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Effects of cyanide and dissolved oxygen concentration on biological Au recovery
Journal of Biotechnology 124 (2006) 545–551
Effects of cyanide and dissolved oxygen concentration on biological Au recovery Yoshito Kita a,∗ , Hiroshi Nishikawa b , Tadashi Takemoto b b
a Graduate School of Engineering, Osaka University, Japan Joining and Welding Research Institute, Osaka University, Japan
Received 4 August 2005; received in revised form 4 January 2006; accepted 13 January 2006
Abstract The number of discarded electric devices containing traces of Au is currently increasing. It is desirable to recover this Au because of its valuable physicochemical properties. Au is usually dissolved with relatively high concentrations of cyanide, which is associated with environmental risk. Chromobacterium violaceum is able to produce and detoxify small amounts of cyanide, and may thus be able to recover Au from discarded electric devices. This study investigated the effects of cyanide and dissolved oxygen concentration on biological Au recovery. Cyanide production by C. violaceum was sufficient to dissolve Au, while maintaining a high cyanide concentration did not enhance Au dissolution. Increased oxygen concentration enhanced Au dissolution from 0.04 to 0.16 mmol/l within the test period of 70 h. Electrochemical measurement clarified this phenomenon; the rest potential of Au in the cyanide solution produced by C. violaceum increased from −400 to −200 mV, while in the sterile cyanide solution, it was constant in cyanide concentrations ranging from 0 to 1.5 mmol/l and increased in dissolved oxygen concentrations ranging from 0 to 0.25 mmol/l. Therefore, it was clarified that dissolved oxygen concentration is the main factor affecting the efficiency of cyanide leaching of gold by using bacteria. © 2006 Elsevier B.V. All rights reserved. Keywords: Gold bioleaching; Chromobacterium violaceum; Anodic polarization curve; Rest potential; Dissolved oxygen; Cyanide
1. Introduction The demand for Au in printed-circuit boards has increased with the advancement of technology. At the same time, the Au present in such boards is decreasing due to advanced plating and joining techniques. ∗
Corresponding author. Fax: +81 6 6879 8691. E-mail address: [email protected] (Y. Kita).
0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2006.01.038
Because Au is a precious metal and is highly valued for its physicochemical properties, it is necessary to recover Au from industrial waste (Fujita and Yen, 2001). At present, scrap-containing Au is generally treated using hydrometallurgical methods such as aqua regia or cyanidation. However, excessive use of cyanide for the dissolution of Au is associated with environmental risk, and thus biological methods for Au dissolution that produce, and subsequently detoxify, small amounts
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of cyanide are being investigated (Norman and Raforth, 1995; Olson, 1994). Chromobacterium violaceum is a mesophilic, motile, Gram-negative, facultative anaerobe (Moss et al., 1981). Strains of this microorganism can produce cyanide (Michaels and Corpe, 1965), and can subsequently detoxify cyanide using -cyanoalanine synthase, when grown in minimal medium (Macadam and Knowles, 1984; Rodgers, 1978, 1982). Therefore, this strain can potentially be used in ecological Au recovery methods (Faramarzia et al., 2004). C. violaceum grown in nutrient broth can dissolve Au powder. Studies of Au dissolution using C. violaceum have investigated the effects of pH (Smith and Hunt, 1985), ore type and content (Lawson et al., 1999), as well as alternative culture conditions (Cambell et al., 2001). In these studies, the Au dissolution rate was found to be almost the same, even though the cyanide concentration varied. Thus, Au inhibitors must be identified in order to increase Au solubility. Au dissolution in cyanide solution consists of an anodic (1) and a cathodic (2) reaction and is summarized by Elsner’s Eq. (3) as follows (Hedley and Tabachnick, 1958). 4Au + 8CN− → 4Au(CN)2 − + 4e−
(1)
O2 + 2H2 O + 4e− → 4OH−
(2)
4Au + 8CN− + O2 + 2H2 O → 4Au(CN)2 − + 4OH−
(3)
The rate of Au dissolution depends on several factors, including cyanide concentration, surface area of Au particles, mixing, solution conditions (temperature and pH), interference from other materials and dissolved oxygen concentration (Haque, 1992). Electrochemically, cyanide and oxygen concentration are important factors for dissolving Au (Heath and Rumball, 1998; Peter and Wesley, 1995; Wadsworth, 1999). For example, Au dissolution from milled Au ores is proportional to the square root of cyanide and oxygen concentrations (Crundwell and Godorr, 1997). However, only sterile cyanide solutions are used in such studies, and thus electrochemical study of Au in bacterial cyanide solution has not yet been investigated. This study aims to investigate the effects of cyanide and dissolved oxygen concentration and to increase the
Au dissolution rate during Au recovery using C. violaceum. The measurement of the anodic polarization curve in the solution was utilized to investigate the effects of cyanide and dissolved oxygen concentration.
