Download An Experimental Study into the Clogging of Leachate Collection

MICROBIAL INTERACTIONS IN MINE
TAILINGS
GEOL 7740 Topic in Environmental Geosciences
Stanislaw Lozecznik
Department of Civil Engineering
University 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)
B
(a) Cells walls of cut
bacteria
(b) ZnS granules
(c) EPS and piece of
wood
A
C
Metal cyanide degrading bacteria from
gold mine tailings Dams
Outline
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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
stain
Gram-positive
Gram negative
Anaerobic, Gram-positive rods
Actinomyces sp.
Anaerobic, Gram-negative rods
Fusobacterium 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)
Temperature
• Psychrophiles T from 0 to 25⁰C
Optimum 10 to 15 ⁰C
• Mesophiles
T from 10 to 40⁰C
Optimum 25 to 40 ⁰C
• Thermophiles T from 50 to 90⁰C
• Optimum 50 to 80 ⁰C
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 eacceptor 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 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).
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- => 6HCO32- + 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)