- Email: [email protected]
Comparative study of external addition of Fe2+ and inoculum on bioleaching of marmatite flotation concentrate using mesophilic and moderate thermophilic bacteria
Hydrometallurgy 93 (2008) 51–56
Contents lists available at ScienceDirect
Hydrometallurgy j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / h yd r o m e t
Comparative study of external addition of Fe2+ and inoculum on bioleaching of marmatite flotation concentrate using mesophilic and moderate thermophilic bacteria Suting Wang a, Guangji Zhang a,⁎, Qiuhong Yuan a, Zhaoheng Fang a, Chao Yang a,b a b
Key Laboratory of Green Process and Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China
A R T I C L E
I N F O
Article history: Received 19 December 2007 Received in revised form 23 February 2008 Accepted 28 February 2008 Available online 18 March 2008 Keywords: Acidithiobacillus ferrooxidans Marmatite concentrate Thermo-acidophilic iron-oxidizing bacteria Inoculum
A B S T R A C T Bioleaching of marmatite flotation concentrate by Acidithiobacillus ferrooxidans and a moderately thermoacidophilic iron-oxidizing bacterial strain (MLY) was carried out in batch experiments. The effects of external addition of Fe2+ and the inoculum of the bacteria were studied. For A. ferrooxidans, the supplementary addition of Fe2+ as a source of Fe3+ to be generated by the bacteria was necessary in the starting of the bioleaching of marmatite. While for MLY, it was not necessary because the higher temperature improved the dissolution of marmatite in sulphuric acid, and thus acidic leaching happened before ferric ion leaching. Leaching rate was enhanced with the increased inoculum, however, this influence decreased at a higher level of inoculum. The evolution of the redox potential and the total concentration of iron in the bioleaching indicated that Eh was more important than the concentration of Fe3+ during the dissolution of the marmatite for both strains. Different affinities for the Fe2+ and S0 substrates led to different trends of leaching rate for the two bacteria. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The interaction between bacteria and sulfide minerals is the key issue in the fundamental study of bioleaching. Since the discovery of bacterium in acidic mine drainage, diversified opinions about the mechanism in bioleaching of sulfides have been put forward. The term ‘direct bioleaching’ was introduced by Silverman and Ehrlich in 1964 to describe a hypothesized enzymatic reaction taking place between attached cells and the underlying mineral surface. The ‘indirect’ mechanism of sulfide oxidation involves non-specific oxidation of surface by Fe3+ ions that are generated by iron-oxidizing microorganisms (Edwards et al., 2001). Sand and his collaborators proposed a new mode by analysis of degradation products occurring in the course of dissolution of metal sulfide. They suggested that ferric ions and/or protons are the only chemical agents which dissolve metal sulfides. The mechanism and chemistry of the degradation are decided by the mineral structure. According to their proposition, sphalerite (ZnS) is degradable by ferric ions and proton attack. The main intermediates are polysulfides and elemental sulphur (Sand et al., 2001). Although the bioleaching of zinc concentrates has been studied extensively (Choi et al., 1993; Deveci et al., 2004; Fowler and Crundwell, 1998; Konishi et al., 1992; Rodriguez et al., 2003), there is no unanimous agreement on the mechanisms, because leaching ⁎ Corresponding author. Tel.: +86 10 62554558; fax: +86 10 62561822. E-mail address: [email protected] (G. Zhang). 0304-386X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.hydromet.2008.02.019
processes are relatively complex and often vary with the composition of the minerals. Marmatite [(Zn,Fe)S] is an important resource of zinc ore in China, which is difficult to be processed effectively by the traditional technologies due to its low content of zinc. But it is more suitable for bioleaching (Shi et al., 2006a,b) and the rate of oxidative dissolution is higher compared with sphalerite due to its high content of iron (Crundwell, 1988). Shi studied the electrochemical behaviors of a marmatite–carbon paste electrode in the presence and absence of bacterial strain and analyzed the surface features of the marmatite particle by a scanning electron microscope (SEM). He concluded that the direct contact leaching by the cells attached on the mineral substrate played an important role on the dissolution of marmatite in addition to the chemical leaching of Fe3+ ions (Shi et al., 2006a,b). As an environmentally benign technology with wide application, bioleaching is featured with low cost for recovering metals from lowgrade ores. The most studied bacterium in bioleaching of sulfide ores is Acidithiobacillus ferrooxidans (abbreviated as A. ferrooxidans), which is a strain of mesophilic bacteria. However, A. ferrooxidans cannot grow at temperatures higher than 40 °C, and this limits its efficiency in leaching. Thermophilic bacteria attract more and more attention due to the potential in improving the kinetics of metal extraction (Brierley and Brierley, 2001; Deveci et al., 2004; Norris et al., 2000; Zhang and Fang, 2005). Based on the indirect mechanism, the IBES process (Indirect Bioleaching with Effects Separation) was developed to improve the kinetics of bioleaching of sulphide concentrates. In the IBES process, the bioleaching process is performed in two separate
52
S. Wang et al. / Hydrometallurgy 93 (2008) 51–56
2. Materials and methods 2.1. Mineral The marmatite flotation concentrate was obtained from a lead– zinc mine in Yunnan province of China. Chemical analysis of the sample revealed the composition of 40.61% Zn; 15.81% Fe; 2.63% Pb; 0.37% Cu; 0.14% Mg and 31.66% S. X-ray diffraction analysis showed the concentrate was mainly composed of marmatite and a small quantity of pyrite. The particle size was −42 μm (over 90%). 2.2. Strains and media
Fig. 1. Comparison of Fe2+ oxidation rate between A. ferrooxidans and MLY.
stages: (1) chemical leaching and (2) biological oxidation of the Fe2+ produced in the chemical stage (Carranza et al., 1993). This process has been applied to chalcopyrite/sphalerite concentrates and copper concentrates (Carranza et al., 1997; Romero et al., 2003). In this study, marmatite flotation concentrate was bioleached by A. ferrooxidans and a moderately thermoacidophilic iron-oxidizing bacterial strain MLY for improving the understanding of the mechanisms of marmatite bioleaching.
