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Adaptation of Sulfolobus metallicus to high pulp densities in the biooxidation of a flotation gold concentrate
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Hydrometallurgy 92 (2008) 11 – 15 www.elsevier.com/locate/hydromet
Adaptation of Sulfolobus metallicus to high pulp densities in the biooxidation of a flotation gold concentrate C. Astudillo ⁎, F. Acevedo School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile Received 5 July 2007; received in revised form 25 September 2007; accepted 6 February 2008 Available online 11 February 2008
Abstract Bioleaching of minerals with thermophilic microorganisms is attracting increasing interest as a promising alternative process. One weak point of thermophilic archaea is their apparent sensitivity to high solids and metallic ions concentrations. The objective of this research was to demonstrate the adaptation capacity of Sulfolobus metallicus to increasingly higher pulp densities in the biooxidation of a highly refractory gold concentrate without significant loss of productivity. A S. metallicus strain was used. The microorganism was cultured in Norris medium with a gold concentrate at pulp densities from 5% to 30% w/v replacing the energy source. The concentrate contained 42 g Au/tonne, 38.4% pyrite (FeS2), 16.4% enargite (Cu3AsS4), 10.6% chalcopyrite (CuFeS2), 10.7% tenantite (Cu12As4 S13) and 23.9% gangue. The 38 to 75 μm particle size fraction was used. Temperature was maintained at 70 °C. pH was not controlled. An adaptation protocol of successive subcultures was adopted. Results show that it is possible to adapt S. metallicus to pulp densities as high as 30% w/v with less than 15% loss of metal productivities. Constant high productivities and Fe2+/Fe3+ ratios under 1.0 were successful as indicators of adaptation. Experimental evidence points at metal toxicity as the cause of incomplete metal extraction. © 2008 Elsevier B.V. All rights reserved. Keywords: Thermophilic archaea; Adaptation strategy; Inhibition phenomenon; Metal toxicity; Metal extraction
1. Introduction Bioleaching of minerals has attracted increasing interest in the past few years as a promising alternative process. It has been pointed out by several authors that this bioprocess presents several attractive features related with low capital cost, the environment and energy requirement (Acevedo, 2000; Olson et al., 2003; Johnson, 2005; Crundwell, 2005). The pyrometallurgical processes used to benefit minerals such as chalcopyrite, arsenopyrite and enargite which are widely distributed in the large copper mines, bring about severe environmental problems as they emit dangerous gaseous contaminants such as sulfur dioxide and arsenic trioxide. ⁎ Corresponding author. Pontificia Universidad Católica de Valparaíso, School of Biochemical Engineering, General Cruz #34, Valparaiso, Valparaiso, Chile. Tel.: +56 32 2925487; fax: +56 32 2273803. E-mail address: [email protected] (C. Astudillo). 0304-386X/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.hydromet.2008.02.003
Mesophilic bacteria have proved to efficiently attack secondary sulfides such as chalcocite, covellite, bornite and, to a less extent, enargite (Muñoz et al., 2006). Nevertheless, they have been less successful in oxidizing chalcopyrite mostly due to passivation of the mineral during its bioleaching (Dew et al., 1999). In order to overcome this difficulty, attention is being paid to the use of hyperthermophilic archaea because they present several positive characteristics and because the high cultivation temperatures (65–80 °C) increase the rate of chemical ferric oxidation of chalcopyrite and other minerals (Kargi and Robinson, 1985; Boogerd et al., 1991). Moreover, the higher operation temperatures decrease the cost of cooling required (Kinnunen et al., 2003) because biooxidation is a highly exothermic reaction (Rossi, 1990; Acevedo and Gentina, 2007). In the operation of a pilot plant for the biooxidation of a zinc sulfide ore with 100 and 500 l reactors, a decrease of 30% in the cooling energy was obtained with Sulfolobus acidocaldarius at 65 °C as compared with Thiobacillus caldus
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at 45 °C (Sandtröm et al., 1997). High metal recoveries can be achieved with thermophiles, a characteristic that has been explored in the European project HIOX® (HIgh-temperature bacterial OXidation) for which the final industrial objective was to propose a completely new method for copper recovery on the basis of an economically and environmentally friendly process (d'Hughes et al., 2001). In order to minimize capital costs in this type of processes pulp density should be as high as possible (Norris, 1997). In the other hand, it has been noted by several researchers that pulp densities higher than 15 to 20% w/v are detrimental to cell viability and oxidizing activity, both working with mesophiles and thermophiles (Torma et al., 1972; Sissing and Harrison, 2003). Groudev (1986) working with mesophilic bacteria observed a proportional increase in productivity with increasing pulp densities up to 12 to 15% w/v after