Table Of ContentF O R E W O R D
This two volume book presents the proceedings of the 31 ht International Symposium
on Biohydrometallurgy held in San Lorenzo de 1E Escorial, Madrid, Spain, on June 20-23,
1999. The International Symposium on Biohydrometallurgy has become the forum where basic
scientists and industrial experts join to interchange not only experimental results but also ideas
about future developments in this interdisciplinary field.
This Symposium is quite different from the previous ones, held in Chile (IBS-95) and in
Australia (IBS-97), where industrial applications had a prominent role. The active practice of
Biohydrometallurgy in both countries justified their selection. In our case we decided to stress
the importance of basic knowledge as the motor of future developments in the field.
Taking into consideration the importance that environmental issues already have in our
society as well as their exponential increase in the coming century, the IBS-99 Organizing
Committee decided to focus the meeting on the environmental aspects of biohydrometallurgy.
The theme of the Symposium is '~iohydrometallurgy and the Environment - toward the
mining of the 12 a century" because we feel that this is the area in which biotechnology will
make its greatest contribution in the next century. The amount of papers from all over the
world, 167 in all, covering most aspects of biohydrometallurgy, is the best way to prove that
the field is ready to face the technological challenges of the next century.
All papers included in these books were previously reviewed by a group of experts
selected from both the International Scientific Committee members and prestigious researchers
in the Biohydrometallurgy field. Therefore, all the papers published have the appropriate
requirements of scientific quality.
The Symposium was organized around four main topics:
- bioleaching
- biosorption
bioremediation, and
-
- biology (micro and molecular)
with invited plenary lectures to review the state of the art and motivate discussion of future
trends in the different areas of biohydrometallurgy, followed by a limited number of oral
presentations selected by the Scientific Committee according to originality, scientific merit and
topic. The rest of the communications were given in well-structured poster sessions organized
according to the topics of the conference, followed by a general discussion of the posters'
contents. Round tables were organized to discuss two important issues in biohydrometaUurgy :
'~Direct versus indirect mechanism in bioleaching" and '~iohydrometallurgy and the
Environment". Preliminary notes of the round table participants have been included in this
iv
proceedings to facilitate its discussion and as a reference for the specialists that could not
attend the meeting.
llA the papers selected for the Symposium have been included in these two volumes:
Part A, contains papers dealing with basic and industrial scale bioleaching of base and precious
metals, followed by contributions addressing the microbiological aspects of the process.
Part B, encompasses papers on the molecular biology of microorganisms responsible
for the different biohydrometallurgical processes, followed by contributions dealing with
environmental aspects of this biotechnology: biosorption and bioremediation.
The editors wish to thank the different Sponsors of the Symposium for their generous
contribution which helped to put the meeting together, especially the Universidad
Complutense de Madrid and the Universidad Aut6noma de Madrid. The editors also wish to
express their appreciation to their colleagues on the International Scientific Committee and
other members of the biohydrometallurgy community for their prompt assistance with the peer
review of the papers and for the selection of the oral presentations, which has not been an easy
task. We also thank our colleagues on the Organizing Committee: Felisa Gonzalez, M. Luisa
Blazquez, Irma Matin and Jesfis .A Mufioz for their valuable assistance and support. Finally,
we want to thank lla the participants to the Symposium and those who contributed with their
work to this book, for without them it would not exist.
Antonio Ballester and Ricardo Amils
Past, present and future of biohydrometallurgy
Henry L. Ehrlich
Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180-3590 USA
The history of bioleaching and its microbiololgical basis are summarized. A possible future
developmental direction is indicated.
