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8 Microbial Leaching of Metals
HELMUT BRANDL
Zürich, Switzerland
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Introduction 192
Terminology 192
Historical Background 192
Principles of Microbial Metal Leaching 194
4.1 Leaching Mechanisms 194
4.2 Models of Leaching Mechanisms 194
4.3 Factors Influencing Bioleaching 197
4.4 Bacterial Attachment on Mineral Surfaces 200
Microbial Diversity in Bioleaching Environments 200
Case Studies of Bioleaching Applications 208
6.1 Commercial-Scale Copper Ore Bioleaching 208
6.2 Reactor Bioleaching of Fly Ash 210
6.3 Shake Flask Bioleaching of Electronic Scrap 212
Economics of Metal Bioleaching 213
Perspectives of Bioleaching Technology 214
8.1 Heterotrophic Leaching 214
8.2 Leaching Under Thermophilic Conditions 215
8.3 Tapping Microbial Diversity 215
8.4 Treatment of Solid Wastes 215
8.5 Bioremediation of Metal-Contaminated Sites 215
Conclusion 217
References 217
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8 Microbial Leaching of Metals
1 Introduction
Future sustainable development requires
measures to reduce the dependence on nonrenewable raw materials and the demand for
primary resources. New resources for metals
must be developed with the aid of novel technologies. in addition, improvement of alredy
existing mining techniques can result in metal
recovery from sources that have not been of
economical interest until today. Metal-winning
processes based on the activity of microorganisms offer a possibility to obtain metals from
mineral resources not accessible by conventional mining (BOSECKER, 1997; BRIERLEY,
1978; BRYNER et al., 1954; TORMA and BANHEGYI, 1984). Microbes such as bacteria and
fungi convert metal compounds into their
water-soluble forms and are biocatalysts of
these leaching processes. Additionally, applying microbiological solubilization processes, it
is possible to recover metal values from industrial wastes which can serve as secondary
raw materials.
2 Terminology
In general, bioleaching is a process described as being “the dissolution of metals
from their mineral source by certain naturally
occurring microorganisms” or “the use of microorganisms to transform elements so that
the elements can be extracted from a material
when water is filtered trough it” (ATLAS and
BARTHA, 1997; PARKER, 1992). Additionally,
the term “biooxidation” is also used (HANSFORD and MILLER, 1993). There are, however,
some small differences by definition (BRIERLEY, 1997): Usually, “bioleaching” is referring
to the conversion of solid metal values into
their water soluble forms using microorganisms. In the case of copper, copper sulfide is
microbially oxidized to copper sulfate and
metal values are present in the aqueous phase.
Remaining solids are discarded. “Biooxidation” describes the microbiological oxidation
of host minerals which contain metal compounds of interest. As a result, metal values remain in the solid residues in a more concen-
trated form. In gold mining operations, biooxidation is used as a pretreatment process to
(partly) remove pyrite or arsenopyrite. This
process is also called “biobeneficiation” where
solid materials are refined and unwanted impurities are removed (GROUDEV, 1999; STRASSER et al., 1993). The terms “biomining”, “bioextraction”, or “biorecovery” are also applied
to describe the mobilization of elements from
solid materials mediated by bacteria and fungi
(HOLMES, 1991; MANDL et al., 1996; RAWLINGS,
1997; WOODS and RAWLINGS, 1989). “Biomining” concerns mostly applications of microbial
metal mobilization processes in large-scale
operations of mining industries for an economical metal recovery.
The area of “biohydrometallurgy” covers
bioleaching or biomining processes (ROSSI,
1990). Biohydrometallurgy represents an interdisciplinary field where aspects of microbiology (especially geomicrobiology), geochemistry, biotechnology, hydrometallurgy, mineralogy, geology, chemical engineering, and mining
engineering are combined. Hydrometallurgy
is defined as the treatment of metals and metal-containing materials by wet processes and
describes “the extraction and recovery of metals from their ores by processes in which aqueous solutions play a predominant role” (PARKER, 1992). Rarely, the term “biogeotechnology” is also used instead of biohydrometallurgy
(FARBISZEWSKA et al., 1994).
3 Historical Background
One of the first reports where leaching
might have been involved in the mobilization
of metals is given by the Roman writer Gaius
Plinius Secundus (23–79 A.D.). In his work on
natural sciences, Plinius describes how copper
minerals are obtained using a leaching process
(KÖNIG, 1989a, b). The translation reads approximately as follows:“Chrysocolla is a liquid
in the before mentioned gold mines running
from the gold vein. In cold weather during the
winter the sludge freezes to the hardness of
pumice. It is known from experience that the
most wanted [chrysocolla] is formed in copper
mines, the following in silver mines. The liquid
3 Historical Background
is also found in lead mines although it is of minor value. In all these mines chrysocolla is also
artificially produced by slowly passing water
through the mine during the winter until the
month of June; subsequently, the water is evaporated in June and July. It is clearly demonstrated that chrysocolla is nothing but a decomposed vein.”
The German physician and mineralogist
Georgius Agricola (1494–1555) describes in
his work de re metallica also techniques for the
recovery of copper that are based on the leaching of copper-containing ores (SCHIFFNER,
1977). A woodcut from his book illustrates
the (manual) transport of metal-containing
leachates from mines and their evaporation in
the sunlight (Fig. 1).
The Rio Tinto mines in south-western Spain
are usually considered the cradle of biohydrometallurgy. These mines have been exploited
since pre-Roman times for their copper, gold,
and silver values. However, with respect to
commercial bioleaching operations on an industrial scale, biohydrometallurgical techniques had been introduced to the Tharsis
Fig. 1. Woodcut from the book de re metallica written by Georgius Agricola (1494–1555) illustrating
the manual recovery of copper-containing mine
effluents which are collected in wooden basins and
concentrated in the sun.
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mine in Spain 10 years earlier (SALKIELD,
1987). As a consequence to the ban of open air
ore roasting and its resulting atmospheric sulfur emissions in 1878 in Portugal, hydrometallurgical metal extraction has been taken into
consideration in other countries more intensely. In addition to the ban, cost savings were another incentive for the development: Heap
leaching techniques were assumed to reduce
transportation costs and to allow the employment of locomotives and wagons for other services (SALKIELD, 1987). From 1900 on, no open
air roasting of low-grade ore was conducted at
the Rio Tinto mines.
