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Bioleaching of pyrite at low pH and low redox potentials by novel mesophilic Gram-positive bacteria
Hydrometallurgy 63 (2002) 181 – 188 www.elsevier.com/locate/hydromet
Bioleaching of pyrite at low pH and low redox potentials by novel mesophilic Gram-positive bacteria Adibah Yahya, D. Barrie Johnson* School of Biological Sciences, University of Wales, Bangor LL57 2UW, UK Received 8 August 2001; received in revised form 13 November 2001; accepted 13 November 2001
Abstract Bioleaching of ground rock pyrite by two novel strains of Sulfobacillus-like acidophilic bacteria was examined in shake flask and bioreactor cultures. The Gram-positive prokaryotes differed from known species of mineral-oxidising Sulfobacillus in being mesophilic and in their tolerance to extreme acidity (pH < 1). The oxidative dissolution of pyrite by one of the novel Sulfobacillus strains was stimulated by yeast extract, though the second strain (L-15) was equally effective in organic-free medium. Pyrite dissolution by Sulfobacillus L-15 was compared with that by the strain type of Acidithiobacillus ferrooxidans and a Leptospirillum ferrooxidans isolate, in media set at different initial pH values. All three bacteria leached the mineral in media set at pH 2.5 and pH 1.5, but at pH 1.2, mineral oxidation (at a rate similar to the maximum recorded for At. ferrooxidans) was observed only in cultures of Sulfobacillus L-15. Pyrite leaching by Sulfobacillus L-15 and At. ferrooxidans was compared in bioreactor cultures, at different and controlled pH values. Lowering bioreactor pH from 1.5 to 1.0 resulted in the cessation of biologically accelerated pyrite oxidation by the At. ferrooxidans culture and rapid mortality of the bacteria. In contrast, Sulfobacillus L-15 continued to leach the mineral when the pH was lowered to 1.0 and (after a short lag period) to pH 0.8. In contrast to the At. ferrooxidans culture, large numbers of viable Sulfobacillus L-15 cells were recovered, regardless of bioreactor pH. Redox potentials and ratios of soluble ferric/ferrous iron in the Sulfobacillus L-15 culture were significantly lower (by about 100 – 150 mV) than values found typically in Gram-negative bacterial leaching cultures, and that recorded in the (active) At. ferrooxidans culture in the present work. The potential advantages of using microorganisms that can leach sulfidic minerals effectively at pH < 1 and at low redox potential are discussed. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Bioleaching; Pyrite; Redox potential; Sulfobacillus; Extreme pH
1. Introduction The use of acidophilic bacteria and archaea to accelerate the oxidative dissolution of sulfidic minerals and ores and, thereby, to facilitate the extraction of occluded gold (‘‘biooxidation’’) or to solubilise base metals such as copper and cobalt (‘‘bioleaching’’), is *
Corresponding author. Tel.: +44-1248-382358; fax: +441248-370731. E-mail address: [email protected] (D.B. Johnson).
now established as an economically important biotechnology (Rawlings, 1997; Brierley and Brierley, 2001). Somewhat surprisingly, reports describing composition of commercial bioleach tanks and heaps are relatively few (e.g. Brierley, 2001; Goebel and Stackebrandt, 1994; Norris et al., 2000). Temperature has an important determinative effect on which acidophiles are active in mineral leaching, with bacteria tending to dominate at lower temperatures (30 –60 C), while at higher temperatures, sulfide mineral oxidation is mediated by acidophilic archaea (e.g. Acidianus spp.,
0304-386X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 3 8 6 X ( 0 1 ) 0 0 2 2 4 - 9
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Metallosphaera spp. and Sulfolobus metallicus; Norris and Johnson, 1998). The mechanism by which bacteria oxidise sulfides has been much debated (e.g. Schippers and Sand, 1999; Fowler et al., 2001). Sulfide minerals may be subdivided into those such as sphalerite that are acidsoluble, and others such as pyrite and chalcopyrite that are acid-insoluble but which can be oxidised by ferric iron in acidic liquors. Iron-oxidising acidophilic prokaryotes have a key role in the biooxidation of this second group of sulfide minerals since they regenerate the oxidant ferric iron, which is reduced to the ferrous state during mineral attack (Eqs. (1) and (2)). þ FeS2 þ 6Fe3þ þ 3H2 O ! 7Fe2þ þ S2 O2 3 þ 6H
ð1Þ 4Fe2þ þ O2 þ 4Hþ ! 4Fe3þ þ 2H2 O
ð2Þ
There are, therefore, two quite separate aspects to sulfide mineral biooxidation (Boon et al., 1999): (i) an anoxic, abiotic reaction (ferric iron attack on the mineral) and (ii) an oxygen-requiring, biological reaction (regeneration of the oxidant, ferric iron). Optimum conditions for these two reactions may be quite different. Temperature and pH optima for regeneration of ferric iron will be determined by the characteristics of the iron-oxidising acidophile(s) present, while the relative slowness of the abiotic oxidation reaction may be improved by leaching minerals at temperatures above those at which biological systems are active. On the other hand, formation of ‘‘passivation layers’’ of ferric iron precipitates (such as jarosites), which deposit on the surfaces of sulfide minerals impeding their oxidation, may be minimised by maintaining reactor conditions at extremely low pH and low redox potentials, though such conditions (in particular solution pH of < 1) may not be conducive to biotic iron oxidation. The oxidative dissolution of sulfide minerals has been studied most extensively with the mesophilic iron-oxidising acidophiles Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans. Both of these develop relatively high redox potentials (Eh) during mineral leaching by maintaining high ratios of ferric to ferrous iron. This is particularly the case with L. ferrooxidans, where Eh values may exceed 900 mV
(Rawlings et al., 1999). The optimum and minimum pH values for the growth of these bacteria are straindependent, though pH 2.5 (optimum) and 1.3 (minimum) for At. ferrooxidans, and 1.7/1.0 for L. ferrooxidans are typical (Norris and Johnson, 1998). In contrast, the moderately thermophilic iron-oxidising bacteria Sulfobacillus thermosulfidooxidans and Sb. acidophilus (temperature optima *47 C) are relatively poor mineral oxidisers when grown in pure culture, though they are more effective in media amended with yeast extract or carbon dioxide, or when grown in mixed culture with other acidophiles (Dopson and Lindstrom, 1999; Norris et al., 1996; Clark and Norris, 1996). Recently, there have been reports of mesophilic, Sulfobacillus-like iron- and sulfur-oxidising acidophiles (Yahya et al., 1999; Crane and Holden, 1999). Two strains (L-15 and Riv-14) isolated from geothermal areas on the Caribbean Island of Montserrat were found to be extremely acidophilic, growing in media of pH < 1.0 (Yahya et al., 1999). These bacteria can grow autotrophically (exclusively using inorganic carbon), heterotrophically (using only organic carbon) or mixotrophically (simultaneously using organic and inorganic carbon), though they require reduced sulfur (in inorganic form, e.g. tetrathionate or pyrite, or in organic form, e.g. cysteine). In this report, we describe the oxidation of ground rock pyrite by these novel Gram-positive mesophilic bacteria, in comparison with the mesophilic iron-oxidising acidophiles At. ferrooxidans and L. ferrooxidans.