.1 PAST
When in 1676, Antonie van Leeuwenhoek first described what has been interpreted to have
been bacteria in a peppercorn infusion, which he examined with his ingeniously fashioned
simple microscope (1), tittle did he or those to whom he revealed his discovery suspect that
other tiny creatures not unlike the ones he saw are able to extract metals from ore. Indeed,
bacteria were not generally recognized as a unique group of organisms until Ferdinand Cohn
did in 1875, who classified them with plants (2,3), and it was not until the 1960's that they
were assigned to a special group of organisms that were distinct from plants and animals, the
prokaryotes. This was based on their unique cell organization (4). What is more, it was not
until 1977 that Carl Woese first recognized that the prokaryotic bacteria could be devided into
two distinct phylogenetic groups, the eubacteria (now bacteria) and the archaebacteria (now
archeota) (5). Both of these groups include members of special importance to
biohydrometallurgy.
In this historical context, it is noteworthy that bioleaching of copper from ores was practiced
many centuries before the discovery of bacteria. It was a purely empirical process then and not
recognized to have a biological connection. It appears to have been performed in China at
least 100-200 years BCE and in Europe at least as far back as the second century CE (6).
According to Hallberg and Rickard, more than 2 million tons of copper have been leached
from the copper deposit of the Falun Mine in central Sweden since 1687 (7). Commercial
copper leaching from partially roasted ore at the Rio Tinto mine in Spain was first recorded in
1752 (6), but to what extent bacteria were involved in this process remains unclear. The
dissolved copper in all these cases was precipitated from pregnant solution on contact with
metallic iron, a process first described by the Chinese (6).
2. PRESENT
First reports demonstrating the involvement of bacteria in copper leaching date back to the
1950's, following reports on the role of bacteria in the formation of acid mine drainage from
pyrite inclusions in bituminous coal deposits (8-10). L.C. Bryner and J.V. Beck and their
students at Brigham Young University in Provo Utah found the same bacteria, Thiobacillus
ferrooxidans dna Thiobacillus thiooxidans, ni copper mine drainage from Kennecott's open-
pit mine in Bingham Canyon, Utah, that had been found in acid coal mine drainage. They
showed in laboratory experiments that .T ferrooxidans was capable of leaching various copper
sulfide minerals as well as molybdenite, but the latter only in the presence of pyrite (11-13).
Demonstration of bioleaching of some other metal sulfides like ZnS, NiS, and PbS soon
followed (14-16).
The chief process in bioleaching of sulfidic ores is the mobilization of metal constituent(s).
This is accomplished through microbially promoted oxidation of the metal sulfide(s).
Silverman and Ehrlich (17) distinguished between two modes of bacterial attack. In one of
these modes (indirect attack), the chief function of .T ferrooxidans, which was the only
organism capable of promoting leaching that was recognized at the time, was to regenerate the
oxidant, ferric ion, in the bulk phase from ferrous ion,
2Fe +2 + 02 + 1-12 + ==> 2Fe +3 + H20 (1)
The ferrous ion resulted from the chemical oxidation of the metal sulfide in the ore by ferric
ion,
2Fe +3 + MS ==> 2Fe +2 + M +2 + S ~ (2)
MS in equation (2) represents a metal sulfide, and M +z the divalent metal ion formed in the
oxidation of MS. In addition to oxidizing Fe ,+2 .T ferrooxidans and/or .T thiooxidans, which
si also detected in bioleach processes, were visualized as oxidizing the S ~ formed in the
chemical oxidation in reaction (2), to 4OS2H (17,18),
(3)
S ~ + 1.502 + HzO ==> H2804
In the other mode of bacterial attack of metal sulfide (direct attack), the bacteria, according to
Silverman and Ehrlich (17) attacked a metal sulfide by attaching to the mineral surface and
oxidizing it enzymatically by conveying electrons from the reduced moiety of the mineral,
usually the sulfide but in the case of CuzS also from the cuprous copper, to 02,
MS + 20 +2H + ==> M +2 + H20 (4)
Clear evidence of the ability of .T ferrooxidans to attach readily to the surface of metal
sulfides was developed subsequently (19-28). In direct attack, electron transfer from sulfide-S,
or from cuprous copper in the case of CuzS, involves Fe(III) bound in the cell envelope and
exopolymer (29-31). This bound Fe acts as an electron shuttle between the electron donor and
the electron transport system of the cell, which conveys a major portion of the electrons to 02
and the rest to 2OC see (32) for more detail. Thus, the Fe(III) bound in the cell envelope
and exopolymer is thought to undergo reversible reduction and oxidation in this electron
transfer. The sites on a metal sulfide particle for bacterial attachment and attack seem to be
limited. Thus, once maximum attachment has been achieved, further multiplication of attached
cells, if it occurs, should result in the displacement into the bulk phase of one of the two
daughter cells of each dividing bacterium.