Efforts to establish bioleaching at the Rio
Tinto mines had been undertaken in the beginning of the 1890s. Heaps (10 m in height) of
low-grade ore (containing 0.75% Cu) were
built and left for one to three years for “natural” decomposition (SALKIELD, 1987). 20 to
25% of the copper left in the heaps were recovered annually. It was calculated that approximately 200,000 t of rough ore could be
treated in 1896. Although industrial leaching
operations were conducted at the Rio Tinto
mines for several decades, the contribution of
bacteria to metal solubilization was confirmed
only in 1961, when Thiobacillus ferrooxidans
was identified in the leachates.
Early reports state that factors affecting bioleaching operations were the height of the
heap, particle size, initial ore washing with
acid, and temperature control to about 50 °C
(SALKIELD, 1987). Another critical factor was
the supply of water for the leaching heaps. Although usually acidic mine waters were used
for ore processing, 4 billion liters of freshwater
were required annually (SALKIELD, 1987).
Although metal leaching from mineral resources has a very long historical record (EHRLICH, 1999; ROSSI, 1990) and although the oxidation of reduced sulfur compounds and elemental sulfur resulting in the formation of sulfuric acid was demonstrated already in the
1880s (WINOGRADSKY, 1887), the oxidation of
metal sulfides was not described until 1922
when mobilization of zinc from zinc sulfide
was investigated (RUDOLFS, 1922; RUDOLFS
and HELBRONNER, 1922). It was found that the
transformation of zinc sulfide to zinc sulfate
was microbially mediated. Based on these results, the economic recovery of zinc from zinc-
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8 Microbial Leaching of Metals
containing ores by biological methods was
proposed. In 1947, Thiobacillus ferrooxidans
was identified as part of the microbial community found in acid mine drainage (COLMER and
HINKLE, 1947). A first patent was granted in
1958 (ZIMMERLEY et al., 1958). The patent describes a cyclic process where a ferric sulfate/
sulfuric acid lixiviant solution is used for metal
extraction, regenerated by aeration (ferrous
iron oxidation by iron-oxidizing organisms),
and reused in a next leaching stage.
4 Principles of Microbial
Metal Leaching
4.1 Leaching Mechanisms
Mineralytic effects of bacteria and fungi on
minerals are based mainly on three principles,
namely acidolysis, complexolysis, and redoxolysis. Microorganisms are able to mobilize metals by (1) the formation of organic or inorganic acids (protons); (2) oxidation and reduction
reactions; and (3) the excretion of complexing
agents. Sulfuric acid is the main inorganic acid
found in leaching environments. It is formed
by sulfur-oxidizing microorganisms such as
thiobacilli.A series of organic acids are formed
by bacterial (as well as fungal) metabolism
resulting in organic acidolysis, complex and
chelate formation (BERTHELIN, 1983). A kinetic model of the coordination chemistry of mineral solubilization has been developed which
describes the dissolution of oxides by the protonation of the mineral surface as well as the
surface concentration of suitable complexforming ligands such as oxalate, malonate,
citrate, and succinate (FURRER and STUMM,
1986). Proton-induced and ligand-induced
mineral solubilization occurs simultaneously
in the presence of ligands under acidic conditions.
4.2 Models of Leaching
Mechanisms
Originally, a model with two types of mechanisms which are involved in the microbial
mobilization of metals has been proposed
(EWART and HUGHES, 1991; SILVERMAN and
EHRLICH, 1964): (1) Microorganisms can oxidize metal sulfides by a “direct” mechanism
obtaining electrons directly from the reduced
minerals. Cells have to be attached to the mineral surface and a close contact is needed. The
adsorption of cells to suspended mineral particles takes place within minutes or hours. This
has been demonstrated using either radioactively labeled Thiobacillus ferrooxidans cells
grown on NaH14CO3 or the oxidative capacity
of bacteria attached to the mineral surface
(ESCOBAR et al., 1996). Cells adhere selectively
to mineral surfaces occupying preferentially irregularities of the surface structure (EDWARDS
et al., 1999; EWART and HUGHES, 1991). In addition, a chemotactic behavior to copper, iron,
or nickel ions has been demonstrated for Leptospirillum ferrooxidans (ACUNA et al., 1992).
Genes involved in the chemotaxis were also
detected in Thiobacillus ferrooxidans and
Thiobacillus thiooxidans (ACUNA et al., 1992).
(2) The oxidation of reduced metals through
the “indirect” mechanism is mediated by ferric
iron (Fe3c) originating from the microbial
oxidation of ferrous iron (Fe2c) compounds
present in the minerals. Ferric iron is an oxidizing agent and can oxidize, e.g., metal sulfides
and is (chemically) reduced to ferrous iron
which, in turn, can be microbially oxidized
again (EWART and HUGHES, 1991). In this case,
iron has a role as electron carrier. It was proposed that no direct physical contact is needed
for the oxidation of iron.
In many cases it was concluded that the “direct” mechanism dominates over the “indirect” mostly due to the fact that “direct” was
equated with “direct physical contact”. This
domination has been observed for the oxidation of covellite or pyrite in studies employing
mesophilic T. ferrooxidans and thermophilic
Acidianus brierleyi in bioreactors which consisted of chambers separated with dialysis
membranes to avoid physical contact (LARSSON et al., 1993; POGLIANI et al., 1990). How-
4 Principles of Microbial Metal Leaching
ever, the attachment of microorganisms on
surfaces is not an indication per se for the existence of a direct mechanism (EDWARDS et al.,
1999). The term “contact leaching” has been
introduced to indicate the importance of bacterial attachment to mineral surfaces (TRIBUTSCH, 1999).
The following equations describe the “direct” and “indirect” mechanism for the oxidation of pyrite (MURR, 1980; SAND et al., 1999):
direct:
2 FeS2c7 O2c2 H2O
c2 H2SO4
thiobacilli
] 2 FeSO4
(1)
indirect:
4 FeSO4cO2
T. ferrooxidans, L. ferrooxidans
]
c2 H2SO4
(2)
2 Fe2(SO4)3c2 H2O
FeS2cFe2(SO4)3
c2 S
2 Sc3 O2cH2O
chemical oxidation
] 3 FeSO4
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around cells of T. ferrooxidans during growth
on synthetic pyrite films (ROJAS et al., 1995).