which a sustained decrease was evident. The author proposed that this effect could be due to limitation in the supply of gaseous nutrients (CO2 and/or O2). It has been reported that working with thermophilic archaea in stirred reactors a maximum of 10% of pulp density could be achieved before the inhibition phenomenon took place (Boogerd et al., 1990). Acevedo et al. (2004) working with Sulfolobus metallicus and a pyritic gold concentrates in shake flasks determined that the optimal pulp density and particle size were 7.8% and 35 μm respectively. Additionally, several authors have pointed out the high adaptation capacity of archaea opening the possibility of overriding this problem (Brierley, 1974; Miller et al., 1992; Rubio et al., 1995; Mier et al., 1996; Rawlings, 2005). Considering the above comments, the objective of this work was to demonstrate the adaptation capacity of S. metallicus to increasingly higher pulp densities in the biooxidation of a highly refractory gold concentrate without significant loss of productivity. 2. Materials and methods 2.1. Microorganism and culture medium
2.3. Analytical methods Ferrous ion was determined colorimetrically by a modified Muir method (Herrera et al., 1989) using a Shimadzu UV-160 double beam spectrophotometer. Interference of ferric ion was avoided by adding NaF. Total soluble iron was determined by reducing ferric ions with hydroxylamine and measuring by the same method. Ferric iron was estimated by difference between total iron and ferrous ion. Soluble copper was measured volumetrically with iodine and sodium thiosulfate (Vogel, 1988). Again ferric iron interference was avoided by adding NaF. Eh was measured with a Schott-Geräte Blue Line electrode (PtAg/AgCl) and a Cole-Parmer model 5997-60 potentiometer. pH was determined with a Hanna Instruments model HI 9321 instrument equipped with a Ag/AgCl electrode.
2.4. Cell population adaptation protocol The strain used in this work had been previously grown on a high chalcopyrite concentrate at low pulp densities (PD) of no more than 1% w/v. In order to adapt it to the new concentrate and to higher PD an experimental program was established. First the cells were adapted to the new substrate composition by successive subcultures al 1% PD. The adaptation criterion was the appearance of significant amounts of soluble iron and copper and a Fe2+/Fe3+ ratio lower than 1.0, indicative of an efficient ferrous iron biooxidation. Successive subcultures were repeated until reproducible results were obtained. This adapted population grown at 1% PD was used as inoculum to new cultures of increasing PD. Again cultivations were carried on until adaptation was achieved using the same adaptation criterion. Pulp densities were 1, 5, 10, 15, 20, 25 and 30% w/v. The adaptation was validated by running parallel cultures with non-adapted cells at 30% PD. Sterile runs were also made in order to account for the abiotic oxidation. In these runs a solution of thymol in methanol (10%) was added (Donati et al., 1992).
3. Results and discussion Fig. 1 shows the volumetric productivities for iron and copper at different PD during the process of adaptation. It can be appreciated that the strain responded well to increasing PD, but it appears that a PD of 25–30% PD is near the adaptation capacity. Figs. 2 and 3 depict the productivities for iron and copper of the adapted population. Sterile runs are also included. A significant difference is evident in the behavior of the sterile, non-adapted and adapted runs. These results also suggest that optimum conditions are in
A S. metallicus strain kindly supplied by Professor A. Ballester from the Universidad Complutense, Madrid, was used. The microorganism was cultured in Norris medium (Norris, 1989) with the gold concentrate at pulp densities from 5% to 30% w/v replacing the energy source. The flotation concentrate was supplied by Minera El Indio, IV Region, Chile, and contained 42 g Au/tonne, 38.4% pyrite (FeS2), 16.4% enargite (Cu3AsS4), 10.6% chalcopyrite (CuFeS2), 10.7% tenantite (Cu12As4 S13) and 23.9% gangue. The 38 to 75 μm particle size fraction that was used was washed three times with acid water (pH 2.0). Temperature was maintained at 70 °C. pH was not controlled.
2.2. Culture conditions Experiments were performed in 1-l flasks with 300 ml of pulp. Flasks were inoculated with 20% v/v of active culture. Initial pH was adjusted to 1.8 with sulfuric acid. Cultures were run in a thermostatic rotary agitator (Innova, 4080 Incubator Shaker, New Brunswick Scientific Co. Inc., Edison, NJ) at 70 °C and 150 rpm. Water loss by evaporation was periodically compensated by distilled water addition. Samples taken periodically were centrifuged for 15 min at 15,000 rpm (Biofuge 15, Heraeus Sepatech, Midland, ON, Canada) and the clear liquid was use to analyze for ferrous ion, total iron and copper. Eh and pH were monitored. Volumetric productivities of soluble iron and copper were calculated after 30 days of cultivation.
Fig. 1. Volumetric productivities of iron and copper during the adaptation of S. metallicus. Experimental conditions: agitated flasks, 150 rpm; initial pH of 1.8; 70 °C.
