Sunteți pe pagina 1din 16

8 Microbial Leaching of Metals

HELMUT BRANDL
Zürich, Switzerland

1 Introduction 192
2 Terminology 192
3 Historical Background 192
4 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
5 Microbial Diversity in Bioleaching Environments 200
6 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
7 Economics of Metal Bioleaching 213
8 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
9 Conclusion 217
10 References 217
192 8 Microbial Leaching of Metals

1 Introduction trated form. In gold mining operations, biooxi-


dation is used as a pretreatment process to
(partly) remove pyrite or arsenopyrite. This
Future sustainable development requires process is also called “biobeneficiation” where
measures to reduce the dependence on non- solid materials are refined and unwanted im-
renewable raw materials and the demand for purities are removed (GROUDEV, 1999; STRAS-
primary resources. New resources for metals SER et al., 1993). The terms “biomining”, “bio-
must be developed with the aid of novel tech- extraction”, or “biorecovery” are also applied
nologies. in addition, improvement of alredy to describe the mobilization of elements from
existing mining techniques can result in metal solid materials mediated by bacteria and fungi
recovery from sources that have not been of (HOLMES, 1991; MANDL et al., 1996; RAWLINGS,
economical interest until today. Metal-winning 1997; WOODS and RAWLINGS, 1989). “Biomin-
processes based on the activity of microorgan- ing” concerns mostly applications of microbial
isms offer a possibility to obtain metals from metal mobilization processes in large-scale
mineral resources not accessible by conven- operations of mining industries for an eco-
tional mining (BOSECKER, 1997; BRIERLEY, nomical metal recovery.
1978; BRYNER et al., 1954; TORMA and BAN- The area of “biohydrometallurgy” covers
HEGYI, 1984). Microbes such as bacteria and bioleaching or biomining processes (ROSSI,
fungi convert metal compounds into their 1990). Biohydrometallurgy represents an in-
water-soluble forms and are biocatalysts of terdisciplinary field where aspects of microbiol-
these leaching processes. Additionally, apply- ogy (especially geomicrobiology), geochemis-
ing microbiological solubilization processes, it try, biotechnology, hydrometallurgy, mineralo-
is possible to recover metal values from in- gy, geology, chemical engineering, and mining
dustrial wastes which can serve as secondary engineering are combined. Hydrometallurgy
raw materials. is defined as the treatment of metals and met-
al-containing materials by wet processes and
describes “the extraction and recovery of met-
als from their ores by processes in which aque-
2 Terminology ous solutions play a predominant role” (PARK-
ER, 1992). Rarely, the term “biogeotechnolo-
In general, bioleaching is a process de- gy” is also used instead of biohydrometallurgy
scribed as being “the dissolution of metals (FARBISZEWSKA et al., 1994).
from their mineral source by certain naturally
occurring microorganisms” or “the use of mi-
croorganisms to transform elements so that
the elements can be extracted from a material
when water is filtered trough it” (ATLAS and 3 Historical Background
BARTHA, 1997; PARKER, 1992). Additionally,
the term “biooxidation” is also used (HANS- One of the first reports where leaching
FORD and MILLER, 1993). There are, however, might have been involved in the mobilization
some small differences by definition (BRIER- of metals is given by the Roman writer Gaius
LEY, 1997): Usually, “bioleaching” is referring Plinius Secundus (23–79 A.D.). In his work on
to the conversion of solid metal values into natural sciences, Plinius describes how copper
their water soluble forms using microorgan- minerals are obtained using a leaching process
isms. In the case of copper, copper sulfide is (KÖNIG, 1989a, b). The translation reads ap-
microbially oxidized to copper sulfate and proximately as follows:“Chrysocolla is a liquid
metal values are present in the aqueous phase. in the before mentioned gold mines running
Remaining solids are discarded. “Biooxida- from the gold vein. In cold weather during the
tion” describes the microbiological oxidation winter the sludge freezes to the hardness of
of host minerals which contain metal com- pumice. It is known from experience that the
pounds of interest. As a result, metal values re- most wanted [chrysocolla] is formed in copper
main in the solid residues in a more concen- mines, the following in silver mines. The liquid
3 Historical Background 193

is also found in lead mines although it is of mi- mine in Spain 10 years earlier (SALKIELD,
nor value. In all these mines chrysocolla is also 1987). As a consequence to the ban of open air
artificially produced by slowly passing water ore roasting and its resulting atmospheric sul-
through the mine during the winter until the fur emissions in 1878 in Portugal, hydrometal-
month of June; subsequently, the water is evap- lurgical metal extraction has been taken into
orated in June and July. It is clearly demon- consideration in other countries more intense-
strated that chrysocolla is nothing but a de- ly. In addition to the ban, cost savings were an-
composed vein.” other incentive for the development: Heap
The German physician and mineralogist leaching techniques were assumed to reduce
Georgius Agricola (1494–1555) describes in transportation costs and to allow the employ-
his work de re metallica also techniques for the ment of locomotives and wagons for other ser-
recovery of copper that are based on the leach- vices (SALKIELD, 1987). From 1900 on, no open
ing of copper-containing ores (SCHIFFNER, air roasting of low-grade ore was conducted at
1977). A woodcut from his book illustrates the Rio Tinto mines.
the (manual) transport of metal-containing Efforts to establish bioleaching at the Rio
leachates from mines and their evaporation in Tinto mines had been undertaken in the begin-
the sunlight (Fig. 1). ning of the 1890s. Heaps (10 m in height) of
The Rio Tinto mines in south-western Spain low-grade ore (containing 0.75% Cu) were
are usually considered the cradle of biohydro- built and left for one to three years for “natu-
metallurgy. These mines have been exploited ral” decomposition (SALKIELD, 1987). 20 to
since pre-Roman times for their copper, gold, 25% of the copper left in the heaps were re-
and silver values. However, with respect to covered annually. It was calculated that ap-
commercial bioleaching operations on an in- proximately 200,000 t of rough ore could be
dustrial scale, biohydrometallurgical tech- treated in 1896. Although industrial leaching
niques had been introduced to the Tharsis 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 bio-
leaching 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. Al-
though usually acidic mine waters were used
for ore processing, 4 billion liters of freshwater
were required annually (SALKIELD, 1987).
Although metal leaching from mineral re-
sources has a very long historical record (EHR-
LICH, 1999; ROSSI, 1990) and although the oxi-
dation of reduced sulfur compounds and ele-
mental sulfur resulting in the formation of sul-
furic 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
Fig. 1. Woodcut from the book de re metallica writ- was investigated (RUDOLFS, 1922; RUDOLFS
ten by Georgius Agricola (1494–1555) illustrating and HELBRONNER, 1922). It was found that the
the manual recovery of copper-containing mine transformation of zinc sulfide to zinc sulfate
effluents which are collected in wooden basins and was microbially mediated. Based on these re-
concentrated in the sun. sults, the economic recovery of zinc from zinc-
194 8 Microbial Leaching of Metals