2. Materials and methods 2.1. Organism and culture conditions C. violaceum NBRC 12614 was grown in flasks containing YP medium (polypepton 10 g, yeast extract 2 g, MgSO4 ·7H2 O 1 g, distilled water 1 l). C. violaceum was incubated at 30 ◦ C with agitation at 110 rpm. Longterm storage was at −80 ◦ C. 2.2. Au recovery from powder Au powder (superficial area, 0.4 m2 /g; average particle diameter, 1.1 m; tap density, 6.4 g/cm3 ) was added to a 250-ml conical flask containing 150 ml of YP medium inoculated with 1.5 ml of actively growing C. violaceum culture. To accelerate the cyanide production of C. violaceum, phosphate buffer (sodium dihydrogen phosphate 50 mmol; disodium hydrogen phosphate, 50 mmol) was added to 1 l of YP medium. Phosphate buffer is able to maintain the culture at pH 7.0, at which C. violaceum cyanide production is maximized (graph not shown). In order to increase the dissolved oxygen concentration, aseptic air was blown into the beaker at 100 ml/min. The solution was then incubated at 30 ◦ C with mixing at 110 rpm. 2.3. Electrochemical measurements The anodic polarization curves of the solution inoculated with C. violaceum and sterile cyanide solution were measured using a potentiostat and a function generator in order to investigate the electrochemical dissolution of Au. Anodic polarization was measured from −800 to 1000 mV, with a sweep rate of 1 mV/s, using an electrode of 99.99% Au. Pt and Ag/AgCl were used as the counter and standard reference electrodes, respectively. Electrode surfaces were polished using emery paper and were finished with 1 m diamond powder. Electrodes were encapsulated in epoxy resin and at one end in order to expose a surface area of 1.0 cm2 . The solution was maintained at 30 ◦ C and was stirred.
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Fig. 1. Schematic diagram of electric chemical experiment.
Sterile cyanide solution was prepared using potassium cyanide and dissolved oxygen was controlled by changing the N2 :O2 ratio of the mixed gas at a flow rate of 75 ml/min. Fig. 1 presents a schematic diagram of the electrochemical experiment.
(0–24 h), a stationary phase (24–120 h) and a declining phase (120–290 h). Cyanide concentration increased after inoculation of C. violaceum and reached a peak (0.65 mmol/l) in the growth phase, before decreas-
2.4. Analysis Organism growth was monitored by plate counts on YP medium agar (YP medium with agar, 15 g). Viable suspended cells were counted using the drop plate method with serial dilution on YP medium. Plates were incubated for 24 h at 37 ◦ C. Total cyanide concentration was analyzed colorimetrically at 520 nm using distillation and the picric acid colorimetric method. Au content was analyzed by atomic absorption analysis. Dissolved oxygen was analyzed using the diaphragm galvanic battery method.
3. Results and discussion 3.1. Au, cyanide and dissolved oxygen concentration during the growth of bacteria C. violaceum grown in YP culture produced cyanide and solubilized Au from Au powder (Fig. 2). Fig. 2(a) shows the C. violaceum cell population and the Au dissolution, while Fig. 2(b) shows the cyanide and dissolved oxygen concentrations. Three phases could be seen for the cell population; a growth phase
Fig. 2. Gold dissolution from Au powder by using cyanide production of Chromobacterium violaceum. (a) Cell population C. violaceum (×) and Au concentration () in the solution. (b) Cyanide concentration () and dissolved oxygen concentration () in the solution.
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ing (0.20 mmol/l) in the stationary and declining phases. In contrast, dissolved oxygen concentration rapidly decreased in the growth phase, remained low (0.05 mmol/l) in the stationary phase and increased in the declining phase. The Au dissolution rate was low in the growth and stationary phases but became higher in the declining phase. C. violaceum actively produces cyanide during the growth phase and converts it into -cyanoalanine during the stationary and declining phases, as reported previously (Rodgers, 1978). Based on the dissolved oxygen concentration, it is clear that C. violaceum consume dissolved oxygen for bacterial respiration during the growth and stationary phases, and that consumption ends after the declining phase (Foucher et al., 2003). The increased rate of Au dissolution after the declining phase implies that the rate of Au dissolution depends on both cyanide concentration and dissolved oxygen concentration. 3.2. Effects of cyanide and dissolved oxygen concentration on bacterial Au dissolution Fig. 3 shows the cyanide and Au concentrations in C. violaceum-inoculated solution with or without 100 mmol/l phosphate buffer. With phosphate buffer, the peak value of cyanide concentration during the growth phase increased from 0.65 to 1.15 mmol/l, and the cyanide concentration during the stationary and declining phases increased from 0.20 to 0.52 mmol/l, as shown in Fig. 3(a). However, the Au dissolution rate remained almost unchanged, as shown in Fig. 3(b). This demonstrates that bacterial Au dissolution rate does not only depend on cyanide concentration. Fig. 4 shows the dissolved oxygen and Au concentrations in C. violaceum-inoculated solution with or without aeration. The dissolved oxygen concentration in both solutions remained below 0.02 mmol/l from 0 to 17 h, but it increased to 0.04 mmol/l without aeration and to 0.10 mmol/l with aeration after 20 h, as shown in Fig. 4(a). Similarly, the Au dissolution rate of both solutions remained around 0 mmol/l from 0 to 17 h and Au dissolution started after 20 h but the dissolution rate with aeration was higher than that without aeration, as shown in Fig. 4(b). It is thus clear that bacterial Au dissolution rate depends on dissolved oxygen concentration.