This model of direct attack is in contrast to the model for indirect attack of metal sulfides in
which Fe +3 in the bulk phase, produced by unattached bacteria from dissolved Fe ,+2 is the
electron acceptor. If these two models for biooxidation of metal sulfides describe the process
correctly, the iron requirement for an optimal rate of metal sulfide oxidation by the direct mode
of attack should be significantly smaller than for the indirect mode. Differences in reaction
kinetics between exclusively direct and indirect modes of attack are also likely.
Sand et al. have recently suggested that because Fe(III) oxidizes metal sulfide in both the
direct and indirect mechanisms, there is no difference between the two mechanisms (33). Their
model emphasizes a similarity in the chemistry of attack of the sulfide moiety by iron, and
makes no distiction between ferric iron in the bulk phase and ferric iron bound in the cell
envelope.
Although initial studies of bioleaching suggested that .T ferrooxidans was the only actor in
bioleaching of metal sulfides, subsequent studies showed that other, phylogenetically unrelated
organisms could also be active. These include not only autotrophs but also heterotrophs (34)
and not only mesophiles but also thermophiles, all of them acidophilic and all of them Fe(II)
oxidizers (34,35). Indeed, in many cases Leptospirillum ferrooxidans, which cannot oxidize
reduced forms of sulfur, seems to dominate the metal-sulfide oxidizing microbial flora (36,37).
Further study of the microbes in pregnant solution from bioleaching operations showed that
the acidophilic iron oxidizers were accompanied by many other kinds of organisms, including
heterotrophic bacteria, fungi, and protozoa (38-40). Indeed, heap, dump, and in-situ leaching
by native microbial flora in the field is probably the result of a consortium of acidophilic
microorganisms including autotrophic and heterotrophic bacteria, fungi, and even protozoa.
The autotrophs can be assumed to be the chief promoters of the actual metal leaching process,
whereas the most important role of the heterotrophs can be assumed to be to limit the
concentration of organics that might otherwise inhibit the autotrophs (41-45).
Some of the heterotrophs can also promote formation of floe, as in the case of .L
Ferrooxidans (37). Protozoans, in addition to aiding in the removal of dissolved organics, may
control the size of the microbial population by preying on it (38). Both autotrophs and
heterotrophs contribute to the weathering of the host rock (gangue) to expose ore mineral that
is encapsulated in the gangue (43). Indeed, sufficient 1A could be mobilized from
aluminosilicates to make its separation desirable (43). The weathering action is due in part to
the sulfiwic acid generated by the autotrophs in attacking pyrite and chalcopyrite minerals,
which causes rupture of Si-O and AI-O bonds in aluminosilicates. Wheathering may also be
promoted by some of the less acidophilic heterotrophs that generate organic acids and/or
ligands that sequester Ca and Mg from the crystal lattice of aluminosilicates in the early stages
of a leaching operation.
With the demonstration of bacterial involvement in bioleaching, four distinct approaches
have been taken in its commercial exploitation. These are heap, dump, in-situ, and reactor
leaching. S.R. Zimmerley, D.G. Wilson and J.D. Prater were issued the first patent on heap
bioleaching on 24 October 1955 (43). They assigned it to Kennecott Copper Corporation.
This patent described a cyclic process of heap leaching of copper-, zinc-, copper-molybdenum-
, chromite- and titanium-ores. The last three ores were meant to be upgraded (beneficiated) by
the process, i.e., the ore was enriched in metal value instead of the metal value being extracted.
Cu recovery from pregnant solution described in this patent was by cementation with metallic
iron.