“Footprints” of organic films containing colloidal sulfur granules are left on the mineral
surface upon detachment of the bacteria.
From the existing data two “indirect” leaching mechanisms have been proposed whereas
no evidence for a “direct” enzymatically mediated process has been found (SAND et al.,
1999).The mineral structure is the determining
factor for the prevailing type of leaching
mechanism. In the “thiosulfate mechanism”
thiosulfate is the main intermediate resulting
from the oxidation of pyrite, molybdenite, or
tungstenite. Polysulfide and elemental sulfur
are the main intermediates in the “polysulfide
mechanism” during the oxidation of galena,
sphalerite, chalcopyrite, hauerite, orpiment, or
realgar. The presence of iron(III) at the beginning of mineral degradation is an important
prerequisite (SAND et al., 1999).
The following equations summarize the oxidation mechanisms (SAND et al., 1999):
(3)
T. thiooxidans
] 2 H2SO4
(4)
However, the model of “direct” and “indirect”
metal leaching is still under discussion. Recently, this model has been revised and replaced by another one which is not dependent
on the differentiation between a “direct” and
an “indirect” leaching mechanisms (SAND et
al., 1995, 1999). All facts have been combined
and a mechanism has been developed which is
characterized by the following features: (1)
cells have to be attached to the minerals and in
physical contact with the surface; (2) cells form
and excrete exopolymers; (3) these exopolymeric cell envelopes contain ferric iron compounds which are complexed to glucuronic
acid residues. These are part of the primary attack mechanism; (4) thiosulfate is formed as
intermediate during the oxidation of sulfur
compounds; (5) sulfur or polythionate granules are formed in the periplasmatic space or
in the cell envelope.
Thiosulfate and traces of sulfite have been
found as intermediates during the oxidation of
sulfur (SHRIHARI et al., 1993). Sulfur granules
(colloidal sulfur) have been identified as energy reserves in the exopolymeric capsule
Thiosulfate mechanism (found for FeS2, MoS2,
WS2):
2c
FeS2c6 Fe3cc3 H2O ] S2O2c
3 P7 Fe
c
c6 H
(5)
3c
2c
S2O2P
c5 H2O ] 2 SO2P
3 c8 Fe
4 c8 Fe
c
c10 H
(6)
Polysulfide mechanism (found for PbS, CuFeS2,
ZnS, MnS2, As2S3, As3S4):
2 MSc2 Fe3cc2 Hc ] 2 M2ccH2Sn
c2 Fe2c
(7)
H2Snc2 Fe3c ] 0.25 S8c2 Fe2cc2 Hc
(8)
c
0.25 S8c3 O2c2 H2O ] 2 SO2P
4 c4 H
(9)
Several biomolecules are involved in the
aerobic respiration on reduced sulfur and iron
compounds. It has been found that up to 5% of
soluble proteins of T. ferrooxidans is made of
an acid stable blue copper protein, called rusticyanin (BLAKE et al., 1993). Additionally, the
iron(II) respiratory system contains a (putative) green copper protein, two types of cytochrome c, one or more types of cytochrome a,
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8 Microbial Leaching of Metals
a porin, and an iron(II)-sulfate chelate (BLAKE
et al., 1993). The acid stability of rusticyanin
suggests that it is located in the periplasmic
space. Figure 2 shows a scheme of the model
which combines the electron transport sequence proposed earlier with concepts stemming from the debate on “direct”/“indirect”
leaching mechanisms (BLAKE and SHUTE,
1994; BLAKE et al., 1993; HAZRA et al., 1992;
SAND et al., 1995).
Some details of the metal mobilization
mechanism, the importance of the presence
and attachment of microorganisms and their
active contribution have been demonstrated
for the leaching of fly ash from municipal
waste incineration (MWI) (BROMBACHER et
al., 1998). Generally, several mechanisms of
metal mobilization can be distinguished: (1)
Contact leaching effect on the release of metals. Stock cultures of Thiobacillus ferrooxidans
and Thiobacillus thiooxidans were added to
ash suspensions and cells were in direct contact with the fly ash. Growth of thiobacilli
might be stimulated by increased energy availability from oxidation of reduced solid particles. (2) Metal solubilization by metabolically
active (enzymatic) compounds in the absence
of bacterial cells. Stock cultures were filtered
to obtain the cell free spent medium. This medium was used for leaching. (3) Metal solubilization by non-enzymatic extracellular metabolic products. Cell free spent medium (see 2)
was autoclaved to obtain a sterile leaching solution without enzymatic activities and to evaluate the leaching ability of acid formed. (4)
Leaching by fresh medium. Fresh non-inoculated and sterile medium was added to the fly
ash suspension and used as control. (5) Chemical leaching due to the preparation of the ash
suspension (acidification to pH 5.4). Certain
elements such as, e.g., Cd or Zn might be chemically mobilized already during acidification.
MWI fly ash contains reduced copper species (chalcocite {Cu2S} or cuprite {Cu2O})
whereas zinc and others are present in their
fully oxidized forms (BROMBACHER et al.,
1998). Therefore, copper release from fly ash is
directly affected and enhanced by T. ferrooxidans, whereas Zn, as well as Al, Cd, Cr, and Ni,
are released primarily due to the acidic environment. Acidification of the fly ash pulp
(chemical mobilization) led already to considerable extraction yields for Cd, Ni, and Zn and
could slightly be increased using non-inoculated sterile medium as lixiviant (Fig. 3). By comparing leached amounts of copper by filtered
cell free spent medium with autoclaved sterile
spent medium, it was concluded that significant amounts of copper were mobilized – in
contrast to other elements – by metabolic
products of T. ferrooxidans. Leaching with cell
free spent medium indicating a solubilizing
mechanism due to extracellular components
was significantly more effective than a leach-
Fig. 2. Schematic mechanistic bioleaching model (after HAZRA et
al., 1992; SAND et al., 1995, 1999;
SCHIPPERS et al., 1996; RAWLINGS,
1999). C: cytoplasm; CM: cell
membrane; PS: periplasmatic
space; OM: outer membrane;
EP: exopolymers; Cyt: cytochrome; RC: rusticyanin;
MeS: metal sulfide
4 Principles of Microbial Metal Leaching
197
Fig. 3. Solubilized metals from fly ash
originating from municipal waste incineration (in suspensions of 40 g LP1) in percent
of the metal amount present with different
lixiviants within 8 d. All samples were
incubated in triplicate. The release of metals due to acidification of the fly ash pulp
is indicated as chemical mobilization (see
text for explanation).
ing with autoclaved spent medium where excreted enzymes had been inactivated. It is
known that several components involved in
the electron transport chain of Thiobacillus
(rusticyanin, cytochromes, iron–sulfur proteins) are located in the periplasmic space
(BLAKE and SHUTE, 1994; SAND et al., 1995)
and might, therefore, also be present in the cell
free spent medium catalyzing oxidation of reduced metal compounds.
