- Email: [email protected]
Bioleaching capacity of an extremely thermophilic culture for chalcopyritic materials
Minerals Engineering 15 (2002) 689–694 This article is also available online at: www.elsevier.com/locate/mineng
Bioleaching capacity of an extremely thermophilic culture for chalcopyritic materials A. Rubio a, F.J. Garcıa Frutos
b,*
a
b
Instituto Geol ogico y Minero de Espa~ na (IGME), La Calera n° 1, 28760 Tres Cantos, Madrid, Spain Centro de Investigaciones Energ eticas, Medioambientales y Tecnol ogicas (CIEMAT), Av. Complutense 22, Edificio 20, 28040 Madrid, Spain Received 9 March 2001; accepted 2 May 2002
Abstract A thermophilic culture specific to bioleaching chalcopyritic materials has been obtained from a typical chalcopyritic copper concentrate of the Spanish Pyritic Belt. This paper shows the effect of pulp density (w/v) on bioleaching culture capacity with respect to this copper concentrate. The results of the batch tests show that it is possible, operating at 10% of pulp density to attain copper extraction of 94% in 10 days and, at higher pulp densities (20%), to attain good copper extraction (80%) in only 14 days. In the same way, the culture has been amply tested with different chalcopyritic ores and copper concentrates. The results obtained with four of these materials, two refractory gold ores and two copper concentrates are also presented. These results show a varying and versatile bioleaching capacity of this bacterial culture. A model of bacterial attack of this culture to leach chalcopyrite is postulated. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Mineral processing; Sulphide ores; Hydrometallurgy; Bioleaching and bacteria
1. Introduction The resources of high ores in the world are becoming more and more scarce making the processing of more complex ores necessary. Conventional mineral processing on complex sulphide ores carried out by differential flotation often produces high grade concentrates but with contaminants that penalise and make its marketing and pyrometallurgical processing difficult. Therefore, a lot of effort has been made in the development of hydrometallurgical process suitable for the treatment of these ores, but most of the proposed methods are complex and expensive, Alvarez (1996). Among these hydrometallurgical processes, biohydrometallurgical techniques appear to be an alternative for the treatment of these concentrates. These methods, that were first applied industrially to copper and uranium productions using bioassisted heap, dump and in situ technologies, are successfully used today in extraction of gold from refractory sulphide-bearing ores and concentrates, Jordan et al. (1996). However, for other * Corresponding author. Tel.: +34-91-3466232; Fax: +34-913466269. E-mail address: [email protected] (F.J. Garcıa Frutos).
metal concentrates, this technology remains as a promising alternative against conventional pyrometallurgical extraction processes. This is the case of the treatment of chalcopyritic concentrates, which represent a more complicated situation, due to the natural refractivity of chalcopyrite. One of the mainly problems of the bioleaching processes applied to copper concentrates and refractory ores are the low kinetics of the reactions, with high residence time that does not permit it to be an economic process. For this reason, current researches into this field are focused on how to increase this bioleaching rate. Apart from improving engineering design of bioreactors, the possibilities to increase bioleaching rates depend on the use of catalyst and isolation and adaptation of new microorganisms with high capacity to leach these ores. Several studies with mesophilic microorganisms as Thiobacillus ferrooxidans and Leptospirillum ferrooxidans had showed very slow copper leaching rates, Mehta (1982) and Sand et al. (1992). However, when thermophilic microorganisms were used leaching rates were considerably increased due to high temperatures, higher metal tolerance capacity and metabolic characteristics of this type of microorganisms, Brierley (1993) and Clark and Norris (1996).
0892-6875/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 8 9 2 - 6 8 7 5 ( 0 2 ) 0 0 1 2 4 - 3
690
A. Rubio, F.J. Garcıa Frutos / Minerals Engineering 15 (2002) 689–694
Many researches have investigated the possibility of using thermophilic microorganisms to improve metal leaching rates instead to mesophilic microorganisms. Future process developments must and will include thermophilic bacteria, which will play an increasingly important role in biooxidations of minerals. Thermophilic Archaea, Sulfolobus species, Acidianus brierleyi and Metallosphaera sedula which grow from 60 to 75 °C, are particularly adept in bioleaching of copper from the highly refractory chalcopyrite, Brierley and Brierley (1999) and Dew et al. (1999). Moreover, the use of these microorganisms that naturally thrive on ore samples and its aqueous environments, could be a good option as they probably have specificity for leaching chalcopyrite and a much higher capacity for adaptation. In this sense, there is a work carried out by Lopez-Archilla et al. (2001), on the microbial ecology of Rio Tinto waters, where they report to have isolated a thermophilic archaeabacteria, Sulfolobus rivotincti, with specificity to bioleach chalcopyrite and therefore with possibilities to be used in chalcopyritic concentrates treatment, Ballester (1996) and G omez et al. (1999). The use of these microorganisms for bioleaching chalcopyrite implies the knowledge of the mechanisms involved in the process. Initially, chalcopyrite can be oxidised by dissolved oxygen according to Eq. (1), Hiroyosi et al. (1997), favoured for acidic solution, and as it is known, two modes of bacterial attack can be distinguished: In indirect attack (Eq. (2)), the role of bacteria is to regenerate the oxidant ferric ion in the bulk phase, from the ferrous iron resulting from the chemical oxidation of the metal sulphide in the ore by ferric iron (Eq. (3)). In the direct attack, the bacteria leach the metal sulphide by attaching to the mineral surface and oxidising it enzimatically by conveying electrons from the reduced moiety of the mineral, Erlich (1999). CuFeS2 þ O2 þ 4Hþ ¼ Cu2þ þ Fe2þ þ 2S0 þ 2H2 O ð1Þ CuFeS2 þ 4Fe 4Fe
2þ
þ
3þ
¼ Cu
2þ
þ 5Fe
þ 4H þ O2 ¼ 4Fe
3þ
2þ
þ 2S
þ 2H2 O
0
ð2Þ ð3Þ
Sand (1997) have suggested that because Fe3þ oxidises metal sulphide with both the direct and indirect mechanisms, there is no difference between the two mechanisms. Their model emphasises a similarity in the chemistry of attack of the sulphide moiety by iron, and makes no distinction between ferric iron in the bulk phase and ferric iron bound in the cell envelope. The authors have obtained a mixed natural thermophilic culture from a typical chalcopyiritic copper concentrate of the Spanish Pyritic Belt, Rubio (1998). This culture has selectivity with respect to leaching chalcopyrite, when this is present together with other sulphides in ores and concentrates.