The strains of A. ferrooxidans and thermoacidophilic bacteria MLY were provided by the Institute of Microbiology, Chinese Academy of Sciences. Both strains were cultured in the Leathen medium (Leathen et al., 1951) with 10 g/L of Fe2+ ions. The iron-free Leathen medium contained 0.15 g/L (NH4)2SO4, 0.05 g/L KCl, 0.05 g/L K2HPO4, 0.5 g/L MgSO4·7H2O, 0.01 g/L Ca(NO3)2. Yeast extract (0.2 g/L) was added particularly to the medium to support the growth of MLY. The optimum pH and temperature for A. ferrooxidans and MLY are 2.0, 35 °C and 1.5, 50 °C, respectively. MLY was isolated from a coal spoil heap in China. It is able to oxidize Fe2+ ions, pyrite and elemental sulphur autotrophically and mixotrophically in the presence of yeast extract. Autotrophic oxidation of elemental sulphur is relatively weak. The comparison of Fe2+ oxidation between MLY and A. ferrooxidans indicated MLY is twice as fast as A. ferrooxidans (Fig. 1) (Li and He, 2001).
Fig. 2. A. ferrooxidans bioleaching of marmatite with 2 g/L added Fe2+ and different % inoculum.
S. Wang et al. / Hydrometallurgy 93 (2008) 51–56
To ensure that the iron in the original medium did not affect the results, cells were harvested in the stationary phase by filtration onto a Millipore filter (pore size 0.22 μm) and re-suspended in iron-free Leathen medium before leaching experiments started. The initial density of cells in the solution was approximately 1 × 107 cells/mL when 10% (v/v) bacteria inoculum was used. 2.3. Experimental procedures Leaching experiments were carried out in 250 mL Erlenmeyer flasks containing 100 mL solution with 5% (w/v) pulp density on an orbital shaker at 160 min− 1. Bioleaching with A. ferrooxidans was carried out at 35 °C and MLY at 50 °C. Each experimental run lasted 7 days. The pH value was measured at each sampling instant first (these data points being plotted in the figures) and then the medium was adjusted back to initial pH. Periodically, samples were taken and the make-up distilled water was added into the flasks to compensate for evaporation loss. In the first part of the experiments, the effects of inoculum of the bacteria were studied. Sterile control was also run to determine the contribution from chemical leaching of zinc. In the second part of the experiments, different concentrations of Fe2+ ions were added to the medium containing 10% (v/v) bacteria inoculum. 2.4. Measurements and analysis The concentration of dissolved zinc in leachate was determined by inductively coupled plasma atomic emission spectroscopy (ICP). The concentrations of Fe2+ ions and total iron were determined by spectrophotometry using the phenanthroline methods (Vogel, 1989). The number of free cells in the liquid samples was determined by direct counting using a hemacytometer. The pH value and redox potential (Eh) in the leaching solution were, respectively, measured with a pH meter and a Pt electrode with reference to a saturated calomel electrode (all potentials in this work were referred to SHE).The solid samples were dried and analyzed by SEM and electron spectroscopy (ES).
53
Elemental sulphur is the main intermediate of ferric ion attack. Some studies have demonstrated that if elemental sulphur was produced in bioleaching, for energetic reasons, the bacteria would prefer sulphur over ferrous ions (Boon et al., 1998; Kuenen et al., 1993; Pronk et al., 1990). In this period, the elemental sulphur could be the main energy resource of A. ferrooxidans, and this led to Eh of the slurry lower than that of the medium containing Fe2+. This presumption is confirmed by Fig. 2b and c. The total iron in the solution decreased due to the formation of jarosite and did not change significantly over the last 3 days and the leaching rate of zinc did not reduce. According to chemometric analysis, complete oxidation of marmatite produces sulphuric acid and oxidation from Fe2+ to Fe3+ by bacteria consumes sulphuric acid, so the whole process is neither acid producing nor acid consuming. Fig. 2d shows the increase of pH value over the first 3 days was due to the oxidation of iron by bacteria and then it decreased. Although pH was adjusted back to 2.0 after each sampling, it was above the initial value at the end of the leaching. A. ferrooxidans can convert part of sulfide to sulphur (H2Sn → S0 + 2H+ + 2e−) instead of sulfate + − (H2Sn + 4H2O → SO2− 4 + 10H + 8e ) (Visser et al., 1997) and the produced sulphur can be stored in the form of sulphur globules, located either inside or outside the cell (Kleinjan et al., 2003). So the incomplete oxidation of sulphur can lead to a rise of pH in the solution. A SEM micrograph (not shown) of the marmatite surface after 7 days of A. ferrooxidans bioleaching also showed elemental sulphur accumulation as determined by ES. Fig. 3a shows that at the same cell density, the growth of Eh slowed as more Fe2+ ions were added into the solution. The Eh in the solution reflects the ratio of Fe3+:Fe2+, so more addition of Fe2+ ions lowers Eh. As Fig. 3a and b shows, a higher leaching rate was achieved at higher
3. Results and discussion 3.1. Bioleaching of marmatite flotation concentrate by A. ferrooxidans Experimental results (data not shown) showed that it was difficult for A. ferrooxidans to survive in the leaching system without the addition of Fe2+ ions even when 40% bacteria inoculum was added. After 120 h, the Eh remained at about 0.55 V indicating no bacterial oxidation occurred. No change of cell number was observed in the bulk solution phase after 48 h. Fig. 2a presents the Eh changes when 2 g/L Fe2+ ions were added into the leaching system with different inoculums. For 40% inoculum, the Eh increased rapidly in the first 80 h, then fluctuated around 0.68 V. For 5% inoculum, the Eh showed an increasing trend. At the end of the bioleaching, similar Eh values and leached Zn (Fig. 2b) were reached under different inoculums except for the 1% inoculum. These results showed the inoculum did not have a serious impact on bioleaching. But the Eh (about 0.68 V) was lower than that of A. ferrooxidans cultured in the Leathen medium containing 10 g/L Fe2+ ions (about 0.8 V). According to the Sand's model, the marmatite bioleaching by A. ferrooxidans could be described as follows (Sand et al., 2001): MS þ Fe3þ þ Hþ →M2þ þ 0:5H2 Sn þ Fe2þ ðn≥2Þ
ð1Þ
0:5H2 Sn þ Fe3þ →0:125S8 þ Fe2þ þ Hþ
ð2Þ
þ 0:125S8 þ 1:5O2 þ H2 O→SO2 4 þ 2H :
ð3Þ
Fig. 3. A. ferrooxidans bioleaching of marmatite with 10% (v/v) bacteria inoculum and 0–20 g/L added Fe2+. a) Changes in Eh; b) concentration of Zn2+.