Fig. 3. Gold dissolution in solution in experiments involving Au powder with and without buffer. (a) Cyanide concentration in solution with () and without () 100 mM phosphate buffer. (b) Au concentration in solution with (䊉) and without () 100 mM phosphate buffer.
3.3. Electrochemical measurement 3.3.1. Anodic behavior of Au in solution with C. violaceum Fig. 5(a) shows the linear sweep voltammograms of solutions in which C. violaceum was incubated for 24, 72, 120, 216 and 358 h (shown in Fig. 2(b)). Current densities on the Au electrode at −800 mV in the solution after 24, 72 and 120 h incubation were smaller than those after 216 and 358 h. In contrast, current densities at +200 mV in the solution after 24, 72, 120 and 216 h were similar, while that after 358 h was slightly smaller. Thus, the cathodic reaction changes, and the anodic reaction does not. Therefore, bacterial Au dissolution is inhibited by the cathodic reaction at 24, 72 and 120 h of incubation (corresponding to the growth and stationary phases) and is accelerated at 216 and 358 h of incubation (corresponding to the declining phase). Fig. 5(b) shows the rest potentials derived from Fig. 5(a). The rest potential decreased from −300
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Fig. 4. Gold dissolution in solution in experiments involving Au powder with and without aeration. (a) Dissolved oxygen concentration in solution with () and without () aeration. (b) Au concentration in solution with (䊉) and without () aeration.
to −400 mV during the growth phase, remained at −400 mV during the stationary phase, and increased to −200 mV during the declining phase. Figs. 2(b) and 5(b) demonstrate that the changes in rest potential are similar to the changes in dissolved oxygen concentration. Ordinary exchange current density is used to determine the metal dissolution rate, but bacterial Au dissolution is not sufficiently large to measure, as shown in Fig. 5(a). Therefore, the rest potential can be substituted for the exchange current density when determining Au dissolution rate. 3.3.2. Effects of cyanide and dissolved oxygen concentration Fig. 6(a) shows the effect of cyanide concentration on Au dissolution in sterile cyanide solution at a dissolved oxygen concentration of 0.25 mmol/l and Fig. 6(b) depicts the rest potential curve derived from Fig. 6(a). As cyanide concentration increased, current density at +200 mV increased, while that at −800 mV
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Fig. 5. (a) Linear sweep voltammograms of Au electrode in the solution in which C. violaceum is incubated at 24, 72, 120, 216 and 358 h as shown in Fig. 2. pH 7.0, 30 ◦ C, 200 rpm rotation speed, 1 mV/s sweep rate. (b) Rest potential taken from linear sweep voltammograms.
remained almost constant. Rest potential was constant at −200 mV when the cyanide concentration is less than 1.0 mmol/l, although it decreased as cyanide concentration increased when the cyanide concentration is more than 1.0 mmol/l. In the bacterial solution cyanide concentrations is from 0.1 to 1.0 mmol/l then rest potential must be −200 mV but it decreased as shown in Fig. 5(b). Electrochemically, it is clear that bacterial Au dissolution rate does not only depend on cyanide concentration. Fig. 7(a) shows the effect of dissolved oxygen concentration on Au dissolution in sterile cyanide solution at a cyanide concentration of 1.0 mmol/l and Fig. 7(b) depicts the rest potential curve derived from Fig. 7(a). As dissolved oxygen increased, current density at −800 mV increased, while that at +200 mV remained almost constant. Rest potential also increased as dissolved oxygen increased. In this experiment, the
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Fig. 6. (a) Linear sweep voltammograms of Au electrode in 0.25 mmol/l oxygen cyanide solutions. pH 7.0, 30 ◦ C, 200 rpm rotation speed, 1 mV/s sweep rate. (b) Rest potential taken from linear sweep voltammograms.