This paper describes the bioleaching capacity of the culture to operate at high pulp density and its versatility for leaching chalcopyritic materials, from ores to concentrates, postulating an ‘‘in situ’’ indirect bacterial attack.
2. Methods 2.1. Microbial inoculum A mixed thermophilic culture (MTC) of native microorganisms, isolated on a typical chalcopyritic concentrate described below (Concentrate 0), was used as inoculum in the initial bioleaching experiments. The methodology of the isolation was the following: The concentrate was strong stirring (360 rpm) at pH higher than 4 and 65 °C of temperature during 2 h for remove the microorganisms attached on the chalcopyrite surface. Once the microorganisms were unattached and free in solution, the solid was removed of the liquid by filtration. The bacteria-laden liquid obtained was filtered through a 0.22 lm Millipore membrane filter to collect the microorganisms, which were suspended in 1:10 9 K medium at pH 1.30. Preliminary comparative bioleaching tests were performed using a bacterial inoculum of Sulfolobus sp. kindly supplied by Dr. F. Carranza, Universidad de Sevilla, Spain. Both thermophilic cultures were maintained on copper concentrate (Concentrate 0) at 1% w/v pulp density medium with 1:10 salts of 9 K medium at 65 °C of temperature and initial pH of 1.30. The cultures were successfully adapted to higher pulp density of copper concentrate until obtaining high copper extractions in short residence times. In these tests, the inoculum was obtained as the culture isolation was made. The final pulp of the bioleching test was filtered, obtaining the leach liquor and the solid residue. The solid was intensively stirring during 2 h with water at pH 4. The pulp obtained was filtered again and the final liquid obtained, which contains most the bacteria, which were attached to the solid residue, was filtered through a 0.22 lm Millipore filter where bacteria were retained. Finally, the bacteria were re-suspended in 50 ml of the leach liquor obtained in the first filtration in order to get the inoculum volume (5% v/v) for the next bioleaching test. 2.2. Ore samples A chalcopyritic copper concentrate obtained from conventional differential flotation was used to develop the mixed thermophilic culture. This concentrate (Concentrate 0) came from the Spanish Pyritic Belt and its
A. Rubio, F.J. Garcıa Frutos / Minerals Engineering 15 (2002) 689–694 Table 1 Chemical composition of copper concentrate Sample
Chemical analysis (wt%) Cu
Zn
Pb
Fe
S
Concentrate 0
23.37
2.58
2.52
32.63
38.00
composition is given in Table 1. The mineralogical composition of this sample shows chalcopyrite and pyrite as main mineralogical species and galena and sphalerite as secondary mineralogical phases. The particle size distribution presents a passing d80 of 20 lm. Concentrate 1, also used in bioleaching tests is essentially the same as Concentrate 0, corresponding to another lot of copper concentrate obtained in the same flotation point at the industrial plant. This sample presents less copper content and higher zinc content than Concentrate 0. The other chalcopyritic samples, refractory gold ores (Ores 1 and 2) and copper concentrates (Concentrate 1 and 2), also came from the Spanish Pyritic Belt, were selected in this work to study the selectivity of the MTC to chalcopyrite. The chemical composition of these chalcopyritic samples is given in Table 2. The copper present in these samples is as chalcopyrite.
691
ished, solids were removed by filtration, and chemically characterised as well as the leachate. Sterile bioleaching tests were carried out with ore sterilised by autoclaving at 121 °C, 30 min and 1atm of pressure, and adding a solution of 10% ethanol to the leaching media. 2.4. Analysis Soluble species of copper, zinc, lead, total iron and minor elements were analysed by ICP, Del Barrio (1992), using a spectrophotometer ICAP-61 Thermo Jarrel Ash. The ferrous iron was analysed by a volumetric method by titration with potassium dichromate, Kolthoff (1979). Copper, zinc, lead and iron content in the ore samples and leaching solid residues were analysed by XRF, Martın Rubı (1998), using a spectrophotometer Philips PW-1404, and minor elements by ICP. Total sulphur was gravimetrically determined and elemental sulphur was analysed by toluene extraction in a Soxhlet apparatus. The pH was measured with a 704 pH-meter Metrohm. The redox potential (Eh) was measured with a platinum electrode with an Ag/AgCl reference electrode. Mineralogical composition was determined by XRD using a difractometer PW-1700 Philips.
2.3. Bioleaching experiments Bioleaching experiments were carried out in 1 l glass cylindrical reactors provided with a cap with four holes to allow mechanical stirring (at 130 rpm), aeration (10– 15 l/h) and sampling. These reactors were placed in a thermostatic bath to keep the temperature constant at 65 °C. During the experiments the pH was kept at 1.30 by the addition of 10N H2 SO4 when were necessary. This 10 N H2 SO4 addition was made to avoid the precipitation of iron in form of jarosites, which damage the bioleaching process. Redox potential and pH were measured daily, while the levels of copper, zinc, and iron in solution were analysed daily or every 2 days, depending on the test. Water was added to the reactors in order to compensate for evaporation losses. Once bioleaching tests were fin-
Table 2 Chemical composition of chalcopyritic samples Sample
Chemical analysis (wt%) Cu
Zn
Pb
Fe
S
Ore 1 Ore 2 Concentrate 1 Concentrate 2
0.69 1.47 20.76 6.70
1.64 1.1 4.65 <0.10
0.71 0.53 2.87 <0.10
39.55 42.15 30.11 24.20
33.35 45.6 37.29 n.a.
n.a: no appear.
3. Results and discussion 3.1. Influence of pulp density A study of bioleaching capacity on copper concentrates by MTC culture was done at different pulp densities such as 1%, 5%, 10%, 15% and 20% (w/v). Preliminary tests carried out at 1% pulp density (w/v) show a higher capacity of MTC culture to bioleach the copper present in the copper concentrate (Concentrate 0) compared to the tests carried out with Sulfolobus sp. culture (Sb) on the same copper concentrate. Both cultures had been maintained and adapted to this copper concentrate by successive sub-culturing for the realisation of these bioleching tests. Fig. 1 shows copper extractions obtained on copper concentrate with MTC and Sb cultures in representative adaptation tests at 1% of pulp density (w/v). In the sterile test, only 11 % copper extraction was obtained in more than 500 h. In bioleaching tests with Sulfolobus sp. culture (Sb), initially a cooper extraction up 98% was reached in 500 h, but after successive adaptation tests, the kinetic of reaction was increasing, obtaining maximum copper extractions (>98%) in 350 h. However, with MTC culture the same copper extraction (more than 98%) was obtained in only 165 h with only two successive adaptive tests.