54
S. Wang et al. / Hydrometallurgy 93 (2008) 51–56
is necessary for the starting of the bioleaching by A. ferrooxidans. Our experiments suggest that without external addition of Fe2+ ions, marmatite cannot be leached by A. ferrooxidans, but further addition of Fe2+ ions slows the leaching rate of the mineral. 3.2. Bioleaching of marmatite flotation concentrate by MLY Fig. 4a shows that in the sterile control, the dissolved iron at 50 °C was nearly as twice as that at 35 °C. At higher temperature and lower pH, the solubility of the marmatite increases, so more Fe2+ is released into the solution. MLY could leach marmatite even with no external addition of Fe2+ ions. When the concentrations of total iron and Fe2+ ions in the leaching solution are compared, with and without bacteria inoculum at 50 °C, after the first day the concentrations of total iron in both leaching solutions were similar. In the sample without bacteria inoculum, all the iron in the solution existed as Fe2+ ion; whilst all the iron in the solution existed as Fe3+ ion with bacteria inoculum. The total iron quantity is significantly larger in bioleaching than that in the sterile control at the end of the experiments. This indicates that all the iron was produced by the dissolution of marmatite in sulphuric acid after the first day and the main role of MLY is to oxidize Fe2+ to Fe3+. It is assumed that at least three processes are involved in MLY bioleaching the marmatite with no addition of Fe2+: namely dissolution of marmatite in sulphuric acid; bacterial oxidation of the dissolved Fe2+; and Fe3+ oxidation of marmatite. Fig. 4b and c shows
Fig. 4. Sterile control and MLY in bioleaching marmatite with no added Fe2+ and different levels of inoculum. a) Concentration of iron in sterile control and 20% MLY at 35 °C and 50 °C; b) changes in Eh with 0–40% MLY; c) concentration of zinc with 0–40% MLY.
Eh, indicating that Eh is more important than the concentration of Fe3+ during the dissolution of marmatite. Marmatite can dissolve in dilute sulphuric acid (Crundwell, 1988), but the leaching rate is very slow. External addition of Fe2+ ions provides the initial energy source for bacteria, which can be oxidized to Fe3+ to accelerate the sulfide oxidation process by oxidizing S2− to S0. Meanwhile, Fe2+ in the marmatite is released and oxidized to Fe3+, so external addition of Fe2+
Fig. 5. MLY bioleaching of marmatite with 10% (v/v) bacteria inoculum and 2–16 g/L added Fe2+. a) Changes in Eh; b) concentration of zinc.
S. Wang et al. / Hydrometallurgy 93 (2008) 51–56
55
of Fe to S in jarosite is about 1.5 to 1, it suggests granules in Fig. 6a were mainly jarosite while in Fig. 6b were mainly elemental sulphur. It reflects the S increase at the end of bioleaching. After the surface coating was cleared away by ultrasonic treatment in trichloromethane, many pits were observed on the surface and some sulphur grains were still accumulated in the pits due to the incomplete cleaning (SEM micrograph not shown). Neither cells nor cell-shaped pits were found on the mineral surface. However, some studies indicated that even the formation of cell-sized and cell-shaped dissolution pits did not require a direct microbially induced surface reaction (Edwards et al., 2001). Several studies reported that yeast extract was consumed in MLY growth and MLY had a weaker oxidizing ability of sulphur under autotrophic condition (Li and He, 2001; Zhang, 2002). In our experiments, because of the consumption of yeast extract at the end of the bioleaching, more elemental sulphur accumulated on the marmatite surface according to the ES analysis. 4. Conclusions For A. ferrooxidans bioleaching, experimental results show that an appropriate amount of Fe2+ ions is needed to trigger marmatite bioleaching. At the end of bioleaching, incomplete oxidation of elemental sulphur leads to the pH rise. For moderate thermophilic bacteria MLY bioleaching, the higher temperature and acidity improve the dissolution of marmatite, thus MLY can leach marmatite without external addition of Fe2+ ions. The main role of MLY in the bioleaching of marmatite concentrate is to oxidize Fe2+. So MLY is suitable to be used in the IBES process. Acknowledgements
Fig. 6. Typical SEM images of leach residues from MLY bioleaching of marmatite with 4 g/L added Fe2+ ions: (a) residue after 2 days; (b) residue after 7 days.
that the increased inoculum contributed to an increase in Eh and helped to reach a higher leaching rate. However, this influence reduced with the lapse of time. When 5% MLY inoculum was added into the solution, the leached zinc was nearly the same as that leached in the sterile control experiment. But when 10% MLY inoculum was added, the leached zinc increased markedly. MLY cannot oxidize Fe2+ effectively at a low cell density but when more bacteria inoculum is added, MLY can oxidize Fe2+ to Fe3+ successfully. At the beginning of the bioleaching, Fe3+ ions play an important role in breaking down the ore to further supply source energy for bacteria multiplication. Fig. 5a shows the pH change in the medium. After the first 3 days, the more Fe2+ that was added to the solution, the slower the increase in pH. This indicates the existence of jarosite because a great deal of Fe3+ was hydrolyzed in the bioleaching process and produces acid, which compensates for the acid consumption when Fe2+ was oxidized. Sampling analysis also showed that small yellow granules were present and suspended in the solution when 16 g/L of Fe2+ ions were added to the solution. Several studies reported dispersed jarosite in the solution had no adverse effect on bacteria growth and on bacterial leaching (Curutchet et al., 1992; Pogliani and Donati, 2000). Fig. 5b shows zinc extraction using MLY is indifferent to Fe2+ addition and that even jarosite precipitating on mineral surfaces might not decrease the leaching rate over the first 3 days. The SEM micrograph (Fig. 6a) shows that the surface of residue particles was covered by a porous layer after MLY bioleaching for 2 days with addition of 4 g/L Fe2+. ES shows that the granules contained S, O, Fe and the atomic ratio of Fe to S was 1.2 to 1. After leaching for 7 days, the surface coating became more compact (Fig. 6b) and ES shows the atomic ratio of Fe to S was 0.7 to 1. Because the atomic ratio
The authors acknowledge the National Natural Science Foundation of China (50574081, 50404009), 973 Program (2004CB619203) and the National High Technology Research and Development Program of China (2007AA060904).