reaction is dominated by the cathodic reaction, and at dissolved oxygen concentrations between 0 and 0.3 mmol/l, which is achieved by aeration, the cathodic reaction is activated as dissolved oxygen increased. It was thus demonstrated electrochemically that dissolved oxygen is a vital factor in bacterial Au dissolution. The rest potential of Au electrode in the bacterial solution shown in Fig. 5 can be explained by the results of that in the sterile cyanide solutions shown in Figs. 6 and 7. Fig. 8 shows the changes in the rest potential in the bacterial solution. The lines between A and B show the cathodic polarization curves as shown in Fig. 7(a) and the line shifts from B to A with the increase of dissolved oxygen concentration. The lines between C and D show the anodic polarization curves as shown in Fig. 6(a) and the line shifts from C to D with the increase of cyanide concentration. The shift range between C and D is smaller than that between A and B because anodic curves are similar when cyanide
Fig. 7. (a) Linear sweep voltammograms of Au electrode in 1.0 mmol/l cyanide solutions containing various oxygen concentrations. pH 7.0, 30 ◦ C, 200 rpm rotation speed, 1 mV/s sweep rate. (b) Rest potential taken from linear sweep voltammograms.
concentration is less than 1.0 mmol/l as shown in Fig. 6(a). As can be seen in Figs. 5(b) and 8, in the growth phase, the rest potential decreases because the anodic
Fig. 8. The change in the rest potential of Au on biological gold leaching shown in Figs. 2 and 5. The lines between A and B symbolize cathodic curves and the lines between C and D symbolize anodic curves.
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line shifts from C to D due to cyanide production by C. violaceum and the cathodic line shifts from A to B due to dissolved oxygen consumption by bacterial respiration. In the stationary phase, it is fairly stable because the anodic line shifts from D to C due to cyanide decomposition by C. violaceum and cathodic line is stable. In the declining phase, it increases because the cathodic reaction is accelerated by the decrease in bacterial respiration. The movement of the rest potential on bacterial Au dissolution was explained electrochemically and it was cleared that oxygen supply is important at growth and stationary phase for high speed Au dissolution. 4. Conclusion C. violaceum, which is able to produce and decompose cyanide, was used to dissolve Au. Bacterial cell population and cyanide/dissolved oxygen concentration were measured in order to investigate the effects of cyanide/dissolved oxygen concentration on dissolution of Au powder. Anodic polarization curves of cyanide solutions with or without C. violaceum were used for electrochemical study of bacterial Au dissolution. Based on the results presented in this paper, the following conclusions can be drawn: 1. In the growth phase, C. violaceum produces cyanide and rapidly consumes dissolved oxygen for bacterial respiration. This decrease in dissolved oxygen concentration inhibits Au dissolution. 2. The increase in cyanide concentration did not affect Au dissolution but the decrease in dissolved oxygen concentration inhibits Au dissolution in the growth phase. 3. Aeration of the bacterial solution effectively increases the Au dissolution rate. 4. Electrochemical study of the sterile cyanide solution confirmed that bacterial Au dissolution rate depends on dissolved oxygen concentration (consumed by C. violaceum). References Cambell, S.C., Olson, G.J., Clark, T.R., McFeters, G., 2001. Biogenic production of cyanide and its application to Au recovery. J. Ind. Microbiol. Biotechnol. 26, 134–139.
