Hydrometallurgy 95 (2009) 302307

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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

Impact of the copper solvent extraction reagent LIX 984N on the growth and activity of selected acidophiles
H.R. Watling a,, F.A. Perrot a, D.W. Shiers a, A. Grosheva b, T.N. Richards c
a b c

Parker Centre for Integrated Hydrometallurgy Solutions, CSIRO Minerals, PO Box 7229, Karawara, Western Australia 6152, Australia Laboratory of Chemical Thermodynamics, Department of Chemistry, M.V. Lomonossov State University, Leninskie Gory 1-3, Moscow 119992, Russia Department of Chemical Engineering, University of Cape Town, Private Bag X3, Rondebosch 7701, Cape Town, South Africa

a r t i c l e

i n f o

a b s t r a c t
The effects of the copper extractant LIX 984N 20% v/v in Shellsol 2046 on the abilities of Acidithiobacillus ferrooxidans and Sulfobacillus thermosuldooxidans to catalyse copper extraction from a chalcopyrite concentrate and to oxidise ferrous ion to ferric ion were compared and the possible role of Acidiphilium cryptum in ameliorating the effects of the SX reagent was examined. The SX reagent up to 250 mg/L was found to have little impact on the extraction of copper from a chalcopyrite concentrate using At. ferrooxidans. In contrast, with S. thermosuldooxidans, copper extraction was reduced to about one third in the presence of 50 mg/L SX reagent and at 250 mg/L SX reagent, was barely more than for an abiotic test. The SX reagent strongly inhibited ferrous ion biooxidation by several bacterial species in contrast to At. ferrooxidans. The presence of 50 mg/L SX reagent caused oxidation rates to drop to between 0 and 12% of those in controls in approximately 40-hour tests. The most toxic component of the SX reagent was found to be 4-nonylphenol. A. cryptum tolerated 250 mg/L SX reagent but did not utilise it as an energy source. Bioleaching of chalcopyrite concentrate was not enhanced signicantly when A. cryptum was added to test inocula. It is proposed that A. cryptum utilises fungal biomass as an energy source in managed heaps with solution recycle via solvent extraction plants. While it shares the environment with iron- and sulfur-oxidising acidophiles, it does not contribute directly to copper extraction from sulde minerals. Crown Copyright 2008 Published by Elsevier B.V. All rights reserved.

Article history: Received 6 June 2008 Received in revised form 15 July 2008 Accepted 15 July 2008 Available online 19 July 2008 Keywords: Bioleaching Ferrous ion oxidation Organic reagents Sulfobacillus Acidiphilium Acidithiobacillus

1. Introduction Heap leaching of copper oxides and secondary copper suldes is responsible for about 20% of world copper production. Copper is recovered from oxide minerals using a dilute sulfuric acid solution. Sulde mineral dissolution requires the presence of an oxidant, such as ferric ion, in the acidic solution and is assisted by the presence of acidophilic bacteria and archaea that utilise sulde minerals as an energy source. About 20 heap bioleaching operations have been commissioned since 1980, initially in Chile but more recently in the Asia-Pacic region (Watling, 2006). In many cases the copper is puried and recovered from solution using solvent extraction and electrowinning technologies. These successful operations demonstrate the robust nature and operational simplicity of mining biotechnology, together with health, safety and environmental benets, and capital and operating cost advantages. Process waters from heap leach solvent extraction electrowinning operations typically contain moderate concentrations of dissolved organic compounds. Some organic compounds become entrained in the aqueous solution during passage of the leach solution
Corresponding author. Tel.: +61 8 9334 8034; fax: +61 8 9334 8001. E-mail address: Helen.Watling@csiro.au (H.R. Watling).