References Boon, M., Snijder, M., Hansford, G.S., Heijnen, J.J., 1998. The oxidation kinetics of zinc sulphide with Thiobacillus ferrooxidans. Hydrometallurgy 48 (2), 171–186. Brierley, J.A., Brierley, C.L., 2001. Present and future commercial applications of biohydrometallurgy. Hydrometallurgy 59 (2–3), 233–239. Carranza, F., Iglesias, N., Romero, R., Palencia, I., 1993. Kinetics improvement of highgrade sulphides bioleaching by effects separation. FEMS Microbiology Reviews 11 (1–3), 129–138. Carranza, F., Palencia, I., Romero, R., 1997. Silver catalyzed IBES process: application to a Spanish copper–zinc sulphide concentrate. Hydrometallurgy 44 (1–2), 29–42. Choi, W.K., Torma, A.E., Ohline, R.W., Ghali, E., 1993. Electrochemical aspects of zinc sulphide leaching by Thiobacillus ferrooxidans. Hydrometallurgy 33 (1–2), 137–152. Crundwell, F.K., 1988. The influence of the electronic structure of solids on the anodic dissolution and leaching of semiconducting sulphide minerals. Hydrometallurgy 21 (2), 155–190. Curutchet, G., Pogliani, C., Donati, E., Tedesco, P., 1992. Effect of iron (IIl) and its hydrolysis products (jarosites) on Thiobacillus ferrooxidans growth and on bacterial leaching. Biotechnology Letters 14 (4), 329–334. Deveci, H., Akcil, A., Alp, I., 2004. Bioleaching of complex zinc sulphides using mesophilic and thermophilic bacteria: comparative importance of pH and iron. Hydrometallurgy 73 (3–4), 293–303. Edwards, K.J., Hu, B., Hamers, R.J., Banfield, J.F., 2001. A new look at microbial leaching patterns on sulfide minerals. FEMS Microbiology Ecology 34 (3), 197–206. Fowler, T.A., Crundwell, F.K., 1998. Leaching of zinc sulfide by Thiobacillus ferrooxidans: experiments with a controlled redox potential indicate no direct bacterial mechanism. Applied and Environmental Microbiology 64 (10), 3570–3575. Kleinjan, W.E., Keizer, A.D., Janssen, A.J.H., 2003. Biologically produced sulfur. Topics in Current Chemistry 230, 167–188. Konishi, Y., Kubo, H., Asai, S., 1992. Bioleaching of zinc sulfide concentrate by Thiobacillus ferrooxidans. Biotechnology and Bioengineering 39, 66–74. Kuenen, J.G., Pronk, J.T., Hazeu, W., Meulenberg, R., Bos, P., 1993. A review of bioenergetics and enzymology of sulfur compound oxidation by acidophilic thiobacilli. Biohydrometallurgical Technologies. Leathen, et al., 1951. A medium for the study of bacterial oxidation of ferrous ion. Science 114, 280–281. Li, Y.Q., He, Z.G., 2001. Studies on the characteristics of a moderately thermoacidophilic iron-oxidizing bacterium. Microbiology 28, 45–47.
56
S. Wang et al. / Hydrometallurgy 93 (2008) 51–56
Norris, P.R., Burton, N.P., Foulis, N.A.M., 2000. Acidophiles in bioreactor mineral processing. Extremophiles 4 (2), 71–76. Pogliani, C., Donati, E., 2000. Immobilisation of Thiobacillus ferrooxidans: importance of jarosite precipitation. Process Biochemistry 35 (9), 997–1004. Pronk, J.T., Meulenberg, R., Hazeu, W., Bos, P., Kuenen, J.G., 1990. Oxidation of reduced inorganic sulphur compounds by acidophilic thiobacilli. FEMS Microbiology Reviews 75, 293–306. Rodriguez, Y., Ballester, A., Blazquez, M.L., Gonzalez, F., Munoz, J.A., 2003. New information on the sphalerite bioleaching mechanism at low and high temperature. Hydrometallurgy 71 (1–2), 57–66. Romero, R., Mazuelos, A., Palencia, I., Carranza, F., 2003. Copper recovery from chalcopyrite concentrates by the BRISA process. Hydrometallurgy 70 (1–3), 205–215. Sand, W., Gehrke, T., Jozsa, P.G., Schippers, A., 2001. (Bio)chemistry of bacterial leaching— direct vs. indirect bioleaching. Hydrometallurgy 59 (2–3), 159–175.
Shi, S.-Y., Fang, Z.-H., Ni, J.-R., 2006a. Comparative study on the bioleaching of zinc sulphides. Process Biochemistry 41 (2), 438–446. Shi, S., Fang, Z., Ni, J., 2006b. Electrochemistry of marmatite–carbon paste electrode in the presence of bacterial strains. Bioelectrochemistry 68 (1), 113–118. Visser, J.M., Robertson, L.A., Verseveld, H.W., Kuenen, J.G., 1997. Sulfur production by obligately chemolithoautotrophic Thiobacillus species. Applied and Environmental Microbiology 63 (6), 2300–2305. Vogel, A., 1989. Vogel's Text Book of Quantitative Chemical Analysis. Longmans Ltd, London. Zhang, G., 2002. Study on mechanism and kinetics of the bioleaching of chalcopyrite. Ph.D. Thesis, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 30–31 pp. Zhang, G., Fang, Z., 2005. The contribution of direct and indirect actions in bioleaching of pentlandite. Hydrometallurgy 80 (1–2), 59–66.