692
A. Rubio, F.J. Garcıa Frutos / Minerals Engineering 15 (2002) 689–694
Fig. 1. Copper extraction in bioleaching adaptive tests at 1% (w/v) pulp density. (Sterile test, Sulfolobus sp. and MTC tests at pH 1.30 and 65 °C). Sample: Concentrate 0. (The sub-indexes 1; 2; 3; . . . ; n correspond to consecutive tests.)
Fig. 3. Comparative copper extraction at the different pulp densities studied (1, 5, 10, 15 and 20% (w/v) at pH 1.30 and 65 °C). Sample: Concentrate 0.
Bioleaching tests carried out at 5% pulp density (w/v) are shown in Fig. 2. Similar ability to preferentially leach copper by MTC thermophilic culture was obtained. The extractions achieved with the MTC were much more significant than Sb culture. It can be observed that in the first test on Concentrate 0 with the culture isolated MTC, a copper extraction of about 90% was obtained in only 21 days. In second test with this culture, in 17 days the copper extraction reached 97%, considerably higher than the extraction attained (80%) after 56 days of bioleaching when Sulfolobus sp. culture was used. From these results, a study in order to define the optimum conditions of this bioleaching process was carried out increasing pulp density (1%, 5%, 10%, 15% and 20% (w/v)). This study is described elsewhere, Rubio (1998). The best conditions obtained from 1% to 20% pulp densities vary only in oxygen uptakes. In Fig. 3 appears the evolution of copper extractions to all tests done at pulp densities studied in its optimum conditions. As it can be observed the results of batch tests show that it is possible to work both with high pulp
density (20%) obtaining good copper extraction (80%) and with lower pulp density (10%) obtaining better copper extraction (94%) in 14 and 10 days respectively. In all tests done at different pulp densities, iron in solution at first stages was mainly as ferrous iron and only the presence of ferric iron was more notable when almost 80% of copper is leached. Fig. 4 shows total iron and ferrous iron during a bioleaching test on Concentrate 0 at 10% of pulp density with MTC culture. As it can be observed, mainly form of iron in solution was as ferrous iron. This ferrous iron in solution decreases at the final stages (from the eighthday on) which corresponds to an increase in the solution redox potential over 450 mV and a copper dissolution of 80%. Redox potential evolution is showed in Fig. 5. As can be seen, Eh in first stages of bioleaching is about 400 mV to reach 425 mV in an intermediate step, confirming the most presence of ferrous iron in solution. The more notable redox potential increase was produced from day 7, when ferrous iron starts to decrease in solution reaching a final Eh of 475 mV. Fig. 6 shows zinc extraction in the same bioleaching test. A fast initial leaching of zinc can be appreciated. At
Fig. 2. Copper extraction in bioleaching adaptive tests at 5% (w/v) pulp density. (Sulfolobus sp. (Sb) and MTC tests at pH 1.30 and 65 °C). Sample: Concentrate 0. (The sub-indexes 1; 2; 3; . . . ; n correspond to consecutive tests.)
Fig. 4. Concentration of iron in leach solution to a representative test at 10% (w/v) pulp density, at pH 1.30 and 65 °C. Sample: Concentrate 0.
A. Rubio, F.J. Garcıa Frutos / Minerals Engineering 15 (2002) 689–694
Fig. 5. Redox potential (Eh) evolution to a representative test at 10% (w/v) pulp density, at pH 1.30 and 65 °C. Sample: Concentrate 0.
Fig. 6. Concentration of zinc in leach solution to a representative test at 10% (w/v) pulp density, at pH 1.30 and 65 °C. Sample: Concentrate 0.
intermediate stage of leaching, as there are not sufficient ferric iron in solution, the zinc dissolution is low, and only an increase in zinc extraction was observed when copper was bioleached (after 8 days of test) coinciding with the increase of ferric iron in solution. The zinc present was almost completely leached only when the duration of tests was prolonged longer after all copper had been leached. X-ray diffraction of solid residues showed elemental sulphur and pyrite as main species present, and anglesite and sphalerite as minor species. 3.2. Bioleaching of chalcopyritic samples In Table 3, appear in short, the results obtained with the two refractory gold ores selected. The tests were
Table 3 Results obtained in bioleaching tests with the refractory gold ores Sample Ore 1 Ore 2
Pulp density (% w/v)
Time (days)
Yield (%) Cu
Zn
Fe
10 10
3 4
90.20 89.16
70.59 59.02
11.52 23.99
693
Fig. 7. Copper extraction in bioleaching tests with copper Concentrate 1 and 2 (10% (w/v) pulp density, pH 1.3 and 65 °C).