2. Materials and methods 2.1. Origin and maintenance of bacteria Sulfobacillus-like isolates L-15 and Riv-14 were isolated from enrichment cultures containing 10 mM ferrous iron and 0.02% (w/v) yeast extract liquid medium (pH 2.0) that had been inoculated with water samples from Galway’s Soufriere and White River areas on Montserrat (Atkinson et al., 2000) and incubated at 30 C. The oxidised cultures were streaked onto ferrous iron/tetrathionate overlay medium (Johnson, 1995) and the ‘‘fried egg’’-morphology colonies were purified by repeated single colony isolation. Bacteria were maintained in ferrous sulfate/yeast extract
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medium and stored at 4 C. Other mesophilic bacteria used in this study were the autotrophs At. ferrooxidans (ATCC 23270; the type strain) and an L. ferrooxidans strain (CF12) that had been isolated from the Cobalt mine, Idaho (Johnson, 2001). These two iron-oxidising acidophiles were maintained in 20 mM ferrous sulfate medium, pH 2.0. 2.2. Pyrite leaching: shake flask cultures Shake flask cultures were prepared with pyrite-rich rock obtained from the Cae Coch mine, North Wales, which contained 80% FeS2, the remaining minerals being predominantly quartz (Ghauri, 1991). The rock was splintered into small fragments with a geological hammer, and ground to < 20 mm. Prior to use, the pyrite was washed with 2 M hydrochloric acid to remove any surface oxidised deposits, rinsed repeatedly with distilled water, and dried at 70 C. One hundred millilitres of basal salts solution (Ghauri, 1991) were put into 250mL Erlenmeyer flasks, 1 –2 g of pyrite added to each, the pH adjusted to desired starting value (described below) and the stoppered flasks autoclaved at 120 C for 20 min. These were inoculated (5%, v/v) when cool, with active cultures of each of the test bacteria. The cultures that were used had been subcultured through several transfers in pyrite medium in order to adapt the bacteria to the experimental conditions. All cultures were incubated, shaken (150 min 1) at 35 C (30 C in one experiment), and samples were taken at regular intervals to determine total soluble iron and ferrous iron, pH, and redox potentials. The effects of organic carbon on pyrite oxidation by Sulfobacillus L-15 and Riv-14 were assessed by comparing iron solubilisation in cultures containing yeast extract (at 0.02%, w/v) and in yeast extract-free medium. The effects of pH on pyrite oxidation by all three iron-oxidising acidophiles were compared by adjusting the initial pH of culture media to 1.2, 1.5, or 2.5 (yeast extract-free medium). All inoculated cultures and uninoculated controls were set up in duplicate. 2.3. Pyrite leaching under controlled pH: bioreactor cultures Leaching of Cae Coch pyrite by Sulfobacillus L-15 and At. ferrooxidans was compared under conditions of controlled pH. A 2% (w/v) suspension of ground
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rock pyrite in basal salts was added to a laboratoryscale bioreactor (LH series 500; working volume 1.5 L) and inoculated (10%, v/v) with an active pure culture of either iron oxidiser. Cultures were stirred at 150 min 1, aerated at 0.5 L/min with sterile air and maintained at 35 C. The pH of the cultures was progressively lowered from pH 1.5 to 1.0 (in the case of At. ferrooxidans) or pH 0.8 (for Sulfobacillus L-15) by the addition of sulfuric acid. Between 11 and 17 days, bacteria were grown under the same conditions at a particular pH value before the pH set point was lowered. Samples were withdrawn daily and analysed for soluble total iron, ferrous iron (soluble ferric iron being calculated from differences between these) and redox potentials. Total planktonic-phase bacteria were enumerated using a Thoma counting chamber, and viable planktonic-phase cells were enumerated by plating onto ferrous iron (At. ferrooxidans) or ferrous iron/tetrathionate (Sulfobacillus L-15) overlay plates (Johnson, 1995). 2.4. Miscellaneous analyses Total soluble iron was determined by atomic absorption spectrophotometry, and ferrous iron using the ferrozine assay (Lovley and Phillips, 1987). Culture pH was determined (shake flask cultures) using a pHase combination electrode fitted to an Accumet 50 meter. Redox potentials were measured using a platinum electrode (combined with a silver/silver chloride reference electrode) and converted to Eh values (i.e. relative to a hydrogen reference electrode) by adding 234 mV to the measured values.