containing ores by biological methods was 4.2 Models of Leaching


proposed. In 1947, Thiobacillus ferrooxidans
was identified as part of the microbial commu- Mechanisms
nity found in acid mine drainage (COLMER and
HINKLE, 1947). A first patent was granted in Originally, a model with two types of mech-
1958 (ZIMMERLEY et al., 1958). The patent de- anisms which are involved in the microbial
scribes a cyclic process where a ferric sulfate/ mobilization of metals has been proposed
sulfuric acid lixiviant solution is used for metal (EWART and HUGHES, 1991; SILVERMAN and
extraction, regenerated by aeration (ferrous EHRLICH, 1964): (1) Microorganisms can oxi-
iron oxidation by iron-oxidizing organisms), dize metal sulfides by a “direct” mechanism
and reused in a next leaching stage. obtaining electrons directly from the reduced
minerals. Cells have to be attached to the min-
eral surface and a close contact is needed. The
adsorption of cells to suspended mineral parti-
cles takes place within minutes or hours. This
has been demonstrated using either radioac-
4 Principles of Microbial tively labeled Thiobacillus ferrooxidans cells
Metal Leaching grown on NaH14CO3 or the oxidative capacity
of bacteria attached to the mineral surface
(ESCOBAR et al., 1996). Cells adhere selectively
4.1 Leaching Mechanisms to mineral surfaces occupying preferentially ir-
regularities of the surface structure (EDWARDS
Mineralytic effects of bacteria and fungi on et al., 1999; EWART and HUGHES, 1991). In ad-
minerals are based mainly on three principles, dition, a chemotactic behavior to copper, iron,
namely acidolysis, complexolysis, and redoxo- or nickel ions has been demonstrated for Lep-
lysis. Microorganisms are able to mobilize met- tospirillum ferrooxidans (ACUNA et al., 1992).
als by (1) the formation of organic or inorgan- Genes involved in the chemotaxis were also
ic acids (protons); (2) oxidation and reduction detected in Thiobacillus ferrooxidans and
reactions; and (3) the excretion of complexing Thiobacillus thiooxidans (ACUNA et al., 1992).
agents. Sulfuric acid is the main inorganic acid (2) The oxidation of reduced metals through
found in leaching environments. It is formed the “indirect” mechanism is mediated by ferric
by sulfur-oxidizing microorganisms such as iron (Fe3c) originating from the microbial
thiobacilli.A series of organic acids are formed oxidation of ferrous iron (Fe2c) compounds
by bacterial (as well as fungal) metabolism present in the minerals. Ferric iron is an oxidiz-
resulting in organic acidolysis, complex and ing agent and can oxidize, e.g., metal sulfides
chelate formation (BERTHELIN, 1983). A kinet- and is (chemically) reduced to ferrous iron
ic model of the coordination chemistry of min- which, in turn, can be microbially oxidized
eral solubilization has been developed which again (EWART and HUGHES, 1991). In this case,
describes the dissolution of oxides by the pro- iron has a role as electron carrier. It was pro-
tonation of the mineral surface as well as the posed that no direct physical contact is needed
surface concentration of suitable complex- for the oxidation of iron.
forming ligands such as oxalate, malonate, In many cases it was concluded that the “di-
citrate, and succinate (FURRER and STUMM, rect” mechanism dominates over the “indi-
1986). Proton-induced and ligand-induced rect” mostly due to the fact that “direct” was
mineral solubilization occurs simultaneously equated with “direct physical contact”. This
in the presence of ligands under acidic condi- domination has been observed for the oxida-
tions. tion of covellite or pyrite in studies employing
mesophilic T. ferrooxidans and thermophilic
Acidianus brierleyi in bioreactors which con-
sisted of chambers separated with dialysis
membranes to avoid physical contact (LARS-
SON et al., 1993; POGLIANI et al., 1990). How-
4 Principles of Microbial Metal Leaching 195

ever, the attachment of microorganisms on around cells of T. ferrooxidans during growth


surfaces is not an indication per se for the exis- on synthetic pyrite films (ROJAS et al., 1995).
tence of a direct mechanism (EDWARDS et al., “Footprints” of organic films containing col-
1999). The term “contact leaching” has been loidal sulfur granules are left on the mineral
introduced to indicate the importance of bac- surface upon detachment of the bacteria.
terial attachment to mineral surfaces (TRI- From the existing data two “indirect” leach-
BUTSCH, 1999). ing mechanisms have been proposed whereas
The following equations describe the “di- no evidence for a “direct” enzymatically medi-
rect” and “indirect” mechanism for the oxida- ated process has been found (SAND et al.,
tion of pyrite (MURR, 1980; SAND et al., 1999): 1999).The mineral structure is the determining
factor for the prevailing type of leaching
direct: mechanism. In the “thiosulfate mechanism”
thiobacilli
thiosulfate is the main intermediate resulting
2 FeS2c7 O2c2 H2O ] 2 FeSO4 from the oxidation of pyrite, molybdenite, or
c2 H2SO4 (1) tungstenite. Polysulfide and elemental sulfur
are the main intermediates in the “polysulfide
indirect: mechanism” during the oxidation of galena,
sphalerite, chalcopyrite, hauerite, orpiment, or
4 FeSO4cO2 T. ferrooxidans, L. ferrooxidans
realgar. The presence of iron(III) at the begin-
c2 H2SO4 ] ning of mineral degradation is an important
2 Fe2(SO4)3c2 H2O (2) prerequisite (SAND et al., 1999).
chemical oxidation
The following equations summarize the oxi-
FeS2cFe2(SO4)3 ] 3 FeSO4 dation mechanisms (SAND et al., 1999):
c2 S (3)
T. thiooxidans
Thiosulfate mechanism (found for FeS2, MoS2,
2 Sc3 O2cH2O ] 2 H2SO4 (4) WS2):