In many leaching environments conditions
(especially iron(II) and iron(III) concentrations) vary with the duration of the leaching.
This makes it difficult to assess the importance
and the effect of the presence of bacteria. Using an experimental setup to maintain constant concentrations of ferrous and ferric iron,
it was possible to show that in the presence of
T. ferrooxidans rates of pyrite or zinc sulfide
leaching are increased (HOLMES et al., 1999;
FOWLER and CRUNDWELL, 1999; FOWLER et al.,
1999).
4.3 Factors Influencing Bioleaching
Standard test methods have been developed
to determine leaching rates of iron from
pyrite mediated by Thiobacillus ferrooxidans
(ASTM, 1991). An active culture of T. ferrooxidans is grown in a defined medium contain-
ing (in g LP1): (NH4)2SO4 (3.0); K2HPO4 (0.5);
MgSO4 · 7 H2O (0.5); KCl (1.0); Ca(NO3)2 ·
4 H2O (0.01); FeSO4 · 7 H2O (44.22); and 1 mL
10 N sulfuric acid (SILVERMAN and LUNDGREN,
1959). Cells are harvested, diluted, and added
to pyrite suspensions with a pulp density of
20 g LP1. Total soluble iron as well as sulfate
formed during oxidation is periodically determined.
Metal bioleaching in acidic environments
is influenced by a series of different factors
(Tab. 1). Physicochemical as well as microbiological factors of the leaching environment are
affecting rates and efficiencies. In addition,
properties of the solids to be leached are of
major importance (ACEVEDO and GENTINA,
1989; BRIERLEY, 1978; DAS et al., 1999; MURR,
1980). As examples, pulp density, pH, and particle size were identified as major factors for
pyrite bioleaching by Sulfolobus acidocaldarius (LINDSTROM et al., 1993). Optimal conditions were 60 g LP1, 1.5, and ~20 µm, respectively. The influence of different parameters
such as activities of the bacteria itself, source
energy, mineralogical composition, pulp density, temperature, and particle size was studied
for the oxidation of sphalerite by T. ferrooxidans (BALLESTER et al., 1989). Best zinc dissolution was obtained at low pulp densities
(50 g LP1), small particle sizes, and temperatures of approximately 35 °C.
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8 Microbial Leaching of Metals
Tab. 1. Factors and Parameters Influencing Bacterial Mineral Oxidation and Metal Mobilization
Factor
Parameter
Physicochemical parameters of a bioleaching environment
temperature
pH
redox potential
water potential
oxygen content and availability
carbon dioxide content
mass transfer
nutrient availability
iron(III) concentration
light
pressure
surface tension
presence of inhibitors
microbial diversity
population density
microbial activities
spatial distribution of microorganisms
metal tolerance
adaptation abilities of microorganisms
mineral type
mineral composition
mineral dissemination
grain size
surface area
porosity
hydrophobicity
galvanic interactions
formation of secondary minerals
leaching mode (in situ, heap, dump, or tank
leaching)
pulp density
stirring rate (in case of tank leaching operations)
heap geometry (in case of heap leaching)
Microbiological parameters of a bioleaching environment
Properties of the minerals to be leached
Processing
Metal oxidation mediated by acidophilic microorganisms can be inhibited by a variety of
factors such as, e.g., organic compounds, surface-active agents, solvents, or specific metals:
The presence of organic compounds (yeast extract) inhibited pyrite oxidation of T. ferrooxidans (BACELAR-NICOLAU and JOHNSON, 1999).
Certain metals present in bioleaching environments can inhibit microbial growth, therefore
reducing leaching efficiencies. For instance, arsenic added to cultures inhibited Sulfolobus
acidocaldarius grown on pyrite and T. ferrooxidans grown on arsenopyrite (HALLBERG et
al., 1996; LAN et al., 1994).Additions of copper,
nickel, uranium, or thorium adversely influ-
enced iron(II) oxidation by T. ferrooxidans
with uranium and thorium showing higher toxicities than copper and nickel (LEDUC et al.,
1997). Silver, mercury, ruthenium, and molybdenum reduced the growth of Sulfolobus
grown on a copper concentrate (MIER et al.,
1996). Industrial biocides such as tetra-n-butyltin, isothiazolinones, N-dimethyl-Nb-phenyl-Nb-(fluorodichloro-methylthio)-sulfamide,
or 2,2b-dihydroxy-5,5b-dichlorophenylmethane
(dichlorophen) reduced the leaching of manganese oxides by heterotrophic microorganisms (ARIEF and MADGWICK, 1992). Biocides
were externally added as selective inhibitors to
suppress unwanted organisms and to improve
4 Principles of Microbial Metal Leaching
manganese leaching efficiencies. At low concentrations of ~5 mg LP1, however, manganese mobilization was increased by 20% (BOUSSIOS and MADGWICK, 1994).
Also gaseous compounds can show inhibitory effects on metal leaching: Aqueous-phase
carbon dioxide at concentration `10 mg LP1
was inhibiting growth of T. ferrooxidans on
pyrite–arsenopyrite–pyrrothite ore (NAGPAL
et al., 1993). Optimal concentrations of carbon
dioxide were found to be in the range of 3 to
7 mg LP1. There are reports on the stimulation
of bacterial leaching and the increase of leaching rates by supplementing leaching fluids with
carbon dioxide (ACEVEDO et al., 1998; BRIERLEY, 1978; TORMA et al., 1972). Concentrations
of 4% (v/v) carbon dioxide in the inlet gas of a
fermenter showed maximum growth rates of
T. ferrooxidans, maximum iron(II), copper,
and arsenic oxidation (ACEVEDO et al., 1998).