The process of heap bioleaching has gone through various improvements over the years, as
will be discussed by Brierley and Brierley in a subsequent chapter (44). Change in the design of
heaps to prevent slumping and optimization of aeration has been a major factor in this
improvement.
Much effort has been expended to design a commercially viable process for bioleaching of
ore concentrate in reactors. Progress has been gradual, with the chief stumbling block having
been slow leaching rates. But as Brierley and Brierley will discuss (44), a breakthrough has
now been achieved, making ore-concentrate bioleaching commercially feasible in certain
instances. Advances in reactor design and, in at least one instance, the use of a moderately
thermophilic acidophile as an agent of leaching (45) have been at the heart of this
breakthrough. A rationale for turning to moderate thermophiles si a more limited cooling
requirement for reactors. Ore concentrate leaching with hyperthermophiles in reactor leaching
has been tried because of observations that leaching rates with such strains were higher than
with mesophiles at ambient temperatures (35). However, more recent studies have shown that
acidophilic hyperthermophiles tested in reactors have much more limited tolerance for high
pulp density than moderate thermophiles or mesophiles (46). The observed accelerating effect
at elevated temperature was probably mostly on indirect leaching. Although commercial
leaching was initially restricted to copper ores, reactor- based processes have recently been
developed for the extraction of other metals such as Co, Ni and Zn (47-50).
Ehrlich reported in 1964 that .T ferrooxidans was capable of oxidizing arsenopyrite (51). In
his study, he measured mob~ation of Fe and As. He did not follow sulfide. The mobilized
iron appeared as Fe(II) and Fe(III). The mobilized arsenic appeared as arsenite As(III) and
as arsenate As(V). Some of the arsenite and arsenate were precipitated by iron. The iron
arsenate compound was later shown to be scorodite FeAsO4.2H20 (52). Although the
possibility suggested itself that the arsenate resulted from oxidation of arsenite by .T
ferrooxidans, this could not be confirmed by direct testing. However, the thermophilic
archeon, Sulfolobus acidocaldarius strain BC, si capable of such oxidation (53). Current
evidence indicates that the arsenate formed in the presence of .T ferrooxidans si the result of
chemical oxidation of arsenite by the bacterially generated Fe(III) (54). Transient formation of
S ~ was also observed in this recent study (54). The ability of .T ferrooxidans to oxidize
arsenopyrite led the late Eric Livsey-Goldblatt in 1983 to propose its use in biobeneficiating
pyritic gold ores in a bioleaching process that he estimated to be significantly more economical
than pyrometallurgical treatment (55), and this has turned out to be the case. In pyritic gold
ores, pyrite and arsenopyrite encapsulate the gold, making it inaccessible to lixiviants such as
cyanide or thiourea. Partial oxidation of the pyrite and arsenopyrite uncover the gold
sufficiently for extraction, and at the same time lessen the non-specific, irreversible
consumption of cyanide during subsequent extraction of the ore.
3. FUTURE
In my opinion, some future fundamental research effort in biohydrometallurgy should be
directed toward leaching and/or beneficiation of oxide, silicate and carbonate ores. Sporadic
research in this area has been done in the past, but it has not led to industrial applications so far
(56-61). Although the metal in some of these ores can be mobilized by sulfuric acid generated
from sulfur by the autotroph .T Thiooxidans (56), organic acids and ligands such as 2-
ketogluconate generated by some heterotrophic bacteria (57), and oxalate and citrate
generated by fungi can also be useful (57,60). In the case of metal oxide ores, anaerobic
bacterial processes in which bacteria reduce the metal oxide and thereby solubilize it may be
the most promising for industrial exploitation (62). In such processes, the bacteria use the
metal oxide as terminal electron acceptor. The electron donor may be organic carbon,
formate, or ,EH depending on the organism (for a review, see (63)). An example of a reaction
in which MnO2 is bacterially reduced to Mn +2 with acetate is the following,
4MnO2 + CH3COO- + 7H + ==> 4Mn +2 + 2HCO3- + 021-14 (5)
For other examples, see Ehrlich (64).