C. Astudillo, F. Acevedo / Hydrometallurgy 92 (2008) 11–15
Fig. 2. Volumetric productivities of copper in runs with adapted and non-adapted cells. A sterile control is included. Experimental conditions: agitated flasks, 150 rpm; initial pH of 1.8; 70 °C.
the range of 10 to 20% PD, with a decreasing tendency at PD of 25 and 30%. In all cases the contribution of the chemical solubilization was low. The degree of extraction of copper and iron in the adapted and nonadapted runs at different PD are shown in Fig. 4. A clear difference in favor of the adapted cells can be seen, as well as declining extractions with increasing PD. Except for the cases of 5 and 10% PD extractions were under 80%, far below complete solubilization. Possible reasons for this behavior could be limitations in oxygen or carbon dioxide transfer, passivation of the mineral surface, and inhibition of microbial action due to the accumulation of soluble iron, copper and/or arsenic. The final pH values in each run are given in Table 1. It may be seen that final values are somewhat higher at higher PD for both adapted and non-adapted cells. This may be due to the less acid generated because of the lower metal extraction at high PD. The results summarized in Fig. 3 are interesting in that the cell population was adapted to high PD with high metal solubilization productivities. As far as the authors' knowledge, similar results have not been published before. Increase in metal extraction productivities when working at low solids concentrations has been reported
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Fig. 4. Extraction degree of iron and copper calculated after 30 days of culture for adapted and non-adapted cells. Experimental conditions: agitated flasks, 150 rpm; initial pH of 1.8; 70 °C.
for Acidithiobacillus ferrooxidans and S. metallicus by authors such as Torma et al. (1970) and Sissing and Harrison (2003). Nevertheless, in those reports the productivity significantly fell at high PD values. In our case after reaching a maximum value at 10% PD, the productivities of the adapted cells runs did not decline abruptly in the range of 15 to 30% PD, while the runs with non-adapted cells showed a considerable decrease. Evidence has been given that PD over 15–18% have adverse effects on bioleaching by Sulfolobus (Sandtröm et al., 1997) and that increases in solids content result in longer lag periods (Norris and Barr, 1988). A number of papers have been published reporting findings on the poor performance or on the low tolerance of thermophiles to high PD (Norris and Barr, 1988; Clark and Norris, 1996; Gericke et al., 2001; Escobar et al., 1993). Le Roux and Wakerly (1988) observed that the productivity of bioleaching of chalcopyrite with Sulfolobus increased with PD reaching a maximum at 15% w/v after which it declined significantly. They also observed that although the productivity decreased at high PD the cell count increased after inoculation, but this population was incapable of maintaining a high leaching rate. Other researchers have found that S. acidocaldarius show a good performance up to 10% PD and can tolerate solids concentration of 20% w/v (Sandtröm et al., 1997). Employing S. metallicus Acevedo et al. (2004) determined that the higher productivities in the bioleaching of a high pyrite gold concentrate in shake flasks was attained in the range of 5 to 10% PD; higher PD had a negative effect on productivity. Working with stirred reactors d'Hughes et al. (2002) could reach stable steady states in continuous operation up to pulp densities of 8%; operation at higher solids concentration resulted in unstable operation. Cell damage was evidenced by a sharp decrease in oxygen consumption. Harrison et al. (2003) and Sissing and Harrison (2003) studied the effect of the presence of solids on the behavior of several microorganisms including Sulfolobus sp. and mesophilic bioleaching bacteria. In order to distinguish between the effects of the substrate and
Table 1 Final pH values of runs with adapted and non-adapted cells Final pH Fig. 3. Volumetric productivities of iron in runs with adapted and non-adapted cells. A sterile control is included. Experimental conditions: agitated flasks, 150 rpm; initial pH of 1.8; 70 °C.
Pulp density (% p/v)
5%
10%
15%
20%
25%
30%
S. metallicus, adapted S. metallicus, non-adapted
0.85 1.15
0.95 1.10
1.19 1.32
1.35 1.53
1.25 1.45
1.43 1.60
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the solids concentrations they used constant mineral concentration and varied the pulp density by adding different amounts of inert quartz. They concluded that PD up to 18% did not affect the cell performance but over that figure decay in the number of cell occurred. It has also been reported that under hydrodynamic stress S. metallicus cell size diminishes and its morphology changes (Nemati et al., 2000). In all the cited publications and others the net result is that the higher limit of bioleaching with Sulfolobus and other thermophiles is in the range of 15 to 20% PD, a limit that seems hard to surpass. In this sense the work of Lawrence and Marchant (1988) could be cited, as they successfully operated with Sulfolobus up to 25% PD. Unfortunately the authors do not describe cell handling and adaptation. In contrast, by applying our adaptation strategy we have been able to operate with PD as high as 30% with high productivities. In respect to the incomplete extraction of the metals one possible reason for this behavior could be the toxicity of high concentrations of iron, copper and arsenic. Concentrations up to 35 g Fe/l and 23 g Cu/l were attained (results not shown), which are in the upper range of toxicity limit (reported Norris and Parrott, 1986; Le Roux and Wakerly, 1988; Rivera-Santillan et al., 1999; Gericke et al., 2001; Rodríguez et al., 2001; d'Hughes et al., 2002; Rubio and Garcia Frutos, 2003). One of the most toxic species that could be present in the liquor is arsenic (III) coming from enargite and tenantite of the concentrate. Although no measurements of soluble arsenic were made, mass balance considerations show that high amounts of arsenic, on the order of 3 to 16 g/l, must have been solubilized. The fact that the cell population was still active can be explained by the oxidation of As(III) to As(V) by the relatively high concentrations of ferric ion present at all times in the liquid (Mateo, 2004). Still, the metal concentrations reached were high enough as to impair the cells and cause incomplete extractions.