Pulp densities of 20 g LP1 delayed the onset
of bioleaching of pyrite derived from coal
(BALDI et al., 1992). Increasing pulp densities
from 30 to 100 g LP1 decreased rates of pyrite
oxidation in Sulfolobus cultures (NGUBANE
and BAECKER, 1990). For fungi such as Aspergillus niger, optimal pulp densities for maximum metal leaching efficiencies were found to
be in the range of 30 to 40 g LP1 (BOSSHARD
et al., 1996). Quartz particles at pulp densities
of 80 g LP1 almost completely inhibited the
oxidation of covellite by T. ferrooxidans especially in the absence of iron(II) (CURUTCHET
et al., 1990).
During bioleaching processes, coprecipitation of metals with mineral phases such as jarosites can reduce leaching efficiencies (HIROYOSHI et al., 1999). In addition, the precipitation of compounds present in the leachates on
the minerals to be leached can make the solid
material inaccessible for bacterial leaching.
Organic solvents such as flotation or solvent
extraction agents, which are added for the
downstream processing of leachates from bioleaching, might also lead to inhibition problems (ACEVEDO and GENTINA, 1989). Isopropylxanthate and LIX 984 (used as flotation
agent and solvent extraction agent, respectively) prevented the oxidation of pyrite and chalcopyrite by T. ferrooxidans (HUERTA et al.,
1995). This fact is of special importance when
spent leaching liquors are recycled for a reuse.
199
It has been demonstrated recently that the
addition of small amounts of amino acids (cysteine in this case) resulted in an increased
pyrite corrosion by T. ferrooxidans as compared to controls without additions (ROJASCHAPANA and TRIBUTSCH, 2000). It is suggested that the microorganisms may profit from
weakening and break up of chemical bonds
mediated by the formation of the cysteine–pyrite complex. This might also be the case under
natural conditions by the excretion of cysteine-containing metabolites. An inexpensive
alternative to increase metal recovery from
ore heaps by the addition of sulfur-containing
amino acids such as cysteine has been suggested (TRIBUTSCH and ROJAS-CHAPANA, 1999).
Other metabolites excreted by Thiobacillus
might also enhance metal leaching efficiencies:
Wetting agents such as mixtures of phospholipids and neutral lipids are formed by Thiobacillus thiooxidans (BEEBE and UMBREIT, 1971).
As a consequence, growth of T. thiooxidans on
sulfur particles is supported by the excretion
of metabolites acting as biosurfactants which
facilitate the oxidation of elemental sulfur. It
was also hypothesized that Thiobacillus caldus
is stimulating the growth of heterotrophic organisms in leaching environments by the excretion of organic compounds and is supporting the solubilization of solid sulfur by the formation of surface-active agents (DOPSON and
LINDSTROM, 1999). Metal solubilization might
also be facilitated by microbial metabolites excreted by organisms other than Thiobacillus
which are part of microbial consortia found in
bioleaching operations. Microbial surfactants,
which show large differences in their chemical
nature, are formed by a wide variety of microorganisms. In the presence of biosurfactants
which lead to changes in the surface tension,
metal desorption from solids might be enhanced resulting in an increased metal mobility in porous media. It has been suggested that
this metabolic potential can be practically used
in the bioremediation of metal-contaminated
soils (MILLER, 1995). However, there is some
evidence that surface-active compounds as
well as organic solvents are inhibitory to bioleaching reactions and prevent bacterial attachment (MURR, 1980). The external addition
of Tween reduced the oxidation of chalcopyrite by T. ferrooxidans (TORMA et al., 1976). It
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8 Microbial Leaching of Metals
was concluded that the need of the microorganisms for surfactants is met by their own formation. In contrast, it was reported that the addition of Tween 80 increased the attachment of
T. ferrooxidans on molybdenite and the oxidation of molybdenum in the absence of iron(II)
(PISTACCIO et al., 1994).
4.4 Bacterial Attachment on
Mineral Surfaces
It is known that the formation of extracellular polymeric substances plays an important
role in the attachment of thiobacilli to mineral
surfaces such as, e.g., sulfur, pyrite, or covellite.
Extraction or loss of these exopolymers prevent cell attachment resulting in decreased
metal leaching efficiencies (ESCOBAR et al.,
1997; GEHRKE et al., 1998; POGLIANI and DONATI, 1992). It was concluded that a direct contact between bacterial cells and solid surfaces
is needed and represents an important prerequisite for an effective metal mobilization
(OSTROWSKI and SKLODOWSKA, 1993). Interactions between microorganisms and the mineral surface occur on two levels (BARRETT et al.,
1993). The first level is a physical sorption because of electrostatic forces. Due to the low
pH usually occurring in leaching environments, microbial cell envelopes are positively
charged leading to electrostatic interactions
with the mineral phase. The second level is
characterized by chemical sorption where
chemical bonds between cells and minerals
might be established (e.g., disulfide bridges).
In addition, extracellular metabolites are
formed and excreted during this phase in the
near vicinity of the attachment site (EWART
and HUGHES, 1991). Low-molecular weight
metabolites excreted by sulfur oxidizers include acids originating from the TCA cycle,
amino acids, or ethanolamine, whereas compounds with relatively high molecular weights
include lipids and phospholipids (BARRETT et
al., 1993). In the presence of elemental sulfur,
sulfur-oxidizing microorganisms from sewage
sludge form a filamentous matrix similar to a
bacterial glycocalyx suggesting the relative importance of these extracellular substances in
the colonization of solid particles (BLAIS et al.,
1994).