Since ores are not sterile and cannot be sterilized on a commercial scale, heterotrophic
leaching presents some process design challenges that autotrophic leaching does not. Aerobic
heterotrophic leaching based on the action of microbially produced acidulants and/or ligands,
would best be operated in a two-reactor system. In such a system, the first reactor would be
the generator in which desired microbes would produce the acidulant/ligand in pure culture
trader optimal growth conditions, preferably in a continuous mode. The spent culture solution
from this reactor would be bled into a second reactor containing the ore to be leached.
Growth of microbes that might destroy the acidulant/ligand in the second reactor could be
controlled by ensuring a very low level of residual nitrogen source in the spent culture medium
and by temperature manipulation.
In anaerobic heterotrophic leaching of metal oxides by a reductive process, the maintenance
of selective growth conditions is extremely important, just as it is in aerobic autotrophic
leaching of metal sulfides. In bioleaching of metal sulfides, the selective growth conditions are
high acidity and the absence of a major organic carbon and energy source, which are readily
established and maintained. In heterotrophic leaching of metal oxides, circumneutral to
moderately acid pH and an adequate supply of a carbon/energy source is essential. However,
by themselves, these conditions are not sufficiently selective, but when combined with
anaerobiosis and use of a very specialized carbon/energy source, the resultant conditions can
be highly selective. In the ideal case, anaerobiosis excludes potentially interfering obligate
aerobes, and the specialized carbon/energy source utilizable only by the leaching organism(s)
prevents overgrowth by anaerobic heterotrophs incapable of leaching a mineral oxide. Phenol
is an example of a specialized carbon/energy source that is toxic to many microorganisms but
can be used as carbon and energy source by some iron oxide- and MnOE-reducers (65).
Acetate is another specialized carbon/energy source. It is non-fermentable except by
acetoclastic methanogens, and it is inadequate as a sole source of carbon for most anaerobes.
But it can be used as a carbon/energy source by some reducers of iron oxide, MnO2, and +22OU
(65,66).
Thus in designing a heterotrophic leaching process, important considerations are selective
conditions in a one-reactor system, or axenic conditions in the first reactor of a two-reactor
system. In choosing a carbon/energy source for commercial heterotrophic leaching, cost
becomes another important consideration. If it has to be sugar, industrial molasses, whether a
byproduct of cane-sugar, beet sugar, or corn-starch hydrolyzate processing, would be a prime
candidate, but it is not very selective. If it is to be an aromatic electron donor, industrial
phenolic waste streams from chemical industry might be worth considering. If it is to be
acetate, production in a reactor with an acetogen like Clostridium thermoaceticum growing on
sugar (glucose or fructose, e.g., invert sugar, or corn starch hydrolyzate, but not sucrose) or
some other feed stocks could take place at the site of the bioleaching plant (67).
4. CONCLUSIONS
From the foregoing brief survey it is apparent that scientifically based biohydrometallurgy
using acidophilic autotrophs has made significant strides in its developent as a commercially
viable technology for the processing of sulfidic ores. The technology for heterotrophic
leaching of metal oxides, carbonates, and silicates on an industrial scale awaits development.
In view of its potential as an environmentally more benign process than pyrometallurgy,
biohydrometallurgy promises to replace many if not all pyrometallurgical ore extraction
methods in the not too distant future.
REFERENCES
.1 Dobell, C. Antony van Leeuwenhoek and his "Little Animals." Dover Publications, New
York, 1960, 109.
2. T. Brock, Milestones in Microbiology. American Society for Microbiology, Washington,
DC, 1961, 210.
3. K.V. Thimann, The Life of Bacteria. 2nd edition. Macmillan, New York, 1963,16.
4. R.Y. Stanier, RY. 1961. Ann. Inst. Past. (Paris) 101 (1961) 297.
.5 C.R. Woese and G.E. Fox. 1977. Proc. Natl. Acad. Sci. USA. 74 (1977) 5088.
