Available online at www.sciencedirect.com Bioresource Technology 99 (2008) 2840–2845 The effects of Fe(II) and Fe(III) concentration and initial pH on microbial leaching of low-grade sphalerite ore in a column reactor S.M. Mousavi a,b,*, S. Yaghmaei a, M. Vossoughi a, R. Roostaazad a, A. Jafari c, M. Ebrahimi d, O. Habibollahnia Chabok d, I. Turunen b a Department of Chemical and Petroleum Engineering, Sharif University of Technology, Tehran, Iran Department of Chemical Engineering, Lappeenranta University of Technology, Lappeenranta, Finland Department of Energy and Environmental Engineering, Lappeenranta University of Technology, Lappeenranta, Finland d Faculty of Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran b c Received 7 May 2007; received in revised form 12 June 2007; accepted 13 June 2007 Available online 14 August 2007 Abstract In this study the effects of initial concentration of Fe(II) and Fe(III) ions as well as initial pH on the bioleaching of a low-grade sphalerite ore in a leaching column over a period of 120 days with and without bacteria were investigated. Four different modifications of medium were used as column feed solutions to investigate the effects of initial concentration of Fe(II) and Fe(III) ions on zinc extraction. The experiments were carried out using a bench-scale, column leaching reactor, which was inoculated with mesophilic iron oxidizing bacteria, Acidithiobacillus ferrooxidans, initially isolated from the Sarcheshmeh chalcopyrite concentrate (Kerman, Iran). The effluent solutions were periodically analyzed for Zn, total Fe, Fe(II) and Fe(III) concentrations as well as pH values. Bacterial population was measured in the solution (free cells). Maximum zinc recovery in the column was achieved about 76% using medium free of initial ferrous ion and 11.4 g/L of ferric ion (medium 2) at pH 1.5. The extent of leaching of sphalerite ore with bacteria was significantly higher than that without bacteria (control) in the presence of ferrous ions. Fe(III) had a strong influence in zinc extraction, and did not adversely affect the growth of the bacteria population.  2007 Elsevier Ltd. All rights reserved. Keywords: Bioleaching; Low-grade sphalerite; Ferrous and ferric ions; Zinc extraction; Column reactor 1. Introduction Since high grade ore deposits are easily accessible, these become rapidly depleted. It thus becomes necessary to recover mineral resources from low-grade ore deposits. However, no appropriate technology is still available for recovery of metals from low-grade deposits. It is encouraging to find some microorganisms that could do it efficiently. * Corresponding author. Address: Department of Chemical and Petroleum Engineering, Sharif University of Technology, Tehran, Iran. Tel.: +98 21 66165494; fax: +98 21 66005417. E-mail address: mousavi@mehr.sharif.edu (S.M. Mousavi). 0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.06.009 The application of biohydrometallurgy to metal recovery is likely to grow because where a suitable mineral deposit is available, to quote Brierley and Brierley (2001), it offers advantages of operational simplicity, low capital and operating cost, and shorter construction times that no other alternative process can provide. Add to that, minimum environmental impact, and the use of this technology in the mining industry is set to increase. The potential benefits of commercial bioleaching of zinc minerals are significant in the exploitation of low-grade ores and in the treatment of zinc concentrates, which are difficult to process using conventional technologies such as direct smelting due to their low metal content. Bioleaching uses microbes to alter the physical or chemical properties of a metallic ore so that the metal can be extracted. S.M. Mousavi et al. / Bioresource Technology 99 (2008) 2840–2845 Metals can be extracted economically from low-grade sulfide or sulfide containing ore by exploiting metabolic activities of thiobacilli, particularly A. ferrooxidans. Common microbes in bioleaching processes are the acidophilic mesophilic bacteria A. ferrooxidans, Acidithiobacillus thiooxidans and Leptospirillum ferrooxidans, which grow optimally at around 30 C and low pH. The acidophilic, thermophilic archaebacteria, Acidianus brierleyi and Sulfolobus acidocaldarius, are also candidate microbes for accomplishing metals leaching. A. brierleyi and S. acidocaldarius