (1970) showed that presence of iron-oxidizing bacteria accelerated the oxidation of Fe 2+ in AMD by a factor larger than 10 6 compared to abiotic conditions Acidithiobacillus ferrooxidans (Gleisner et al., 2006)

3. Gram-negative SRB have been detected in-situ, supporting the important role of these microorganisms in selective ZnS precipitation (Tennyson, Wisconsin, USA) Desulfobacteriase (Labrenz et al., 2004) (a)Cells walls of cut bacteria (b) ZnS granules (c) EPS and piece of wood B A C

4. Metal cyanide degrading bacteria from gold mine tailings Dams

5. Outline General Intro to Bacteria (Prokaryotes) Cell distribution Cell wall composition Metabolic classification Redox reactions Microbial ecology of AMD Role of microorganisms in the treatment of AMD

6. Introduction to Bacteria Eukaryotic cell structure

7. Cell Size Prokaryotic cells are generally smaller than eukaryotic cell Small cells have a higher growth rate than larger cells Small cells have a “higher surface-to-volume” ratio than larger cells The higher metabolic activity of small cells is due to additional membrane surface available for transport of nutrients into the cell.

8. Cell wall Chemical cross biological membranes by diffusion, active transport, and endocytosis. Cell wall in bacteria maintains their characteristic shape, and protecting it from osmotic pressure. Identification of type of bacteria by its cell wall stain  Gram-positive  Gram negative

9. Anaerobic, Gram-positive rods Actinomyces sp. Anaerobic, Gram-negative rods Fusobacterium sp.

10. Metabolic classification Carbon and energy source Effects on pH on growth Temperature Oxygen Aerobic and Anaerobic

11. Carbon and energy source Heterotrophic bacteria Can use either simple or complex organic compounds as a main carbon source, and obtain energy by oxidizing organic compounds Autotrophic bacteria –Grow solely on inorganic compounds, with carbon dioxide as the carbon source and photosynthesis or oxidation of inorganic compounds as the energy source.

12. Effects of pH on growth Each bacterial specie has a range of pH value over which growth is possible –Acidophilic Moderate Extremely (Iron mountain)

13. Psychrophiles T from 0 to 25 ⁰ C Optimum 10 to 15 ⁰ C Mesophiles T from 10 to 40 ⁰ C Optimum 25 to 40 ⁰ C Thermophiles T from 50 to 90 ⁰ C Optimum 50 to 80 ⁰ C Temperature

14. Oxygen Aerobic: Requires oxygen for respiration, although some bacteria are able to use alternative electron acceptors Anaerobic: Grow only in the absence of oxygen and some of them are able to ferment sugars and amino acids to organic acids and alcohols. Facultative : can grow either way Microaerophilic: Are able to grow in very low concentrations of oxygen

15. REDOX and Bacteria involvement (1) FeS 2 + 14Fe 3+ + 8H 2 O -> 15Fe 2+ + 2SO 4 2- + 16H + Oxidation of pyrite with ferric iron (Fe 3+ ) (2) 14Fe 2+ + 3.5O 2 + 14H + -> 14Fe 3+ + 7H 2 O Fe 2+ oxidation by O 2 at low pH is kinetically slow, thus this rate may limit the rate for pyrite dissolution

16. Iron-oxidizing prokaryotes catalyze reaction (2), primarily biological in acidic environment (pH<4). These organisms accelerate pyrite dissolution by re-generating ferric iron (Fe 3+ ). Acidithiobacillus ferrooxidans, L. ferrooxidans

17. Iron Reduction AMD solutions are iron rich because ferric and ferrous iron are very soluble at low pH ( pH < 2.5) In some cases, Fe 3+ may exceed O 2 concentrations by several orders of magnitude

18. Johnson and McGuiness (1991) showed the ability to reduce soluble Fe 3+ among heterotrophic acidophiles Some species are able to reduce Fe 3+ even if it is not in solution. S acidophilus is capable of anaerobic dissolution of iron hydroxide, jarosite and goethite.

19. Sulfur oxidation Most bacterial community of AMD that can oxidize sulfur also can fix CO 2. A variety of sulfur compounds with oxidation states intermediate between 2- to 6+ form during metal sulfide oxidation. A. ferroxidans also can grow under anoxic conditions using Fe 3+ as the e - acceptor and S o the e - donor

20. Biofilm It is a layer of slime made up of EPS, often negatively charged polysaccharides, that surrounds and is excreted by the organisms Water (often > 90%) EPS (up to 90% of organic matter) Cells Entrapped particles and precipitates Sorbed ions and polar and apolar organic molecules

21. Microbial community Individual species of the consortium are arranged within this slime layer so each type of metabolism contributes most efficiently to the whole biofilm ecosystem. Microbial communities affect the pH and Redox of natural waters, determine the form of the iron solution, as well as the iron compounds that are precipitated.