6. G. Rossi. Biohydrometallurgy. McGraw-Hill, Hamburg, Germany, 1990, .1
.7 R.O. Hallberg, and D.T. Rickard. De smh "gruvarbetarna" i Falun. S/irtryck ur Svensk
Naturvetenskap 1973. Swedish Natural Science Research Council, Stockholm, 1973, 102.
.8 A.R. Colmer, K.L. Temple, and H.E. Hinkle. .J Bacteriol. 59 (1950) 317.
.9 K.L. Temple and A.R. Colmer J. Bacteriol. 62 (1951) 605.
.01 W.W. Leathen, S.A. Braley Sr., and L.D. Mclntyre. Appl. Microbiol. 1 (1953) 65.
.11 L.C. Bryner and R. Anderson. 1957. Ind. Eng. Chem. 49 (1957) 1721.
.21 L.C. Bryner and A.K. Jameson 1958. Appl. Microbiol. 6 (1958) 281.
.31 L.C. Bryner, J.V Beck, B.B. Davis and D.G. Wilson., Ind. Eng. Chem. 46 (1954) 2587.
.41 E.E.Malouf and J.D. Prater., .J Metals. 31 (1961) 353.
.51 W.E. Razzell and P.C. Trussell., Appl. Microbiol. 11 (1963) 105.
.61 M. Silver and A.E. Torma. Can. .J Microbiol., 20 (1974) 141.
.71 M.P. Silverman and H.L. Ehrlich. Adv. Appl. Microbiol. 6 (1964) 153.
.81 H.L. Ehrlich and S.I. Fox. Biotechnol. Bioeng. 9 (1967) 471.
.91 C.J.M. McGoran, D.W. Duncan and C.C. Walden. Can. .J Microbiol. 51 (1969) 135.
20. H. Tributsch. Naturwissenschat~en 36 (1976) .88
21. J.C. Bennett and H. Tributsch. J. Bacteriol. 431 (1978) 310.
22. R.M. Bagdigian and A.S. Myerson. Biotechnol. Bioeng. 28 (1986) 467.
23. C.-Y. Chen and D.R. Skidmore. Biotech. Lett. 9 (1987) 191.
24. C.-Y. Chen and D.R. Skidmore. Biotechnol. Progr. 4 (1988) 25.
25. M. Rodriguez-Leiva and H. Tributsch. Arch. Microbiol. 941 (1988) 401.
25a. P.F. Norman and C.P. Snyman. Geomicrobiol. .J 6 (1988) .1
26. C.C. Pogliani, Curutchet, Donati E, Tedesco PH. Biotechnol. Lett. 21 (1990) 515.
27. .C Mustin, J. Berthelin, P. Marion and P. de Donato. Appl. Environ. Microbiol. 58 (1992)
.5711
28. K.S.N. Murthy and K.A. Natarajan. Minerals and Metallurgical Processing, Feb. (1992)
20.
29. W.J. Ingledew and J.G. Cobley. Biochim. Biophys. Acta 590 (1980) 141.
30. W. Sand, T. Gehrke, P.-G. Jozsa and A. Schippers. Novel mechanism for bioleaching of
metal sulfides. Biotechnology Comes of Age. International Biohydrometallurgy
Symposium IBS97 BIOMINE97. Australian Mineral Foundation, Glenside, SA, 1997,
.1.2PQ
31. T. Gehrke , R. Hallmann and W. Sand. Importance of exopolymers from sullicaboihT
ferrooxidans dna Leptospirillum ferrooxidans for bioleaching. Vargas T, Jerez CA.
Wiertz KV. Toledo H (eds.). Biohydrometallurgical Processsing, Vol. .1 University of
Chile, Santiago, 1995, .1
32. H. L. Ehrlich, W. J. Ingledew and J. .C Salerno. Iron- and manganese-oxidizing bacteria.
.J M. Shively and L.L. Barton (eds.). Variations in Autrophic Life. Academic Press,
London, 1991, 147.