through the solvent extraction plant. Bioleaching acidophiles are generally sensitive to organic matter in their environments but the effects of solvent extraction reagents on bacterial growth have not been widely reported, possibly because early results indicated that At. ferrooxidans was not greatly affected by them. Acidithiobacillus ferrooxidans is the most studied of the bioleaching acidophiles and was for many years thought to be the most important contributor to enhanced bioleaching, largely because it ourished in laboratory cultures. It grows well in the pH range 1.82.5 and temperature range 3035 C with an upper limit at about 40 C, is tolerant of high base metal concentrations in its environment and oxidises both iron(II) and reduced sulfur compounds to iron(III) and sulfate, respectively (Rawlings, 1997). However, its ability to oxidise ferrous ion and/or sulfur is repressed to different degrees by the addition of naturally-occurring organic compounds in growth media, among them the relatively simple monocarboxylic acids (Frattini et al., 2000; Fang and Zhou, 2006). The effects of 19 organic reagents used in the copper and uranium industries on the ability of At. ferrooxidans to enhance chalcopyrite oxidation were compared at their saturation concentrations (Torma and Itzkovitch, 1976). In general, the LIX reagents used in solvent extraction, were only moderately deleterious to At. ferrooxidans activity. More recently, Gentina et al. (1987) reported that two LIX

0304-386X/$ see front matter. Crown Copyright 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.hydromet.2008.07.004

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reagents and a diluent at their saturation concentrations were not particularly inhibitory to ferrous ion biooxidation by At. ferrooxidans or to bio-assisted copper extraction from a low-grade sulde ore. Mazuelos et al. (1999) showed that a 15% mix of LIX 64 in kerosene caused a proportional decrease in ferrous ion oxidation rate at concentrations up to 60 mg/L, but had no greater effect at higher concentrations, for a mixed microbial culture containing At. ferrooxidans, Leptospirillum ferrooxidans and some heterotrophic bacteria (mainly Acidiphilium spp.). In another study, the inhibitory concentration of an SX reagent (6% extractant, 94% diluent) contacted with a mixed culture of iron oxidation bacteria, nominally At. ferrooxidans, was 16 mg/L (Rusin et al., 1995). Sulfobacillus thermosuldooxidans is a moderately thermophilic, aerobic mixotroph (requires both inorganic and organic energy sources for growth) that has a broad geographical distribution in suldic environments and is often isolated from sulde heaps (Watling et al., 2008). Its optimum growth conditions are 50 C and pH 1.8 but it grows well at lower temperatures and it can oxidise both reduced iron and sulfur species. The presence of complex organic compounds such as yeast extract or peptone enhanced growth, compared with growth on inorganic substrates alone (Tsaplina et al., 1991). However, mixotrophic growth does not necessarily confer greater tolerance to all organic compounds. For example, carboxylates and sugars did not enhance S. thermosuldooxidans growth rates and may have slightly inhibited them. Reports on the impacts of solvent extraction reagents on Sulfobacillus spp. have not been found but a Sulfobacillus acidophilus isolate (strain NC) was shown to be sensitive to a selection of otation reagents (Okibe and Johnson, 2002). Acidiphilium cryptum (Harrison, 1981), often found in association with At. ferrooxidans (Harrison et al., 1980), has been isolated from acidic coal spoil drainage (Harrison, 1978), acidic drainage on a volcanically active island (Donachie et al., 2002) and from the efuents of a copper sulde mine dump (Banerjee et al., 1996) and heaps (Goebel and Stackebrandt, 1994; this laboratory). This species grows in media between pH 1.9 and 5.9 and between 35 and 40 C. Small amounts of organic compounds are required for growth but large amounts inhibit growth. It does not oxidise iron(II) or sulfur as sole energy substrates (Harrison, 1983). Berthelot et al. (1997) and Johnson and Roberto (1997) have discussed the possibility that heterotrophs, such as Acidiphilium acidophilum (previously Thiobacillus acidophilus Hiraishi et al., 1998), may assist bioleaching environments by metabolising organic matter and thus detoxifying the growth environment for other acidophiles. This cooperative effect was investigated by Paiment et al. (2001) using a coppernickel sulde ore. In 21-day bioleaching experiments, using At. ferrooxidans alone or a mixture of At. ferrooxidans and A. acidophilum, it was shown that the recovery of copper increased from 1.7% to 2.5% with the mixed culture but that nickel recovery (4%) was not improved. Bacelar-Nicolau and Johnson, (1999) showed that pyrite leaching was enhanced when mixed cultures containing iron oxidisers and A. acidophilum were used. However, contrasting results were reported by Marchand and Silverstein (2002), who found that the presence of A. acidophilum in mixed culture with At. ferrooxidans inhibited pyrite oxidation. In this paper, the impacts of LIX 984N 20% v/v in Shellsol 2046 (subsequently referred to as the SX reagent) on copper extraction from a chalcopyrite concentrate were examined. Three bacterial species, S. thermosuldooxidans DSM 9293T, A. cryptum DSM 2389T and At. ferrooxidans DSM 14882T were used in bioleaching and other tests. Bacterial species closely related to these three have been isolated from the copper bioleaching heaps at Whim Creek, north Western Australia (unpublished data), where the SX reagent is used in the solvent extraction plant (MFM DeSouza, personal communication). In addition to the bioleaching studies, growth in the presence of the SX reagent by A. cryptum and its possible utilisation were investigated, and the effect of the SX reagent on ferrous ion oxidation

