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Biomobilization of silver, gold, and platinum from solid wastematerials by HCN-forming microorganisms

Brandl, H; Lehmann, S; Faramarzi, M A; Martinelli, D

Brandl, H; Lehmann, S; Faramarzi, M A; Martinelli, D (2008). Biomobilization of silver, gold, and platinum fromsolid waste materials by HCN-forming microorganisms. Hydrometallurgy, 94(1-4):14-17.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:Hydrometallurgy 2008, 94(1-4):14-17.

Brandl, H; Lehmann, S; Faramarzi, M A; Martinelli, D (2008). Biomobilization of silver, gold, and platinum fromsolid waste materials by HCN-forming microorganisms. Hydrometallurgy, 94(1-4):14-17.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:Hydrometallurgy 2008, 94(1-4):14-17.

Biomobilization of silver, gold, and platinum from solid wastematerials by HCN-forming microorganisms

Abstract

Cyanogenic Chromobacterium violaceum, Pseudomonas fluorescens, and P. plecoglossicida were ableto mobilize silver, gold, and platinum when grown in the presence of various metalcontaining solidssuch as gold-containing electronic scrap, silver-containing jewelry waste, or platinum-containingautomobile catalytic converters. Five percent of silver was microbially mobilized from powderedjewelry waste as dicyanoargentate after one day, although complete dissolution was obtained whennon-biological cyanide leaching was applied. Dicyanoargentate inhibited growth at concentrations of>20 mg/L. Gold was bacterially solubilized from shredded printed circuit boards. Maximumdicyanoaurate concentration corresponded to 68.5% dissolution of the total gold added. Additionally,cyanide-complexed copper was detected during treatment of electronic scrap due to its high coppercontent of approximately 100 g/kg scrap. However, only small amounts of platinum (0.2%) weremobilized from spent automobile catalytic converter after 10 days probably due to passivation of thesurface by an oxide film. In summary, all findings demonstrate the potential of microbial mobilizationof metals as cyanide complex from solid materials and represent a novel type of microbial metalmobilization (termed "biocyanidation") which might find industrial application.

Biomobilization of silver, gold, and platinum from solid waste materials by HCN-forming microorganisms

Helmut Brandla*, Stefan Lehmanna,b, Mohammad A. Faramarzia,c, Daniel Martinellia

aUniversity of Zurich, Institute of Environmental Sciences, Zurich, Switzerland bZurich University of Applied Sciences, Wadenswil, Switzerland cDepartment of Pharmaceutical Biotechnology, Faculty of Pharmacy, Medical Sciences, University of Tehran, Tehran, Iran ARTICLE INFO Keywords HCN Cyanide Cyanogenic microorganisms Pseudomonas Chromobacterium Bioleaching Biocyanidation

ABSTRACT Cyanogenic Chromobacterium violaceum, Pseudomonas fluorescens, and P. plecoglossicida were able to mobilize silver, gold, and platinum when grown in the presence of various metal-containing solids such as gold-containing electronic scrap, silver-containing jewelry waste, or platinum-containing automobile catalytic converters. Five percent of silver was microbially mobilized from powdered jewelry waste as dicyanoargentate after one day, although complete dissolution was obtained when non-biological cyanide leaching was applied. Dicyanoargentate inhibited growth at concentrations of >20 mg/L. Gold was bacterially solubilized from shredded printed circuit boards. Maximum dicyanoaurate concentration corresponded to 68.5% disso-lution of the total gold added. Additionally, cyanide-complexed copper was detected during treatment of electronic scrap due to its high copper content of approximately 100 g/kg scrap. However, only small amounts of platinum (0.2%) were mobilized from spent automobile catalytic converter after 10 days probably due to passivation of the surface by an oxide film. In summary, all findings demonstrate the potential of microbial mobilization of metals as cyanide complex from solid materials and represent a novel type of microbial metal mobilization (termed “biocyanidation”) which might find industrial application.

1. Introduction

In the presence of cyanide, many metals and metalloids (such as Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ge, Mo, Tc Ru, Rh, Pd, Ag, Cd, W, Re, Os, Ir, Pt, Au, Hg, Tl, Po, and U) form well-defined cyanides complexes which show often very good water solubility and exhibit high chemical stability (Ford-Smith, 1964; Chadwick and Sharpe, 1966). This principle is industrially used mainly in mining operations where gold from low-grade ores is converted to a water-soluble compound by adding cyanide “from the bottle”. Dicyanoaurate is subsequently leached from solid mineral sources and recovered e.g. by cementation with zinc powder (Dechênes et al., 2003; Habashi, 2007; McInnes et al., 1994).