01
.33 .W Sand, .T Gerke, .R Hallmann and .A Schippers. Appl. Microbiol. Biotechnol. 34
(1995) 961.
.43 D.B. Johnson, .P Bacelar-Nicolau , D.F. Bruhn and F.F. Roberto. Iron oxidizing
heterotrophic acidophiles: ubiquitous novel bacteria ni leaching environments. .T Vargas,
C.A. Jerez, K.V. Wiertz and .H Toledo (eds.). Biohydrometallurgical Processing, Vol. .1
University of ,elihC Santiago, 1995, .74
.53 .P .R Norris. Acidophilic bacteria and their activity ni mineral sulfide oxidation. H.L.
Ehrlich aand C.L. Brierley (eds.). Microbial Mineral Recovery. McGraw-Hill, New York,
(1990), .3
.63 .W Sand, .K RoMe, .B Sobotke and .C Zenneck. Appl. Environ. Microbiol. 85 (1992) .58
.73 .W Sand, .T Gehrke, .R Hallmann, .K Rohde, B. Sobotke and .S Wentzien. In-situ
gnihcaeloib of metal :sediflus ehT importance of mulliripsotpeL ferrooxidans. Torma AE,
Wey JE, Lakshmanan .LV Biohydrometallurgical Technologies. Vol. .1 The Minerals,
Metals & Materials Society, Warrendale, PA, 1993, .51
.83 H.L. Ehrlich. .J Bacteriol. 68 (1963) 350.
.93 A.I. L6pez-Archilla, .I Marin and .R Amils. Microbial ecology of an acidic river:
Biotechnological applications. .T Vargas, C.A. Jerez, K.V. Wiertz and H. Toledo
Biohydrometallurgical Processing, Vol. ,2 1995, .36
40. D.B. Johnson and F.F. Roberto. Heterotrophic acidophiles and their roles ni bioleaching of
sulfide minerals. D.E. Rawlings (ed.). Biomining. Theory Microbes and Industrial
Processes. Springer-Verlag, Berlin, 1997, 259.
.14 J.H. Tuttle dna P.R. Dugan. Can. .J Microbiol. 22 (1976) 719.
42. G.J.M.W. Arkesteyn dna J.A.M. De Bont. Can. .J Microbiol. 62 (1980) .7501
.34 S.R. Zimmerley, D.G. Wilson, and J.D. Prater. Cyclic leaching process employing iron
gnizidixo bacteria. U.S. Patent No. 2,829,964 (1958).
.44 J.A. Brierley and C.L. Brierley. Present and future commercial applications of
biohydrometallurgy. .A Ballester and .R Amils (eds.). International Biohydrometallugy
Symposium IBS '99. Elsevier, Amsterdam, .9991
.54 P.C. Miller. The Design and olperating practice of bacterial oxidation plant using
moderate thermophiles (The BacTech Process). D.E. Rawlings (ed.). Biomining. Theory
Microbes and Industrial Processes. Springer-Verlag, Berlin, 1997, .18
.64 P.R. Norris. Thermophiles and bioleaching. D.E. Rawlings (ed.). Biomining. Theory
Microbes and Industrial Processes. Springer-Verlag, Berlin, 1997, 247.
.74 .A Briggs dna .M Millard. Cobalt recovery using bacterial leaching at the Kasese project,
Uganda. Biotechnology Comes of Age. International Biohydrometallurgy Symposium
IBS97 BIOMINE97. Australian Mineral Foundation, Glenside, ,AS 1997, M.2.4.1.
.84 .D Dew and D. Miller. The BioNIC process: Bioleaching of mineral sulfide concentrates
for recovery of nickel. Biotechnology Comes of Age. International Biohydrometallurgy
Symposium IBS97 BIOMINE97. Australian Mineral Foundation, Glenside, ,AS 1997,
M7.1.1.
.94 M.L. Steemson, F.S. Wong, and .B Gobel. The intergration of zinc bioleaching with
solvent extaction for the productio of zinc metal from zinc concentrates. Biotechnology