Contents lists available at ScienceDirect
Hydrometallurgy j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / h yd r o m e t
Comparative study of external addition of Fe2+ and inoculum on bioleaching of marmatite flotation concentrate using mesophilic and moderate thermophilic bacteria Suting Wang a, Guangji Zhang a,⁎, Qiuhong Yuan a, Zhaoheng Fang a, Chao Yang a,b a b
Key Laboratory of Green Process and Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China
A R T I C L E
I N F O
Article history: Received 19 December 2007 Received in revised form 23 February 2008 Accepted 28 February 2008 Available online 18 March 2008 Keywords: Acidithiobacillus ferrooxidans Marmatite concentrate Thermo-acidophilic iron-oxidizing bacteria Inoculum
A B S T R A C T Bioleaching of marmatite flotation concentrate by Acidithiobacillus ferrooxidans and a moderately thermoacidophilic iron-oxidizing bacterial strain (MLY) was carried out in batch experiments. The effects of external addition of Fe2+ and the inoculum of the bacteria were studied. For A. ferrooxidans, the supplementary addition of Fe2+ as a source of Fe3+ to be generated by the bacteria was necessary in the starting of the bioleaching of marmatite. While for MLY, it was not necessary because the higher temperature improved the dissolution of marmatite in sulphuric acid, and thus acidic leaching happened before ferric ion leaching. Leaching rate was enhanced with the increased inoculum, however, this influence decreased at a higher level of inoculum. The evolution of the redox potential and the total concentration of iron in the bioleaching indicated that Eh was more important than the concentration of Fe3+ during the dissolution of the marmatite for both strains. Different affinities for the Fe2+ and S0 substrates led to different trends of leaching rate for the two bacteria. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The interaction between bacteria and sulfide minerals is the key issue in the fundamental study of bioleaching. Since the discovery of bacterium in acidic mine drainage, diversified opinions about the mechanism in bioleaching of sulfides have been put forward. The term ‘direct bioleaching’ was introduced by Silverman and Ehrlich in 1964 to describe a hypothesized enzymatic reaction taking place between attached cells and the underlying mineral surface. The ‘indirect’ mechanism of sulfide oxidation involves non-specific oxidation of surface by Fe3+ ions that are generated by iron-oxidizing microorganisms (Edwards et al., 2001). Sand and his collaborators proposed a new mode by analysis of degradation products occurring in the course of dissolution of metal sulfide. They suggested that ferric ions and/or protons are the only chemical agents which dissolve metal sulfides. The mechanism and chemistry of the degradation are decided by the mineral structure. According to their proposition, sphalerite (ZnS) is degradable by ferric ions and proton attack. The main intermediates are polysulfides and elemental sulphur (Sand et al., 2001). Although the bioleaching of zinc concentrates has been studied extensively (Choi et al., 1993; Deveci et al., 2004; Fowler and Crundwell, 1998; Konishi et al., 1992; Rodriguez et al., 2003), there is no unanimous agreement on the mechanisms, because leaching ⁎ Corresponding author. Tel.: +86 10 62554558; fax: +86 10 62561822. E-mail address: [email protected] (G. Zhang). 0304-386X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.hydromet.2008.02.019
processes are relatively complex and often vary with the composition of the minerals. Marmatite [(Zn,Fe)S] is an important resource of zinc ore in China, which is difficult to be processed effectively by the traditional technologies due to its low content of zinc. But it is more suitable for bioleaching (Shi et al., 2006a,b) and the rate of oxidative dissolution is higher compared with sphalerite due to its high content of iron (Crundwell, 1988). Shi studied the electrochemical behaviors of a marmatite–carbon paste electrode in the presence and absence of bacterial strain and analyzed the surface features of the marmatite particle by a scanning electron microscope (SEM). He concluded that the direct contact leaching by the cells attached on the mineral substrate played an important role on the dissolution of marmatite in addition to the chemical leaching of Fe3+ ions (Shi et al., 2006a,b). As an environmentally benign technology with wide application, bioleaching is featured with low cost for recovering metals from lowgrade ores. The most studied bacterium in bioleaching of sulfide ores is Acidithiobacillus ferrooxidans (abbreviated as A. ferrooxidans), which is a strain of mesophilic bacteria. However, A. ferrooxidans cannot grow at temperatures higher than 40 °C, and this limits its efficiency in leaching. Thermophilic bacteria attract more and more attention due to the potential in improving the kinetics of metal extraction (Brierley and Brierley, 2001; Deveci et al., 2004; Norris et al., 2000; Zhang and Fang, 2005). Based on the indirect mechanism, the IBES process (Indirect Bioleaching with Effects Separation) was developed to improve the kinetics of bioleaching of sulphide concentrates. In the IBES process, the bioleaching process is performed in two separate
52
S. Wang et al. / Hydrometallurgy 93 (2008) 51–56
2. Materials and methods 2.1. Mineral The marmatite flotation concentrate was obtained from a lead– zinc mine in Yunnan province of China. Chemical analysis of the sample revealed the composition of 40.61% Zn; 15.81% Fe; 2.63% Pb; 0.37% Cu; 0.14% Mg and 31.66% S. X-ray diffraction analysis showed the concentrate was mainly composed of marmatite and a small quantity of pyrite. The particle size was −42 μm (over 90%). 2.2. Strains and media
Fig. 1. Comparison of Fe2+ oxidation rate between A. ferrooxidans and MLY.
stages: (1) chemical leaching and (2) biological oxidation of the Fe2+ produced in the chemical stage (Carranza et al., 1993). This process has been applied to chalcopyrite/sphalerite concentrates and copper concentrates (Carranza et al., 1997; Romero et al., 2003). In this study, marmatite flotation concentrate was bioleached by A. ferrooxidans and a moderately thermoacidophilic iron-oxidizing bacterial strain MLY for improving the understanding of the mechanisms of marmatite bioleaching.