5 Microbial Diversity in
Bioleaching Environments
A variety of microorganisms is found in
leaching environments and has been isolated
from leachates and acidic mine drainage. Although environmental conditions are usually
described (from an anthropocentric view!) as
being extreme and harsh due to pH values (as
low as P3.6; NORDSTROM et al., 2000) and high
metal concentrations (as high as 200 g LP1;
NORDSTROM et al., 2000), these systems can
show high levels of microbial biodiversity including bacteria, fungi, and algae (LOPEZ-ARCHILLA et al., 1993). It has long been known
that bacteria (Thiobacillus sp.), yeasts (Rhodotorula sp., Trichosporon sp.), flagellates (Eutrepia sp.), amoebes and protozoa are part of
the microbial biocenosis found in acidic waters
of a copper mine (EHRLICH, 1963). Recent detailed investigations based on molecular methods such as DNA–DNA hybridization, 16S
rRNA sequencing, RCR-based methods with
primers derived from rRNA sequencing, fluorescence in situ hybridization (FISH), or immunological techniques revealed that microbial bioleaching communities are composed of
a vast variety of microorganisms resulting in
complex microbial interactions and nutrient
flows (such as synergism, mutualism, competition, predation) (AMARO et al., 1992; DE
WULF-DURAND et al., 1997; EHRLICH, 1997;
JOHNSON, 1998; EDWARDS et al., 1999). Selected
organisms of these communities are given in
Table 2.The composition of these communities
is usually subjected to seasonal fluctuations
and may vary between different mining locations (EDWARDS et al., 1999; GROUDEV and
GROUDEVA, 1993). In addition, organisms are
not homogeneously distributed over the whole
leaching environment (CERDÁ et al., 1993).
The organism studied most is Thiobacillus
ferrooxidans. Although this is the best known
organism from acidic habitats, one may not
conclude that this organism is dominant in
these ecosystems. It has been found that under
specific environmental conditions Leptospirillum sp. is even more abundant than T. ferrooxidans suggesting an important ecological
role in the microbial community structure of
5 Microbial Diversity in Bioleaching Environments
bioleaching habitats (SAND, 1992; SCHRENK et
al., 1998). Thiobacilli are members of the division of Proteobacteria close to the junction
between the and subdivision whereas
leptospirilli are placed in the Nitrospira division (RAWLINGS, 1999). Genetic studies revealed that the role of T. ferrooxidans in leaching operations has probably been overestimated. Excellent reviews on the genetics of Thiobacilli and leptospirilli have been published
recently (RAWLINGS, 1999; RAWLINGS and KUSANO, 1994).
Thiobacillus ferrooxidans belongs to the
group of chemolithotrophic organisms. The organism is rod-shaped (usually single or in
pairs), non-spore forming, gram-negative, motile, and single-pole flagellated (HORAN, 1999;
KELLY and HARRISON, 1984; LEDUC and FERRONI, 1994; MURR, 1980). As carbon source,
carbon dioxide is utilized. Ferrous iron is oxidized. Ammonium is used as nitrogen source.
Although T. ferrooxidans has been characterized as being a strictly aerobic organism, it can
also grow on elemental sulfur or metal sulfides
under anoxic conditions using ferric iron as
electron acceptor (DONATI et al., 1997; PRONK
et al., 1992).
The genus Thiobacillus represents a versatile group of chemolithoautotrophic organisms. Optimum pH values for growth vary
between 2 and 8 (Fig. 4). It has been demonstrated that sulfur-oxidizing bacteria are capable of reducing the pH of highly alkaline fly
ash suspensions amended with elemental sulfur from approximately 9 to 0.5 (KREBS et al.,
1999) (Fig. 5). It is likely that thiobacilli contribute to increasing acidification of leaching
ecosystems in a successive mode: In the initial
stages the growth of less acidophilic strains
(e.g., Thiobacillus thioparus) is stimulated
whereas during prolonged leaching the pH decreases gradually supporting growth of more
acidophilic strains. This has already been observed in metal leaching from wastewater sewage sludge (BLAIS et al., 1993).
A variety of thermophilic microorganisms
(especially Sulfolobus species) has been enriched and isolated from bioleaching environments (BRIERLEY, 1990; NEMATI et al., 2000;
NORRIS and OWEN, 1993). Temperature optima
for growth and metal leaching were in the
range between 65 and 85 °C. Although copper
201
extraction from mine tailings is more efficient
using thermophilic instead of mesophilic organisms, extremely thermophilic microorganisms show a higher sensitivity to copper and to
high pulp densities in agitated systems limiting, therefore, some practical applications
(DUARTE et al., 1993; NORRIS and OWEN,
1993).
Although environmental conditions in
leaching operations favor the growth and development of mesophilic, moderately thermophilic, and extremely thermophilic microbial
communities, metal leaching at low temperatures has also been observed. Copper and
nickel were leached from pyritic ore samples
in significant amounts at 4 °C (AHONEN and
TUOVINEN, 1992). However, leaching rates
were lower by a factor of 30 to 50 as compared
to experiments conducted at 37 °C. T. ferrooxidans recovered from mine waters was able to
grow at 2 °C with a generation time of approximately 250 h suggesting a psychrotrophic nature of the organism (FERRONI et al., 1986).
Bacterial iron mobilization has also been observed at 0 °C in ore samples obtained from
Greenland (LANGDAHL and INGVORSEN, 1997).
Solubilization rates at these low temperatures
were still approximately 25 to 30% of the maximum values observed at 21 °C. All these findings may have a potential for practical applications in geographical areas where field operations are subjected to low temperature regimes.
A series of heterotrophic microorganisms
(bacteria, fungi) is also part of microbial bioleaching communities (Tab. 2). This group of
organisms uses extracellular metabolites and
cell lysates from autotrophs as carbon source
resulting in the removal of an inhibitory excess
of carbon and stimulating, therefore, growth
and iron oxidation of thiobacilli (BUTLER and
KEMPTON, 1987; FOURNIER et al., 1998). In addition, several heterotrophs can also contribute to metal solubilization by the excretion
of organic acids such as citrate, gluconate, oxalate, or succinate.
Nutrition Type
Main
Leaching
Agent
Archaea Acidianus ambivalens
Acidianus brierleyi
Acidianus infernus
Ferroplasma acidiphilum
Metallosphaera prunae
facult. heterotrophic
facult. heterotrophic
facult. heterotrophic
chemolithoautotrophic
chemolithoautotrophic
sulfuric acid
sulfuric acid acidophilic 1.5–3.0
sulfuric acid
ferric iron
1.3–2.2
1.7
ferric iron,
sulfuric acid
ferric iron, acidophilic
sulfuric acid
Metallosphaera sedula
chemolithoautotrophic
Picrophilus oshimae
Picrophilus torridus
Sulfolobus acidocaldarius
chemolithoautotrophic
Sulfolobus ambivalens
chemolithoautotrophic
Sulfolobus brierleyi
chemolithoautotrophic
Sulfolobus hakonensis
Sulfolobus metallicus
Sulfolobus solfataricus
chemolithoautotrophic
chemolithoautotrophic
chemolithoautotrophic
Sulfolobus thermosulfidooxidans
chemolithoautotrophic
Sulfolobus yellowstonii
chemolithoautotrophic
Sulfurococcus mirabilis
mixotrophic
Sulfurococcus yellowstonii
mixotrophic
pH Range pH
Opt.
ferric iron, 0.9–5.8
sulfuric acid
ferric iron,
sulfuric acid
ferric iron,
sulfuric acid
ferric iron,
sulfuric acid
ferric iron,
sulfuric acid
ferric iron,
sulfuric acid
ferric iron, acidophilic
sulfuric acid
ferric iron,
sulfuric acid
45–75
15– 45
JOHNSON (1998)
MUÑOZ et al. (1995)
JOHNSON (1998)
GOLYSHINA et al. (2000)
JOHNSON (1998)
extr.