4. Conclusions It is possible to adapt S. metallicus to pulp densities up to 30% w/v with less than 15% loss of metal productivities by a strategy of successive subcultures at increasing PD. Constant high productivities and Fe2+ /Fe3+ ratios under 1.0 were successful as indicators of adaptation. Experimental evidence points to metal toxicity as the cause of incomplete metal extraction. Acknowledgement This work was supported by the National Commission for Scientific and Technological Research (CONICYT-Chile) through the FONDECYT project 1020768. References Acevedo, F., 2000. The use of reactors in biomining processes. Electronic J. Biotechnol. 3 (2), 184–194. Available from http://www.ejbiotechnology. info. Acevedo, F., Gentina, J.C., 2007. Bioreactor design fundamentals and their application to gold mining. In: Donati, E.R., Sand, W. (Eds.), Microbial Processing of Metal Sulfides. Springer, Dordrecht, The Netherlands, pp. 151–168. Acevedo, F., Gentina, J.C., Valencia, P., 2004. Optimisation of pulp density and particle size in the biooxidation of a pyritic gold concentrate by Sulfolobus metallicus. World J. Microbiol. Biotechnol. 20, 865–869.
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C. Astudillo, F. Acevedo / Hydrometallurgy 92 (2008) 11–15 Le Roux, N.W., Wakerley, D.S., 1988. Leaching of chalcopyrite (CuFeS2) at 70 °C using Sulfolobus. In: Norris, P.R., Kelly, D.P. (Eds.), Biohydrometallurgy. Proceedings of the International Biohydrometallurgy Symposium. Science and Technology Letters. Warwick, U.K., pp. 305–317. Mateo, M., 2004. Oxidación de arsénico (III) en soluciones de biooxidación con Acidithiobacillus ferrooxidans y Sulfolobus metallicus. PhD thesis, School of Biochemical Engineering, Catholic University of Valparaíso, Valparaíso, Chile. Mier, J., Ballester, A., González, F., Blázquez, M., Gómez, E., 1996. The influence of metallic ions on the activity of Sulfolobus BC. Chem. Technol. Biotechnol. 65, 272–280. Miller, K., Risanico, S., Risatti, J., 1992. Differential tolerance of Sulfolobus strains to transition metals. FEMS Microbiol. Lett. 93, 69–74. Muñoz, J.A., Blázquez, M.L., González, F., Ballester, A., Acevedo, F., Gentina, J.C., González, P., 2006. Electrochemical study of enargite bioleaching by mesophilic and thermophilic microorganisms. Hydrometallurgy 84, 175–186. Nemati, M., Lowenadler, J., Harrison, S.T.L., 2000. Particle size effects in bioleaching of pyrite by acidophilic thermophile Sulfolobus metallicus (BC). Appl. Microbiol. Biotechnol. 53, 173–179. Norris, P.R., 1989. Factors affecting bacterial oxidation: the example of the carbon dioxide in the context of bacterial diversity. In: Salley, J., McCready, R.G.L., Wichlacz, P.L. (Eds.), Proceedings of the International Biohydrometallurgy Symposium. CANMET, Canada, pp. 3–14. Norris, P.R., 1997. Thermophiles and bioleaching. In: Rawlings, D.E. (Ed.), Biomining—Theory, Microbes and Industrial Processes. Springer Verlag, Berlin, pp. 247–258. Norris, P.R., Barr, D.W., 1988. Bacterial oxidation of pyrite in high temperature reactors. In: Norris, P.R., Kelly, D.P. (Eds.), Biohydrometallurgy. Proceedings of the International Biohydrometallurgy Symposium. Science and Technology Letters. Warwick, U.K., pp. 532–536. Norris, P.R., Parrott, L., 1986. High temperature, mineral concentrate dissolution with Sulfolobus. In: Lawrance, R., Branion, R., Ebner, H. (Eds.), Fundamental and Applied Biohydrometallurgy. Proceedings of the Sixth International Symposium on Biohydrometallurgy. Elsevier, Vancouver B.C., pp. 355–365. Olson, G.J., Brierley, J.A., Brierley, C.L., 2003. Bioleaching review part B: Progress in bioleaching: applications of microbial processes by the minerals industries. Appl. Microbiol. Biotechnol. 63, 249–257.