done at 10% pulp density. The copper present in these ores was quickly leached in 3 and 4 days, and the zinc was only extracted when copper is almost leached and the redox potential was over 475 mV. Fig. 7 shows the copper extraction obtained in bioleaching tests carried out at 10% pulp density with the other copper concentrates used (Concentrate 1 and 2). Concentrate 2 with less initial copper content than Concentrate 1, was more easily bioleached by MTC culture, obtaining more than 90% of copper extraction in 6 days. With Concentrate 1 (similar to Concentrate 0) and without a period of adaptation, more than 70% of copper extraction was obtained in 12 days. In the sterile tests, copper extraction obtained was 14% for Concentrate 1 and 21% for Concentrate 2. From these tests it can be observed that the leaching behaviour of this culture with respect to chalcopyrite present in these samples, was similar to the initial copper concentrate, leaching in a selective way this mineralogical species. 3.3. Mechanism of bioleaching Three significant facts were observed in all bioleaching tests carried out with the MTC culture: 1. Iron in solution was mainly found as ferrous iron. 2. Only when copper extraction was significant the presence of ferric iron in solution was more notable. 3. Zinc extractions were only about 60%, being almost constant along the leaching time. In addition to the high temperature of leaching, these characteristics may have a significant incidence on the high bioleaching rate obtained with MTC culture. From these facts the following ‘‘in situ’’ indirect leaching model may be considered: In acidic solution, chalcopyrite is oxidised by dissolved oxygen according to the reaction in Eq. (1), Hiroyosi et al. (1997). In addition to this reaction, the Eq. (3) occurs in the presence of ferrous ions. If ferric ions
694
A. Rubio, F.J. Garcıa Frutos / Minerals Engineering 15 (2002) 689–694
were produced by the reaction in Eq. (3), chalcopyrite oxidation by ferric ions would be taking place in addition to reaction in Eq. (1) through the reaction in Eq. (2). Considering the final products obtained in this case, an indirect leaching mechanism could be postulated. However, ferric iron in solution was low and MTC culture have very low capacity to oxidise ferrous iron in solution, Rubio (1998). Besides, according to rest potentials, sphalerite should be easier to be leached but this is not true, in this case. Because of that, the authors believe that probably, ferrous ions are adsorbed first onto chalcopyrite surface, being the culture able to catalyse ferrous oxidation to ferric iron at the surface. Then, ferric ions produced on the surface oxidise chalcopyrite according to reaction of Eq. (2) being simultaneously reduced to ferrous ions. When the chalcopyrite leaching is almost completed, ferric ion concentration in solution is already high (10 g/l) and then the Fe3þ is able to leach sphalerite by ferric leaching through reaction in Eq. (4). ZnS þ 2Fe3þ ¼ Zn2þ þ 2Fe2þ þ S0
ð4Þ
This model is in accordance with the results obtained by the authors in a study carried out improving the rate of bioleaching of MTC culture on this copper concentrate when initial additions of ferrous ions were made. When initial additions of 1.8 g/l of ferrous iron were done, the rate increased from 60 mg/lh to 96 mg/lh, Rubio (1998).
4. Conclusions A natural MTC was obtained from a chalcopyritic copper concentrate with an ability to preferentially leach chalcopyrite in concentrates against other mineralogical species less refractory as sphalerite. After a period of adaptation of the mixed thermophilic culture to increasing pulp densities (1, 5, 10, 15 and 20% w/v), high copper extractions (>94%) and high leaching rates (2.3 g/l per day at 10% of pulp density) can be obtained. From the results observed in all bioleaching tests, it was proposed an ‘‘in situ’’ indirect leaching mechanism of chalcopyrite. From these laboratory results this culture can be considered as a promising advance in the biohydrometallurgical treatment of chalcopyritic concentrates and its potential use on an industrial scale.
Acknowledgements We would like to thank A. Ilarri, researchers and technicians of IGME Laboratory where this study was
carried out for your assistance in the analyses and experiments.
References Alvarez, J.L., 1996. Revisi on crıtica de los procesos de beneficio de sulfuros complejos. Simposio sulfuros polimetalicos de la Faja Pirıtica Iberica, Huelva. Ballester, A., 1996. Biohidrometalurgia y sulfuros complejos. Investigaciones actuales. Simposio sulfuros polimetalicos de la Faja Pirıtica Iberica, Huelva, 1996. Brierley, C.L., 1993. Practical role of thermophilic bacteria in bioleaching and biooxidation. In: International Conference and Workshop Application of Biotechnology to the Mineral Industry, Australian Mineral Foundation, pp. 2.1–2.7. Brierley, J.A., Brierley, C.L., 1999. Present and future commercial applications of biohydrometallurgy. In: Amils, R., Ballester, A. (Eds.), International Biohydrometallurgy Symposium IBSÕ99. Part A. Elsevier, pp. 81–89. Clark, D.A., Norris, P.R., 1996. Oxidation of minerals sulfides by thermophilic microorganisms. Minerals Engineering 9 (11), 1119–1125. Del Barrio, S., 1992. Estudio e investigaci on de un espectrofot ometro de emisi on plasma ICP aplicado a la prospecci on geoquımica multielemental. Boletın Geologico y Minero (Publicaci on especial). Dew van Buuren, McEwan, Bowker, 1999. Bioleaching of base metal sulphide concentrates: a comparison of mesophile and thermophile bacterial cultures. In: Amils, R., Ballester, A. (Eds.), International Biohydrometallurgy Symposium IBSÕ99. Part A. Elsevier, pp. 229– 238. Erlich, H., 1999. Past, present and future of biohydrometallurgy. In: Amils, R., Ballester, A. (Eds.), International Biohydrometallurgy Symposium IBSÕ99. Part A. Elsevier, pp. 3–12. G omez, E., Ballester, A., Gonzalez, F., Blazquez, M.L., 1999. Leaching capacity of a new extremely thermophilic microorganism. Sulfolobus rivotincti. Hydrometallurgy 52, 349–366. Hiroyosi, N., Hirota, M., Hirajima, T., Ysunekawa, M., 1997. A case of ferrous sulfate addition enhancing chalcopyrite leaching. Hydrometallurgy 47, 37–45. Jordan, M.A., McGuiness, S., Philips, C.V., 1996. Acidophilic bacteria-their potential mining and environmental applications. Minerals Engineering 9 (2), 169–181. Kolthoff, I., 1979. M. Analisis quımico cuantitativo, Ed.Niger SRL. Lopez-Archilla, A.I., Marın, I., Amils, R., 2001. Microbial community composition and ecology of an acidic aquatic environment: the Tinto river, Spain. Microbial Ecology 41, 20–35. Martın Rubı, J.A., 1998. Analisis por fluorescencia de Rayos X de los sulfuros masivos en perlas de fusi on. In: IV Simposio Internacional de Sulfuretos Polimetalicos de la Faixa Piritosa Iberica, C10, pp. 1–13. Mehta, A.P., Murr, L.E., 1982. Kinetics study of sulfide leaching by galvanic interactions between chalcopyrite, pyrite and sphalerite in the presence of T. ferrooxidans (30 °C) and a thermophilic microorganism (55 °C). Biotechnology and Bioengineering XXIV, 919–940. Rubio, A., 1998. Investigaci on en la mejora de la cinetica de biolixiviaci on de concentrados calcopirıticos. Utilizaci on de microorganismos term ofilos, PhD Thesis, Dpto. Biologıa Molecular, Universidad Aut onoma de Madrid. Sand, W., Rhode, K., Sobotke, B., Zenneck, C., 1992. Evaluation of leptospirillum ferrooxidans for leaching. Applied and Environmental Microbiology 58, 85–92. Sand, W., 1997. Novel mechanism for bioleaching of metal sulfides. In: Biotechnology Comes of Age. International Biohydrometallurgy Symposium IBS97 BIOMINE97. Australian Mineral Foundation, Glenside, SA.