3. Results The two Montserrat Sulfobacillus isolates showed differing responses to the inclusion of yeast extract in the mineral medium; rates of pyrite oxidation were far greater in yeast extract-amended medium in the case of strain Riv-14, whereas they were very similar in cultures of strain L-15 (Fig. 1). This difference was reflected in differences in culture pH values, which were consistently lower in yeast extract-amended cultures (compared to yeast extract-free cultures) of strain Riv-14, but not those of strain L-15. Sulfobacillus L-15 was therefore selected for further exper-
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Fig. 1. Leaching of 1% (w/v) ground rock pyrite by cultures of mesophilic Gram-positive bacteria amended, or not, with yeast extract (at 0.02%, w/v). Key: E, Sulfobacillus L-15 (with yeast extract); D, Sulfobacillus L-15 (without yeast extract); x, Sulfobacillus Riv-14 (with yeast extract); w, Sulfobacillus Riv-14 (without yeast extract); +, uninoculated control. All data points are the mean of replicate cultures.
imental work, where organic-free media were used routinely. Culture pH was found to have a major and differential impact on pyrite leaching by Sulfobacillus L15, At. ferrooxidans, and L. ferrooxidans (Fig. 2). All three bacteria oxidised pyrite in media set initially at either pH 2.5 or 1.5. At. ferroxidans was the most efficient metal-mobilising bacterium, and Sulfobacillus L-15 the least efficient, at pH 2.5 (Fig. 2a). However, in cultures set at pH 1.2, pyrite oxidation was observed only in cultures inoculated with Sulfobacillus L-15 (Fig. 2c). The pH of the cultures set initially at 2.5 fell during mineral oxidation, presumably as a consequence of biotic and abiotic oxidation of RISCs to sulfuric acid. The cultures set at pH 1.5 and 1.2 were, however, more strongly buffered, due to the influence of the SO42 /HSO4 couple (the pKa2 of sulfuric acid is 1.92) and these cultures (particularly those at pH 1.2) displayed smaller changes in pH during mineral oxidation.
Fig. 2. Leaching of 2% (w/v) pyrite in media set at initial values of (a) 2.5, (b) 1.5, and (c) 1.2. Key: 6, At. ferrooxidans; w, L. ferrooxidans; n, Sulfobacillus L-15; +, uninoculated control. All data points are the mean of replicate cultures.
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Mean mineral leaching rates (calculated from the main linear phases of mineral oxidation) by the three iron oxidisers are shown in Table 1. Sulfobacillus L-15 was effective at oxidising pyrite only in cultures set at pH 1.5 and 1.2. It is interesting to note that the rate of pyrite oxidation by Sulfobacillus L-15 in media that were poised initially at pH 2.5 increased at day 44, by which time the pH had fallen to 1.4. Leaching of pyrite by Sulfobacillus L-15 and the type strain of At. ferrooxidans under controlled pH are shown in Figs. 3 and 4, respectively. No secondary deposits or precipitates were observed in these bioreactor cultures. During the first 12-day phase of the experiment, pH was maintained at 1.5, and pyrite oxidation was observed with both cultures. However, although concentrations of total soluble iron increased in both, there were notable differences in iron speciation, with higher ferric/ferrous ratios occurring in the At. ferrooxidans bioreactor. This trend was confirmed by the measured Eh values, which increased from + 780 to + 900 mV in the At. ferrooxidans culture during this period, while the maximum value recorded for the Sulfobacillus L-15 culture was + 799 mV. Overall, Sulfobacillus L-15 displayed superior pyrite leaching during this first phase; total soluble iron was 1.6 g/L in the Sulfobacillus L-15 bioreactor, and 1.2 g/L in the At. ferrooxidans culture at day 12. Numbers of total and viable bacteria increased in both cultures during this first phase (Fig. 5). At day 12, the pH was lowered and fixed at 1.0 for both cultures. In the case of At. ferrooxidans, this caused a temporary (3 days) cessation of pyrite leaching (as evidenced by total soluble iron concentrations), though this resumed at day 15 before halting again at day 23. This period of pyrite dissolution corresponded
Table 1 Comparison of rates of pyrite oxidation by mesophilic iron-oxidising acidophiles in shake flask cultures adjusted to different initial pH values Bacteria
At. ferrooxidans L. ferrooxidans Sulfobacillus L-15
Pyrite oxidation rates (mg Fe solubilised per day) pH 1.2
pH 1.5
pH 2.5
n.o n.o. 204
223 57 230
240 136 68
n.o., no ferrous iron oxidation.