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Rawlings, D.E., 2005. Characteristics and adaptability of iron-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates. Microb. Cell Fact. 4, 13–28. Rivera-Santillan, R.E., Ballester, A., Blázquez, M.L., González, F., 1999. Bioleaching of a copper sulphide flotation concentrate using mesophilic and thermophilic microorganisms. In: Ballester, A., Amils, R. (Eds.), Biohydrometallurgy and the Environment Toward the Mining of the 21st Century. Elsevier, Amsterdam, pp. 149–158. Rodríguez, Y., Ballester, A., Blázquez, M.L., González, F., Muñoz, J.A., 2001. Mecanismos de biolixiviación de sulfuros metálicos. Rev. Metal. Madrid 37, 665–672. Rossi, G., 1990. Biohydrometallurgy. McGraw-Hill Book Co., Hamburg. Rubio, A., Jimeno, A., García-Frutos, F.J., 1995. Influence of the ions (Bi+3, Ag+) in the process of bioleaching of a copper concentrate form the Spanish pyritic belt by thermophilic microorganims. In: Jerez, C.A., Vargas, T., Toledo, H., Wiertz, J.V. (Eds.), Biohydrometallurgical Processing, Vol III. University of Chile. Santiago, Chile, pp. 317–327. Rubio, A., Garcia Frutos, F.J., 2003. Enhancement of chalcopyrite bioleaching capacity of an extremely thermophilic culture by adition of ferrous sulfate. Proceedings of the 15th International Biohydrometallurgy Symposium. Athens, Greece. paper 24. Sandtröm, Å., Sundkvist, J.E., Peterson, S., 1997. Bio-oxidation of a complex zinc sulphide ore: a study performed in continuous bench and pilot scale. Proceedings Biohydrometallurgy International Symposium. Sydney, Australia, pp. M 1.1.1–M1.1.11. Sissing, A., Harrison, S.T.L., 2003. Thermophilic mineral bioleaching performances: a compromise between maximizing mineral loading and maximizing microbial growth and activity. J. S. Afr. I. Min. Metall. 103 (Part 2), 139–142. Torma, A., Walden, C., Duncan, D., Branion, R., 1970. Microbiological leaching of a zinc sulphide concentrate. Biotechol. Bioeng. 12, 501–517. Torma, A., Walden, C., Duncan, D., Branion, R., 1972. The effect of carbon dioxide and particle surface area on the microbiological leaching of zinc sulphide concentrate. Biotechnol. Bioeng. 14, 777–786. Vogel, A., 1988. Química analítica cuantitativa. Tomo I. Volumetría y Gravimetría. Editorial Kapeluz S.A., Buenos Aires, Argentina.
Hydrometallurgy 92 (2008) 11 – 15 www.elsevier.com/locate/hydromet
Adaptation of Sulfolobus metallicus to high pulp densities in the biooxidation of a flotation gold concentrate C. Astudillo ⁎, F. Acevedo School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile Received 5 July 2007; received in revised form 25 September 2007; accepted 6 February 2008 Available online 11 February 2008
Abstract Bioleaching of minerals with thermophilic microorganisms is attracting increasing interest as a promising alternative process. One weak point of thermophilic archaea is their apparent sensitivity to high solids and metallic ions concentrations. The objective of this research was to demonstrate the adaptation capacity of Sulfolobus metallicus to increasingly higher pulp densities in the biooxidation of a highly refractory gold concentrate without significant loss of productivity. A S. metallicus strain was used. The microorganism was cultured in Norris medium with a gold concentrate at pulp densities from 5% to 30% w/v replacing the energy source. The concentrate contained 42 g Au/tonne, 38.4% pyrite (FeS2), 16.4% enargite (Cu3AsS4), 10.6% chalcopyrite (CuFeS2), 10.7% tenantite (Cu12As4 S13) and 23.9% gangue. The 38 to 75 μm particle size fraction was used. Temperature was maintained at 70 °C. pH was not controlled. An adaptation protocol of successive subcultures was adopted. Results show that it is possible to adapt S. metallicus to pulp densities as high as 30% w/v with less than 15% loss of metal productivities. Constant high productivities and Fe2+/Fe3+ ratios under 1.0 were successful as indicators of adaptation. Experimental evidence points at metal toxicity as the cause of incomplete metal extraction. © 2008 Elsevier B.V. All rights reserved. Keywords: Thermophilic archaea; Adaptation strategy; Inhibition phenomenon; Metal toxicity; Metal extraction