However, the model of “direct” and “indirect” FeS2c6 Fe3cc3 H2O ] S2O2c
3 P7 Fe
2c

metal leaching is still under discussion. Re- c6 H c


(5)
cently, this model has been revised and re-
placed by another one which is not dependent S2O2P
3 c8 Fe
3c
c5 H2O ] 2 SO2P
4 c8 Fe
2c

on the differentiation between a “direct” and c10 H c


(6)
an “indirect” leaching mechanisms (SAND et
al., 1995, 1999). All facts have been combined Polysulfide mechanism (found for PbS, CuFeS2,
and a mechanism has been developed which is ZnS, MnS2, As2S3, As3S4):
characterized by the following features: (1)
cells have to be attached to the minerals and in 2 MSc2 Fe3cc2 Hc ] 2 M2ccH2Sn
physical contact with the surface; (2) cells form c2 Fe2c (7)
and excrete exopolymers; (3) these exopoly-
meric cell envelopes contain ferric iron com- H2Snc2 Fe3c ] 0.25 S8c2 Fe2cc2 Hc (8)
pounds which are complexed to glucuronic
acid residues. These are part of the primary at- 0.25 S8c3 O2c2 H2O ] 2 SO2P
4 c4 H
c
(9)
tack mechanism; (4) thiosulfate is formed as
intermediate during the oxidation of sulfur Several biomolecules are involved in the
compounds; (5) sulfur or polythionate gran- aerobic respiration on reduced sulfur and iron
ules are formed in the periplasmatic space or compounds. It has been found that up to 5% of
in the cell envelope. soluble proteins of T. ferrooxidans is made of
Thiosulfate and traces of sulfite have been an acid stable blue copper protein, called rus-
found as intermediates during the oxidation of ticyanin (BLAKE et al., 1993). Additionally, the
sulfur (SHRIHARI et al., 1993). Sulfur granules iron(II) respiratory system contains a (puta-
(colloidal sulfur) have been identified as ener- tive) green copper protein, two types of cyto-
gy reserves in the exopolymeric capsule chrome c, one or more types of cytochrome a,
196 8 Microbial Leaching of Metals

a porin, and an iron(II)-sulfate chelate (BLAKE was autoclaved to obtain a sterile leaching so-
et al., 1993). The acid stability of rusticyanin lution without enzymatic activities and to eval-
suggests that it is located in the periplasmic uate the leaching ability of acid formed. (4)
space. Figure 2 shows a scheme of the model Leaching by fresh medium. Fresh non-inocul-
which combines the electron transport se- ated and sterile medium was added to the fly
quence proposed earlier with concepts stem- ash suspension and used as control. (5) Chem-
ming from the debate on “direct”/“indirect” ical leaching due to the preparation of the ash
leaching mechanisms (BLAKE and SHUTE, suspension (acidification to pH 5.4). Certain
1994; BLAKE et al., 1993; HAZRA et al., 1992; elements such as, e.g., Cd or Zn might be chem-
SAND et al., 1995). ically mobilized already during acidification.
Some details of the metal mobilization MWI fly ash contains reduced copper spe-
mechanism, the importance of the presence cies (chalcocite {Cu2S} or cuprite {Cu2O})
and attachment of microorganisms and their whereas zinc and others are present in their
active contribution have been demonstrated fully oxidized forms (BROMBACHER et al.,
for the leaching of fly ash from municipal 1998). Therefore, copper release from fly ash is
waste incineration (MWI) (BROMBACHER et directly affected and enhanced by T. ferrooxi-
al., 1998). Generally, several mechanisms of dans, whereas Zn, as well as Al, Cd, Cr, and Ni,
metal mobilization can be distinguished: (1) are released primarily due to the acidic envi-
Contact leaching effect on the release of met- ronment. Acidification of the fly ash pulp
als. Stock cultures of Thiobacillus ferrooxidans (chemical mobilization) led already to consid-
and Thiobacillus thiooxidans were added to erable extraction yields for Cd, Ni, and Zn and
ash suspensions and cells were in direct con- could slightly be increased using non-inoculat-
tact with the fly ash. Growth of thiobacilli ed sterile medium as lixiviant (Fig. 3). By com-
might be stimulated by increased energy avail- paring leached amounts of copper by filtered
ability from oxidation of reduced solid parti- cell free spent medium with autoclaved sterile
cles. (2) Metal solubilization by metabolically spent medium, it was concluded that signifi-
active (enzymatic) compounds in the absence cant amounts of copper were mobilized – in
of bacterial cells. Stock cultures were filtered contrast to other elements – by metabolic
to obtain the cell free spent medium. This me- products of T. ferrooxidans. Leaching with cell
dium was used for leaching. (3) Metal solubil- free spent medium indicating a solubilizing
ization by non-enzymatic extracellular meta- mechanism due to extracellular components
bolic products. Cell free spent medium (see 2) was significantly more effective than a leach-

Fig. 2. Schematic mechanistic bio-


leaching 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: cyto-
chrome; RC: rusticyanin;
MeS: metal sulfide
4 Principles of Microbial Metal Leaching 197

Fig. 3. Solubilized metals from fly ash


originating from municipal waste incinera-
tion (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 me-
tals due to acidification of the fly ash pulp
is indicated as chemical mobilization (see
text for explanation).