rates was compared for selected bioleaching organisms using an electrochemical monitoring method. 2. Materials and methods 2.1. Bacteria and growth media In this study, both At. ferrooxidans and S. thermosuldooxidans were grown in basal salts medium (BSM) with yeast extract: 1.5 g (NH4)2SO4, 0.25 g KH2PO4 and 0.25 g MgSO47H2O were dissolved in 1 L deionised water. While not required for At. ferrooxidans cultures, yeast extract was added to all media for consistency. The pH was adjusted to pH 1.8 by dropwise addition of sulfuric acid. The solution was autoclaved for 20 min at 121 C. For iron(II) oxidation tests, metals tolerance tests, and organics tolerance tests, 10 g FeSO47H2O were dissolved in 25 mL BSM removed from a 1 L stock solution. This solution was lter-sterilised back into the BSM stock solution, followed by the addition of 10 mL of 10% sterile yeast extract solution. The nal concentration of iron(II) was 2.01 g/L. A. cryptum was grown in DSM medium M269: 2 g (NH4)2SO4, 0.1 g KCl, 0.5 g K2HPO4, 0.5 g MgSO47H2O were dissolved in 1 L deionised water and the pH adjusted to pH 3 by dropwise addition of sulfuric acid. The solution was autoclaved for 20 min at 121 C after which 10 mL each of sterile 10% glucose and 3% yeast extract solutions were added. Final concentrations of glucose and yeast extract were 1 g/L and 0.3 g/L, respectively. Solid M269 plates were prepared by adding 2% Gelrite Gellan Gum (Sigma) to the medium, autoclaving, and then pouring into petri dishes. Plates were incubated at 30 C. 2.2. The SX reagent The copper extractant in use at the Whim Creek copper heap leach operation and used in this study is LIX 984N. The reagent is a 1:1 v/v blend of LIX 84 (2-hydroxy-5-nonylacetophenone oxime) and LIX 860N (5-nonylsalicylaldoxime) at a concentration of 20% v/v in Shellsol 2046. The 4-nonylphenol is probably a residual of the organic synthesis of LIX 984N (b 6%) but acts as a modier, facilitating the stripping of copper from the strong copper extractants. Shellsol 2046 is comprised of about 18% aromatic hydrocarbons, 16% naphthenic hydrocarbons and 55% parafnic hydrocarbons. 2.3. Chalcopyrite bioleaching The composition of the concentrate used for bioleaching tests was 23.6% Cu, 29.6% Fe and 32.9% S. It contained chalcopyrite (88%), pyrite (4%) and quartz (8%). Cell concentrations in prepared inocula were S. thermosuldooxidans, 5.7 107 cells/mL, At. ferrooxidans, 5.2 107 cells/mL, and A. cryptum, 2.8 108 cells/mL. One gram portions of chalcopyrite concentrate were mixed with 100 mL aliquots of BSM in 250 mL Erlenmeyer asks. The mass of each ask was recorded and asks were autoclaved for 20 min at 121 C. The mass of cooled asks was recorded and lost water replaced by the aseptic addition of sterile water (pH 1.8). Triplicate asks were inoculated (10% inoculum) with autotrophs and/or heterotrophs with or without the SX reagent. Appropriate additional asks were prepared but not inoculated to provide abiotic controls. Flasks were incubated at 35 C in an environmental shaking incubator (150 RPM). Water lost to evaporation was replaced with deionised water and samples were collected periodically for elemental analysis, pH and solution potential measurements. Cells in solution were examined using phase contrast microscopy. An aliquot (1.0 mL) of each sample was ltered through a 0.45 m pore-size membrane, acidied with 0.5 mL concentrated hydrochloric acid, and analysed for total copper and iron using inductively-coupled plasma-atomic emission spectrometry (ICP-AES).