Bioleaching capacity of an extremely thermophilic culture for chalcopyritic materials A. Rubio a, F.J. Garcıa Frutos
b,*
a
b
Instituto Geol ogico y Minero de Espa~ na (IGME), La Calera n° 1, 28760 Tres Cantos, Madrid, Spain Centro de Investigaciones Energ eticas, Medioambientales y Tecnol ogicas (CIEMAT), Av. Complutense 22, Edificio 20, 28040 Madrid, Spain Received 9 March 2001; accepted 2 May 2002
Abstract A thermophilic culture specific to bioleaching chalcopyritic materials has been obtained from a typical chalcopyritic copper concentrate of the Spanish Pyritic Belt. This paper shows the effect of pulp density (w/v) on bioleaching culture capacity with respect to this copper concentrate. The results of the batch tests show that it is possible, operating at 10% of pulp density to attain copper extraction of 94% in 10 days and, at higher pulp densities (20%), to attain good copper extraction (80%) in only 14 days. In the same way, the culture has been amply tested with different chalcopyritic ores and copper concentrates. The results obtained with four of these materials, two refractory gold ores and two copper concentrates are also presented. These results show a varying and versatile bioleaching capacity of this bacterial culture. A model of bacterial attack of this culture to leach chalcopyrite is postulated. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Mineral processing; Sulphide ores; Hydrometallurgy; Bioleaching and bacteria
1. Introduction The resources of high ores in the world are becoming more and more scarce making the processing of more complex ores necessary. Conventional mineral processing on complex sulphide ores carried out by differential flotation often produces high grade concentrates but with contaminants that penalise and make its marketing and pyrometallurgical processing difficult. Therefore, a lot of effort has been made in the development of hydrometallurgical process suitable for the treatment of these ores, but most of the proposed methods are complex and expensive, Alvarez (1996). Among these hydrometallurgical processes, biohydrometallurgical techniques appear to be an alternative for the treatment of these concentrates. These methods, that were first applied industrially to copper and uranium productions using bioassisted heap, dump and in situ technologies, are successfully used today in extraction of gold from refractory sulphide-bearing ores and concentrates, Jordan et al. (1996). However, for other * Corresponding author. Tel.: +34-91-3466232; Fax: +34-913466269. E-mail address: [email protected] (F.J. Garcıa Frutos).
metal concentrates, this technology remains as a promising alternative against conventional pyrometallurgical extraction processes. This is the case of the treatment of chalcopyritic concentrates, which represent a more complicated situation, due to the natural refractivity of chalcopyrite. One of the mainly problems of the bioleaching processes applied to copper concentrates and refractory ores are the low kinetics of the reactions, with high residence time that does not permit it to be an economic process. For this reason, current researches into this field are focused on how to increase this bioleaching rate. Apart from improving engineering design of bioreactors, the possibilities to increase bioleaching rates depend on the use of catalyst and isolation and adaptation of new microorganisms with high capacity to leach these ores. Several studies with mesophilic microorganisms as Thiobacillus ferrooxidans and Leptospirillum ferrooxidans had showed very slow copper leaching rates, Mehta (1982) and Sand et al. (1992). However, when thermophilic microorganisms were used leaching rates were considerably increased due to high temperatures, higher metal tolerance capacity and metabolic characteristics of this type of microorganisms, Brierley (1993) and Clark and Norris (1996).
0892-6875/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 8 9 2 - 6 8 7 5 ( 0 2 ) 0 0 1 2 4 - 3
690
A. Rubio, F.J. Garcıa Frutos / Minerals Engineering 15 (2002) 689–694
Many researches have investigated the possibility of using thermophilic microorganisms to improve metal leaching rates instead to mesophilic microorganisms. Future process developments must and will include thermophilic bacteria, which will play an increasingly important role in biooxidations of minerals. Thermophilic Archaea, Sulfolobus species, Acidianus brierleyi and Metallosphaera sedula which grow from 60 to 75 °C, are particularly adept in bioleaching of copper from the highly refractory chalcopyrite, Brierley and Brierley (1999) and Dew et al. (1999). Moreover, the use of these microorganisms that naturally thrive on ore samples and its aqueous environments, could be a good option as they probably have specificity for leaching chalcopyrite and a much higher capacity for adaptation. In this sense, there is a work carried out by Lopez-Archilla et al. (2001), on the microbial ecology of Rio Tinto waters, where they report to have isolated a thermophilic archaeabacteria, Sulfolobus rivotincti, with specificity to bioleach chalcopyrite and therefore with possibilities to be used in chalcopyritic concentrates treatment, Ballester (1996) and G omez et al. (1999). The use of these microorganisms for bioleaching chalcopyrite implies the knowledge of the mechanisms involved in the process. Initially, chalcopyrite can be oxidised by dissolved oxygen according to Eq. (1), Hiroyosi et al. (1997), favoured for acidic solution, and as it is known, two modes of bacterial attack can be distinguished: In indirect attack (Eq. (2)), the role of bacteria is to regenerate the oxidant ferric ion in the bulk phase, from the ferrous iron resulting from the chemical oxidation of the metal sulphide in the ore by ferric iron (Eq. (3)). In the direct attack, the bacteria leach the metal sulphide by attaching to the mineral surface and oxidising it enzimatically by conveying electrons from the reduced moiety of the mineral, Erlich (1999). CuFeS2 þ O2 þ 4Hþ ¼ Cu2þ þ Fe2þ þ 2S0 þ 2H2 O ð1Þ CuFeS2 þ 4Fe 4Fe
2þ
þ
3þ
¼ Cu
2þ
þ 5Fe
þ 4H þ O2 ¼ 4Fe
3þ
2þ
þ 2S
þ 2H2 O
0
ð2Þ ð3Þ
Sand (1997) have suggested that because Fe3þ oxidises metal sulphide with both the direct and indirect mechanisms, there is no difference between the two mechanisms. Their model emphasises a similarity in the chemistry of attack of the sulphide moiety by iron, and makes no distinction between ferric iron in the bulk phase and ferric iron bound in the cell envelope. The authors have obtained a mixed natural thermophilic culture from a typical chalcopyiritic copper concentrate of the Spanish Pyritic Belt, Rubio (1998). This culture has selectivity with respect to leaching chalcopyrite, when this is present together with other sulphides in ores and concentrates.