ing with autoclaved spent medium where ex- ing (in g LP1): (NH4)2SO4 (3.0); K2HPO4 (0.5);
creted enzymes had been inactivated. It is MgSO4 · 7 H2O (0.5); KCl (1.0); Ca(NO3)2 ·
known that several components involved in 4 H2O (0.01); FeSO4 · 7 H2O (44.22); and 1 mL
the electron transport chain of Thiobacillus 10 N sulfuric acid (SILVERMAN and LUNDGREN,
(rusticyanin, cytochromes, iron–sulfur pro- 1959). Cells are harvested, diluted, and added
teins) are located in the periplasmic space to pyrite suspensions with a pulp density of
(BLAKE and SHUTE, 1994; SAND et al., 1995) 20 g LP1. Total soluble iron as well as sulfate
and might, therefore, also be present in the cell formed during oxidation is periodically deter-
free spent medium catalyzing oxidation of re- mined.
duced metal compounds. Metal bioleaching in acidic environments
In many leaching environments conditions is influenced by a series of different factors
(especially iron(II) and iron(III) concentra- (Tab. 1). Physicochemical as well as microbio-
tions) vary with the duration of the leaching. logical factors of the leaching environment are
This makes it difficult to assess the importance affecting rates and efficiencies. In addition,
and the effect of the presence of bacteria. Us- properties of the solids to be leached are of
ing an experimental setup to maintain con- major importance (ACEVEDO and GENTINA,
stant concentrations of ferrous and ferric iron, 1989; BRIERLEY, 1978; DAS et al., 1999; MURR,
it was possible to show that in the presence of 1980). As examples, pulp density, pH, and par-
T. ferrooxidans rates of pyrite or zinc sulfide ticle size were identified as major factors for
leaching are increased (HOLMES et al., 1999; pyrite bioleaching by Sulfolobus acidocalda-
FOWLER and CRUNDWELL, 1999; FOWLER et al., rius (LINDSTROM et al., 1993). Optimal condi-
1999). tions were 60 g LP1, 1.5, and ~20 µm, respec-
tively. The influence of different parameters
such as activities of the bacteria itself, source
4.3 Factors Influencing Bioleaching energy, mineralogical composition, pulp den-
sity, temperature, and particle size was studied
Standard test methods have been developed for the oxidation of sphalerite by T. ferrooxi-
to determine leaching rates of iron from dans (BALLESTER et al., 1989). Best zinc dis-
pyrite mediated by Thiobacillus ferrooxidans solution was obtained at low pulp densities
(ASTM, 1991). An active culture of T. ferro- (50 g LP1), small particle sizes, and tempera-
oxidans is grown in a defined medium contain- tures of approximately 35 °C.
198 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
Microbiological parameters of a bioleaching environment microbial diversity
population density
microbial activities
spatial distribution of microorganisms
metal tolerance
adaptation abilities of microorganisms
Properties of the minerals to be leached mineral type
mineral composition
mineral dissemination
grain size
surface area
porosity
hydrophobicity
galvanic interactions
formation of secondary minerals
Processing leaching mode (in situ, heap, dump, or tank
leaching)
pulp density
stirring rate (in case of tank leaching opera-
tions)
heap geometry (in case of heap leaching)

Metal oxidation mediated by acidophilic mi- enced iron(II) oxidation by T. ferrooxidans


croorganisms can be inhibited by a variety of with uranium and thorium showing higher tox-
factors such as, e.g., organic compounds, sur- icities than copper and nickel (LEDUC et al.,
face-active agents, solvents, or specific metals: 1997). Silver, mercury, ruthenium, and molyb-
The presence of organic compounds (yeast ex- denum reduced the growth of Sulfolobus
tract) inhibited pyrite oxidation of T. ferrooxi- grown on a copper concentrate (MIER et al.,
dans (BACELAR-NICOLAU and JOHNSON, 1999). 1996). Industrial biocides such as tetra-n-bu-
Certain metals present in bioleaching environ- tyltin, isothiazolinones, N-dimethyl-Nb-phe-
ments can inhibit microbial growth, therefore nyl-Nb-(fluorodichloro-methylthio)-sulfamide,
reducing leaching efficiencies. For instance, ar- or 2,2b-dihydroxy-5,5b-dichlorophenylmethane
senic added to cultures inhibited Sulfolobus (dichlorophen) reduced the leaching of man-
acidocaldarius grown on pyrite and T. ferro- ganese oxides by heterotrophic microorgan-
oxidans grown on arsenopyrite (HALLBERG et isms (ARIEF and MADGWICK, 1992). Biocides
al., 1996; LAN et al., 1994).Additions of copper, were externally added as selective inhibitors to
nickel, uranium, or thorium adversely influ- suppress unwanted organisms and to improve
4 Principles of Microbial Metal Leaching 199