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DSM-M269 plus yeast extract and glucose in the presence of 250 mg/L SX reagent was inoculated onto solid M269 plates. 3. Results and discussion Most heap leaching operations that process low-grade copper suldes use solvent extraction electrowinning technologies to concentrate, purify and separate the copper from heap leachates. Aldoxime/ketoxime mixed extractants, which take advantage of the rapid kinetics and good copper extraction properties of the aldoximes and the stability and physical performance of the ketoximes, have proved to be very useful for copper solvent extraction from leach and other liquors. The effect of the SX reagent at two concentrations, 50 mg/L and 250 mg/L, on the leaching and bioleaching of the chalcopyrite concentrate were investigated. The upper concentration was chosen because it was thought to approximate the saturation concentration of the reagent in aqueous solution. Product information sheets report an aqueous solubility of b 1000 mg/L for LIX 984N and each of its components. 3.1. Acid leaching of chalcopyrite In the abiotic controls, copper extraction during acid leaching of the chalcopyrite concentrate was impacted by the presence of the SX reagent to a greater extent than was iron (Fig.1). The maximum standard deviation % determined for triplicate data points was 5% and the average was 3% for iron and 2% for copper. The error bars were not displayed as they were largely masked by the symbols. The initial copper concentrations ( 50 mg/L) and iron concentrations ( 120 mg/L) reected the rapid dissolution of oxidised phases on the mineral surfaces. More iron than copper was extracted, due to partial leaching of the small (4%) pyrite content in the concentrate. During the leach, ferrous ion concentrations were generally about 65% of the total iron concentrations for all tests, consistent with the measured low redox potentials (b 400 mV versus Ag/AgCl). All leachates were pH b 2 and no bacteria were observed at any time during the experiments. While copper extraction was low, 13%, as anticipated for these abiotic acid-leaching tests conducted at 35 C, results from replicate tests showed that the leaching method was reproducible (0.1%). 3.2. Bioleaching of chalcopyrite The impact of the SX reagent on the bioleaching of chalcopyrite concentrate was compared for tests inoculated with At. ferrooxidans alone or in mixed culture with A. cryptum. Control copper concentrations

Fig. 1. Impact of the SX reagent on the extraction of Cu (closed symbols) and Fe (open symbols) during the abiotic leaching of chalcopyrite concentrate. no added organic; 50 mg/L and 250 mg/L SX reagent.

