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

MICROBIAL INTERACTIONS IN MINE TAILINGS

GEOL 7740 Topic in Environmental GeosciencesStanislaw Lozecznik

Department of Civil EngineeringUniversity of Manitoba

Bacterial Activity • Singer and Stumm (1970) showed that presence of iron-

oxidizing bacteria accelerated the oxidation of Fe2+ in AMD by a factor larger than 106 compared to abiotic conditions

Acidithiobacillus ferrooxidans(Gleisner et al., 2006)

• 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

Metal cyanide degrading bacteria from gold mine tailings Dams

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

Introduction to Bacteria

Eukaryotic cell structure

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.

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 stainGram-positiveGram negative

Anaerobic, Gram-positive rodsActinomyces sp.

Anaerobic, Gram-negative rodsFusobacterium sp.

Metabolic classification

• Carbon and energy source

• Effects on pH on growth

• Temperature

• Oxygen

• Aerobic and Anaerobic

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.

Effects of pH on growth

• Each bacterial specie has a range of pH value over which growth is possible

– Acidophilic • Moderate• Extremely (Iron mountain)

• Psychrophiles T from 0 to 25 C⁰Optimum 10 to 15 C⁰

• Mesophiles T from 10 to 40 C⁰Optimum 25 to 40 C⁰

• Thermophiles T from 50 to 90 C⁰• Optimum 50 to 80 C⁰

Temperature

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

REDOX and Bacteria involvement

(1) FeS2 + 14Fe3+ + 8H2O -> 15Fe2+ + 2SO42- + 16H+

Oxidation of pyrite with ferric iron (Fe3+)

(2) 14Fe2+ + 3.5O2 + 14H+ -> 14Fe3+ + 7H2O

Fe2+ oxidation by O2 at low pH is kinetically slow, thus this rate may limit the rate for pyrite dissolution

• Iron-oxidizing prokaryotes catalyze reaction (2), primarily biological in acidic environment (pH<4).

• These organisms accelerate pyrite dissolution by re-generating ferric iron (Fe3+).

• Acidithiobacillus ferrooxidans, L. ferrooxidans

Iron Reduction

• AMD solutions are iron rich because ferric and ferrous iron are very soluble at low pH ( pH < 2.5)

• In some cases, Fe3+ may exceed O2 concentrations by several orders of magnitude

• Johnson and McGuiness (1991) showed the ability to reduce soluble Fe3+ among heterotrophic acidophiles

• Some species are able to reduce Fe3+

even if it is not in solution. S acidophilus is capable of anaerobic dissolution of iron hydroxide, jarosite and goethite.

Sulfur oxidation

• Most bacterial community of AMD that can oxidize sulfur also can fix CO2.

• 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 Fe3+ as the e- acceptor and So the e- donor

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

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.

Microbial ecology of acid environmentsThis environment contain a variety of acidophilic

microorganisms

Fe oxidizersS oxidizersFacultative 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).

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)

• 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)

• Molecular biology: Identify bacteria that are not culturable by conventional means

1.DNA extraction

2.Reverse sample genome probe (RSGP)

3.FISH

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.

• 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.

• 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

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

• SRB : Sulfate reduction produces HCO3- and HS-.

HS- leads to permanent alkalinity when sulfide escapes as H2S gas. The lower the pH, more H2S gas is released from the system.

2CH3CHOHCOO- + 3SO42- => 6HCO3

2- + 3HS- + H+

SRB play one of the most important role in AMD

mitigation

• 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

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 CO2.

• 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).

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 !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

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.

• 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)