1. Introduction Bioleaching of minerals has attracted increasing interest in the past few years as a promising alternative process. It has been pointed out by several authors that this bioprocess presents several attractive features related with low capital cost, the environment and energy requirement (Acevedo, 2000; Olson et al., 2003; Johnson, 2005; Crundwell, 2005). The pyrometallurgical processes used to benefit minerals such as chalcopyrite, arsenopyrite and enargite which are widely distributed in the large copper mines, bring about severe environmental problems as they emit dangerous gaseous contaminants such as sulfur dioxide and arsenic trioxide. ⁎ Corresponding author. Pontificia Universidad Católica de Valparaíso, School of Biochemical Engineering, General Cruz #34, Valparaiso, Valparaiso, Chile. Tel.: +56 32 2925487; fax: +56 32 2273803. E-mail address: [email protected] (C. Astudillo). 0304-386X/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.hydromet.2008.02.003
Mesophilic bacteria have proved to efficiently attack secondary sulfides such as chalcocite, covellite, bornite and, to a less extent, enargite (Muñoz et al., 2006). Nevertheless, they have been less successful in oxidizing chalcopyrite mostly due to passivation of the mineral during its bioleaching (Dew et al., 1999). In order to overcome this difficulty, attention is being paid to the use of hyperthermophilic archaea because they present several positive characteristics and because the high cultivation temperatures (65–80 °C) increase the rate of chemical ferric oxidation of chalcopyrite and other minerals (Kargi and Robinson, 1985; Boogerd et al., 1991). Moreover, the higher operation temperatures decrease the cost of cooling required (Kinnunen et al., 2003) because biooxidation is a highly exothermic reaction (Rossi, 1990; Acevedo and Gentina, 2007). In the operation of a pilot plant for the biooxidation of a zinc sulfide ore with 100 and 500 l reactors, a decrease of 30% in the cooling energy was obtained with Sulfolobus acidocaldarius at 65 °C as compared with Thiobacillus caldus
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at 45 °C (Sandtröm et al., 1997). High metal recoveries can be achieved with thermophiles, a characteristic that has been explored in the European project HIOX® (HIgh-temperature bacterial OXidation) for which the final industrial objective was to propose a completely new method for copper recovery on the basis of an economically and environmentally friendly process (d'Hughes et al., 2001). In order to minimize capital costs in this type of processes pulp density should be as high as possible (Norris, 1997). In the other hand, it has been noted by several researchers that pulp densities higher than 15 to 20% w/v are detrimental to cell viability and oxidizing activity, both working with mesophiles and thermophiles (Torma et al., 1972; Sissing and Harrison, 2003). Groudev (1986) working with mesophilic bacteria observed a proportional increase in productivity with increasing pulp densities up to 12 to 15% w/v after which a sustained decrease was evident. The author proposed that this effect could be due to limitation in the supply of gaseous nutrients (CO2 and/or O2). It has been reported that working with thermophilic archaea in stirred reactors a maximum of 10% of pulp density could be achieved before the inhibition phenomenon took place (Boogerd et al., 1990). Acevedo et al. (2004) working with Sulfolobus metallicus and a pyritic gold concentrates in shake flasks determined that the optimal pulp density and particle size were 7.8% and 35 μm respectively. Additionally, several authors have pointed out the high adaptation capacity of archaea opening the possibility of overriding this problem (Brierley, 1974; Miller et al., 1992; Rubio et al., 1995; Mier et al., 1996; Rawlings, 2005). Considering the above comments, the objective of this work was to demonstrate the adaptation capacity of S. metallicus to increasingly higher pulp densities in the biooxidation of a highly refractory gold concentrate without significant loss of productivity. 2. Materials and methods 2.1. Microorganism and culture medium
2.3. Analytical methods Ferrous ion was determined colorimetrically by a modified Muir method (Herrera et al., 1989) using a Shimadzu UV-160 double beam spectrophotometer. Interference of ferric ion was avoided by adding NaF. Total soluble iron was determined by reducing ferric ions with hydroxylamine and measuring by the same method. Ferric iron was estimated by difference between total iron and ferrous ion. Soluble copper was measured volumetrically with iodine and sodium thiosulfate (Vogel, 1988). Again ferric iron interference was avoided by adding NaF. Eh was measured with a Schott-Geräte Blue Line electrode (PtAg/AgCl) and a Cole-Parmer model 5997-60 potentiometer. pH was determined with a Hanna Instruments model HI 9321 instrument equipped with a Ag/AgCl electrode.