JOHNSON (1998)
thermophilic
JOHNSON (1998)
JOHNSON (1998)
2.0–3.0 55–85
AMARO et al. (1992)
extr.
ROSSI (1990)
thermophilic
extr.
BRIERLEY (1977)
thermophilic
ROSSI (1990)
ROSSI (1990)
extr.
JOHNSON (1998)
thermophilic
extr.
JOHNSON (1998)
thermophilic
extr.
JOHNSON (1998)
thermophilic
extr.
BARRETT et al. (1993),
thermophilic JOHNSON (1998)
JOHNSON (1998)
JOHNSON (1998)
JOHNSON (1998)
Thermoplasma acidophilum
Thermoplasma volcanicum
Bacteria Acetobacter methanolicus
Acidimicrobium ferrooxidans
Temperature Reference
[°C]
heterotrophic
gluconate
acidophilic
GLOMBITZA et al. (1988)
JOHNSON (1998),
EDWARDS et al. (1999)
8 Microbial Leaching of Metals
Domain Organism
202
Tab. 2. Microbial Diversity of Acidic Bioleaching Environments and Acidic Mine Drainage: Selection of Microorganisms known to Mediate Metal Bioleaching Reactions from Ores and Minerals or known to be Part of the Microbial Consortia Found in Bioleaching Habitats
Tab. 2. Continued
Domain Organism
Main
Leaching
Agent
Acidiphilium angustum
Acidiphilium cryptum
heterotrophic
organic acids 2.0–6.0
Acidiphilium symbioticum
heterotrophic
organic acids
Acidobacterium capsulatum
Acidocella sp.
Acidomonas methanolica
Arthrobacter sp.
Aureobacterium liquifaciens
Bacillus sp.
chemoorganotrophic
Bacillus coagulans
Bacillus licheniformis
heterotrophic
heterotrophic
Bacillus megaterium
Bacillus polymyxa
Chromobacterium violaceum
Comamonas testosteroni
Crenothrix sp.
Enterobacter agglomerans
Enterobacter cloacae
Gallionella sp.
Kingella kingae
Lactobacillus acidophilus
Leptospirillum ferrooxidans
heterotrophic
heterotrophic
heterotrophic
heterotrophic
facult. autotrophic
heterotrophic
heterotrophic
autotrophic
Temperature Reference
[°C]
mesophilic
3.0
mesophilic
3.0–6.0
mesophilic
5.4–6.0
22
37
heterotrophic
heterotrophic
heterotrophic
heterotrophic
heterotrophic
chemolithoautotrophic
Leptospirillum thermoferrooxidans chemolithoautotrophic
Leptothrix discophora
facult. autotrophic
heterotrophic
heterotrophic
heterotrophic
heterotrophic
citrate
cyanide
ferric iron
5.5–6.2
5.4–6.0
ferric iron
6.4–6.8
18–24
22
22
6–25
37
2.5–3.0 30
ferric iron
ferric iron
ferric iron, 5.8–7.8
sulfuric acid
ferric iron
3.5–6.8
5.4–6.0
1.7–1.9 45–50
5–40
4.1
37
22
EDWARDS et al. (1999)
GOEBEL and
STACKEBRANDT (1994)
BHATTACHARYYA et al.
(1991)
KISHIMOTO et al. (1991)
JOHNSON (1998)
JOHNSON (1998)
BOSECKER (1993)
EDWARDS et al. (1999)
CERDÁ et al. (1993),
GROUDEV and
GROUDEVA (1993)
BAGLIN et al. (1992)
MOHANTY and MISHRA
(1993)
KREBS et al. (1997)
LAWSON et al. (1999)
EDWARDS et al. (1999)
ROSSI (1990)
BAGLIN et al. (1992)
BAGLIN et al. (1992)
ROSSI (1990)
EDWARDS et al. (1999)
ACHARYA et al. (1998)
SAND (1992),
RAWLINGS et al. (1999)
BARRETT et al. (1993)
EDWARDS et al. (1999)
ROSSI (1990)
EDWARDS et al. (1999)
ACHARYA et al. (1998)
BAGLIN et al. (1992)
203
Metallogenium sp.
Ochrobacterium anthropi
Propionibacterium acnes
Pseudomonas cepacia
pH Range pH
Opt.
5 Microbial Diversity in Bioleaching Environments
Nutrition Type
204
Tab. 2. Continued
Domain Organism
Main
Leaching
Agent
pH Range pH
Opt.
Temperature Reference
[°C]
Pseudomonas putida
heterotrophic
citrate,
gluconate
Psychrobacter glacincola
Serratia ficaria
Siderocapsa sp.