have optimum pH of 1–2 and also optimum temperature of 60–70 C for activity. These acidophilic thermophiles are capable of oxidizing sulfide minerals and ferrous iron in the presence of dissolved heavy metals which are leached from metal sulfides (Konishi et al., 1998). Bacterial leaching of high-grade sulfide ores and concentrates is in use commercially and offers many cost advantages over other techniques such as pressure oxidation. Base metal bioleaching is concurrent with the recovery of the metals from a bleed solution stream by conventional downstream processes such as solvent extraction and electrowinning. The principal bioleaching techniques in use for the treatment of sulfide minerals include stirred tank leaching (Kinnunen et al., 2006), heap and dump leaching (Renman et al., 2006), and concentrate heap leaching (Sampson et al., 2005). Heap leaching provides both operating and capital cost advantages. However, the use of heap leaching is limited to cases in which the temperature can be maintained within the heap at a suitable temperature without external heating (Sampson et al., 2005). Recently, zinc sulfide bioleaching investigations have been carried out by some researchers in columns or in shake flasks (Mousavi et al., 2007, 2006a, b; Deveci et al., 2004; Pani et al., 2003; Rodrıguez et al., 2003), though the leaching effects reported were different in each study due to the different conditions and distinct properties of the mineral specimens used in the various experiments. Leaching in columns, with or without the recirculation of the leaching liquid, simulates percolation leaching because the conditions are very similar to those in the heap. Since results obtained in the laboratory can be extrapolated, with slight correction, to the real situation they will help show whether bacterial leaching is possible under acceptable conditions (Lizama and Suzuki, 1989). In this sense, we might consider the columns as the heart of the heap, with the same degree of access for the leaching solution and the circulating gases in both. In other words, a column experiment simulates the flow or, at least, one of the possible paths of a liquid percolating through a mass of material by gravity. The objective of this work was to investigate the effects of initial concentration of Fe(II) and Fe(III) ions as well as initial pH of feed solution on the leaching of a low-grade sphalerite ore in a leaching column. Four different modifications of medium were used as column feed solutions to assess the effects of initial concentration of ferrous and ferric ions on zinc extraction. 2841 2. Methods 2.1. Bacterial culture and nutrients The mesophilic iron oxidizing bacterium used in this work was A. ferrooxidans which was originally isolated from the Sarcheshmeh chalcopyrite concentrate (Kerman, Iran). To culture the bacteria were grown by the multiple transfer technique to the fresh medium which was contained the following components (g/L): FeSO4 Æ 7H2O, 33.4; (NH4)2SO4, 0.4; MgSO4 Æ 7H2O, 0.4 and K2HPO4, 0.4 (Atlas, 1997). The culture of A. ferrooxidans was incubated in 500 ml Erlenmeyer flask containing 200 ml of the medium and 10% (v/ v) inoculum, on a rotary shaker at 180 rpm at constant temperature of 33 C. The initial pH of the culture was adjusted to 1.5 using 1 N H2SO4. The cells toward the end of the logarithmic phase were harvested by centrifugation at 15,000·g for 10 min at 4 C. The cells were washed with acidified distilled water pH 1.5 and were finally resuspended in the same solution. The stock and pre-inoculum culture were maintained in the same medium under similar conditions. The cultures that were used had been subcultured through several transfers in sphalerite medium without ferrous sulfate to adapt the bacteria to the experimental conditions. The stock cultures were subcultured at two week intervals. To investigate the effects of Fe(II) and Fe(III) concentration on the bioleaching of a low-grade sphalerite ore in the column four different media containing different quantities of Fe(II) and Fe(III) ions were used. The concentration of ferrous and ferric ions in the medium 1, 2, 3, and 4 was 0,0; 0,11.4; 6.7,0 and 6.7,11.4 g/L, respectively. 