22. Microbial ecology of acid environments This environment contain a variety of acidophilic microorganisms  Fe oxidizers  S oxidizers  Facultative S-oxidizing and obligate heterotrophs More recent work has shown extremely acid ecosystems (Iron mountain, CA) support a diverse and unusual suite of organisms (extreme acidophiles – many of the Archae type).

23. Methods for the study of microorganisms in tailings environment Isolation of bacteria: Culturing a sample in a enrichment or selective liquid medium (e.g. solid agar medium depending on the type of organism isolated). The medium contains nutrients (e.g. ferrous iron and inorganic nutrients for A. ferrooxidans)

24. Enumeration: Direct colony count or by a statistical technique know as most probable number (MPN) Based on dilution and estimation of single cells from a homogenous suspension (e.g. sample bioluminescent)

25. Molecular biology: Identify bacteria that are not culturable by conventional means 1.DNA extraction 2.Reverse sample genome probe (RSGP) 3.FISH

26. The DNA is extracted and specific segments of DNA corresponding to the 16S rRNA are amplified using the polymerase chain reaction (PCR). The segments are cloned, and the cloned fragments are then sequenced. This sequence is compared to clone libraries of 6S rRNA.

27. The RSGP involves extracting DNA from a sample and spotting it on a filter containing bacterial DNA from pure isolates. Various portions of the DNA sample will hybridize with the DNA of either identical or closely related bacterial. The microbial isolates on the filter composition of the sample can be estimated from the bacteria isolates on the filter that show hybridization.

28. FISH involves adding a fluorescently labeled oligunocleotide probe to a sample fixed on a microscope slide. The fluorescent nucleotide probes can be designed to hybridize with (1) one specie of bacteria, (2) small number of related species, or (3) a large group of related bacteria Nitrifiers (red) autotrophs Denitrifiers (green) heterotrophs

29. Treatment of existing AMD Lime is a common method but it produces large quantities of sludge. Tailings can also generate acid for a long period of time (100 years), making expensive its use. Biological treatment offer the possibility of treatment process that are inexpensive and potentially self-sustaining

30. SRB : Sulfate reduction produces HCO 3 - and HS -. HS - leads to permanent alkalinity when sulfide escapes as H 2 S gas. The lower the pH, more H 2 S gas is released from the system. 2CH 3 CHOHCOO - + 3SO 4 2- => 6HCO 3 2- + 3HS - + H + SRB play one of the most important role in AMD mitigation

31. Algae: Can sequester metal ions by cation exchange, chelation, adsorption, modification of chemical environment around the cell, or by acting as nucleation center for metal precipitation. Iron and Mn-reducing bacteria

32. Passive treatment of AMD in bioreactors AMD contaminated waters contain low concentrations of dissolved organic carbon that can limit microbial activity Not all SRB species are capable of oxidizing lactate and ethanol to CO 2. Natural organic materials such as wastes from agricultural and food processing industry have also been assessed for their potential to promote and sustain sulphate-reduction. They are divided in two groups: cellulosic wastes and organic wastes. The most efficient mixtures usually contain relatively easily biodegradable sources (animal manure or sludge) and recalcitrant ones (sawdust, hay, alfalfa or wood chips).

33. Existing pilot and field scale passive reactors First generation bioreactors generally use substrates consisting of composted animal manure or mushroom compost because they provide significant. New generation of bioreactors use a combination of limestone, sawdust, and alfalfa instead of animal manure because it provides alkalinity, a significantly higher hydraulic conductivity, and appears to be a better energy source for bacterial community. ASSIGNMENT !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

34. References Baker, B.J. and Banfield, J.F. (2003). Microbial communities in acid mine drainage FEMS Microbiology Ecology (44) pp 139-152. Bitton, G. (1999). Wastewater Microbiology: Second Edition. Wiley-Liss, US, pp 578. Brown, D.A., Sherriff, B.L., Sawicki, J.A and Sparling, R. (1999). Precipitation of iron minerals by a natural microbial consortium. 63(15) pp 2163-2169. Gould, W.D. and Kapoor, A. Chapter 10. The microbiology of acid mine drainage pp.203-226 In: Jambor, J.L., Blowes, D.W. and Ritchie, A.I.M. Environmental Aspects of Mine Wastes, Vancouver.

35. Johnson, D.B. and Hallberg, K.B. (2003). The microbiology of acidic mine waters. Research in Microbiology. 14 pp 466-473. Johnson, D.B. and McGuiness, S. (1991). Ferric Iron reduction by acidophilic heterothrophic bacteria. Appl. Environ. Microbiol. 57,207-211. Rittmann, B., McCarty, P. (2001). Environmental Biotechnology: Principles and applications: McGraw and Hill, New York, pp 754. Southam, G.,.(2000). Bacterial Surface-Mediated Mineral Formation pp. 257-276 In: Lovley D.R., editor. Environmental Microbe-Metal Interactions, Washington, Asm Press. Zaguri, G.J. and Neculita,C. (2007) Passive treatment of AMD in bioreactors: Short review, applications, and research needed. Proceedings of OttawaGeo 2007, Ontario (1439-1446)