The strains of A. ferrooxidans and thermoacidophilic bacteria MLY were provided by the Institute of Microbiology, Chinese Academy of Sciences. Both strains were cultured in the Leathen medium (Leathen et al., 1951) with 10 g/L of Fe2+ ions. The iron-free Leathen medium contained 0.15 g/L (NH4)2SO4, 0.05 g/L KCl, 0.05 g/L K2HPO4, 0.5 g/L MgSO4·7H2O, 0.01 g/L Ca(NO3)2. Yeast extract (0.2 g/L) was added particularly to the medium to support the growth of MLY. The optimum pH and temperature for A. ferrooxidans and MLY are 2.0, 35 °C and 1.5, 50 °C, respectively. MLY was isolated from a coal spoil heap in China. It is able to oxidize Fe2+ ions, pyrite and elemental sulphur autotrophically and mixotrophically in the presence of yeast extract. Autotrophic oxidation of elemental sulphur is relatively weak. The comparison of Fe2+ oxidation between MLY and A. ferrooxidans indicated MLY is twice as fast as A. ferrooxidans (Fig. 1) (Li and He, 2001).
Fig. 2. A. ferrooxidans bioleaching of marmatite with 2 g/L added Fe2+ and different % inoculum.
S. Wang et al. / Hydrometallurgy 93 (2008) 51–56
To ensure that the iron in the original medium did not affect the results, cells were harvested in the stationary phase by filtration onto a Millipore filter (pore size 0.22 μm) and re-suspended in iron-free Leathen medium before leaching experiments started. The initial density of cells in the solution was approximately 1 × 107 cells/mL when 10% (v/v) bacteria inoculum was used. 2.3. Experimental procedures Leaching experiments were carried out in 250 mL Erlenmeyer flasks containing 100 mL solution with 5% (w/v) pulp density on an orbital shaker at 160 min− 1. Bioleaching with A. ferrooxidans was carried out at 35 °C and MLY at 50 °C. Each experimental run lasted 7 days. The pH value was measured at each sampling instant first (these data points being plotted in the figures) and then the medium was adjusted back to initial pH. Periodically, samples were taken and the make-up distilled water was added into the flasks to compensate for evaporation loss. In the first part of the experiments, the effects of inoculum of the bacteria were studied. Sterile control was also run to determine the contribution from chemical leaching of zinc. In the second part of the experiments, different concentrations of Fe2+ ions were added to the medium containing 10% (v/v) bacteria inoculum. 2.4. Measurements and analysis The concentration of dissolved zinc in leachate was determined by inductively coupled plasma atomic emission spectroscopy (ICP). The concentrations of Fe2+ ions and total iron were determined by spectrophotometry using the phenanthroline methods (Vogel, 1989). The number of free cells in the liquid samples was determined by direct counting using a hemacytometer. The pH value and redox potential (Eh) in the leaching solution were, respectively, measured with a pH meter and a Pt electrode with reference to a saturated calomel electrode (all potentials in this work were referred to SHE).The solid samples were dried and analyzed by SEM and electron spectroscopy (ES).
53
Elemental sulphur is the main intermediate of ferric ion attack. Some studies have demonstrated that if elemental sulphur was produced in bioleaching, for energetic reasons, the bacteria would prefer sulphur over ferrous ions (Boon et al., 1998; Kuenen et al., 1993; Pronk et al., 1990). In this period, the elemental sulphur could be the main energy resource of A. ferrooxidans, and this led to Eh of the slurry lower than that of the medium containing Fe2+. This presumption is confirmed by Fig. 2b and c. The total iron in the solution decreased due to the formation of jarosite and did not change significantly over the last 3 days and the leaching rate of zinc did not reduce. According to chemometric analysis, complete oxidation of marmatite produces sulphuric acid and oxidation from Fe2+ to Fe3+ by bacteria consumes sulphuric acid, so the whole process is neither acid producing nor acid consuming. Fig. 2d shows the increase of pH value over the first 3 days was due to the oxidation of iron by bacteria and then it decreased. Although pH was adjusted back to 2.0 after each sampling, it was above the initial value at the end of the leaching. A. ferrooxidans can convert part of sulfide to sulphur (H2Sn → S0 + 2H+ + 2e−) instead of sulfate + − (H2Sn + 4H2O → SO2− 4 + 10H + 8e ) (Visser et al., 1997) and the produced sulphur can be stored in the form of sulphur globules, located either inside or outside the cell (Kleinjan et al., 2003). So the incomplete oxidation of sulphur can lead to a rise of pH in the solution. A SEM micrograph (not shown) of the marmatite surface after 7 days of A. ferrooxidans bioleaching also showed elemental sulphur accumulation as determined by ES. Fig. 3a shows that at the same cell density, the growth of Eh slowed as more Fe2+ ions were added into the solution. The Eh in the solution reflects the ratio of Fe3+:Fe2+, so more addition of Fe2+ ions lowers Eh. As Fig. 3a and b shows, a higher leaching rate was achieved at higher
3. Results and discussion 3.1. Bioleaching of marmatite flotation concentrate by A. ferrooxidans Experimental results (data not shown) showed that it was difficult for A. ferrooxidans to survive in the leaching system without the addition of Fe2+ ions even when 40% bacteria inoculum was added. After 120 h, the Eh remained at about 0.55 V indicating no bacterial oxidation occurred. No change of cell number was observed in the bulk solution phase after 48 h. Fig. 2a presents the Eh changes when 2 g/L Fe2+ ions were added into the leaching system with different inoculums. For 40% inoculum, the Eh increased rapidly in the first 80 h, then fluctuated around 0.68 V. For 5% inoculum, the Eh showed an increasing trend. At the end of the bioleaching, similar Eh values and leached Zn (Fig. 2b) were reached under different inoculums except for the 1% inoculum. These results showed the inoculum did not have a serious impact on bioleaching. But the Eh (about 0.68 V) was lower than that of A. ferrooxidans cultured in the Leathen medium containing 10 g/L Fe2+ ions (about 0.8 V). According to the Sand's model, the marmatite bioleaching by A. ferrooxidans could be described as follows (Sand et al., 2001): MS þ Fe3þ þ Hþ →M2þ þ 0:5H2 Sn þ Fe2þ ðn≥2Þ
ð1Þ
0:5H2 Sn þ Fe3þ →0:125S8 þ Fe2þ þ Hþ
ð2Þ
þ 0:125S8 þ 1:5O2 þ H2 O→SO2 4 þ 2H :
ð3Þ
Fig. 3. A. ferrooxidans bioleaching of marmatite with 10% (v/v) bacteria inoculum and 0–20 g/L added Fe2+. a) Changes in Eh; b) concentration of Zn2+.