2.4. Cell population adaptation protocol The strain used in this work had been previously grown on a high chalcopyrite concentrate at low pulp densities (PD) of no more than 1% w/v. In order to adapt it to the new concentrate and to higher PD an experimental program was established. First the cells were adapted to the new substrate composition by successive subcultures al 1% PD. The adaptation criterion was the appearance of significant amounts of soluble iron and copper and a Fe2+/Fe3+ ratio lower than 1.0, indicative of an efficient ferrous iron biooxidation. Successive subcultures were repeated until reproducible results were obtained. This adapted population grown at 1% PD was used as inoculum to new cultures of increasing PD. Again cultivations were carried on until adaptation was achieved using the same adaptation criterion. Pulp densities were 1, 5, 10, 15, 20, 25 and 30% w/v. The adaptation was validated by running parallel cultures with non-adapted cells at 30% PD. Sterile runs were also made in order to account for the abiotic oxidation. In these runs a solution of thymol in methanol (10%) was added (Donati et al., 1992).
3. Results and discussion Fig. 1 shows the volumetric productivities for iron and copper at different PD during the process of adaptation. It can be appreciated that the strain responded well to increasing PD, but it appears that a PD of 25–30% PD is near the adaptation capacity. Figs. 2 and 3 depict the productivities for iron and copper of the adapted population. Sterile runs are also included. A significant difference is evident in the behavior of the sterile, non-adapted and adapted runs. These results also suggest that optimum conditions are in
A S. metallicus strain kindly supplied by Professor A. Ballester from the Universidad Complutense, Madrid, was used. The microorganism was cultured in Norris medium (Norris, 1989) with the gold concentrate at pulp densities from 5% to 30% w/v replacing the energy source. The flotation concentrate was supplied by Minera El Indio, IV Region, Chile, and contained 42 g Au/tonne, 38.4% pyrite (FeS2), 16.4% enargite (Cu3AsS4), 10.6% chalcopyrite (CuFeS2), 10.7% tenantite (Cu12As4 S13) and 23.9% gangue. The 38 to 75 μm particle size fraction that was used was washed three times with acid water (pH 2.0). Temperature was maintained at 70 °C. pH was not controlled.
2.2. Culture conditions Experiments were performed in 1-l flasks with 300 ml of pulp. Flasks were inoculated with 20% v/v of active culture. Initial pH was adjusted to 1.8 with sulfuric acid. Cultures were run in a thermostatic rotary agitator (Innova, 4080 Incubator Shaker, New Brunswick Scientific Co. Inc., Edison, NJ) at 70 °C and 150 rpm. Water loss by evaporation was periodically compensated by distilled water addition. Samples taken periodically were centrifuged for 15 min at 15,000 rpm (Biofuge 15, Heraeus Sepatech, Midland, ON, Canada) and the clear liquid was use to analyze for ferrous ion, total iron and copper. Eh and pH were monitored. Volumetric productivities of soluble iron and copper were calculated after 30 days of cultivation.
Fig. 1. Volumetric productivities of iron and copper during the adaptation of S. metallicus. Experimental conditions: agitated flasks, 150 rpm; initial pH of 1.8; 70 °C.
C. Astudillo, F. Acevedo / Hydrometallurgy 92 (2008) 11–15
Fig. 2. Volumetric productivities of copper in runs with adapted and non-adapted cells. A sterile control is included. Experimental conditions: agitated flasks, 150 rpm; initial pH of 1.8; 70 °C.
the range of 10 to 20% PD, with a decreasing tendency at PD of 25 and 30%. In all cases the contribution of the chemical solubilization was low. The degree of extraction of copper and iron in the adapted and nonadapted runs at different PD are shown in Fig. 4. A clear difference in favor of the adapted cells can be seen, as well as declining extractions with increasing PD. Except for the cases of 5 and 10% PD extractions were under 80%, far below complete solubilization. Possible reasons for this behavior could be limitations in oxygen or carbon dioxide transfer, passivation of the mineral surface, and inhibition of microbial action due to the accumulation of soluble iron, copper and/or arsenic. The final pH values in each run are given in Table 1. It may be seen that final values are somewhat higher at higher PD for both adapted and non-adapted cells. This may be due to the less acid generated because of the lower metal extraction at high PD. The results summarized in Fig. 3 are interesting in that the cell population was adapted to high PD with high metal solubilization productivities. As far as the authors' knowledge, similar results have not been published before. Increase in metal extraction productivities when working at low solids concentrations has been reported
13
Fig. 4. Extraction degree of iron and copper calculated after 30 days of culture for adapted and non-adapted cells. Experimental conditions: agitated flasks, 150 rpm; initial pH of 1.8; 70 °C.