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Fig. 3. Leaching of 2% (w/v) pyrite by Sulfobacillus L-15 under controlled pH in a stirred bioreactor (phase 1, pH 1.5; phase 2, pH 1.0; phase 3, pH 0.8). Key: n, total soluble iron; w, soluble ferrous iron; E, soluble ferric iron; , redox potentials (Eh).
with changes in ferrous/ferric ratios (and redox potentials) which indicated that abiotic, ferric iron-catalysed oxidation of the mineral was occurring, but not bacterial regeneration of ferric iron. The rate of pyrite leaching in the At. ferrooxidans culture was also less than when the culture was maintained at pH 1.5 (47 mg, compared with 81 mg iron solubilised per day). During this second leaching phase, viable At. ferrooxidans cells were detected only on day 14, and were fewer than those counted at the end of phase 1 of the experiment (Fig. 5). Most, if not all, of the bacteria counted under the microscope (total counts) were therefore nonviable during phase 2. In contrast, pyrite leaching continued uninterrupted in the Sulfobacillus L-15 culture, and similar ferrous/ferric ratios were maintained, when the pH was lowered from pH 1.5 to 1.0 (Fig. 4). Numbers of total and viable Sulfobacillus L-15 both increased and remained similar to each other during leaching phase 2 (Fig. 5). The third phase of the fixed pH pyrite leaching experiment was different for the two cultures, in view of the differential patterns that had emerged during phase 2. In the case of Sulfobacillus L-15, the culture pH was lowered further to 0.8. This caused a temporary halt in pyrite leaching, a decline in the ferric/ferrous iron ratio and a corresponding decrease in redox potential. However, after 10 days had elapsed, the
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Fig. 4. Leaching of 2% (w/v) pyrite by At. ferrooxidans under controlled pH in a stirred bioreactor (phase 1, pH 1.5; phase 2, pH 1.0; phase 3, pH 1.5). Key: n, total soluble iron; w, soluble ferrous iron; E, soluble ferric iron; , redox potentials (Eh).
Fig. 5. Cell counts of Sulfobacillus L-15 and At. ferrooxidans during the leaching of 2% (w/v) pyrite under controlled pH in stirred bioreactor cultures. Total numbers of cells were estimated microscopically using a Thoma cell counting chamber, and numbers of viable bacteria using overlaid solid media. Phase 1, pH 1.5; phase 2, pH 1.2; phase 3, pH 0.8 (Sulfobacillus L-15) or pH 1.5 (At. ferrooxidans).
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culture appeared to recover and the subsequent rate of pyrite oxidation (238 mg iron solubilised per day) appeared to be greater than rates recorded at pH 1.5 or 1.0, though this was calculated from fewer data points. While numbers of both total and viable Sulfobacillus L-15 were less than those counted at pH 1.0, the ratio of viable/total cells was similar to that found during the earlier phases of the experiment (Fig. 5). In the case of the At. ferrooxidans culture, it was decided, in phase 3, to raise the pH to 1.5 in an attempt to restart microbial leaching. However, this resulted in no additional leaching of pyrite or isolation of viable bacteria (Figs. 3 and 5).
4. Discussion It is now recognised that a wide range of acidophilic bacteria and archaea can accelerate the oxidative dissolution of sulfidic minerals due to their abilities to oxidise reduced iron and/or sulfur. The two known Sulfobacillus species that catalyse pyrite oxidation (Sb. thermosulfidooxidans and Sb. acidophilus) are both ‘‘moderate thermophiles’’. In contrast, while the two Montserrat Sulfobacillus isolates (L-15 and Riv-14) described in this paper have a number of physiological characteristics that are in common with these classified species (e.g. in being Gram-positive, spore-forming iron-oxidisers), they differ from them in being mesophilic (neither strain Riv-14 nor L-15 can grow at 40 C), and in their tolerance of extreme acidity (Sb. thermosulfidooxidans and Sb. acidophilus do not grow below pH 1.5; Norris and Johnson, 1998). Sulfobacillus strains L-15 and Riv-14 displayed different responses to the addition of yeast extract to mineral medium. The greatly enhanced leaching displayed by strain Riv-14 in yeast extract-amended cultures was similar to the response of the moderately thermophilic Sulfobacillus spp., while the negligible effect of yeast extract on pyrite leaching by strain L-15 suggests that this bacterium has a superior capacity for autotrophic growth. Commercial mineral leaching processes operate without addition of organic substrates, and this would tend to select chemoautotrophic bacteria and archaea rather than heterotrophic acidophiles that require extraneous fixed carbon for optimum leaching activity. However, commensal interactions, whereby heterotrophs scavenge organic compounds (lysates and
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exudates) originating from CO2-fixing acidophiles is known to occur (Johnson, 1998). Shake flask leaching studies, using media set at different pH values, showed that only Sulfobacillus L15 (of the three iron oxidisers that were tested) was able to oxidise pyrite at pH 1.2. In general, L. ferrooxidans tends to be slightly more tolerant of low pH than At. ferrooxidans (Norris and Johnson, 1998), though strains of both of these bacteria vary in this respect. The rate of pyrite oxidation displayed by Sulfobacillus L-15 at pH 1.2 was only slightly less than at pH 1.5 (the pH optimum of this iron oxidiser is 1.6; Yahya et al., 1999). This, in turn, was similar to the maximum rate displayed by At. ferrooxidans in cultures adjusted to the initial pH of 2.5. The different abilities of Sulfobacillus L-15 and At. ferrooxidans to leach pyrite at extremely low pH values was even more clearly illustrated in batch cultures grown in pH-statted bioreactors. Whereas Sulfobacillus L-15 leached pyrite when the culture was maintained at pH 1.5, 1.0, and (after a period of adaptation) 0.8, At. ferrooxidans was active only at pH 1.5. Comparison of total and plate counts of these bacteria showed that Sulfobacillus L-15 remained viable throughout the experiment. In contrast, lowering the pH of the leach liquor to 1.0 caused rapid mortality of At. ferrooxidans, and the bacterial population did not recover when the pH was raised subsequently back to 1.5. While it is possible