54
S. Wang et al. / Hydrometallurgy 93 (2008) 51–56
is necessary for the starting of the bioleaching by A. ferrooxidans. Our experiments suggest that without external addition of Fe2+ ions, marmatite cannot be leached by A. ferrooxidans, but further addition of Fe2+ ions slows the leaching rate of the mineral. 3.2. Bioleaching of marmatite flotation concentrate by MLY Fig. 4a shows that in the sterile control, the dissolved iron at 50 °C was nearly as twice as that at 35 °C. At higher temperature and lower pH, the solubility of the marmatite increases, so more Fe2+ is released into the solution. MLY could leach marmatite even with no external addition of Fe2+ ions. When the concentrations of total iron and Fe2+ ions in the leaching solution are compared, with and without bacteria inoculum at 50 °C, after the first day the concentrations of total iron in both leaching solutions were similar. In the sample without bacteria inoculum, all the iron in the solution existed as Fe2+ ion; whilst all the iron in the solution existed as Fe3+ ion with bacteria inoculum. The total iron quantity is significantly larger in bioleaching than that in the sterile control at the end of the experiments. This indicates that all the iron was produced by the dissolution of marmatite in sulphuric acid after the first day and the main role of MLY is to oxidize Fe2+ to Fe3+. It is assumed that at least three processes are involved in MLY bioleaching the marmatite with no addition of Fe2+: namely dissolution of marmatite in sulphuric acid; bacterial oxidation of the dissolved Fe2+; and Fe3+ oxidation of marmatite. Fig. 4b and c shows
Fig. 4. Sterile control and MLY in bioleaching marmatite with no added Fe2+ and different levels of inoculum. a) Concentration of iron in sterile control and 20% MLY at 35 °C and 50 °C; b) changes in Eh with 0–40% MLY; c) concentration of zinc with 0–40% MLY.
Eh, indicating that Eh is more important than the concentration of Fe3+ during the dissolution of marmatite. Marmatite can dissolve in dilute sulphuric acid (Crundwell, 1988), but the leaching rate is very slow. External addition of Fe2+ ions provides the initial energy source for bacteria, which can be oxidized to Fe3+ to accelerate the sulfide oxidation process by oxidizing S2− to S0. Meanwhile, Fe2+ in the marmatite is released and oxidized to Fe3+, so external addition of Fe2+
Fig. 5. MLY bioleaching of marmatite with 10% (v/v) bacteria inoculum and 2–16 g/L added Fe2+. a) Changes in Eh; b) concentration of zinc.
S. Wang et al. / Hydrometallurgy 93 (2008) 51–56
55
of Fe to S in jarosite is about 1.5 to 1, it suggests granules in Fig. 6a were mainly jarosite while in Fig. 6b were mainly elemental sulphur. It reflects the S increase at the end of bioleaching. After the surface coating was cleared away by ultrasonic treatment in trichloromethane, many pits were observed on the surface and some sulphur grains were still accumulated in the pits due to the incomplete cleaning (SEM micrograph not shown). Neither cells nor cell-shaped pits were found on the mineral surface. However, some studies indicated that even the formation of cell-sized and cell-shaped dissolution pits did not require a direct microbially induced surface reaction (Edwards et al., 2001). Several studies reported that yeast extract was consumed in MLY growth and MLY had a weaker oxidizing ability of sulphur under autotrophic condition (Li and He, 2001; Zhang, 2002). In our experiments, because of the consumption of yeast extract at the end of the bioleaching, more elemental sulphur accumulated on the marmatite surface according to the ES analysis. 4. Conclusions For A. ferrooxidans bioleaching, experimental results show that an appropriate amount of Fe2+ ions is needed to trigger marmatite bioleaching. At the end of bioleaching, incomplete oxidation of elemental sulphur leads to the pH rise. For moderate thermophilic bacteria MLY bioleaching, the higher temperature and acidity improve the dissolution of marmatite, thus MLY can leach marmatite without external addition of Fe2+ ions. The main role of MLY in the bioleaching of marmatite concentrate is to oxidize Fe2+. So MLY is suitable to be used in the IBES process. Acknowledgements
Fig. 6. Typical SEM images of leach residues from MLY bioleaching of marmatite with 4 g/L added Fe2+ ions: (a) residue after 2 days; (b) residue after 7 days.
that the increased inoculum contributed to an increase in Eh and helped to reach a higher leaching rate. However, this influence reduced with the lapse of time. When 5% MLY inoculum was added into the solution, the leached zinc was nearly the same as that leached in the sterile control experiment. But when 10% MLY inoculum was added, the leached zinc increased markedly. MLY cannot oxidize Fe2+ effectively at a low cell density but when more bacteria inoculum is added, MLY can oxidize Fe2+ to Fe3+ successfully. At the beginning of the bioleaching, Fe3+ ions play an important role in breaking down the ore to further supply source energy for bacteria multiplication. Fig. 5a shows the pH change in the medium. After the first 3 days, the more Fe2+ that was added to the solution, the slower the increase in pH. This indicates the existence of jarosite because a great deal of Fe3+ was hydrolyzed in the bioleaching process and produces acid, which compensates for the acid consumption when Fe2+ was oxidized. Sampling analysis also showed that small yellow granules were present and suspended in the solution when 16 g/L of Fe2+ ions were added to the solution. Several studies reported dispersed jarosite in the solution had no adverse effect on bacteria growth and on bacterial leaching (Curutchet et al., 1992; Pogliani and Donati, 2000). Fig. 5b shows zinc extraction using MLY is indifferent to Fe2+ addition and that even jarosite precipitating on mineral surfaces might not decrease the leaching rate over the first 3 days. The SEM micrograph (Fig. 6a) shows that the surface of residue particles was covered by a porous layer after MLY bioleaching for 2 days with addition of 4 g/L Fe2+. ES shows that the granules contained S, O, Fe and the atomic ratio of Fe to S was 1.2 to 1. After leaching for 7 days, the surface coating became more compact (Fig. 6b) and ES shows the atomic ratio of Fe to S was 0.7 to 1. Because the atomic ratio
The authors acknowledge the National Natural Science Foundation of China (50574081, 50404009), 973 Program (2004CB619203) and the National High Technology Research and Development Program of China (2007AA060904).