manganese leaching efficiencies. At low con- It has been demonstrated recently that the
centrations of ~5 mg LP1, however, mangan- addition of small amounts of amino acids (cys-
ese mobilization was increased by 20% (BOUS- teine in this case) resulted in an increased
SIOS and MADGWICK, 1994). pyrite corrosion by T. ferrooxidans as com-
Also gaseous compounds can show inhibito- pared to controls without additions (ROJAS-
ry effects on metal leaching: Aqueous-phase CHAPANA and TRIBUTSCH, 2000). It is suggest-
carbon dioxide at concentration `10 mg LP1 ed that the microorganisms may profit from
was inhibiting growth of T. ferrooxidans on weakening and break up of chemical bonds
pyrite–arsenopyrite–pyrrothite ore (NAGPAL mediated by the formation of the cysteine–py-
et al., 1993). Optimal concentrations of carbon rite complex. This might also be the case under
dioxide were found to be in the range of 3 to natural conditions by the excretion of cys-
7 mg LP1. There are reports on the stimulation teine-containing metabolites. An inexpensive
of bacterial leaching and the increase of leach- alternative to increase metal recovery from
ing rates by supplementing leaching fluids with ore heaps by the addition of sulfur-containing
carbon dioxide (ACEVEDO et al., 1998; BRIER- amino acids such as cysteine has been suggest-
LEY, 1978; TORMA et al., 1972). Concentrations ed (TRIBUTSCH and ROJAS-CHAPANA, 1999).
of 4% (v/v) carbon dioxide in the inlet gas of a Other metabolites excreted by Thiobacillus
fermenter showed maximum growth rates of might also enhance metal leaching efficiencies:
T. ferrooxidans, maximum iron(II), copper, Wetting agents such as mixtures of phospho-
and arsenic oxidation (ACEVEDO et al., 1998). lipids and neutral lipids are formed by Thioba-
Pulp densities of 20 g LP1 delayed the onset cillus thiooxidans (BEEBE and UMBREIT, 1971).
of bioleaching of pyrite derived from coal As a consequence, growth of T. thiooxidans on
(BALDI et al., 1992). Increasing pulp densities sulfur particles is supported by the excretion
from 30 to 100 g LP1 decreased rates of pyrite of metabolites acting as biosurfactants which
oxidation in Sulfolobus cultures (NGUBANE facilitate the oxidation of elemental sulfur. It
and BAECKER, 1990). For fungi such as Asper- was also hypothesized that Thiobacillus caldus
gillus niger, optimal pulp densities for maxi- is stimulating the growth of heterotrophic or-
mum metal leaching efficiencies were found to ganisms in leaching environments by the ex-
be in the range of 30 to 40 g LP1 (BOSSHARD cretion of organic compounds and is support-
et al., 1996). Quartz particles at pulp densities ing the solubilization of solid sulfur by the for-
of 80 g LP1 almost completely inhibited the mation of surface-active agents (DOPSON and
oxidation of covellite by T. ferrooxidans espe- LINDSTROM, 1999). Metal solubilization might
cially in the absence of iron(II) (CURUTCHET also be facilitated by microbial metabolites ex-
et al., 1990). creted by organisms other than Thiobacillus
During bioleaching processes, coprecipita- which are part of microbial consortia found in
tion of metals with mineral phases such as ja- bioleaching operations. Microbial surfactants,
rosites can reduce leaching efficiencies (HI- which show large differences in their chemical
ROYOSHI et al., 1999). In addition, the precipita- nature, are formed by a wide variety of micro-
tion of compounds present in the leachates on organisms. In the presence of biosurfactants
the minerals to be leached can make the solid which lead to changes in the surface tension,
material inaccessible for bacterial leaching. metal desorption from solids might be en-
Organic solvents such as flotation or solvent hanced resulting in an increased metal mobil-
extraction agents, which are added for the ity in porous media. It has been suggested that
downstream processing of leachates from bio- this metabolic potential can be practically used
leaching, might also lead to inhibition prob- in the bioremediation of metal-contaminated
lems (ACEVEDO and GENTINA, 1989). Isopro- soils (MILLER, 1995). However, there is some
pylxanthate and LIX 984 (used as flotation evidence that surface-active compounds as
agent and solvent extraction agent, respective- well as organic solvents are inhibitory to bio-
ly) prevented the oxidation of pyrite and chal- leaching reactions and prevent bacterial at-
copyrite by T. ferrooxidans (HUERTA et al., tachment (MURR, 1980). The external addition
1995). This fact is of special importance when of Tween reduced the oxidation of chalcopyr-
spent leaching liquors are recycled for a reuse. ite by T. ferrooxidans (TORMA et al., 1976). It
200 8 Microbial Leaching of Metals

was concluded that the need of the microor- 5 Microbial Diversity in


ganisms for surfactants is met by their own for-
mation. In contrast, it was reported that the ad- Bioleaching Environments
dition of Tween 80 increased the attachment of
T. ferrooxidans on molybdenite and the oxida-
tion of molybdenum in the absence of iron(II) A variety of microorganisms is found in
(PISTACCIO et al., 1994). leaching environments and has been isolated
from leachates and acidic mine drainage. Al-
though environmental conditions are usually
4.4 Bacterial Attachment on described (from an anthropocentric view!) as
Mineral Surfaces being extreme and harsh due to pH values (as
low as P3.6; NORDSTROM et al., 2000) and high
It is known that the formation of extracellu- metal concentrations (as high as 200 g LP1;
lar polymeric substances plays an important NORDSTROM et al., 2000), these systems can
role in the attachment of thiobacilli to mineral show high levels of microbial biodiversity in-
surfaces such as, e.g., sulfur, pyrite, or covellite. cluding bacteria, fungi, and algae (LOPEZ-AR-
Extraction or loss of these exopolymers pre- CHILLA et al., 1993). It has long been known
vent cell attachment resulting in decreased that bacteria (Thiobacillus sp.), yeasts (Rho-
metal leaching efficiencies (ESCOBAR et al., dotorula sp., Trichosporon sp.), flagellates (Eu-
1997; GEHRKE et al., 1998; POGLIANI and DO- trepia sp.), amoebes and protozoa are part of
NATI, 1992). It was concluded that a direct con- the microbial biocenosis found in acidic waters
tact between bacterial cells and solid surfaces of a copper mine (EHRLICH, 1963). Recent de-
is needed and represents an important prere- tailed investigations based on molecular meth-
quisite for an effective metal mobilization ods such as DNA–DNA hybridization, 16S
(OSTROWSKI and SKLODOWSKA, 1993). Interac- rRNA sequencing, RCR-based methods with
tions between microorganisms and the miner- primers derived from rRNA sequencing, fluor-
al surface occur on two levels (BARRETT et al., escence in situ hybridization (FISH), or im-
1993). The first level is a physical sorption be- munological techniques revealed that micro-
cause of electrostatic forces. Due to the low bial bioleaching communities are composed of
pH usually occurring in leaching environ- a vast variety of microorganisms resulting in
ments, microbial cell envelopes are positively complex microbial interactions and nutrient
charged leading to electrostatic interactions flows (such as synergism, mutualism, competi-
with the mineral phase. The second level is tion, predation) (AMARO et al., 1992; DE
characterized by chemical sorption where WULF-DURAND et al., 1997; EHRLICH, 1997;
chemical bonds between cells and minerals JOHNSON, 1998; EDWARDS et al., 1999). Selected
might be established (e.g., disulfide bridges). organisms of these communities are given in
In addition, extracellular metabolites are Table 2.The composition of these communities
formed and excreted during this phase in the is usually subjected to seasonal fluctuations
near vicinity of the attachment site (EWART and may vary between different mining loca-
and HUGHES, 1991). Low-molecular weight tions (EDWARDS et al., 1999; GROUDEV and
metabolites excreted by sulfur oxidizers in- GROUDEVA, 1993). In addition, organisms are
clude acids originating from the TCA cycle, not homogeneously distributed over the whole
amino acids, or ethanolamine, whereas com- leaching environment (CERDÁ et al., 1993).
pounds with relatively high molecular weights The organism studied most is Thiobacillus
include lipids and phospholipids (BARRETT et ferrooxidans. Although this is the best known
al., 1993). In the presence of elemental sulfur, organism from acidic habitats, one may not
sulfur-oxidizing microorganisms from sewage conclude that this organism is dominant in
sludge form a filamentous matrix similar to a these ecosystems. It has been found that under
bacterial glycocalyx suggesting the relative im- specific environmental conditions Leptospiril-
portance of these extracellular substances in lum sp. is even more abundant than T. ferro-
the colonization of solid particles (BLAIS et al., oxidans suggesting an important ecological
1994). role in the microbial community structure of
5 Microbial Diversity in Bioleaching Environments 201