that the plating technique might have enumerated spores of Sulfobacillus L-15 rather than active vegetative cells, endospores were rarely seen when the culture was viewed under the microscope, at any phase of the experiment, and it was concluded that plate counts recorded numbers of viable vegetative cells. During pyrite leaching, Sulfobacillus L-15 maintained a redox potential of between 760 and 800 mV when the bioreactor was set at pH 1.5 and 1.0, though this fell to < 650 mV when the culture pH was adjusted to 0.8. The redox potential in pyrite leaching systems is determined by the ratio of ferric to ferrous iron in solution; the E0 of the ferrous/ferric couple is 770 mV at pH 2.0. The ferrous/ferric iron ratios and measured redox potentials showed good agreement in both bioreactor cultures. Concentrations of soluble ferrous and ferric iron in the Sulfobacillus L-15 culture increased during leaching but remained very similar to each other up to the time that the culture pH was low-
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ered to 0.8. In contrast, the redox potential in the At. ferrooxidans bioreactor increased rapidly to 890 mV in phase 1 (pH 1.5) of the experiment, and > 99% of the soluble iron was present as ferric iron, which is typical of pyrite leaching by this iron oxidiser. However, a major disadvantage of high Eh values in leachate liquors, so far as commercial leaching operations are concerned, is that elevated concentrations of ferric iron promote the formation of secondary ferric iron-containing minerals, such as jarosites. In this regard, it would be advantageous, particularly with some ores and concentrates (e.g. chalcopyrite), to utilise microorganisms such as Sulfobacillus L-15 that are able to leach sulfide minerals effectively while maintaining relatively low redox potentials and correspondingly low concentrations of soluble ferric iron. Formation of jarosites is also precluded at extremely low pH (these are basic ferric sulphates, e.g. natrojarosite: NaFe3(SO4)2(OH)6) where Sulfobacillus L-15, unlike other mesophilic iron-oxidising bacteria, is active. These physiological characteristics, together with its capacity for chemolithotrophic growth on sulfide minerals (and sulfur), suggest that this novel mesophilic, Grampositive bacterium could have considerable potential for bioprocessing of ores and concentrates. Acknowledgements Adibah Yahya is grateful to the University Technology Malaysia for providing a research studentship. References Atkinson, T., Cairns, S., Cowan, D.A., Danson, M.J., Hough, D.W., Johnson, D.B., Norris, P.R., Raven, N., Robson, R., Robinson, C., Sharp, R.J., 2000. A microbiological survey of Montserrat island hydrothermal biotopes. Extremophiles 4, 305 – 313. Boon, M., Brasser, H.J., Hansford, G.S., Heijnen, J.J., 1999. Comparison of the oxidation kinetics of different pyrites in the presence of Thiobacillus ferrooxidans or Leptospirillum ferrooxidans. Hydrometallurgy 53, 57 – 72. Brierley, C.L., 2001. Bacterial succession in bioheap leaching. Hydrometallurgy 59, 249 – 256. Brierley, J.A., Brierley, C.L., 2001. Present and future commercial applications in biohydrometallurgy. Hydrometallurgy 59, 233 – 240. Clark, D.A., Norris, P.R., 1996. Acidimicrobium ferrooxidans gen.
nov., sp. nov.: mixed-culture ferrous iron oxidation with Sulfobacillus species. Microbiology 141, 785 – 790. Crane, A.G., Holden, P.J., 1999. Leaching of harbour sediments by estuarine iron-oxidising bacteria. In: Amils, R., Ballester, A. (Eds.), Biohydrometallurgy and the Environment Toward the Mining of the 21st Century, Process Microbiology 9A. Elsevier, Amsterdam, pp. 347 – 356. Dopson, M., Lindstrom, E.B., 1999. Potential role of Thiobacillus caldus in arsenopyrite leaching. Appl. Environ. Microbiol. 65, 36 – 40. Fowler, T.A., Holmes, P.R., Crundwell, F.K., 2001. On the kinetics and mechanism of the dissolution of pyrite in the presence of Thiobacillus ferrooxidans. Hydrometallurgy 59, 257 – 270. Ghauri, M.A., 1991. A study of the diversity of acidophilic bacteria. PhD Thesis, University of Wales. Goebel, B.M., Stackebrandt, E., 1994. Cultural and phylogenetic analysis of mixed microbial populations found in natural and commercial bioleaching environments. Appl. Environ. Microbiol. 60, 1614 – 1621. Johnson, D.B., 1995. Selective solid media for isolating and enumerating acidophilic bacteria. J. Microbiol. Methods, 205 – 218. Johnson, D.B., 1998. Biodiversity and ecology of acidophilic microorganisms. FEMS Microbiol. Ecol. 27, 307 – 317. Johnson, D.B., 2001. Genus Leptospirillum. In: Boone, D.R., Castenholz, R.W., Garrity, G.M. (Eds.), Bergey’s Manual of Systematic Bacteriology (2nd edn.), vol. 1. Springer, New York, pp. 453 – 456. Lovley, D.R., Phillips, E.J.P., 1987. Rapid assay for microbially reducible ferric iron in aquatic sediments. Appl. Environ. Microbiol. 53, 1536 – 1540. Norris, P.R., Johnson, D.B., 1998. Acidophilic microorganisms. In: Horikoshi, K., Grant, W.D. (Eds.), Extremophiles: Microbial Life in Extreme Environments. Wiley, New York, pp. 133 – 154. Norris, P.R., Clark, D.A., Owen, J.P., Waterhouse, S., 1996. Characteristics of Sulfobacillus acidophilus so. nov. and other moderately thermophilic mineral sulphide-oxidizing bacteria. Microbiology 141, 775 – 783. Norris, P.R., Burton, N.P., Foulis, N.A.M., 2000. Acidophiles in bioreactor mineral processing. Extremophiles 4, 71 – 76. Rawlings, D.E. (Ed.), 1997. Biomining: Theory, Microbes and Industrial Processes. Springer-Verlag/Landes Bioscience, Georgetown, TX. Rawlings, D.E., Tributsch, H., Hansford, G.S., 1999. Reasons why ‘‘Leptospirillum-like’’ species rather than Thiobacillus ferrooxidans are the dominant iron-oxidizing bacteria in many commercial processes for the biooxidation of pyrite and related ores. Microbiology 145, 5 – 13. Schippers, A., Sand, W., 1999. Bacterial leaching of metal sulfides proceeds by two indirect mechanisms via thiosulfate or via polysulfides and sulfur. Appl. Environ. Microbiol. 65, 319 – 321. Yahya, A., Roberto, F.F., Johnson, D.B., 1999. Novel mineral-oxidising bacteria from Montserrat (W.I.): physiological and phylogenetic characteristics. In: Amils, R., Ballester, A. (Eds.), Biohydrometallurgy and the Environment Toward the Mining of the 21st Century Process Microbiology 9A. Elsevier, Amsterdam, pp. 72 – 740.