2.4. Impact of the SX reagent on ferrous ion biooxidation rates The method used to monitor ferrous ion oxidation is based on that described by Pesic et al. (1989). The ferrous ion concentration in medium prior to inoculation was determined via titration using standard potassium dichromate solution. The total iron was determined by ICP-AES to be about 5% greater than the ferrous ion concentration. Solution potentials were measured using a calibrated EH electrode (Ionode model IP1306, Pt Ag/AgCl(satKCl)). Experimental Nernstian constants of RT / nF (0.0292 0.001 V) and E (0.498 0.024 V), determined at 35 C, were used to calculate ferrous ion concentrations from experimental EH values using the Nernst equation (Eq. (1)) and ferric ion concentrations were calculated by difference. EE


2:3RT Fe3 log 2 : nF Fe

Typical ferrous ion oxidation data for S. thermosuldooxidans, obtained at 35 C during method development indicated acceptably small variability between replicate experiments but strong dependence on initial cell density (Watling et al., 2008). The experimental method was therefore standardised as follows. Flasks containing 90 mL BSM with yeast extract and 10 g/L FeSO47H2O were inoculated with 10 mL S. thermosuldooxidans inoculum (1 107 cells/mL) with/without the SX reagent or one of its components and incubated at 35 C using an environmental shaking incubator. Each ask was equipped with an EH electrode connected to a data-taker with data collection frequency of 15 min. Similar tests were undertaken using At. ferrooxidans, Sulfobacillus sibiricus, S. acidophilus, Sulfobacillus thermotolerans and Acidimicrobium ferrooxidans maintained in laboratory culture media similar to that described for At. ferrooxidans and S. thermosuldooxidans. 2.5. Heterotrophic growth on the SX reagent Aliquots (100 mL) of DSM-M269 medium with added yeast extract (1 g/L) and glucose (0.3 g/L) were inoculated with 10 mL A. cryptum (1.6 108 cells/mL). Triplicate asks with/without added SX reagent (250 mg/L) were incubated at 35 C in an environmental shaker. In a second experiment, A. cryptum (2 108 cells/mL) was inoculated into DSM-M269 without added yeast extract and glucose but with/without added SX reagent (250 mg/L). For these experiments, each ask was subcultured weekly into an identical, freshly-prepared medium. Samples were collected periodically from asks and cell concentrations estimated using a Helber Bacteria Counting Chamber (Thoma ruling, 0.02 mm cell depth). In a subsidiary test, A. cryptum grown on

Fig. 2. Impact of the SX reagent on Cu extraction from a chalcopyrite concentrate using At. ferrooxidans (closed symbols) or a mixed culture of At. ferrooxidans and A. acidiphilium (open symbols). no added organic; 50 mg/L and 250 mg/L SX reagent.

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Fig. 3. Impact of the SX reagent on Cu extraction from a chalcopyrite concentrate using S. thermosuldooxidans (closed symbols) or a mixed culture of A. cryptum with S. thermosuldooxidans (open symbols). no added organic; 50 mg/L and 250 mg/L SX reagent.

(abiotic leaching) were subtracted from the bioleaching results to represent more clearly the impact of the SX reagent on the bacterial contribution during bioleaching. While there was a short lag time, the SX reagent had little effect on the ability of At. ferrooxidans to enhance copper extraction from the concentrate (Fig. 2). Total copper extractions ranged from 10% with no added SX reagent to 7% in the presence of 250 mg/L SX reagent. The results for the single culture and the mixed culture for each treatment were not signicantly different. Solutions remained below pH 2.1 at all times. Redox potentials were moderately high in the inoculated tests (N 550 mV versus Ag/AgCl), consistent with low ferrous ion concentrations in all inoculated tests, b 5% of the total soluble iron. The results obtained are consistent with those reported by Torma and Itzkovitch (1976) for the limited impacts of several LIX reagents on oxygen uptake by At. ferrooxidans during the bioleaching of a chalcopyrite concentrate. Similarly Gentina et al. (1987) reported that the LIX reagents tested did not impact greatly on either At. ferrooxidans growth or the extraction of copper from a low-grade copper sulde ore. Similarly, the impact of the SX reagent on the bioleaching of chalcopyrite concentrate was compared for tests inoculated with only S. thermosuldooxidans or with S. thermosuldooxidans and A. cryptum in mixed culture. Copper extraction decreased with increasing SX reagent concentration (Fig. 3). Total copper extraction was reduced from 11% with no added SX reagent to 2.5% in the presence of 250 mg/L. The results for the single culture and the mixed culture were not signicantly different. Typically, with no added SX reagent, ferrous ions accounted for only 23% of the total soluble iron, consistent with high redox potentials (N 600 mV versus Ag/AgCl). However, with 50 mg/L and 250 mg/L SX reagent, ferrous ion concentrations made up from 4065% of the total soluble iron and the redox potentials were 400 mV. Bacterial cell numbers increased from 106 to 108 cells/mL with no added SX reagent, but remained relatively constant at about 106 cells/mL with 50 mg/L SX reagent; few viable cells were observed with 250 mg/L SX reagent after one week. No comparative data on the effects of LIX extractants on bioleaching with S. thermosuldooxidans have been found. While overall leaching efciency was low because of the low experimental temperature (35 C), the results indicated clearly that S. thermosuldooxidans was signicantly less tolerant of the SX reagent in its environment than was At. ferrooxidans. The main discernible effect was the inhibition of ferrous ion biooxidation. The presence of the heterotroph A. acidiphilium in the inoculum did not assist bioleaching, as assessed by copper extraction. 3.3. Impact of the SX reagent on ferrous ion oxidation The results of the bioleaching experiments, particularly the relationship between ferric and ferrous ion in leachates, indicated