bioleaching habitats (SAND, 1992; SCHRENK et extraction from mine tailings is more efficient
al., 1998). Thiobacilli are members of the divi- using thermophilic instead of mesophilic or-
sion of Proteobacteria close to the junction ganisms, extremely thermophilic microorgan-
between the  and  subdivision whereas isms show a higher sensitivity to copper and to
leptospirilli are placed in the Nitrospira divi- high pulp densities in agitated systems limit-
sion (RAWLINGS, 1999). Genetic studies re- ing, therefore, some practical applications
vealed that the role of T. ferrooxidans in leach- (DUARTE et al., 1993; NORRIS and OWEN,
ing operations has probably been overestimat- 1993).
ed. Excellent reviews on the genetics of Thio- Although environmental conditions in
bacilli and leptospirilli have been published leaching operations favor the growth and de-
recently (RAWLINGS, 1999; RAWLINGS and KU- velopment of mesophilic, moderately thermo-
SANO, 1994). philic, and extremely thermophilic microbial
Thiobacillus ferrooxidans belongs to the communities, metal leaching at low tempera-
group of chemolithotrophic organisms. The or- tures has also been observed. Copper and
ganism is rod-shaped (usually single or in nickel were leached from pyritic ore samples
pairs), non-spore forming, gram-negative, mo- in significant amounts at 4 °C (AHONEN and
tile, and single-pole flagellated (HORAN, 1999; TUOVINEN, 1992). However, leaching rates
KELLY and HARRISON, 1984; LEDUC and FER- were lower by a factor of 30 to 50 as compared
RONI, 1994; MURR, 1980). As carbon source, to experiments conducted at 37 °C. T. ferrooxi-
carbon dioxide is utilized. Ferrous iron is oxi- dans recovered from mine waters was able to
dized. Ammonium is used as nitrogen source. grow at 2 °C with a generation time of approx-
Although T. ferrooxidans has been character- imately 250 h suggesting a psychrotrophic na-
ized as being a strictly aerobic organism, it can ture of the organism (FERRONI et al., 1986).
also grow on elemental sulfur or metal sulfides Bacterial iron mobilization has also been ob-
under anoxic conditions using ferric iron as served at 0 °C in ore samples obtained from
electron acceptor (DONATI et al., 1997; PRONK Greenland (LANGDAHL and INGVORSEN, 1997).
et al., 1992). Solubilization rates at these low temperatures
The genus Thiobacillus represents a versa- were still approximately 25 to 30% of the max-
tile group of chemolithoautotrophic organ- imum values observed at 21 °C. All these find-
isms. Optimum pH values for growth vary ings may have a potential for practical applica-
between 2 and 8 (Fig. 4). It has been demon- tions in geographical areas where field opera-
strated that sulfur-oxidizing bacteria are ca- tions are subjected to low temperature regimes.
pable of reducing the pH of highly alkaline fly A series of heterotrophic microorganisms
ash suspensions amended with elemental sul- (bacteria, fungi) is also part of microbial bio-
fur from approximately 9 to 0.5 (KREBS et al., leaching communities (Tab. 2). This group of
1999) (Fig. 5). It is likely that thiobacilli con- organisms uses extracellular metabolites and
tribute to increasing acidification of leaching cell lysates from autotrophs as carbon source
ecosystems in a successive mode: In the initial resulting in the removal of an inhibitory excess
stages the growth of less acidophilic strains of carbon and stimulating, therefore, growth
(e.g., Thiobacillus thioparus) is stimulated and iron oxidation of thiobacilli (BUTLER and
whereas during prolonged leaching the pH de- KEMPTON, 1987; FOURNIER et al., 1998). In ad-
creases gradually supporting growth of more dition, several heterotrophs can also con-
acidophilic strains. This has already been ob- tribute to metal solubilization by the excretion
served in metal leaching from wastewater sew- of organic acids such as citrate, gluconate, oxa-
age sludge (BLAIS et al., 1993). late, or succinate.
A variety of thermophilic microorganisms
(especially Sulfolobus species) has been en-
riched and isolated from bioleaching environ-
ments (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
202
Tab. 2. Microbial Diversity of Acidic Bioleaching Environments and Acidic Mine Drainage: Selection of Microorganisms known to Mediate Metal Bio-
leaching Reactions from Ores and Minerals or known to be Part of the Microbial Consortia Found in Bioleaching Habitats

Domain Organism Nutrition Type Main pH Range pH Temperature Reference

8 Microbial Leaching of Metals


Leaching Opt. [°C]
Agent

Archaea Acidianus ambivalens facult. heterotrophic sulfuric acid JOHNSON (1998)