Bioleaching of pyrite at low pH and low redox potentials by novel mesophilic Gram-positive bacteria Adibah Yahya, D. Barrie Johnson* School of Biological Sciences, University of Wales, Bangor LL57 2UW, UK Received 8 August 2001; received in revised form 13 November 2001; accepted 13 November 2001
Abstract Bioleaching of ground rock pyrite by two novel strains of Sulfobacillus-like acidophilic bacteria was examined in shake flask and bioreactor cultures. The Gram-positive prokaryotes differed from known species of mineral-oxidising Sulfobacillus in being mesophilic and in their tolerance to extreme acidity (pH < 1). The oxidative dissolution of pyrite by one of the novel Sulfobacillus strains was stimulated by yeast extract, though the second strain (L-15) was equally effective in organic-free medium. Pyrite dissolution by Sulfobacillus L-15 was compared with that by the strain type of Acidithiobacillus ferrooxidans and a Leptospirillum ferrooxidans isolate, in media set at different initial pH values. All three bacteria leached the mineral in media set at pH 2.5 and pH 1.5, but at pH 1.2, mineral oxidation (at a rate similar to the maximum recorded for At. ferrooxidans) was observed only in cultures of Sulfobacillus L-15. Pyrite leaching by Sulfobacillus L-15 and At. ferrooxidans was compared in bioreactor cultures, at different and controlled pH values. Lowering bioreactor pH from 1.5 to 1.0 resulted in the cessation of biologically accelerated pyrite oxidation by the At. ferrooxidans culture and rapid mortality of the bacteria. In contrast, Sulfobacillus L-15 continued to leach the mineral when the pH was lowered to 1.0 and (after a short lag period) to pH 0.8. In contrast to the At. ferrooxidans culture, large numbers of viable Sulfobacillus L-15 cells were recovered, regardless of bioreactor pH. Redox potentials and ratios of soluble ferric/ferrous iron in the Sulfobacillus L-15 culture were significantly lower (by about 100 – 150 mV) than values found typically in Gram-negative bacterial leaching cultures, and that recorded in the (active) At. ferrooxidans culture in the present work. The potential advantages of using microorganisms that can leach sulfidic minerals effectively at pH < 1 and at low redox potential are discussed. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Bioleaching; Pyrite; Redox potential; Sulfobacillus; Extreme pH
1. Introduction The use of acidophilic bacteria and archaea to accelerate the oxidative dissolution of sulfidic minerals and ores and, thereby, to facilitate the extraction of occluded gold (‘‘biooxidation’’) or to solubilise base metals such as copper and cobalt (‘‘bioleaching’’), is *
Corresponding author. Tel.: +44-1248-382358; fax: +441248-370731. E-mail address: [email protected] (D.B. Johnson).
now established as an economically important biotechnology (Rawlings, 1997; Brierley and Brierley, 2001). Somewhat surprisingly, reports describing composition of commercial bioleach tanks and heaps are relatively few (e.g. Brierley, 2001; Goebel and Stackebrandt, 1994; Norris et al., 2000). Temperature has an important determinative effect on which acidophiles are active in mineral leaching, with bacteria tending to dominate at lower temperatures (30 –60 C), while at higher temperatures, sulfide mineral oxidation is mediated by acidophilic archaea (e.g. Acidianus spp.,
0304-386X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 3 8 6 X ( 0 1 ) 0 0 2 2 4 - 9
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Metallosphaera spp. and Sulfolobus metallicus; Norris and Johnson, 1998). The mechanism by which bacteria oxidise sulfides has been much debated (e.g. Schippers and Sand, 1999; Fowler et al., 2001). Sulfide minerals may be subdivided into those such as sphalerite that are acidsoluble, and others such as pyrite and chalcopyrite that are acid-insoluble but which can be oxidised by ferric iron in acidic liquors. Iron-oxidising acidophilic prokaryotes have a key role in the biooxidation of this second group of sulfide minerals since they regenerate the oxidant ferric iron, which is reduced to the ferrous state during mineral attack (Eqs. (1) and (2)). þ FeS2 þ 6Fe3þ þ 3H2 O ! 7Fe2þ þ S2 O2 3 þ 6H
ð1Þ 4Fe2þ þ O2 þ 4Hþ ! 4Fe3þ þ 2H2 O
ð2Þ
There are, therefore, two quite separate aspects to sulfide mineral biooxidation (Boon et al., 1999): (i) an anoxic, abiotic reaction (ferric iron attack on the mineral) and (ii) an oxygen-requiring, biological reaction (regeneration of the oxidant, ferric iron). Optimum conditions for these two reactions may be quite different. Temperature and pH optima for regeneration of ferric iron will be determined by the characteristics of the iron-oxidising acidophile(s) present, while the relative slowness of the abiotic oxidation reaction may be improved by leaching minerals at temperatures above those at which biological systems are active. On the other hand, formation of ‘‘passivation layers’’ of ferric iron precipitates (such as jarosites), which deposit on the surfaces of sulfide minerals impeding their oxidation, may be minimised by maintaining reactor conditions at extremely low pH and low redox potentials, though such conditions (in particular solution pH of < 1) may not be conducive to biotic iron oxidation. The oxidative dissolution of sulfide minerals has been studied most extensively with the mesophilic iron-oxidising acidophiles Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans. Both of these develop relatively high redox potentials (Eh) during mineral leaching by maintaining high ratios of ferric to ferrous iron. This is particularly the case with L. ferrooxidans, where Eh values may exceed 900 mV