References Boon, M., Snijder, M., Hansford, G.S., Heijnen, J.J., 1998. The oxidation kinetics of zinc sulphide with Thiobacillus ferrooxidans. Hydrometallurgy 48 (2), 171–186. Brierley, J.A., Brierley, C.L., 2001. Present and future commercial applications of biohydrometallurgy. Hydrometallurgy 59 (2–3), 233–239. Carranza, F., Iglesias, N., Romero, R., Palencia, I., 1993. Kinetics improvement of highgrade sulphides bioleaching by effects separation. FEMS Microbiology Reviews 11 (1–3), 129–138. Carranza, F., Palencia, I., Romero, R., 1997. Silver catalyzed IBES process: application to a Spanish copper–zinc sulphide concentrate. Hydrometallurgy 44 (1–2), 29–42. Choi, W.K., Torma, A.E., Ohline, R.W., Ghali, E., 1993. Electrochemical aspects of zinc sulphide leaching by Thiobacillus ferrooxidans. Hydrometallurgy 33 (1–2), 137–152. Crundwell, F.K., 1988. The influence of the electronic structure of solids on the anodic dissolution and leaching of semiconducting sulphide minerals. Hydrometallurgy 21 (2), 155–190. Curutchet, G., Pogliani, C., Donati, E., Tedesco, P., 1992. Effect of iron (IIl) and its hydrolysis products (jarosites) on Thiobacillus ferrooxidans growth and on bacterial leaching. Biotechnology Letters 14 (4), 329–334. Deveci, H., Akcil, A., Alp, I., 2004. Bioleaching of complex zinc sulphides using mesophilic and thermophilic bacteria: comparative importance of pH and iron. Hydrometallurgy 73 (3–4), 293–303. Edwards, K.J., Hu, B., Hamers, R.J., Banfield, J.F., 2001. A new look at microbial leaching patterns on sulfide minerals. FEMS Microbiology Ecology 34 (3), 197–206. Fowler, T.A., Crundwell, F.K., 1998. Leaching of zinc sulfide by Thiobacillus ferrooxidans: experiments with a controlled redox potential indicate no direct bacterial mechanism. Applied and Environmental Microbiology 64 (10), 3570–3575. Kleinjan, W.E., Keizer, A.D., Janssen, A.J.H., 2003. Biologically produced sulfur. Topics in Current Chemistry 230, 167–188. Konishi, Y., Kubo, H., Asai, S., 1992. Bioleaching of zinc sulfide concentrate by Thiobacillus ferrooxidans. Biotechnology and Bioengineering 39, 66–74. Kuenen, J.G., Pronk, J.T., Hazeu, W., Meulenberg, R., Bos, P., 1993. A review of bioenergetics and enzymology of sulfur compound oxidation by acidophilic thiobacilli. Biohydrometallurgical Technologies. Leathen, et al., 1951. A medium for the study of bacterial oxidation of ferrous ion. Science 114, 280–281. Li, Y.Q., He, Z.G., 2001. Studies on the characteristics of a moderately thermoacidophilic iron-oxidizing bacterium. Microbiology 28, 45–47.
56
S. Wang et al. / Hydrometallurgy 93 (2008) 51–56
Norris, P.R., Burton, N.P., Foulis, N.A.M., 2000. Acidophiles in bioreactor mineral processing. Extremophiles 4 (2), 71–76. Pogliani, C., Donati, E., 2000. Immobilisation of Thiobacillus ferrooxidans: importance of jarosite precipitation. Process Biochemistry 35 (9), 997–1004. Pronk, J.T., Meulenberg, R., Hazeu, W., Bos, P., Kuenen, J.G., 1990. Oxidation of reduced inorganic sulphur compounds by acidophilic thiobacilli. FEMS Microbiology Reviews 75, 293–306. Rodriguez, Y., Ballester, A., Blazquez, M.L., Gonzalez, F., Munoz, J.A., 2003. New information on the sphalerite bioleaching mechanism at low and high temperature. Hydrometallurgy 71 (1–2), 57–66. Romero, R., Mazuelos, A., Palencia, I., Carranza, F., 2003. Copper recovery from chalcopyrite concentrates by the BRISA process. Hydrometallurgy 70 (1–3), 205–215. Sand, W., Gehrke, T., Jozsa, P.G., Schippers, A., 2001. (Bio)chemistry of bacterial leaching— direct vs. indirect bioleaching. Hydrometallurgy 59 (2–3), 159–175.
Shi, S.-Y., Fang, Z.-H., Ni, J.-R., 2006a. Comparative study on the bioleaching of zinc sulphides. Process Biochemistry 41 (2), 438–446. Shi, S., Fang, Z., Ni, J., 2006b. Electrochemistry of marmatite–carbon paste electrode in the presence of bacterial strains. Bioelectrochemistry 68 (1), 113–118. Visser, J.M., Robertson, L.A., Verseveld, H.W., Kuenen, J.G., 1997. Sulfur production by obligately chemolithoautotrophic Thiobacillus species. Applied and Environmental Microbiology 63 (6), 2300–2305. Vogel, A., 1989. Vogel's Text Book of Quantitative Chemical Analysis. Longmans Ltd, London. Zhang, G., 2002. Study on mechanism and kinetics of the bioleaching of chalcopyrite. Ph.D. Thesis, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 30–31 pp. Zhang, G., Fang, Z., 2005. The contribution of direct and indirect actions in bioleaching of pentlandite. Hydrometallurgy 80 (1–2), 59–66.