for Acidithiobacillus ferrooxidans and S. metallicus by authors such as Torma et al. (1970) and Sissing and Harrison (2003). Nevertheless, in those reports the productivity significantly fell at high PD values. In our case after reaching a maximum value at 10% PD, the productivities of the adapted cells runs did not decline abruptly in the range of 15 to 30% PD, while the runs with non-adapted cells showed a considerable decrease. Evidence has been given that PD over 15–18% have adverse effects on bioleaching by Sulfolobus (Sandtröm et al., 1997) and that increases in solids content result in longer lag periods (Norris and Barr, 1988). A number of papers have been published reporting findings on the poor performance or on the low tolerance of thermophiles to high PD (Norris and Barr, 1988; Clark and Norris, 1996; Gericke et al., 2001; Escobar et al., 1993). Le Roux and Wakerly (1988) observed that the productivity of bioleaching of chalcopyrite with Sulfolobus increased with PD reaching a maximum at 15% w/v after which it declined significantly. They also observed that although the productivity decreased at high PD the cell count increased after inoculation, but this population was incapable of maintaining a high leaching rate. Other researchers have found that S. acidocaldarius show a good performance up to 10% PD and can tolerate solids concentration of 20% w/v (Sandtröm et al., 1997). Employing S. metallicus Acevedo et al. (2004) determined that the higher productivities in the bioleaching of a high pyrite gold concentrate in shake flasks was attained in the range of 5 to 10% PD; higher PD had a negative effect on productivity. Working with stirred reactors d'Hughes et al. (2002) could reach stable steady states in continuous operation up to pulp densities of 8%; operation at higher solids concentration resulted in unstable operation. Cell damage was evidenced by a sharp decrease in oxygen consumption. Harrison et al. (2003) and Sissing and Harrison (2003) studied the effect of the presence of solids on the behavior of several microorganisms including Sulfolobus sp. and mesophilic bioleaching bacteria. In order to distinguish between the effects of the substrate and
Table 1 Final pH values of runs with adapted and non-adapted cells Final pH Fig. 3. Volumetric productivities of iron in runs with adapted and non-adapted cells. A sterile control is included. Experimental conditions: agitated flasks, 150 rpm; initial pH of 1.8; 70 °C.
Pulp density (% p/v)
5%
10%
15%
20%
25%
30%
S. metallicus, adapted S. metallicus, non-adapted
0.85 1.15
0.95 1.10
1.19 1.32
1.35 1.53
1.25 1.45
1.43 1.60
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the solids concentrations they used constant mineral concentration and varied the pulp density by adding different amounts of inert quartz. They concluded that PD up to 18% did not affect the cell performance but over that figure decay in the number of cell occurred. It has also been reported that under hydrodynamic stress S. metallicus cell size diminishes and its morphology changes (Nemati et al., 2000). In all the cited publications and others the net result is that the higher limit of bioleaching with Sulfolobus and other thermophiles is in the range of 15 to 20% PD, a limit that seems hard to surpass. In this sense the work of Lawrence and Marchant (1988) could be cited, as they successfully operated with Sulfolobus up to 25% PD. Unfortunately the authors do not describe cell handling and adaptation. In contrast, by applying our adaptation strategy we have been able to operate with PD as high as 30% with high productivities. In respect to the incomplete extraction of the metals one possible reason for this behavior could be the toxicity of high concentrations of iron, copper and arsenic. Concentrations up to 35 g Fe/l and 23 g Cu/l were attained (results not shown), which are in the upper range of toxicity limit (reported Norris and Parrott, 1986; Le Roux and Wakerly, 1988; Rivera-Santillan et al., 1999; Gericke et al., 2001; Rodríguez et al., 2001; d'Hughes et al., 2002; Rubio and Garcia Frutos, 2003). One of the most toxic species that could be present in the liquor is arsenic (III) coming from enargite and tenantite of the concentrate. Although no measurements of soluble arsenic were made, mass balance considerations show that high amounts of arsenic, on the order of 3 to 16 g/l, must have been solubilized. The fact that the cell population was still active can be explained by the oxidation of As(III) to As(V) by the relatively high concentrations of ferric ion present at all times in the liquid (Mateo, 2004). Still, the metal concentrations reached were high enough as to impair the cells and cause incomplete extractions.
4. Conclusions It is possible to adapt S. metallicus to pulp densities up to 30% w/v with less than 15% loss of metal productivities by a strategy of successive subcultures at increasing PD. Constant high productivities and Fe2+ /Fe3+ ratios under 1.0 were successful as indicators of adaptation. Experimental evidence points to metal toxicity as the cause of incomplete metal extraction. Acknowledgement This work was supported by the National Commission for Scientific and Technological Research (CONICYT-Chile) through the FONDECYT project 1020768. References Acevedo, F., 2000. The use of reactors in biomining processes. Electronic J. Biotechnol. 3 (2), 184–194. Available from http://www.ejbiotechnology. info. Acevedo, F., Gentina, J.C., 2007. Bioreactor design fundamentals and their application to gold mining. In: Donati, E.R., Sand, W. (Eds.), Microbial Processing of Metal Sulfides. Springer, Dordrecht, The Netherlands, pp. 151–168. Acevedo, F., Gentina, J.C., Valencia, P., 2004. Optimisation of pulp density and particle size in the biooxidation of a pyritic gold concentrate by Sulfolobus metallicus. World J. Microbiol. Biotechnol. 20, 865–869.
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