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Crundwell, F., Godorr, S., 1997. A mathematical model of the leaching of Au in cyanide solutions. Hydrometallurgy 44, 147– 162. Faramarzia, M.A., Stagarsa, M., Pensinib, E., Krebsb, W., Brandl, H., 2004. Metal solubilization from metal-containing solid materials by cyanogenic Chromobacterium violaceum. J. Biotech. 113 (1–3), 321–326. Foucher, S., Battaglia-Brunet, F., Hugues, P., 2003. Evolution of the bacterial population during the batch bioleaching of a cobaltiferous pyrite in a suspended-solids bubble column and comparison with a mechanically agitated reactor. Hydrometallurgy 71, 5–12. Fujita, T., Yen, W.T., 2001. Au leaching from electric machine part scraps using thiosulfate or hypochlorite. In: Symposium on Actions for Environmental Concern in Electronics Assembly, pp. 118–127. Haque, K., 1992. The role of oxygen in cyanide leaching of Au ore. CIM Bull. 85-963, 31–38. Heath, A.R., Rumball, J.A., 1998. Optimising cyanide oxygen ratios in Au CIP/CIL circuit. Min. Eng. 11, 999–1010. Hedley, H., Tabachnick, H., 1958. Chemistry of cyanidation. In: Mineral Dressing Notes, No.23. American Cyanamid Co. Lawson, E.N., Barkhuizen, M., Dew, D.W., 1999. Au solubilization by the cyanide producing bacteria Chromobacterium violaceum. In: Amils, R., Ballester, A. (Eds.), Biohydrometallurgy and the Environment Toward the Mining of 21st Century, pp. 239– 246. Macadam, A.M., Knowles, C.J., 1984. Furification and properties of -cyano-l-alanine synthase from the cyanide-producing bacterium, Chromobacterium violaceum. Biochem. Biophys. Acta 786, 123–132. Michaels, R., Corpe, W., 1965. Cyanide formation by Chromobacterium violaceum. J. Bacteriol. 89 (1), 106–112. Moss, M., Ryall, Cn, 1981. The genus chromobacterium. In: The Prokaryotes, II, second ed. Springer-Verlag, New York, NY, pp. 1356–1364. Norman, D., Raforth, R., 1995. Cyanide heap leaching—the process, environmental problems, and regulation in Washington. Wash. Geol. 23 (1), 30–41. Olson, G., 1994. Microbial oxidation of Au ores and Au bioleaching. FEMS Microbiol. Lett. 119, 1–6. Peter, D.K., Wesley, F.G., 1995. The use of oxygen in Au cyanidation. Can. Metull. Quart. 35, 39–45. Rodgers, P.B., 1978. Cyanide production and degradation during growth of Chromobacterium violaceum. J. Gen. Microbiol. 108, 261–267. Rodgers, P.B., 1982. Cyanide metabolism and -cyanoalanine formation by washd, nonproliferating cultures of Chromobacterium violaceum. J. Gen. Microbiol. 128, 2983– 2989. Smith, A., Hunt, R., 1985. Solubilisation of Au by Chromobacterium violaceum. J. Chem. Technol. Biotechnol. B 35, 110– 116. Wadsworth, M.E., 1999. Surface processes in silver and Au cyanidation. Int. J. Miner. Process 58, 351–368.
Effects of cyanide and dissolved oxygen concentration on biological Au recovery Yoshito Kita a,∗ , Hiroshi Nishikawa b , Tadashi Takemoto b b
a Graduate School of Engineering, Osaka University, Japan Joining and Welding Research Institute, Osaka University, Japan
Received 4 August 2005; received in revised form 4 January 2006; accepted 13 January 2006
Abstract The number of discarded electric devices containing traces of Au is currently increasing. It is desirable to recover this Au because of its valuable physicochemical properties. Au is usually dissolved with relatively high concentrations of cyanide, which is associated with environmental risk. Chromobacterium violaceum is able to produce and detoxify small amounts of cyanide, and may thus be able to recover Au from discarded electric devices. This study investigated the effects of cyanide and dissolved oxygen concentration on biological Au recovery. Cyanide production by C. violaceum was sufficient to dissolve Au, while maintaining a high cyanide concentration did not enhance Au dissolution. Increased oxygen concentration enhanced Au dissolution from 0.04 to 0.16 mmol/l within the test period of 70 h. Electrochemical measurement clarified this phenomenon; the rest potential of Au in the cyanide solution produced by C. violaceum increased from −400 to −200 mV, while in the sterile cyanide solution, it was constant in cyanide concentrations ranging from 0 to 1.5 mmol/l and increased in dissolved oxygen concentrations ranging from 0 to 0.25 mmol/l. Therefore, it was clarified that dissolved oxygen concentration is the main factor affecting the efficiency of cyanide leaching of gold by using bacteria. © 2006 Elsevier B.V. All rights reserved. Keywords: Gold bioleaching; Chromobacterium violaceum; Anodic polarization curve; Rest potential; Dissolved oxygen; Cyanide
1. Introduction The demand for Au in printed-circuit boards has increased with the advancement of technology. At the same time, the Au present in such boards is decreasing due to advanced plating and joining techniques. ∗
Corresponding author. Fax: +81 6 6879 8691. E-mail address: [email protected] (Y. Kita).