(Rawlings et al., 1999). The optimum and minimum pH values for the growth of these bacteria are straindependent, though pH 2.5 (optimum) and 1.3 (minimum) for At. ferrooxidans, and 1.7/1.0 for L. ferrooxidans are typical (Norris and Johnson, 1998). In contrast, the moderately thermophilic iron-oxidising bacteria Sulfobacillus thermosulfidooxidans and Sb. acidophilus (temperature optima *47 C) are relatively poor mineral oxidisers when grown in pure culture, though they are more effective in media amended with yeast extract or carbon dioxide, or when grown in mixed culture with other acidophiles (Dopson and Lindstrom, 1999; Norris et al., 1996; Clark and Norris, 1996). Recently, there have been reports of mesophilic, Sulfobacillus-like iron- and sulfur-oxidising acidophiles (Yahya et al., 1999; Crane and Holden, 1999). Two strains (L-15 and Riv-14) isolated from geothermal areas on the Caribbean Island of Montserrat were found to be extremely acidophilic, growing in media of pH < 1.0 (Yahya et al., 1999). These bacteria can grow autotrophically (exclusively using inorganic carbon), heterotrophically (using only organic carbon) or mixotrophically (simultaneously using organic and inorganic carbon), though they require reduced sulfur (in inorganic form, e.g. tetrathionate or pyrite, or in organic form, e.g. cysteine). In this report, we describe the oxidation of ground rock pyrite by these novel Gram-positive mesophilic bacteria, in comparison with the mesophilic iron-oxidising acidophiles At. ferrooxidans and L. ferrooxidans.
2. Materials and methods 2.1. Origin and maintenance of bacteria Sulfobacillus-like isolates L-15 and Riv-14 were isolated from enrichment cultures containing 10 mM ferrous iron and 0.02% (w/v) yeast extract liquid medium (pH 2.0) that had been inoculated with water samples from Galway’s Soufriere and White River areas on Montserrat (Atkinson et al., 2000) and incubated at 30 C. The oxidised cultures were streaked onto ferrous iron/tetrathionate overlay medium (Johnson, 1995) and the ‘‘fried egg’’-morphology colonies were purified by repeated single colony isolation. Bacteria were maintained in ferrous sulfate/yeast extract
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medium and stored at 4 C. Other mesophilic bacteria used in this study were the autotrophs At. ferrooxidans (ATCC 23270; the type strain) and an L. ferrooxidans strain (CF12) that had been isolated from the Cobalt mine, Idaho (Johnson, 2001). These two iron-oxidising acidophiles were maintained in 20 mM ferrous sulfate medium, pH 2.0. 2.2. Pyrite leaching: shake flask cultures Shake flask cultures were prepared with pyrite-rich rock obtained from the Cae Coch mine, North Wales, which contained 80% FeS2, the remaining minerals being predominantly quartz (Ghauri, 1991). The rock was splintered into small fragments with a geological hammer, and ground to < 20 mm. Prior to use, the pyrite was washed with 2 M hydrochloric acid to remove any surface oxidised deposits, rinsed repeatedly with distilled water, and dried at 70 C. One hundred millilitres of basal salts solution (Ghauri, 1991) were put into 250mL Erlenmeyer flasks, 1 –2 g of pyrite added to each, the pH adjusted to desired starting value (described below) and the stoppered flasks autoclaved at 120 C for 20 min. These were inoculated (5%, v/v) when cool, with active cultures of each of the test bacteria. The cultures that were used had been subcultured through several transfers in pyrite medium in order to adapt the bacteria to the experimental conditions. All cultures were incubated, shaken (150 min 1) at 35 C (30 C in one experiment), and samples were taken at regular intervals to determine total soluble iron and ferrous iron, pH, and redox potentials. The effects of organic carbon on pyrite oxidation by Sulfobacillus L-15 and Riv-14 were assessed by comparing iron solubilisation in cultures containing yeast extract (at 0.02%, w/v) and in yeast extract-free medium. The effects of pH on pyrite oxidation by all three iron-oxidising acidophiles were compared by adjusting the initial pH of culture media to 1.2, 1.5, or 2.5 (yeast extract-free medium). All inoculated cultures and uninoculated controls were set up in duplicate. 2.3. Pyrite leaching under controlled pH: bioreactor cultures Leaching of Cae Coch pyrite by Sulfobacillus L-15 and At. ferrooxidans was compared under conditions of controlled pH. A 2% (w/v) suspension of ground
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rock pyrite in basal salts was added to a laboratoryscale bioreactor (LH series 500; working volume 1.5 L) and inoculated (10%, v/v) with an active pure culture of either iron oxidiser. Cultures were stirred at 150 min 1, aerated at 0.5 L/min with sterile air and maintained at 35 C. The pH of the cultures was progressively lowered from pH 1.5 to 1.0 (in the case of At. ferrooxidans) or pH 0.8 (for Sulfobacillus L-15) by the addition of sulfuric acid. Between 11 and 17 days, bacteria were grown under the same conditions at a particular pH value before the pH set point was lowered. Samples were withdrawn daily and analysed for soluble total iron, ferrous iron (soluble ferric iron being calculated from differences between these) and redox potentials. Total planktonic-phase bacteria were enumerated using a Thoma counting chamber, and viable planktonic-phase cells were enumerated by plating onto ferrous iron (At. ferrooxidans) or ferrous iron/tetrathionate (Sulfobacillus L-15) overlay plates (Johnson, 1995). 2.4. Miscellaneous analyses Total soluble iron was determined by atomic absorption spectrophotometry, and ferrous iron using the ferrozine assay (Lovley and Phillips, 1987). Culture pH was determined (shake flask cultures) using a pHase combination electrode fitted to an Accumet 50 meter. Redox potentials were measured using a platinum electrode (combined with a silver/silver chloride reference electrode) and converted to Eh values (i.e. relative to a hydrogen reference electrode) by adding 234 mV to the measured values.