Regarding biological cyanide formation, cyanide is formed by a variety of bacteria as secondary metabolite, e.g. Chromobacterium violaceum, Pseudomonas fluorescens, or P. aeruginosa, many of them belonging to the soil microflora (Knowles and Bunch, 1986. Although bacterial cyanide formation is known for many years (Clawson and Young, 1913), quantitative data on the ability of HCN production of many species are often missing. Glycine is a direct precursor of cyanide which is formed by oxidative decarbo-xylation. Typically, cyanide is mainly formed during a short growth period in the early stationary phase (Knowles and Bunch, 1986). It is assumed that the formation of cyanide gives the organ-ism a selective advantage by out-competing other microorganisms in natural systems such as soil. * Corresponding author. E-mail address: hbrandl@uwinst.uzh.ch (H. Brandl)

However, until today a combination of chemical knowled-

ge (interaction of metals and cyanide) with microbiological principles (biological cyanide formation) regarding metal so-lubilization from metal-containing solids and the formation of water-soluble cyanide complexes has been considered margi-nally. Only very few reports can be found in the literature describing the biological solublization of gold by C. violace-um and microbe-mediated formation of gold cyanide (see e.g. Kita et al., 2006). However, none of these showed the forma-tion of cyanide-complexed gold (dicyanoaurate), but only the increase of solubilized gold during incubation.

The objective of this study was to evaluate the potential of cyanogenic microorganisms such as C. violaceum, P. fluores-cens, and P. plecoglossicida to solubilize metals from metal-containing solids; in particular, the biological mobilization of silver, gold, and platinum as cyano complexes which were identified by high pressure liquid chromatography. 2. Materials and methods

C. violaceum was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ strain 30191). P. fluorescens CHA0 was kindly provided by Dr. Monika Maurhofer of the Swiss Federal Institute of Technology (ETH Zurich). P. plecoglossicida (a pseudomonad formerly un-known to form HCN) was an own isolate from soil collected in Tehran, Iran (main campus of Medical Sciences, Univer-sity of Tehran) (Faramarzi and Brandl, 2006).

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Fig. 1. Growth and formation of dicyanoaurate by C. violaceum (●) and P. fluorescens (○) when grown in the presence of gold-containing electronic scrap. (a) optical density at 660 and 450 nm for C.violaceum and P. fluorescens, respectively; (b) pH; (c) free cyanide; (d) dicyanoaurate (mg/l).

Luria-Bertani (LB) broth containing tryptone (10.0), yeast extract (5.0), sodium chloride (10.0) was used for growth experi-ments. The pH was adjusted to 7.2. Glycine and metal-containing solids were additionally supplied in different amounts according to the experimental setup. Duplicate cultures were grown in 250 ml baffled Erlenmeyer flasks in 100 ml of medium and incubated at 30°C on a rotary shaker at 150 rpm. Optical density and pH was recorded routinely. For metal mobilization experiments, different amounts (up to 75 g/l) of solid materials (e.g. powdered metal-containing materials, shredded electronic scrap) were added to the medium.

Free cyanide was quantitatively analyzed applying the picric acid colorimetric method (Drochioiu et al., 2003). Alternatively, cyanide was determined by the methemoglobin method (Baumeis-ter and Schievelbein, 1971). Analyses of metal cyano complexes were performed by reversed phase high pressure liquid chro-matography (rP-HPLC; Metrohm AG, Herisau, Switzerland) at their corresponding maximum UV absorption values (Faramarzi et al., 2004). Commercially available metal cyanides were used as standards. The method was slightly modified: Instead of 60 mM tetrabutylammoniumhydroxide (TBAOH), the eluent contained 90 mM TBAOH leading to decreased retention times.

Bacterial oxygen consumption was monitored using the Degra-mat® system (Baumann, 1994). One-liter reaction vessels were filled with 200 ml of sterile LB medium (supplemented with glycine; 5 g/l), inoculated with 2 ml from a stock culture of P. plecoglossicida, and tightly covered with a piezoelectric pressure sensitive measuring head. Together with the measuring head a test tube filled with NaOH was placed inside the reaction vessels to absorb carbon dioxide formed. Vessels were incubated on a magnetic stirrer (400 rpm) for 42 h at 30°C. Pressure decrease was recorded automatically every hour and the oxygen consumed was calculated. Non-inoculated media served as control to compensate pressure fluctuations and non-biological gas consumption and for-mation during incubation.