2.2. Mineral characteristics The tests were performed on a low-grade sphalerite ore, which was the natural mineral obtained from the Kooshk lead–zinc mine (Yazd, Iran). The natural mineral was ground to obtain a sample in the size range of 10–20 mm. X-ray diffraction (XRD) analysis of the sample showed that the major components present were sphalerite (ZnS) (13%); pyrite (FeS2) (14%), and quartz (SiO2) (18%), while calcite (CaCO3) (5%) and galena (PbS) were the minor minerals. Chalcopyrite (CuFeS2) was present in trace amounts. The elemental composition of ore samples is based on averaged results obtained by X-ray fluorescence spectrometry (XRF). The chemical composition of the ore revealed S 22.8%, Fe 8%, Si 9.32%, Zn 8.9%, Pb 2.9%, CaO 2.3% and Cu less than 0.2%. 2.3. Apparatus and procedure A 120 cm high, 5 mm thick Plexiglas column with an internal diameter of 15 cm was used and loaded with around 55 kg of the ore. A HDPE support plate with multiple 10 mm holes was placed on 100 mm high supports allowing air to be injected below the plate and dispersed uniformly over the ores in the column. The column was 2842 S.M. Mousavi et al. / Bioresource Technology 99 (2008) 2840–2845 fed with different acidic solutions using a peristaltic pump at the rate of 5 L/(m2 h). Solution was applied to the surface of the column charge using a simple garden sprinkler head, of the type used in drip irrigation systems. The leach solution was passed through the ore sample by gravity and recirculated through a side loop with a peristaltic pump. In order to maintain feed at 30 C a water bath was used. A container with a capacity of 20 L collected the pregnant leach solution (PLS) draining from the column. The solution level was maintained at a sufficient height to provide a seal, forcing the air upwards through the column charge. In the leaching experiments the column system was comprised 12 L of medium solution (contained 10% inoculum with cell density of about 107 cells/mL). The column was aerated at a rate of 20 L/(m2 min). Sampling ports were taken at 30-cm intervals starting from the top of the column. Feed and PLS were sampled and analyzed to determine solution concentrations and metal dissolution. For all of the tests (except one test to assess the effects of initial pH) initial temperature and pH of column feed were 30 C and 1.5, respectively. Acid consumption is an important factor in the bacterial leaching processes. The amount of consumed acid by the sample ore during the column experiments was measured about 120 g acid/kg ore. Control tests were carried out by the addition of 5 ml of 0.5% (v/v) formaline in ethanol to the same medium. In order to analyze, the control samples were taken from the port 4, which was provided on the bottom of the column. 2.4. Analytical techniques Free bacteria in solution were counted by direct counting, using a Thoma chamber of 0.1 mm depth and 0.0025 mm2 area with an optical microscope (·1000). Soluble zinc and iron in the leached solutions were measured by an atomic absorption spectrophotometer (German, model AAS 5EA) after the filtration of the suspension through a 0.22 lm membrane filter to remove biomass. The solid residues were air dried and samples were taken for chemical analysis and X-ray diffraction (XRD). The ferric iron concentration in the solution was determined by 5-sulfosalicylic acid spectroscopy method (Varian Techtron UV– VIS spectrophotometer, model 635) (Karamanev et al., 2002). The ferrous iron concentration was ascertained by a volumetric method by titration with potassium dichromate. The pH of the cultural suspension and zinc extractive solutions were monitored at room temperature with a pH meter (Metrohm, model 691) calibrated with a low pH buffer. For all of experiments, chemical grade reagents and distilled water were used, with the exception of the chemical analysis in which double distilled water was used. 3. Results and discussion 3.1. Effects of Fe(II) and Fe(III) concentrations Fig. 1a–d depicts zinc dissolution in the presence of A. ferrooxidans with media containing different initial 80 Control 14 Port 1 12 Port 2 10 Port 3 8 Port 4 Control 70 Zn Recovery (%) Zn Recovery (%) 16 6 Port 1 60 Port 2 50 Port 3 40 Port 4 30 4 20 2 10 0 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 0 10 20 30 40 50 60 70 80 Time (Day) 90 100 110 120 130 40 50 60 70 80 Time (Day) 90 100 110 120 130 Time (Day) 60 70 Control 60 Port 1 Port 2 50 Port 3 40 Control 50 Zn Recovery (%) Zn Recovery (%) 80 Port 4 30 Port 1 Port 2 40 Port 3 30 Port 4 20 20 10 10 0 0 0 10 20 30 40 50 60 70 Time (Day) 80 90 100 110 120 130 0 10 20 30 Fig. 1. Zinc recovery using different media on bioleaching time: (a) free of ferric and ferrous ions (medium 1); (b) free of ferrous ion and 11.4 g/L of ferric ion (medium 2); (c) 6.7 g/L of ferrous ion and free of ferric ion (medium 3); (d) 6.7 g/L of ferrous ion and 11.4 g/L ferric ion (medium 4). 