0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2006.01.038
Because Au is a precious metal and is highly valued for its physicochemical properties, it is necessary to recover Au from industrial waste (Fujita and Yen, 2001). At present, scrap-containing Au is generally treated using hydrometallurgical methods such as aqua regia or cyanidation. However, excessive use of cyanide for the dissolution of Au is associated with environmental risk, and thus biological methods for Au dissolution that produce, and subsequently detoxify, small amounts
546
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of cyanide are being investigated (Norman and Raforth, 1995; Olson, 1994). Chromobacterium violaceum is a mesophilic, motile, Gram-negative, facultative anaerobe (Moss et al., 1981). Strains of this microorganism can produce cyanide (Michaels and Corpe, 1965), and can subsequently detoxify cyanide using -cyanoalanine synthase, when grown in minimal medium (Macadam and Knowles, 1984; Rodgers, 1978, 1982). Therefore, this strain can potentially be used in ecological Au recovery methods (Faramarzia et al., 2004). C. violaceum grown in nutrient broth can dissolve Au powder. Studies of Au dissolution using C. violaceum have investigated the effects of pH (Smith and Hunt, 1985), ore type and content (Lawson et al., 1999), as well as alternative culture conditions (Cambell et al., 2001). In these studies, the Au dissolution rate was found to be almost the same, even though the cyanide concentration varied. Thus, Au inhibitors must be identified in order to increase Au solubility. Au dissolution in cyanide solution consists of an anodic (1) and a cathodic (2) reaction and is summarized by Elsner’s Eq. (3) as follows (Hedley and Tabachnick, 1958). 4Au + 8CN− → 4Au(CN)2 − + 4e−
(1)
O2 + 2H2 O + 4e− → 4OH−
(2)
4Au + 8CN− + O2 + 2H2 O → 4Au(CN)2 − + 4OH−
(3)
The rate of Au dissolution depends on several factors, including cyanide concentration, surface area of Au particles, mixing, solution conditions (temperature and pH), interference from other materials and dissolved oxygen concentration (Haque, 1992). Electrochemically, cyanide and oxygen concentration are important factors for dissolving Au (Heath and Rumball, 1998; Peter and Wesley, 1995; Wadsworth, 1999). For example, Au dissolution from milled Au ores is proportional to the square root of cyanide and oxygen concentrations (Crundwell and Godorr, 1997). However, only sterile cyanide solutions are used in such studies, and thus electrochemical study of Au in bacterial cyanide solution has not yet been investigated. This study aims to investigate the effects of cyanide and dissolved oxygen concentration and to increase the
Au dissolution rate during Au recovery using C. violaceum. The measurement of the anodic polarization curve in the solution was utilized to investigate the effects of cyanide and dissolved oxygen concentration.
2. Materials and methods 2.1. Organism and culture conditions C. violaceum NBRC 12614 was grown in flasks containing YP medium (polypepton 10 g, yeast extract 2 g, MgSO4 ·7H2 O 1 g, distilled water 1 l). C. violaceum was incubated at 30 ◦ C with agitation at 110 rpm. Longterm storage was at −80 ◦ C. 2.2. Au recovery from powder Au powder (superficial area, 0.4 m2 /g; average particle diameter, 1.1 m; tap density, 6.4 g/cm3 ) was added to a 250-ml conical flask containing 150 ml of YP medium inoculated with 1.5 ml of actively growing C. violaceum culture. To accelerate the cyanide production of C. violaceum, phosphate buffer (sodium dihydrogen phosphate 50 mmol; disodium hydrogen phosphate, 50 mmol) was added to 1 l of YP medium. Phosphate buffer is able to maintain the culture at pH 7.0, at which C. violaceum cyanide production is maximized (graph not shown). In order to increase the dissolved oxygen concentration, aseptic air was blown into the beaker at 100 ml/min. The solution was then incubated at 30 ◦ C with mixing at 110 rpm. 2.3. Electrochemical measurements The anodic polarization curves of the solution inoculated with C. violaceum and sterile cyanide solution were measured using a potentiostat and a function generator in order to investigate the electrochemical dissolution of Au. Anodic polarization was measured from −800 to 1000 mV, with a sweep rate of 1 mV/s, using an electrode of 99.99% Au. Pt and Ag/AgCl were used as the counter and standard reference electrodes, respectively. Electrode surfaces were polished using emery paper and were finished with 1 m diamond powder. Electrodes were encapsulated in epoxy resin and at one end in order to expose a surface area of 1.0 cm2 . The solution was maintained at 30 ◦ C and was stirred.
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547
Fig. 1. Schematic diagram of electric chemical experiment.
Sterile cyanide solution was prepared using potassium cyanide and dissolved oxygen was controlled by changing the N2 :O2 ratio of the mixed gas at a flow rate of 75 ml/min. Fig. 1 presents a schematic diagram of the electrochemical experiment.
(0–24 h), a stationary phase (24–120 h) and a declining phase (120–290 h). Cyanide concentration increased after inoculation of C. violaceum and reached a peak (0.65 mmol/l) in the growth phase, before decreas-
2.4. Analysis Organism growth was monitored by plate counts on YP medium agar (YP medium with agar, 15 g). Viable suspended cells were counted using the drop plate method with serial dilution on YP medium. Plates were incubated for 24 h at 37 ◦ C. Total cyanide concentration was analyzed colorimetrically at 520 nm using distillation and the picric acid colorimetric method. Au content was analyzed by atomic absorption analysis. Dissolved oxygen was analyzed using the diaphragm galvanic battery method.