Staphylococcus lactis
Stenotrophomonas maltophila
Sulfobacillus thermosulfidooxidans
heterotrophic
heterotrophic
heterotrophic
heterotrophic
heterotrophic
chemolithoautotrophic
Thermothrix thiopara
Thiobacillus acidophilus
chemolithoautotrophic
mixotrophic
ferric iron,
sulfuric acid
sulfuric acid
sulfuric acid
Thiobacillus albertis
Thiobacillus caldus
chemolithoautotrophic
chemolithoautotrophic
sulfuric acid 2.0–4.5
sulfuric acid
Thiobacillus capsulatus
chemolithoautotrophic
sulfuric acid
Thiobacillus concretivorus
Thiobacillus delicatus
Thiobacillus denitrificans
chemolithoautotrophic
mixotrophic
chemolithoautotrophic
sulfuric acid 0.5–6.0
sulfuric acid
sulfuric acid 5.0–7.0
Thiobacillus ferrooxidans
chemolithoautotrophic
1.4–6.0
2.4
Thiobacillus intermedius
Thiobacillus kabobis
Thiobacillus neapolitanus
facult. heterotrophic
mixotrophic
chemolithoautotrophic
ferric iron,
sulfuric acid
sulfuric acid
sulfuric acid
sulfuric acid
1.9–7.0
1.8–6.0
3.0–8.5
6.8
30
3.0
28
6.2–7.0 28
Thiobacillus novellus
Thiobacillus organoparus
Thiobacillus perometabolis
Thiobacillus prosperus
chemolithoautotrophic
mixotrophic
chemolithoheterotrophic
chemolithoautotrophic
sulfuric acid
sulfuric acid
sulfuric acid
sulfuric acid
5.0–9.0
1.5–5.0
2.6–6.8
1.0–4.5
7.8–9.0 30
2.5–3.0 27–30
6.9
30
23–41
Thiobacillus pumbophilus
Thiobacillus rubellus
chemolithoautotrophic
chemolithoautotrophic
sulfuric acid 4.0–6.5
sulfuric acid
27
5.0–7.0 25–30
KREBS et al. (1997)
ferric iron
37
extr.
acidoph.
neutral
1.5–6.0
50
3.0
60–75
25–30
3.5–4.0 28–30
45
5.0–7.0 25–30
30
28–35
EDWARDS et al. (1999)
EDWARDS et al. (1999)
ROSSI (1990)
ACHARYA et al. (1998)
EDWARDS et al. (1999)
JOHNSON (1998)
BRIERLEY (1977)
CERDÁ et al. (1993),
JOHNSON (1998)
JOHNSON (1998)
AMARO et al. (1992),
DOPSON and LINDSTROM
(1999)
EWART and HUGHES
(1991)
ROSSI (1990)
ROSSI (1990)
GROUDEV and
GROUDEVA (1993)
SAND (1992)
ROSSI (1990)
ROSSI (1990)
GROUDEV and
GROUDEVA (1993)
ROSSI (1990)
ROSSI (1990)
ROSSI (1990)
HUBER and STETTER
(1989)
DROBNER et al. (1992)
BARRETT et al. (1993)
8 Microbial Leaching of Metals
Nutrition Type
Tab. 2. Continued
Domain Organism
Nutrition Type
Main
Leaching
Agent
pH Range pH
Opt.
Temperature Reference
[°C]
Thiobacillus tepidarius
chemolithoautotrophic
sulfuric acid
Thiobacillus thiooxidans
Thiobacillus thioparus
Thiobacillus versutus
Thiomonas cuprinus
chemolithoautotrophic
chemolithoautotrophic
chemolithoautotrophic
facult. heterotrophic
sulfuric acid 0.5–6.0
sulfuric acid 4.5–10.0
sulfuric acid
sulfuric acid
Eukarya Actinomucor sp.
heterotrophic
succinate
27
MÜLLER and FÖRSTER
(1964)
Fungi
Alternaria sp.
heterotrophic
citrate,
oxalate
32
Aspergillus awamori
Aspergillus fumigatus
Aspergillus niger
heterotrophic
heterotrophic
heterotrophic
KOVALENKO and
MALAKHOVA (1990)
OGURTSOVA et al. (1989)
BOSECKER (1989)
DAVE et al. (1981),
BOSECKER (1987)
Aspergillus ochraceus
Aspergillus sp.
heterotrophic
heterotrophic
Cladosporium resinae
Cladosporium sp.
heterotrophic
heterotrophic
Coriolus versicolor
Fusarium sp.
heterotrophic
heterotrophic
2.0–3.5 10–37
6.6–7.2 11–25
8.0–9.0
3.0–4.0 30–36
oxalate,
citrate,
gluconate,
malate,
tartrate,
succinate
citrate
citrate,
oxalate
oxalate
oxalate,
malate,
pyruvate,
oxalacetate
30
28
30
OGURTSOVA et al. (1989)
TZEFERIS (1994)
28
OGURTSOVA et al. (1989)
KOVALENKO and
MALAKHOVA (1990)
SAYER et al. (1999)
BOSECKER (1989)
5 Microbial Diversity in Bioleaching Environments
28
HUGHES and POOLE
(1989)
SAND (1992)
BLOWES et al. (1998)
ROSSI (1990)
HUBER and STETTER
(1990)
205
206
Tab. 2. Continued
Domain Organism
Algae
Main
Leaching
Agent
pH Range pH
Opt.
Temperature Reference
[°C]
Mucor racemosus
heterotrophic
Paecilomyces variotii
heterotrophic
citrate,
succinate
citrate,
oxalate
Penicillium sp.
heterotrophic
Penicillium chrysogenum
Penicillium funiculosum
Penicillium notatum
Penicillium simplicissimum
heterotrophic
heterotrophic
heterotrophic
heterotrophic
Rhizopus japonicus
Trichoderma lignorum
Trichoderma viride
heterotrophic
heterotrophic
heterotrophic
Candida lipolytica
Rhodotorula sp.
heterotrophic
heterotrophic
30
Saccharomyces cerevisiae
Torulopsis sp.
Trichosporon
heterotrophic
heterotrophic
heterotrophic
28
27
MÜLLER and FÖRSTER
(1964)
DAVE et al. (1981)
25
GUPTA and EHRLICH
(1989)
OGURTSOVA et al. (1989)
BOSECKER (1989)
KARAVAIKO et al. (1980)
TARASOVA et al. (1993),
SILVERMAN and MUNOZ
(1971)
OGURTSOVA et al. (1989)
AVAKYAN et al. (1981)
BOROVEC (1990)
28
citrate
citrate,
oxalate,
gluconate
26
22–30
24–26
32
GROUDEV (1987)
EHRLICH (1963),
CERDÁ et al. (1993)
OGURTSOVA et al. (1989)
CERDÁ et al. (1993)
EHRLICH (1963)
not identified
GROUDEV and
GROUDEVA (1993)
Protozoa not identified
GROUDEV and
GROUDEVA (1993)
Amoebae not identified
EHRLICH (1963)
8 Microbial Leaching of Metals
Yeasts
Nutrition Type