2843 S.M. Mousavi et al. / Bioresource Technology 99 (2008) 2840–2845 ZnS þ Fe2 ðSO4 Þ3 ! ZnSO4 þ S0 þ 2FeSO4 0 ZnS þ 1=2O2 þ H2 SO4 ! ZnSO4 þ S þ H2 O 2FeS2 þ 15=2O2 þ H2 O ! Fe2 ðSO4 Þ3 þ H2 SO4 : ð1Þ ð2Þ ð3Þ The percentages of iron precipitation have been estimated as the difference between the expected soluble iron concentration and the iron value at the end of leaching experiments divided by the expected soluble iron. Fig. 2 shows the greater jarosite precipitation occurred in the presence of bacteria perhaps because of the rapid oxidation of iron and the consequent increase of pH. In the presence of iron, according to the analysis, sulfur was detected on the sulfide surface as a product of the dissolution reaction from A. ferrooxidans culture suggesting that A. ferrooxidans in the pure culture can not oxidize the sulfur formed on the surface of the sphalerite to sulfate. This result is in good agreement with Sand et al. (1999). 3.2. Effect of initial pH 50 Percentage of iron precipitated (%) A. ferrooxidans 40 Control 30 20 10 0 medium 1 medium 2 medium 3 medium 4 Fig. 2. Percentages of iron precipitated during the bioleaching experiments in four different media. ing, range from 1.25 to 2.25 in a medium containing 6.7 g/L (medium 3) as initial Fe(II) concentration. Fig. 3 indicates that the best pH for sphalerite dissolution in the column was 1.5 at which the maximum bioleaching of zinc was 76%. Zinc extraction in solution was lower in the experiments carried out at pH 1.75, and 2 than that observed at pH 1.5. In the experiments carried out at pH 2 and higher, the reduction of zinc extraction can be credited to Fe(III) precipitation which probably took place under the jarosite form. Jarosite starts to precipitate at pH 2.0, thus reducing the oxidant (Fe(III)) concentration (Pina et al., 2005; Malhotra et al., 2002). There was also a reduction in the cell population in solution for the experiments carried out at pH 1.75 and 2. pHs below 1.5 are not recommend due to the slowing effect on the microorganism metabolism. The tolerance of acidophiles to most metals in low pH media probably results from effective competition by H+ ions for negatively-charged sites at the cell surface (Sharma and Verma, 1991). However, it is needless to mention that A. ferrooxidans is a gram-negative, acidophilic, mesophilic and chemoautotroph bacteria. In bioleaching of Zn from concentrates, pH sharply decreases with time which results in suppression of bacterial activity (Falco 80 pH=1.25 70 pH=1.5 60 Zn Recovery (%) concentrations of Fe(II) and Fe(III). Fig. 1a shows the extraction of zinc using medium free of initial Fe(II) and Fe(III) ions. In the experiment using medium 1, zinc extraction and bacterial population were lower than those where Fe(II) was presented. This is because there is a lack of substrate for bacterial growth and consequently of oxidant (ferric iron) for the sulfide oxidation. In the beginning of the experiments with media 2 and 4, because of the existence of Fe(III), sphalerite leaching accelerates (Figs. 1b and d). Since the oxidation occurs by a chemical mechanism and the rate of bacterial oxidation of ferrous ion is low in the initial stage of experiments. In the first 40 days, the zinc extraction obtained in the experiment with 11.4 g/L Fe(III) was around 24% (from port 4). This value is five times higher than that achieved when there was no Fe(III) addition (5%). High Fe(III) concentrations may affect A. ferrooxidans activity. Actually, the inhibitory effect of iron (III) on bacterial growth depends on the type of strain studied. According to Kawabe et al. (2003) the smallest value which the oxidation is completely inhibited is 16.75 g/L Fe(III), well above the values studied in this work. Zinc extraction was low in the absence of microorganisms (control) as compared with the experiments carried out with bacteria. Note that the results obtained from the control test should be compared with the results of port 4 because control samples were taken from mentioned port and in the column