3. Results and discussion 3.1. Au, cyanide and dissolved oxygen concentration during the growth of bacteria C. violaceum grown in YP culture produced cyanide and solubilized Au from Au powder (Fig. 2). Fig. 2(a) shows the C. violaceum cell population and the Au dissolution, while Fig. 2(b) shows the cyanide and dissolved oxygen concentrations. Three phases could be seen for the cell population; a growth phase
Fig. 2. Gold dissolution from Au powder by using cyanide production of Chromobacterium violaceum. (a) Cell population C. violaceum (×) and Au concentration () in the solution. (b) Cyanide concentration () and dissolved oxygen concentration () in the solution.
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ing (0.20 mmol/l) in the stationary and declining phases. In contrast, dissolved oxygen concentration rapidly decreased in the growth phase, remained low (0.05 mmol/l) in the stationary phase and increased in the declining phase. The Au dissolution rate was low in the growth and stationary phases but became higher in the declining phase. C. violaceum actively produces cyanide during the growth phase and converts it into -cyanoalanine during the stationary and declining phases, as reported previously (Rodgers, 1978). Based on the dissolved oxygen concentration, it is clear that C. violaceum consume dissolved oxygen for bacterial respiration during the growth and stationary phases, and that consumption ends after the declining phase (Foucher et al., 2003). The increased rate of Au dissolution after the declining phase implies that the rate of Au dissolution depends on both cyanide concentration and dissolved oxygen concentration. 3.2. Effects of cyanide and dissolved oxygen concentration on bacterial Au dissolution Fig. 3 shows the cyanide and Au concentrations in C. violaceum-inoculated solution with or without 100 mmol/l phosphate buffer. With phosphate buffer, the peak value of cyanide concentration during the growth phase increased from 0.65 to 1.15 mmol/l, and the cyanide concentration during the stationary and declining phases increased from 0.20 to 0.52 mmol/l, as shown in Fig. 3(a). However, the Au dissolution rate remained almost unchanged, as shown in Fig. 3(b). This demonstrates that bacterial Au dissolution rate does not only depend on cyanide concentration. Fig. 4 shows the dissolved oxygen and Au concentrations in C. violaceum-inoculated solution with or without aeration. The dissolved oxygen concentration in both solutions remained below 0.02 mmol/l from 0 to 17 h, but it increased to 0.04 mmol/l without aeration and to 0.10 mmol/l with aeration after 20 h, as shown in Fig. 4(a). Similarly, the Au dissolution rate of both solutions remained around 0 mmol/l from 0 to 17 h and Au dissolution started after 20 h but the dissolution rate with aeration was higher than that without aeration, as shown in Fig. 4(b). It is thus clear that bacterial Au dissolution rate depends on dissolved oxygen concentration.
Fig. 3. Gold dissolution in solution in experiments involving Au powder with and without buffer. (a) Cyanide concentration in solution with () and without () 100 mM phosphate buffer. (b) Au concentration in solution with (䊉) and without () 100 mM phosphate buffer.
3.3. Electrochemical measurement 3.3.1. Anodic behavior of Au in solution with C. violaceum Fig. 5(a) shows the linear sweep voltammograms of solutions in which C. violaceum was incubated for 24, 72, 120, 216 and 358 h (shown in Fig. 2(b)). Current densities on the Au electrode at −800 mV in the solution after 24, 72 and 120 h incubation were smaller than those after 216 and 358 h. In contrast, current densities at +200 mV in the solution after 24, 72, 120 and 216 h were similar, while that after 358 h was slightly smaller. Thus, the cathodic reaction changes, and the anodic reaction does not. Therefore, bacterial Au dissolution is inhibited by the cathodic reaction at 24, 72 and 120 h of incubation (corresponding to the growth and stationary phases) and is accelerated at 216 and 358 h of incubation (corresponding to the declining phase). Fig. 5(b) shows the rest potentials derived from Fig. 5(a). The rest potential decreased from −300
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Fig. 4. Gold dissolution in solution in experiments involving Au powder with and without aeration. (a) Dissolved oxygen concentration in solution with () and without () aeration. (b) Au concentration in solution with (䊉) and without () aeration.
to −400 mV during the growth phase, remained at −400 mV during the stationary phase, and increased to −200 mV during the declining phase. Figs. 2(b) and 5(b) demonstrate that the changes in rest potential are similar to the changes in dissolved oxygen concentration. Ordinary exchange current density is used to determine the metal dissolution rate, but bacterial Au dissolution is not sufficiently large to measure, as shown in Fig. 5(a). Therefore, the rest potential can be substituted for the exchange current density when determining Au dissolution rate. 3.3.2. Effects of cyanide and dissolved oxygen concentration Fig. 6(a) shows the effect of cyanide concentration on Au dissolution in sterile cyanide solution at a dissolved oxygen concentration of 0.25 mmol/l and Fig. 6(b) depicts the rest potential curve derived from Fig. 6(a). As cyanide concentration increased, current density at +200 mV increased, while that at −800 mV
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Fig. 5. (a) Linear sweep voltammograms of Au electrode in the solution in which C. violaceum is incubated at 24, 72, 120, 216 and 358 h as shown in Fig. 2. pH 7.0, 30 ◦ C, 200 rpm rotation speed, 1 mV/s sweep rate. (b) Rest potential taken from linear sweep voltammograms.