that the SX reagent impacted directly on bacterial ferrous ion oxidation. Preliminary ask tests with periodic sampling showed that the SX reagent caused decreased ferrous ion biooxidation rates and increased lag times, the latter making it difcult to predict optimum periodic sampling points. The dual impact has previously been reported for S. metallicus (Dopson et al., 2006) and for a mixed culture containing At. ferrooxidans, L. ferrooxidans and Acidiphilium species (Mazuelos et al., 1999). An automated electrochemical method was therefore developed to quantify the impact of the SX reagent on ferrous ion biooxidation and generate reliable, comparable results (Section 2.3). The growth rate of iron-oxidising bacterial strains such as S. thermosuldooxidans and At. ferrooxidans is approximately proportional to the rate of ferric ion production (Eq. (2)) as long as the number of bacteria per mass of ferric ion produced is constant in the whole range of iron concentrations tested (Silverman and Lundgren, 1959). The modest total iron concentration of 2000 mg/L used in these tests meets that requirement. According to Franzmann et al. (2005), ferrous ion oxidation follows rst-order reaction kinetics for both the test species. Thus a ferric ion doubling time can be estimated from that portion of a plot of the natural logarithm of ferric ion concentration versus time that follows a rst-order reaction rate. Ferric ion doubling times are roughly equivalent to cell generation times if iron oxidation and growth are coupled. Data used to estimate ferric ion concentrations, rate constants and ferric ion doubling times in hours (=ln2/Fe where Fe is the rate constant for ferric ion production (h 1)) are molar concentrations; the initial ferrous ion concentration of 2000 mg/L is equivalent to 0.036 M. 4Fe2 4H O2 4Fe3 2H2 Obio assisted oxidation 2

Example data for S. thermosuldooxidans and At. ferrooxidans (Fig. 4A and B) showed clearly that the SX reagent impacted more

Fig. 4. Impact of the SX reagent on ferrous ion oxidation by (A) S. thermosuldooxidans and (B) At. ferrooxidans.

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H.R. Watling et al. / Hydrometallurgy 95 (2009) 302307 Table 1 Ferric ion doubling times (h) estimated from ferrous ion biooxidation rates At. ferrooxidans SX reagent (mg/L)
a

0 5.1 0.5 0 5.8 1.3 0 6.1 0 7.3 0 7.1 0 6.5

25 5.8 0.4 10 5.6 0.8 8 6.3 0.625 8.8 0.625 8.5 0.4 7.0

50 6.1 0.8 25 5.9 1 20 6.3 1.56 9.4 1.56 13.5 1.0 9.2

75 7.0 0.6 50 51 16 40 7.1 3.125 10.0 3.125 14.7 2.0 140

S. thermosuldooxidans SX reagent (mg/L) a Shellsol (mg/L) LIX 860 (mg/L) LIX 84 (mg/L) Nonyl phenol (mg/L)

Fig. 5. The impact of the SX reagent on ferrous ion oxidation rates, Fe as a percentage of their respective controls for selected iron-oxidising bacteria.