3. Results and discussion

Cyanide formation by C. violaceum, P. fluorescens, and P. plecoglossicida was observed within a short incubation time (Fig. 1c, Fig 2). C. violaceum and P. plecoglossicida proved to be very effective in forming cyanide. Characteristically, cyanide concentration decreased with time after fast genera-tion in the beginning possibly due to sorption processes, the re-use as carbon and nitrogen source by the organisms, or outgassing. This decrease was already observed in earlier experiments (Faramarzi and Brandl, 2006). In all experiments carried out, pH increased to values of 9 to 9.5 during incubation. In contrast to the application of autotrophic sulfur oxidizing microorganisms forming sulfuric acid (Krebs at al., 2001) or heterotrophic microorganisms forming organic acids (Brandl et al., 2001), the use of cyanogenic bacteria allows the mobilization of metals from solids under alkaline condi-tions. This might be of industrial interest since metal chemis-try (e.g. solubility, mobility, sorption, precipitation, formation of secondary minerals) compared to acidic conditions might change completely under alkaline conditions (especially in the presence of cyanide) and metal values might be recovered more easily, e.g. by sorption onto activated carbon (Ibragi-mova et al., 2007). Additionally, leachates might be less cor-rosive at alkaline pH. Generally, metal bioleaching has been studied and industrially applied mainly under acidic condi-tions (Brandl, 2001). Leaching under alkaline conditions (pH 8.4) using ammonifying and basophilic chemolithotrophic bacteria has been reported only very rarely (Groudeva et al., 2007). However, copper mobilization from bornite, covellite, and chalcopyrite in carbonate-rich rocks was rather low (approx. 10%), even after incubation times of 30 days. From a chemical point of view, it has been shown that metal mobi-lization from e.g. fly ash was significant only at pH values of <4.5 and >11 (Mitzutani et al., 1997).

Gold was mobilized from shredded electronic scrap by C. violeacum and P. fluorescens as dicyanoaurate, [Au(CN)2]-, after a lag-phase of 3 d (Fig. 1d). Especially when P. fluo-rescens was applied, dicyanoaurate does not remain stable in solution with prolonged incubation times. This was already observed in earlier investigations (Brandl et al., 2003) and might by due to sorption processes onto biomass or biodegra-dation because metal cyanides serve as carbon or nitrogen source (Baxter & Cummings, 2006; Patil & Paknikar, 1999). Fig. 2. Growth and formation dicyanoargentate by. P. pleco-glossicida when grown on LB medium and powdered silver-con-taining jewelry waste (286 mg of Ag per liter; supplemented with glycine, 5 g/l). OD at 660 nm (●); free cyanide ( ); silver mobilized as dicyanoargenate ( ).

H. Brandl et al / Hydrometallurgy 94 (2008) 14-17 16

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C. violaceum proved to be more efficient regarding gold mobi-lization, i.e. yielded higher concentrations of dicyanoaurate. In addition to dicyanoaurate, cyanide-complexed copper was also detected during treatment of electronic scrap probably due to the high copper content of the scrap and its rapid reaction with cyanide. This has also been observed in the mining of copper-containing gold ores. However, the presence of copper might pose a major problem due to its cyanicidic properties (Rees and van Deventer, 1999). In the presence of copper higher amounts of cyanide are needed to dissolve gold (Brierley and Brierley, 2001).

Fast mobilization of silver was observed when P. pleco-glossicida was grown in the presence of powdered jewelry waste (suspension of 286 mg/l). After one day of cultivation appro-ximately 5% of silver was mobilized as dicyanoargentate, [Ag(CN)2]-, (Fig. 2). After this fast initial growth phase cyanide-complexed silver concentration increased only slightly until the end of the incubation period. Although silver was completely mobilized when non-biological cyanide leaching using 10 mM KCN solution was applied, we found that dicyanoargentate at concentrations >20 mg/L inhibited growth of P. plecoglossicida resulting in a low mobilization efficiency (Fig. 3). Observed half maximal effective concentration (EC50) was in the range of 12 to 15 mg/l.

In spite of the toxicity of dicyanoargentate, mobilization of silver by cyanogenic microorganisms proved to be promising. However, the process has to be optimized by reducing inhibitory effects, e.g. by trapping the dicyanoargentate formed or by applying a sequential two-step process where biological cyanide formation is separated from metal mobilization. In analogy, this has already been proposed to reduce the toxicity of arsenic during the biological treatment of gold-containing arsenopyrite by Sulfolobus metallicus (Lindström et al., 2003).

From an industrial point of view it might be of importance to note that P. plecoglossicida is able to grow in NaCl solutions of up to 50 g/l (data not shown). Firstly, metal mobilization using P. plecoglossicida might still be carried out in solutions with high salt loads resulting from mineral processing. Secondly, increased salinity might inhibit microorganisms competitively growing under the same conditions, therefore allowing metal mobilization without the need of growth media sterilization.