Acidianus brierleyi facult. heterotrophic sulfuric acid acidophilic 1.5–3.0 45–75 MUÑOZ et al. (1995)
Acidianus infernus facult. heterotrophic sulfuric acid JOHNSON (1998)
Ferroplasma acidiphilum chemolithoautotrophic ferric iron 1.3–2.2 1.7 15– 45 GOLYSHINA et al. (2000)
Metallosphaera prunae chemolithoautotrophic ferric iron, JOHNSON (1998)
sulfuric acid
Metallosphaera sedula chemolithoautotrophic ferric iron, acidophilic extr. JOHNSON (1998)
sulfuric acid thermophilic
Picrophilus oshimae JOHNSON (1998)
Picrophilus torridus JOHNSON (1998)
Sulfolobus acidocaldarius chemolithoautotrophic ferric iron, 0.9–5.8 2.0–3.0 55–85 AMARO et al. (1992)
sulfuric acid
Sulfolobus ambivalens chemolithoautotrophic ferric iron, extr. ROSSI (1990)
sulfuric acid thermophilic
Sulfolobus brierleyi chemolithoautotrophic ferric iron, extr. BRIERLEY (1977)
sulfuric acid thermophilic
Sulfolobus hakonensis chemolithoautotrophic ROSSI (1990)
Sulfolobus metallicus chemolithoautotrophic ROSSI (1990)
Sulfolobus solfataricus chemolithoautotrophic ferric iron, extr. JOHNSON (1998)
sulfuric acid thermophilic
Sulfolobus thermosulfidooxidans chemolithoautotrophic ferric iron, extr. JOHNSON (1998)
sulfuric acid thermophilic
Sulfolobus yellowstonii chemolithoautotrophic ferric iron, extr. JOHNSON (1998)
sulfuric acid thermophilic
Sulfurococcus mirabilis mixotrophic ferric iron, acidophilic extr. BARRETT et al. (1993),
sulfuric acid thermophilic JOHNSON (1998)
Sulfurococcus yellowstonii mixotrophic ferric iron, JOHNSON (1998)
sulfuric acid
Thermoplasma acidophilum JOHNSON (1998)
Thermoplasma volcanicum JOHNSON (1998)

Bacteria Acetobacter methanolicus heterotrophic gluconate acidophilic GLOMBITZA et al. (1988)


Acidimicrobium ferrooxidans JOHNSON (1998),
EDWARDS et al. (1999)
Tab. 2. Continued

Domain Organism Nutrition Type Main pH Range pH Temperature Reference


Leaching Opt. [°C]
Agent

Acidiphilium angustum EDWARDS et al. (1999)


Acidiphilium cryptum heterotrophic organic acids 2.0–6.0 mesophilic GOEBEL and
STACKEBRANDT (1994)
Acidiphilium symbioticum heterotrophic organic acids 3.0 mesophilic BHATTACHARYYA et al.
(1991)
Acidobacterium capsulatum chemoorganotrophic 3.0–6.0 mesophilic KISHIMOTO et al. (1991)
Acidocella sp. JOHNSON (1998)
Acidomonas methanolica heterotrophic JOHNSON (1998)
Arthrobacter sp. heterotrophic BOSECKER (1993)
Aureobacterium liquifaciens heterotrophic EDWARDS et al. (1999)
Bacillus sp. heterotrophic CERDÁ et al. (1993),
GROUDEV and

5 Microbial Diversity in Bioleaching Environments


GROUDEVA (1993)
Bacillus coagulans heterotrophic 5.4–6.0 22 BAGLIN et al. (1992)
Bacillus licheniformis heterotrophic 37 MOHANTY and MISHRA
(1993)
Bacillus megaterium heterotrophic citrate KREBS et al. (1997)
Bacillus polymyxa heterotrophic
Chromobacterium violaceum heterotrophic cyanide LAWSON et al. (1999)
Comamonas testosteroni heterotrophic EDWARDS et al. (1999)
Crenothrix sp. facult. autotrophic ferric iron 5.5–6.2 18–24 ROSSI (1990)
Enterobacter agglomerans heterotrophic 5.4–6.0 22 BAGLIN et al. (1992)
Enterobacter cloacae heterotrophic 22 BAGLIN et al. (1992)
Gallionella sp. autotrophic ferric iron 6.4–6.8 6–25 ROSSI (1990)
Kingella kingae EDWARDS et al. (1999)
Lactobacillus acidophilus heterotrophic 37 ACHARYA et al. (1998)
Leptospirillum ferrooxidans chemolithoautotrophic ferric iron 2.5–3.0 30 SAND (1992),
RAWLINGS et al. (1999)
Leptospirillum thermoferrooxidans chemolithoautotrophic ferric iron 1.7–1.9 45–50 BARRETT et al. (1993)
Leptothrix discophora facult. autotrophic ferric iron, 5.8–7.8 5–40 EDWARDS et al. (1999)
sulfuric acid
Metallogenium sp. heterotrophic ferric iron 3.5–6.8 4.1 ROSSI (1990)
Ochrobacterium anthropi heterotrophic EDWARDS et al. (1999)
Propionibacterium acnes heterotrophic 37 ACHARYA et al. (1998)

203
Pseudomonas cepacia heterotrophic 5.4–6.0 22 BAGLIN et al. (1992)
204
Tab. 2. Continued

Domain Organism Nutrition Type Main pH Range pH Temperature Reference


Leaching Opt. [°C]

