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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
Fe
2+

in AMD by a factor larger than 10
6

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
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)






Psychrophiles T from 0 to 25

C


Optimum 10 to 15

C



Mesophiles


T from 10 to 40

C


Optimum 25 to 40

C



Thermophiles


T from 50 to 90

C


Optimum 50 to 80


C

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) 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



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

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


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.


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

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 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


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
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).

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)