P. plecoglossicida was used to mobilize platinum from spent platinum-containing automobile catalytic converter (1.06 g Pt / kg) as tetracyanoplatinate, [Pt(CN)4]2-. Pulp density has a major influence on bacterial growth and activity (Fig. 4). Oxygen con-sumption (used as indicator of metabolic activity) was signi-ficantly reduced at pulp densities of >50 g/l. Suspended solids at a concentration of 25 g/l did not significantly affect metabolic activities and was hence used for further experiments. This is in agreement with leaching operations under acidic conditions. Pulp Fig. 3. Growth of P. plecoglossicida in LB medium supplemented with different amounts of dicyanoargentate. OD at 660 nm was determined 28 h after inoculation (n=4).

Fig. 4. Oxygen consumption after 30 h of cultivation (mg oxygen consumed per liter of culture) as indication of biological activity in the presence of different amounts of powdered spent automobile catalytic converter. Points represent single measurements. densities of 20 g/l delayed the onset of bioleaching of pyrite derived from coal (Baldi et al., 1992). Quartz particles at pulp densities of 80 g/l almost completely inhibited the oxidation of covellite by Thiobacillus ferrooxidans (Curutchet at al., 1990).

In order to improve bacterial growth as well as cyanide generation and subsequent formation of cyanide-complexed metals, a process was applied for the cultivation of P. pleco-glossicida where fresh medium was added after a certain time to boost cyanide formation and subsequent metal mobili-zation (Fig. 5). Cyanide concentration decreased after fast ini-tial formation. However, resulting water-soluble tetracyano platinate was detected only in small amounts. Approximately 0.2% of the total Pt present in the converter was mobilized (Fig. 5b). It is assumed that Pt mobilization is prevented by a passivating oxide film which might be overcome by pre-treatment of the solid material, e.g. roasting at <600ºC (Muir & Ariti, 1991). It is also known from mining of low-grade ores that at pH 9.5 only limited amounts of platinum can be Fig. 5. Growth and cyanide formation by P. plecoglossicida in LB medium. (a) Cultivation in the absence of automobile catalytic con-verter. After 4 days (dashed line) fresh medium was added to stimu-late cyanide formation. OD at 660 nm (○); free cyanide ( ). (b) Cul-tivation in the presence of automobile catalytic converter (25 g/l). Cell counts were determined instead of OD because of background turbidity. Cell count per ml (●); free cyanide ( ); platinum mobi-lized as tetracyanoplatinate ( ).

H. Brandl et al / Hydrometallurgy 94 (2008) 14-17 17

mobilized by cyanidation using NaCN (0.083%), independent of pretreatments such as the addition of sodium chloride, sodium sul-fite, sodium thiosulfate, lead salts, and hydrogen peroxide (McInnes et al., 1994). Regarding biological Pt mobilization by cyanogens, a process under reduced or anoxic conditions might be considered, because it is known that cyanide is formed most effi-ciently under limited oxygen availability (Mascher et al., 2003). 4. Conclusions

In summary, our results demonstrate the potential of microbial mobilization of metals (in our case silver, gold, and platinum) as cyanide complex from solid materials applying C. violaceum, P. fluorescens, and P. plecoglossicida. Silver and gold was easily mobilized as their corresponding cyanide complex. However, di-cyanoargentate showed toxic effects on P. plecoglossicida de-creasing bioleaching efficiency. Platinum proved to be very re-sistant to mobilization by cyanogenic microorganisms.

Our findings represent a novel type of microbial metal mobi-lization (termed “biocyanidation”) which might find industrial ap-plication provided that mobilization efficiencies (especially for Pt) can be improved (Brandl and Faramarzi, 2006). Nevertheless, our results show that microbial mobilization of metals from solid ma-terials as cyanide complex is possible. So far, only gold mobiliza-tion by C. violeceum has been addressed in earlier reports (Camp-bell et al., 2001; Kita at al, 2006; Lawson et al., 1999; Smith and Hunt, 1985), but cyanogenic microorganisms are also able to mo-bilize iron, copper, nickel, silver, gold, and platinum (Faramarzi et al., 2004; Faramarzi and Brandl, 2006). In contrast to other bio-leaching techniques (Brandl, 2001), metal mobilization by cy-anogens occurs under alkaline conditions. Regarding metal reco-very from solutions, cyanide-complexed metals might be easily separated by chromatographic methods and sorbed onto activated carbon. Acknowledgement P. fluorescens was kindly provided by Dr. M. Maurhofer (Swiss Federal Institute of Technology/ETH, Zurich, Switzerland). References Baldi, F., Clark, T., Pollack, S.S., Olson, G.J., 1992. Leaching of pyrites of

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