experiments, height of column is an important factor which has effect on the efficiency of zinc extraction, as it can be seen in Fig. 1a–d. The expected iron concentration without any precipitation of iron in the solution was calculated by adding the theoretical amount of iron leached from sphalerite based on the concentration of zinc in the solution to the iron concentration at the beginning of the experiments, according to the following reactions: pH=1.75 50 pH=2 40 pH=2.25 30 20 10 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Time (Day) One experimental run was performed testing the effect of the initial pH of the column feed over the sphalerite leach- Fig. 3. Effect of initial pH over the sphalerite leaching with A. ferrooxidans in a medium containing 6.7 g/L Fe(II) (medium 3). 2844 S.M. Mousavi et al. / Bioresource Technology 99 (2008) 2840–2845 4. Conclusion 1.E+09 20 days 60 days Cell density (cell/mL) 1.E+08 110 days 1.E+07 1.E+06 1.E+05 1.E+04 1.25 1.5 1.75 2 2.25 pH Fig. 4. Effects of initial pH and time on growth of A. ferrooxidans using medium 2. et al., 2003). In the experiments carried out in the present work negative effect due to the presence of calcite was not observed. In some experiments, attempts were made to enumerate bacteria by microscopic counts of effluent samples during batch experiments. Higher bacterial counts were observed at the lower pH ranges. This fact is in keeping with the dynamics of bioleaching, whereby active bacteria account for the formation of oxidized chemical species in the solution; the oxidation being coupled to growth, and thus to cell density. Fig. 4 indicates that the A. ferrooxidans population increased as a consequence of Fe(II) oxidation (in pHs 1.5 and 1.75). At the beginning of jarosite precipitation (pH 2), the free bacterial population started to decrease due to attachment to the deposits (Pogliani and Donati, 2000). It is known that Fe(II) oxidation by A. ferrooxidans decreases at a pH higher than 2. This is partially due to the reduction of the mass transfer on the cell surface by the formation of ferric iron precipitates. However, in the case of the Fe(II) oxidation by A. ferrooxidans, the proton is one of the reactants. Therefore, the decrease in the growth rate with an increase in pH cannot be avoided (Kai et al., 2007). The bacterial counts are based only on free bacteria in solution and no attempt was made to determine bacterial densities either qualitatively or quantitatively on mineral surfaces. At present, there is not sufficient information to evaluate the relative role of cell attachment in contributing to bacterial counts in active bioleaching systems. 3.3. Effect of time on bioleaching of sphalerite As can be seen in Fig. 1a–d for A. ferrooxidans the suitable time of leaching of low-grade zinc sulfide ore in the column is about 90 days. According to this figure about 40 days are required by the bacteria for the adaptation on the solid substrate (ore) system. This period is the initial lag-phase. The maximum Zn bioleaching was observed 76%. In the present attempt the effects of initial ferrous and ferric ions concentration as well as initial pH of feed solution on bacterial leaching of low-grade sphalerite ore using a column bioreactor were investigated. Bioleaching of zinc sulfide ore by an indigenous A. ferrooxidans was shown to be an effective bacterial leaching process. The maximum bioleaching of Zn from low-grade sphalerite ore in a column was obtained 76% using a medium free of ferrous ion and 11.4 g/L of ferric ion after 120 days. Experiments in this study confirm that ferric iron is a key component in sphalerite bioleaching. In the medium without initial addition of Fe(II), small amounts of iron were detected in the presence of iron-oxidizing bacteria. Under these conditions, zinc extractions were similar to those found in the medium supplemented with Fe(II), indicating that the role of iron-oxidizing bacteria was the oxidation of ferrous iron. pH is also a significant operating parameter governing the oxidative activity of bacteria and solubility of ferric iron. The suitable pH for leaching was observed 1.5. pHs below 1.5 are not recommended due to the slowing effect on the microorganism metabolism. Values above 2.0 are not suggested due to the precipitation of Fe(III) compounds such as jarosite. 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