This paper describes the bioleaching capacity of the culture to operate at high pulp density and its versatility for leaching chalcopyritic materials, from ores to concentrates, postulating an ‘‘in situ’’ indirect bacterial attack.
2. Methods 2.1. Microbial inoculum A mixed thermophilic culture (MTC) of native microorganisms, isolated on a typical chalcopyritic concentrate described below (Concentrate 0), was used as inoculum in the initial bioleaching experiments. The methodology of the isolation was the following: The concentrate was strong stirring (360 rpm) at pH higher than 4 and 65 °C of temperature during 2 h for remove the microorganisms attached on the chalcopyrite surface. Once the microorganisms were unattached and free in solution, the solid was removed of the liquid by filtration. The bacteria-laden liquid obtained was filtered through a 0.22 lm Millipore membrane filter to collect the microorganisms, which were suspended in 1:10 9 K medium at pH 1.30. Preliminary comparative bioleaching tests were performed using a bacterial inoculum of Sulfolobus sp. kindly supplied by Dr. F. Carranza, Universidad de Sevilla, Spain. Both thermophilic cultures were maintained on copper concentrate (Concentrate 0) at 1% w/v pulp density medium with 1:10 salts of 9 K medium at 65 °C of temperature and initial pH of 1.30. The cultures were successfully adapted to higher pulp density of copper concentrate until obtaining high copper extractions in short residence times. In these tests, the inoculum was obtained as the culture isolation was made. The final pulp of the bioleching test was filtered, obtaining the leach liquor and the solid residue. The solid was intensively stirring during 2 h with water at pH 4. The pulp obtained was filtered again and the final liquid obtained, which contains most the bacteria, which were attached to the solid residue, was filtered through a 0.22 lm Millipore filter where bacteria were retained. Finally, the bacteria were re-suspended in 50 ml of the leach liquor obtained in the first filtration in order to get the inoculum volume (5% v/v) for the next bioleaching test. 2.2. Ore samples A chalcopyritic copper concentrate obtained from conventional differential flotation was used to develop the mixed thermophilic culture. This concentrate (Concentrate 0) came from the Spanish Pyritic Belt and its
A. Rubio, F.J. Garcıa Frutos / Minerals Engineering 15 (2002) 689–694 Table 1 Chemical composition of copper concentrate Sample
Chemical analysis (wt%) Cu
Zn
Pb
Fe
S
Concentrate 0
23.37
2.58
2.52
32.63
38.00
composition is given in Table 1. The mineralogical composition of this sample shows chalcopyrite and pyrite as main mineralogical species and galena and sphalerite as secondary mineralogical phases. The particle size distribution presents a passing d80 of 20 lm. Concentrate 1, also used in bioleaching tests is essentially the same as Concentrate 0, corresponding to another lot of copper concentrate obtained in the same flotation point at the industrial plant. This sample presents less copper content and higher zinc content than Concentrate 0. The other chalcopyritic samples, refractory gold ores (Ores 1 and 2) and copper concentrates (Concentrate 1 and 2), also came from the Spanish Pyritic Belt, were selected in this work to study the selectivity of the MTC to chalcopyrite. The chemical composition of these chalcopyritic samples is given in Table 2. The copper present in these samples is as chalcopyrite.
691
ished, solids were removed by filtration, and chemically characterised as well as the leachate. Sterile bioleaching tests were carried out with ore sterilised by autoclaving at 121 °C, 30 min and 1atm of pressure, and adding a solution of 10% ethanol to the leaching media. 2.4. Analysis Soluble species of copper, zinc, lead, total iron and minor elements were analysed by ICP, Del Barrio (1992), using a spectrophotometer ICAP-61 Thermo Jarrel Ash. The ferrous iron was analysed by a volumetric method by titration with potassium dichromate, Kolthoff (1979). Copper, zinc, lead and iron content in the ore samples and leaching solid residues were analysed by XRF, Martın Rubı (1998), using a spectrophotometer Philips PW-1404, and minor elements by ICP. Total sulphur was gravimetrically determined and elemental sulphur was analysed by toluene extraction in a Soxhlet apparatus. The pH was measured with a 704 pH-meter Metrohm. The redox potential (Eh) was measured with a platinum electrode with an Ag/AgCl reference electrode. Mineralogical composition was determined by XRD using a difractometer PW-1700 Philips.
2.3. Bioleaching experiments Bioleaching experiments were carried out in 1 l glass cylindrical reactors provided with a cap with four holes to allow mechanical stirring (at 130 rpm), aeration (10– 15 l/h) and sampling. These reactors were placed in a thermostatic bath to keep the temperature constant at 65 °C. During the experiments the pH was kept at 1.30 by the addition of 10N H2 SO4 when were necessary. This 10 N H2 SO4 addition was made to avoid the precipitation of iron in form of jarosites, which damage the bioleaching process. Redox potential and pH were measured daily, while the levels of copper, zinc, and iron in solution were analysed daily or every 2 days, depending on the test. Water was added to the reactors in order to compensate for evaporation losses. Once bioleaching tests were fin-
Table 2 Chemical composition of chalcopyritic samples Sample
Chemical analysis (wt%) Cu
Zn
Pb
Fe
S
Ore 1 Ore 2 Concentrate 1 Concentrate 2
0.69 1.47 20.76 6.70
1.64 1.1 4.65 <0.10
0.71 0.53 2.87 <0.10
39.55 42.15 30.11 24.20
33.35 45.6 37.29 n.a.
n.a: no appear.
3. Results and discussion 3.1. Influence of pulp density A study of bioleaching capacity on copper concentrates by MTC culture was done at different pulp densities such as 1%, 5%, 10%, 15% and 20% (w/v). Preliminary tests carried out at 1% pulp density (w/v) show a higher capacity of MTC culture to bioleach the copper present in the copper concentrate (Concentrate 0) compared to the tests carried out with Sulfolobus sp. culture (Sb) on the same copper concentrate. Both cultures had been maintained and adapted to this copper concentrate by successive sub-culturing for the realisation of these bioleching tests. Fig. 1 shows copper extractions obtained on copper concentrate with MTC and Sb cultures in representative adaptation tests at 1% of pulp density (w/v). In the sterile test, only 11 % copper extraction was obtained in more than 500 h. In bioleaching tests with Sulfolobus sp. culture (Sb), initially a cooper extraction up 98% was reached in 500 h, but after successive adaptation tests, the kinetic of reaction was increasing, obtaining maximum copper extractions (>98%) in 350 h. However, with MTC culture the same copper extraction (more than 98%) was obtained in only 165 h with only two successive adaptive tests.