remained almost constant. Rest potential was constant at −200 mV when the cyanide concentration is less than 1.0 mmol/l, although it decreased as cyanide concentration increased when the cyanide concentration is more than 1.0 mmol/l. In the bacterial solution cyanide concentrations is from 0.1 to 1.0 mmol/l then rest potential must be −200 mV but it decreased as shown in Fig. 5(b). Electrochemically, it is clear that bacterial Au dissolution rate does not only depend on cyanide concentration. Fig. 7(a) shows the effect of dissolved oxygen concentration on Au dissolution in sterile cyanide solution at a cyanide concentration of 1.0 mmol/l and Fig. 7(b) depicts the rest potential curve derived from Fig. 7(a). As dissolved oxygen increased, current density at −800 mV increased, while that at +200 mV remained almost constant. Rest potential also increased as dissolved oxygen increased. In this experiment, the
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Fig. 6. (a) Linear sweep voltammograms of Au electrode in 0.25 mmol/l oxygen cyanide solutions. pH 7.0, 30 ◦ C, 200 rpm rotation speed, 1 mV/s sweep rate. (b) Rest potential taken from linear sweep voltammograms.
reaction is dominated by the cathodic reaction, and at dissolved oxygen concentrations between 0 and 0.3 mmol/l, which is achieved by aeration, the cathodic reaction is activated as dissolved oxygen increased. It was thus demonstrated electrochemically that dissolved oxygen is a vital factor in bacterial Au dissolution. The rest potential of Au electrode in the bacterial solution shown in Fig. 5 can be explained by the results of that in the sterile cyanide solutions shown in Figs. 6 and 7. Fig. 8 shows the changes in the rest potential in the bacterial solution. The lines between A and B show the cathodic polarization curves as shown in Fig. 7(a) and the line shifts from B to A with the increase of dissolved oxygen concentration. The lines between C and D show the anodic polarization curves as shown in Fig. 6(a) and the line shifts from C to D with the increase of cyanide concentration. The shift range between C and D is smaller than that between A and B because anodic curves are similar when cyanide
Fig. 7. (a) Linear sweep voltammograms of Au electrode in 1.0 mmol/l cyanide solutions containing various oxygen concentrations. pH 7.0, 30 ◦ C, 200 rpm rotation speed, 1 mV/s sweep rate. (b) Rest potential taken from linear sweep voltammograms.
concentration is less than 1.0 mmol/l as shown in Fig. 6(a). As can be seen in Figs. 5(b) and 8, in the growth phase, the rest potential decreases because the anodic
Fig. 8. The change in the rest potential of Au on biological gold leaching shown in Figs. 2 and 5. The lines between A and B symbolize cathodic curves and the lines between C and D symbolize anodic curves.
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line shifts from C to D due to cyanide production by C. violaceum and the cathodic line shifts from A to B due to dissolved oxygen consumption by bacterial respiration. In the stationary phase, it is fairly stable because the anodic line shifts from D to C due to cyanide decomposition by C. violaceum and cathodic line is stable. In the declining phase, it increases because the cathodic reaction is accelerated by the decrease in bacterial respiration. The movement of the rest potential on bacterial Au dissolution was explained electrochemically and it was cleared that oxygen supply is important at growth and stationary phase for high speed Au dissolution. 4. Conclusion C. violaceum, which is able to produce and decompose cyanide, was used to dissolve Au. Bacterial cell population and cyanide/dissolved oxygen concentration were measured in order to investigate the effects of cyanide/dissolved oxygen concentration on dissolution of Au powder. Anodic polarization curves of cyanide solutions with or without C. violaceum were used for electrochemical study of bacterial Au dissolution. Based on the results presented in this paper, the following conclusions can be drawn: 1. In the growth phase, C. violaceum produces cyanide and rapidly consumes dissolved oxygen for bacterial respiration. This decrease in dissolved oxygen concentration inhibits Au dissolution. 2. The increase in cyanide concentration did not affect Au dissolution but the decrease in dissolved oxygen concentration inhibits Au dissolution in the growth phase. 3. Aeration of the bacterial solution effectively increases the Au dissolution rate. 4. Electrochemical study of the sterile cyanide solution confirmed that bacterial Au dissolution rate depends on dissolved oxygen concentration (consumed by C. violaceum). References Cambell, S.C., Olson, G.J., Clark, T.R., McFeters, G., 2001. Biogenic production of cyanide and its application to Au recovery. J. Ind. Microbiol. Biotechnol. 26, 134–139.
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