8 Microbial Leaching of Metals


Agent

Pseudomonas putida heterotrophic citrate, KREBS et al. (1997)


gluconate
Psychrobacter glacincola heterotrophic EDWARDS et al. (1999)
Serratia ficaria heterotrophic EDWARDS et al. (1999)
Siderocapsa sp. heterotrophic ferric iron ROSSI (1990)
Staphylococcus lactis heterotrophic 37 ACHARYA et al. (1998)
Stenotrophomonas maltophila heterotrophic EDWARDS et al. (1999)
Sulfobacillus thermosulfidooxidans chemolithoautotrophic ferric iron, extr. 50 JOHNSON (1998)
sulfuric acid acidoph.
Thermothrix thiopara chemolithoautotrophic sulfuric acid neutral 60–75 BRIERLEY (1977)
Thiobacillus acidophilus mixotrophic sulfuric acid 1.5–6.0 3.0 25–30 CERDÁ et al. (1993),
JOHNSON (1998)
Thiobacillus albertis chemolithoautotrophic sulfuric acid 2.0–4.5 3.5–4.0 28–30 JOHNSON (1998)
Thiobacillus caldus chemolithoautotrophic sulfuric acid 45 AMARO et al. (1992),
DOPSON and LINDSTROM
(1999)
Thiobacillus capsulatus chemolithoautotrophic sulfuric acid EWART and HUGHES
(1991)
Thiobacillus concretivorus chemolithoautotrophic sulfuric acid 0.5–6.0 ROSSI (1990)
Thiobacillus delicatus mixotrophic sulfuric acid 5.0–7.0 25–30 ROSSI (1990)
Thiobacillus denitrificans chemolithoautotrophic sulfuric acid 5.0–7.0 30 GROUDEV and
GROUDEVA (1993)
Thiobacillus ferrooxidans chemolithoautotrophic ferric iron, 1.4–6.0 2.4 28–35 SAND (1992)
sulfuric acid
Thiobacillus intermedius facult. heterotrophic sulfuric acid 1.9–7.0 6.8 30 ROSSI (1990)
Thiobacillus kabobis mixotrophic sulfuric acid 1.8–6.0 3.0 28 ROSSI (1990)
Thiobacillus neapolitanus chemolithoautotrophic sulfuric acid 3.0–8.5 6.2–7.0 28 GROUDEV and
GROUDEVA (1993)
Thiobacillus novellus chemolithoautotrophic sulfuric acid 5.0–9.0 7.8–9.0 30 ROSSI (1990)
Thiobacillus organoparus mixotrophic sulfuric acid 1.5–5.0 2.5–3.0 27–30 ROSSI (1990)
Thiobacillus perometabolis chemolithoheterotrophic sulfuric acid 2.6–6.8 6.9 30 ROSSI (1990)
Thiobacillus prosperus chemolithoautotrophic sulfuric acid 1.0–4.5 23–41 HUBER and STETTER
(1989)
Thiobacillus pumbophilus chemolithoautotrophic sulfuric acid 4.0–6.5 27 DROBNER et al. (1992)
Thiobacillus rubellus chemolithoautotrophic sulfuric acid 5.0–7.0 25–30 BARRETT et al. (1993)
Tab. 2. Continued

Domain Organism Nutrition Type Main pH Range pH Temperature Reference


Leaching Opt. [°C]
Agent

Thiobacillus tepidarius chemolithoautotrophic sulfuric acid HUGHES and POOLE


(1989)
Thiobacillus thiooxidans chemolithoautotrophic sulfuric acid 0.5–6.0 2.0–3.5 10–37 SAND (1992)
Thiobacillus thioparus chemolithoautotrophic sulfuric acid 4.5–10.0 6.6–7.2 11–25 BLOWES et al. (1998)
Thiobacillus versutus chemolithoautotrophic sulfuric acid 8.0–9.0 ROSSI (1990)
Thiomonas cuprinus facult. heterotrophic sulfuric acid 3.0–4.0 30–36 HUBER and STETTER
(1990)

Eukarya Actinomucor sp. heterotrophic succinate 27 MÜLLER and FÖRSTER


(1964)

Fungi Alternaria sp. heterotrophic citrate, 32 KOVALENKO and

5 Microbial Diversity in Bioleaching Environments


oxalate MALAKHOVA (1990)
Aspergillus awamori heterotrophic 28 OGURTSOVA et al. (1989)
Aspergillus fumigatus heterotrophic BOSECKER (1989)
Aspergillus niger heterotrophic oxalate, 30 DAVE et al. (1981),
citrate, BOSECKER (1987)
gluconate,
malate,
tartrate,
succinate
Aspergillus ochraceus heterotrophic citrate 28 OGURTSOVA et al. (1989)
Aspergillus sp. heterotrophic citrate, 30 TZEFERIS (1994)
oxalate
Cladosporium resinae heterotrophic 28 OGURTSOVA et al. (1989)
Cladosporium sp. heterotrophic KOVALENKO and
MALAKHOVA (1990)
Coriolus versicolor heterotrophic oxalate SAYER et al. (1999)
Fusarium sp. heterotrophic oxalate, BOSECKER (1989)
malate,
pyruvate,
oxalacetate

205
206
Tab. 2. Continued

Domain Organism Nutrition Type Main pH Range pH Temperature Reference


Leaching Opt. [°C]

8 Microbial Leaching of Metals


Agent

Mucor racemosus heterotrophic citrate, 27 MÜLLER and FÖRSTER


succinate (1964)
Paecilomyces variotii heterotrophic citrate, DAVE et al. (1981)
oxalate
Penicillium sp. heterotrophic 25 GUPTA and EHRLICH
(1989)
Penicillium chrysogenum heterotrophic 28 OGURTSOVA et al. (1989)
Penicillium funiculosum heterotrophic citrate BOSECKER (1989)
Penicillium notatum heterotrophic 26 KARAVAIKO et al. (1980)
Penicillium simplicissimum heterotrophic citrate, 22–30 TARASOVA et al. (1993),
oxalate, SILVERMAN and MUNOZ
gluconate (1971)
Rhizopus japonicus heterotrophic OGURTSOVA et al. (1989)
Trichoderma lignorum heterotrophic 24–26 AVAKYAN et al. (1981)
Trichoderma viride heterotrophic 32 BOROVEC (1990)

Yeasts Candida lipolytica heterotrophic 30 GROUDEV (1987)


Rhodotorula sp. heterotrophic EHRLICH (1963),
CERDÁ et al. (1993)
Saccharomyces cerevisiae heterotrophic 28 OGURTSOVA et al. (1989)
Torulopsis sp. heterotrophic CERDÁ et al. (1993)
Trichosporon heterotrophic EHRLICH (1963)

Algae not identified GROUDEV and


GROUDEVA (1993)

Protozoa not identified GROUDEV and


GROUDEVA (1993)

Amoebae not identified EHRLICH (1963)

S-ar putea să vă placă și