692
A. Rubio, F.J. Garcıa Frutos / Minerals Engineering 15 (2002) 689–694
Fig. 1. Copper extraction in bioleaching adaptive tests at 1% (w/v) pulp density. (Sterile test, Sulfolobus sp. and MTC tests at pH 1.30 and 65 °C). Sample: Concentrate 0. (The sub-indexes 1; 2; 3; . . . ; n correspond to consecutive tests.)
Fig. 3. Comparative copper extraction at the different pulp densities studied (1, 5, 10, 15 and 20% (w/v) at pH 1.30 and 65 °C). Sample: Concentrate 0.
Bioleaching tests carried out at 5% pulp density (w/v) are shown in Fig. 2. Similar ability to preferentially leach copper by MTC thermophilic culture was obtained. The extractions achieved with the MTC were much more significant than Sb culture. It can be observed that in the first test on Concentrate 0 with the culture isolated MTC, a copper extraction of about 90% was obtained in only 21 days. In second test with this culture, in 17 days the copper extraction reached 97%, considerably higher than the extraction attained (80%) after 56 days of bioleaching when Sulfolobus sp. culture was used. From these results, a study in order to define the optimum conditions of this bioleaching process was carried out increasing pulp density (1%, 5%, 10%, 15% and 20% (w/v)). This study is described elsewhere, Rubio (1998). The best conditions obtained from 1% to 20% pulp densities vary only in oxygen uptakes. In Fig. 3 appears the evolution of copper extractions to all tests done at pulp densities studied in its optimum conditions. As it can be observed the results of batch tests show that it is possible to work both with high pulp
density (20%) obtaining good copper extraction (80%) and with lower pulp density (10%) obtaining better copper extraction (94%) in 14 and 10 days respectively. In all tests done at different pulp densities, iron in solution at first stages was mainly as ferrous iron and only the presence of ferric iron was more notable when almost 80% of copper is leached. Fig. 4 shows total iron and ferrous iron during a bioleaching test on Concentrate 0 at 10% of pulp density with MTC culture. As it can be observed, mainly form of iron in solution was as ferrous iron. This ferrous iron in solution decreases at the final stages (from the eighthday on) which corresponds to an increase in the solution redox potential over 450 mV and a copper dissolution of 80%. Redox potential evolution is showed in Fig. 5. As can be seen, Eh in first stages of bioleaching is about 400 mV to reach 425 mV in an intermediate step, confirming the most presence of ferrous iron in solution. The more notable redox potential increase was produced from day 7, when ferrous iron starts to decrease in solution reaching a final Eh of 475 mV. Fig. 6 shows zinc extraction in the same bioleaching test. A fast initial leaching of zinc can be appreciated. At
Fig. 2. Copper extraction in bioleaching adaptive tests at 5% (w/v) pulp density. (Sulfolobus sp. (Sb) and MTC tests at pH 1.30 and 65 °C). Sample: Concentrate 0. (The sub-indexes 1; 2; 3; . . . ; n correspond to consecutive tests.)
Fig. 4. Concentration of iron in leach solution to a representative test at 10% (w/v) pulp density, at pH 1.30 and 65 °C. Sample: Concentrate 0.
A. Rubio, F.J. Garcıa Frutos / Minerals Engineering 15 (2002) 689–694
Fig. 5. Redox potential (Eh) evolution to a representative test at 10% (w/v) pulp density, at pH 1.30 and 65 °C. Sample: Concentrate 0.
Fig. 6. Concentration of zinc in leach solution to a representative test at 10% (w/v) pulp density, at pH 1.30 and 65 °C. Sample: Concentrate 0.
intermediate stage of leaching, as there are not sufficient ferric iron in solution, the zinc dissolution is low, and only an increase in zinc extraction was observed when copper was bioleached (after 8 days of test) coinciding with the increase of ferric iron in solution. The zinc present was almost completely leached only when the duration of tests was prolonged longer after all copper had been leached. X-ray diffraction of solid residues showed elemental sulphur and pyrite as main species present, and anglesite and sphalerite as minor species. 3.2. Bioleaching of chalcopyritic samples In Table 3, appear in short, the results obtained with the two refractory gold ores selected. The tests were
Table 3 Results obtained in bioleaching tests with the refractory gold ores Sample Ore 1 Ore 2
Pulp density (% w/v)
Time (days)
Yield (%) Cu
Zn
Fe
10 10
3 4
90.20 89.16
70.59 59.02
11.52 23.99
693
Fig. 7. Copper extraction in bioleaching tests with copper Concentrate 1 and 2 (10% (w/v) pulp density, pH 1.3 and 65 °C).
done at 10% pulp density. The copper present in these ores was quickly leached in 3 and 4 days, and the zinc was only extracted when copper is almost leached and the redox potential was over 475 mV. Fig. 7 shows the copper extraction obtained in bioleaching tests carried out at 10% pulp density with the other copper concentrates used (Concentrate 1 and 2). Concentrate 2 with less initial copper content than Concentrate 1, was more easily bioleached by MTC culture, obtaining more than 90% of copper extraction in 6 days. With Concentrate 1 (similar to Concentrate 0) and without a period of adaptation, more than 70% of copper extraction was obtained in 12 days. In the sterile tests, copper extraction obtained was 14% for Concentrate 1 and 21% for Concentrate 2. From these tests it can be observed that the leaching behaviour of this culture with respect to chalcopyrite present in these samples, was similar to the initial copper concentrate, leaching in a selective way this mineralogical species. 3.3. Mechanism of bioleaching Three significant facts were observed in all bioleaching tests carried out with the MTC culture: 1. Iron in solution was mainly found as ferrous iron. 2. Only when copper extraction was significant the presence of ferric iron in solution was more notable. 3. Zinc extractions were only about 60%, being almost constant along the leaching time. In addition to the high temperature of leaching, these characteristics may have a significant incidence on the high bioleaching rate obtained with MTC culture. From these facts the following ‘‘in situ’’ indirect leaching model may be considered: In acidic solution, chalcopyrite is oxidised by dissolved oxygen according to the reaction in Eq. (1), Hiroyosi et al. (1997). In addition to this reaction, the Eq. (3) occurs in the presence of ferrous ions. If ferric ions
694
A. Rubio, F.J. Garcıa Frutos / Minerals Engineering 15 (2002) 689–694
were produced by the reaction in Eq. (3), chalcopyrite oxidation by ferric ions would be taking place in addition to reaction in Eq. (1) through the reaction in Eq. (2). Considering the final products obtained in this case, an indirect leaching mechanism could be postulated. However, ferric iron in solution was low and MTC culture have very low capacity to oxidise ferrous iron in solution, Rubio (1998). Besides, according to rest potentials, sphalerite should be easier to be leached but this is not true, in this case. Because of that, the authors believe that probably, ferrous ions are adsorbed first onto chalcopyrite surface, being the culture able to catalyse ferrous oxidation to ferric iron at the surface. Then, ferric ions produced on the surface oxidise chalcopyrite according to reaction of Eq. (2) being simultaneously reduced to ferrous ions. When the chalcopyrite leaching is almost completed, ferric ion concentration in solution is already high (10 g/l) and then the Fe3þ is able to leach sphalerite by ferric leaching through reaction in Eq. (4). ZnS þ 2Fe3þ ¼ Zn2þ þ 2Fe2þ þ S0
ð4Þ
This model is in accordance with the results obtained by the authors in a study carried out improving the rate of bioleaching of MTC culture on this copper concentrate when initial additions of ferrous ions were made. When initial additions of 1.8 g/l of ferrous iron were done, the rate increased from 60 mg/lh to 96 mg/lh, Rubio (1998).