a Ferric ion doubling times with standard deviations for replicate tests using the SX reagent.

strongly on S. thermosuldooxidans. Replicate test data for S. thermosuldooxidans (Fe = 0.122 0.022) and At. ferrooxidans (Fe = 0.133 0.018) indicated that calculated oxidation rates were reproducible for particular inocula. In order to minimise differences in growth rates between freshlyprepared inocula, normalised rate data (rate constants as a percentage of their respective controls) were compared for six iron-oxidising bacteria (Fig. 5). The results indicated that 10 mg/L SX reagent was tolerable to all strains, and that 50 mg/L was strongly inhibitory to all test species except At. ferrooxidans. 3.4. Impacts of the SX reagent components on ferrous ion oxidation Given the marked inhibitory effect of the SX reagent on both bioleaching and ferrous ion oxidation by S. thermosuldooxidans, the components were tested individually at concentrations appropriate to their presence in the mixed reagent (Fig. 6). Shellsol 2046, the diluent and major component in the reagent, had very little impact on ferrous ion biooxidation (data not shown) or ferric ion doubling times (Table 1). [Standard deviations for replicate tests using the SX reagent are included to indicate method precision and variations in growth rates between different cultures of each species obtained over a 6 month period.] At 40 mg/L Shellsol 2046, Fe was 85% of that in the control. Both the aldoxime and the ketoxime had moderately inhibitory effects and longer ferric ion doubling times as the organic test concentration was increased. 4-Nonylphenol, the minor constituent, had the strongest impact, with minimal ferrous ion oxidation by S. thermosuldooxidans at 2 mg/L. Interestingly, the LIX reagents LIX 973, which contains 4nonylphenol, and LIX 622, which contains 4-dodecylphenol, were

among the most toxic of a wide range of chemicals tested on the archaeon Sulfolobus metallicus (Dopson et al., 2006). The two LIX reagents impacted strongly on both ferrous ion and tetrathionate oxidation and on chalcopyrite bioleaching. Similarly, LIX65N which contains the nonylphenol group (5-nonyl-2-hydroxybenzaldoxime), 4-nonylphenol, and Kelex 120 (80% 4-nonylphenol) were moderate to strong inhibitors of oxygen uptake by At. ferrooxidans in 90-minute tests (Torma and Itzkovitch, 1976). These authors reiterated the recommendation of Eliasen and Edmunds (1974) that, where solvent extraction is to be used to treat bioleaching liquors, the aqueous stream should be treated with activated charcoal. LIX 64 (which is 99% LIX 65, an orthohydroxy benzophenone oxime) 15% v/v in kerosene was shown to cause ferrous ion oxidation rates to decrease and the lag phase to increase with increasing concentrations up to 60 mg/L (Mazuelos et al., 1999). These authors also recommended the use of activated charcoal to remove the inhibitory effects on bacterial activity. The results obtained in the present study together with these reported data indicate that phenols are particularly toxic to ironand sulfur-oxidising acidophiles. 3.5. Tolerance and utilisation of the SX reagent by A. cryptum A. cryptum grew well on yeast extract and glucose medium in the presence of 250 mg/L SX reagent. Growth was rapid, cell numbers increasing by at least an order of magnitude in a few days, comparable with growth in the absence of the SX reagent. Growth was sustained through two subcultures in fresh medium in tests spanning 25 days. In addition, A. cryptum retained its ability to grow on solid-medium plates and was tolerant of the SX reagent in that environment. However, few A. cryptum cells remained after one week in medium with the SX reagent but without yeast extract and glucose, and no cells survived a further week in freshly-prepared medium, indicating that A. cryptum could not utilise the SX reagent as an energy source. The result is consistent with the general nding that growth on large molecular weight substrates by heterotrophic acidophiles is limited but, at the same time has not been studied to any great extent (Johnson and Roberto, 1997). Nevertheless, A. cryptum grows in managed copper sulde heaps and requires a carbon source for growth, whether or not its activity is benecial to the iron- and sulfur-oxidising organisms that inhabit the same environment. Arguably, the greatest biomass in managed sulde heaps, at least near the top, is comprised of the acidophilic fungi and bacteria that colonise the solvent extraction plant and, subsequently the ore via solution recycle to the heap. No reports on the identities or growth substrates of the fungal species in managed sulde heaps have been found but there is a large body of literature pertaining to hydrocarbon utilisation by soil microorganisms (Tortella and Diez, 2005). The possibility that A. cryptum can utilise the