4. Conclusions A natural MTC was obtained from a chalcopyritic copper concentrate with an ability to preferentially leach chalcopyrite in concentrates against other mineralogical species less refractory as sphalerite. After a period of adaptation of the mixed thermophilic culture to increasing pulp densities (1, 5, 10, 15 and 20% w/v), high copper extractions (>94%) and high leaching rates (2.3 g/l per day at 10% of pulp density) can be obtained. From the results observed in all bioleaching tests, it was proposed an ‘‘in situ’’ indirect leaching mechanism of chalcopyrite. From these laboratory results this culture can be considered as a promising advance in the biohydrometallurgical treatment of chalcopyritic concentrates and its potential use on an industrial scale.
Acknowledgements We would like to thank A. Ilarri, researchers and technicians of IGME Laboratory where this study was
carried out for your assistance in the analyses and experiments.
References Alvarez, J.L., 1996. Revisi on crıtica de los procesos de beneficio de sulfuros complejos. Simposio sulfuros polimetalicos de la Faja Pirıtica Iberica, Huelva. Ballester, A., 1996. Biohidrometalurgia y sulfuros complejos. Investigaciones actuales. Simposio sulfuros polimetalicos de la Faja Pirıtica Iberica, Huelva, 1996. Brierley, C.L., 1993. Practical role of thermophilic bacteria in bioleaching and biooxidation. In: International Conference and Workshop Application of Biotechnology to the Mineral Industry, Australian Mineral Foundation, pp. 2.1–2.7. Brierley, J.A., Brierley, C.L., 1999. Present and future commercial applications of biohydrometallurgy. In: Amils, R., Ballester, A. (Eds.), International Biohydrometallurgy Symposium IBSÕ99. Part A. Elsevier, pp. 81–89. Clark, D.A., Norris, P.R., 1996. Oxidation of minerals sulfides by thermophilic microorganisms. Minerals Engineering 9 (11), 1119–1125. Del Barrio, S., 1992. Estudio e investigaci on de un espectrofot ometro de emisi on plasma ICP aplicado a la prospecci on geoquımica multielemental. Boletın Geologico y Minero (Publicaci on especial). Dew van Buuren, McEwan, Bowker, 1999. Bioleaching of base metal sulphide concentrates: a comparison of mesophile and thermophile bacterial cultures. In: Amils, R., Ballester, A. (Eds.), International Biohydrometallurgy Symposium IBSÕ99. Part A. Elsevier, pp. 229– 238. Erlich, H., 1999. Past, present and future of biohydrometallurgy. In: Amils, R., Ballester, A. (Eds.), International Biohydrometallurgy Symposium IBSÕ99. Part A. Elsevier, pp. 3–12. G omez, E., Ballester, A., Gonzalez, F., Blazquez, M.L., 1999. Leaching capacity of a new extremely thermophilic microorganism. Sulfolobus rivotincti. Hydrometallurgy 52, 349–366. Hiroyosi, N., Hirota, M., Hirajima, T., Ysunekawa, M., 1997. A case of ferrous sulfate addition enhancing chalcopyrite leaching. Hydrometallurgy 47, 37–45. Jordan, M.A., McGuiness, S., Philips, C.V., 1996. Acidophilic bacteria-their potential mining and environmental applications. Minerals Engineering 9 (2), 169–181. Kolthoff, I., 1979. M. Analisis quımico cuantitativo, Ed.Niger SRL. Lopez-Archilla, A.I., Marın, I., Amils, R., 2001. Microbial community composition and ecology of an acidic aquatic environment: the Tinto river, Spain. Microbial Ecology 41, 20–35. Martın Rubı, J.A., 1998. Analisis por fluorescencia de Rayos X de los sulfuros masivos en perlas de fusi on. In: IV Simposio Internacional de Sulfuretos Polimetalicos de la Faixa Piritosa Iberica, C10, pp. 1–13. Mehta, A.P., Murr, L.E., 1982. Kinetics study of sulfide leaching by galvanic interactions between chalcopyrite, pyrite and sphalerite in the presence of T. ferrooxidans (30 °C) and a thermophilic microorganism (55 °C). Biotechnology and Bioengineering XXIV, 919–940. Rubio, A., 1998. Investigaci on en la mejora de la cinetica de biolixiviaci on de concentrados calcopirıticos. Utilizaci on de microorganismos term ofilos, PhD Thesis, Dpto. Biologıa Molecular, Universidad Aut onoma de Madrid. Sand, W., Rhode, K., Sobotke, B., Zenneck, C., 1992. Evaluation of leptospirillum ferrooxidans for leaching. Applied and Environmental Microbiology 58, 85–92. Sand, W., 1997. Novel mechanism for bioleaching of metal sulfides. In: Biotechnology Comes of Age. International Biohydrometallurgy Symposium IBS97 BIOMINE97. Australian Mineral Foundation, Glenside, SA.