Fig. 6. The impact of LIX 84, LIX 860 and 4-nonyl phenol on rates of ferrous ion oxidation (Fe) by S. thermosuldooxidans.

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products of lysed fungal cells as growth substrates may be worth exploring. In addition, it has been noted that Acidiphilium species have the ability to catalyse the reductive dissolution of iron(III) oxides, hydroxides and hydroxy sulfates, with rates of dissolution tending to be faster for less crystalline compounds (Bridge and Johnson, 2000; Kusel et al., 2000). Conditions for these reactions to take place do not have to be anoxic but can be oxygen-limited, a condition that very likely exists in localised zones of large bioleaching heaps. However, when jarosite and other insoluble iron(III) compounds are formed in heaps, the net reactions tend to be acid generating; their re-dissolution, therefore, will be acid consuming. Thus, while ferrous ions are produced in the bio-assisted dissolution reactions and can be utilised by iron-oxidising bacteria such as At. ferrooxidans and S. thermosuldooxidans, the sulde heap that the bacteria inhabit is likely to be of sufciently high pH as to slow ferrous ion oxidation signicantly and to limit the availability of ferric ions for further sulde oxidation. The absence of sulfur oxidation by A. cryptum indicates that it will not assist bioleaching to any great extent through the removal of sulfur reaction products from leached sulde surfaces. Thus it is proposed that A. cryptum shares the managed heap environment with the sulfur- and iron-oxidising bacteria, possibly utilising the degraded products of the available fungal biomass for growth, without contributing directly to metals extraction to any signicant extent. 4. Conclusions The solvent extraction reagent LIX 984N 20% v/v in the diluent Shellsol 2046 had little impact on At. ferrooxidans at concentrations up to 250 mg/L but signicantly inhibited S. thermosuldooxidans growth, ferrous ion oxidation and ability to enhance the extraction of copper from a chalcopyrite concentrate. Selected iron-oxidising bacteria other than At. ferrooxidans were similarly inhibited. The presence of 50 mg/L SX reagent caused oxidation rates to drop to between 0 and 12% of that in the control in approximately 40-hour tests. The most toxic component of the SX reagent was found to be 4-nonylphenol. A. cryptum grew well in the presence of 250 mg/L SX reagent but did not utilise the reagent and did not enhance chalcopyrite bioleaching in mixed culture. It is proposed that A. cryptum grows on fungal biomass in managed heaps with solution recycle via solvent extraction plants. While it shares the environment with iron- and sulfur-oxidising acidophiles, it does not contribute directly to copper extraction. Acknowledgements Keith Barnard is thanked for providing the SX reagents used in these tests and for helpful discussions about their properties. The nancial support of the Australian Government through the A.J. Parker Cooperative Research Centre for Integrated Hydrometallurgy Solutions is gratefully acknowledged. Anna Grosheva and Tracey Richards thank BHP-Billiton Innovation Pty Ltd and Moscow State University, and Anglo Research and the University of Cape Town, respectively, for the opportunity to participate in the Parker Centre's Student-Industry Research Programme and gain research experience at the CSIRO Minerals laboratories. References
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