3. Results The two Montserrat Sulfobacillus isolates showed differing responses to the inclusion of yeast extract in the mineral medium; rates of pyrite oxidation were far greater in yeast extract-amended medium in the case of strain Riv-14, whereas they were very similar in cultures of strain L-15 (Fig. 1). This difference was reflected in differences in culture pH values, which were consistently lower in yeast extract-amended cultures (compared to yeast extract-free cultures) of strain Riv-14, but not those of strain L-15. Sulfobacillus L-15 was therefore selected for further exper-
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Fig. 1. Leaching of 1% (w/v) ground rock pyrite by cultures of mesophilic Gram-positive bacteria amended, or not, with yeast extract (at 0.02%, w/v). Key: E, Sulfobacillus L-15 (with yeast extract); D, Sulfobacillus L-15 (without yeast extract); x, Sulfobacillus Riv-14 (with yeast extract); w, Sulfobacillus Riv-14 (without yeast extract); +, uninoculated control. All data points are the mean of replicate cultures.
imental work, where organic-free media were used routinely. Culture pH was found to have a major and differential impact on pyrite leaching by Sulfobacillus L15, At. ferrooxidans, and L. ferrooxidans (Fig. 2). All three bacteria oxidised pyrite in media set initially at either pH 2.5 or 1.5. At. ferroxidans was the most efficient metal-mobilising bacterium, and Sulfobacillus L-15 the least efficient, at pH 2.5 (Fig. 2a). However, in cultures set at pH 1.2, pyrite oxidation was observed only in cultures inoculated with Sulfobacillus L-15 (Fig. 2c). The pH of the cultures set initially at 2.5 fell during mineral oxidation, presumably as a consequence of biotic and abiotic oxidation of RISCs to sulfuric acid. The cultures set at pH 1.5 and 1.2 were, however, more strongly buffered, due to the influence of the SO42 /HSO4 couple (the pKa2 of sulfuric acid is 1.92) and these cultures (particularly those at pH 1.2) displayed smaller changes in pH during mineral oxidation.
Fig. 2. Leaching of 2% (w/v) pyrite in media set at initial values of (a) 2.5, (b) 1.5, and (c) 1.2. Key: 6, At. ferrooxidans; w, L. ferrooxidans; n, Sulfobacillus L-15; +, uninoculated control. All data points are the mean of replicate cultures.
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Mean mineral leaching rates (calculated from the main linear phases of mineral oxidation) by the three iron oxidisers are shown in Table 1. Sulfobacillus L-15 was effective at oxidising pyrite only in cultures set at pH 1.5 and 1.2. It is interesting to note that the rate of pyrite oxidation by Sulfobacillus L-15 in media that were poised initially at pH 2.5 increased at day 44, by which time the pH had fallen to 1.4. Leaching of pyrite by Sulfobacillus L-15 and the type strain of At. ferrooxidans under controlled pH are shown in Figs. 3 and 4, respectively. No secondary deposits or precipitates were observed in these bioreactor cultures. During the first 12-day phase of the experiment, pH was maintained at 1.5, and pyrite oxidation was observed with both cultures. However, although concentrations of total soluble iron increased in both, there were notable differences in iron speciation, with higher ferric/ferrous ratios occurring in the At. ferrooxidans bioreactor. This trend was confirmed by the measured Eh values, which increased from + 780 to + 900 mV in the At. ferrooxidans culture during this period, while the maximum value recorded for the Sulfobacillus L-15 culture was + 799 mV. Overall, Sulfobacillus L-15 displayed superior pyrite leaching during this first phase; total soluble iron was 1.6 g/L in the Sulfobacillus L-15 bioreactor, and 1.2 g/L in the At. ferrooxidans culture at day 12. Numbers of total and viable bacteria increased in both cultures during this first phase (Fig. 5). At day 12, the pH was lowered and fixed at 1.0 for both cultures. In the case of At. ferrooxidans, this caused a temporary (3 days) cessation of pyrite leaching (as evidenced by total soluble iron concentrations), though this resumed at day 15 before halting again at day 23. This period of pyrite dissolution corresponded
Table 1 Comparison of rates of pyrite oxidation by mesophilic iron-oxidising acidophiles in shake flask cultures adjusted to different initial pH values Bacteria
At. ferrooxidans L. ferrooxidans Sulfobacillus L-15
Pyrite oxidation rates (mg Fe solubilised per day) pH 1.2
pH 1.5
pH 2.5
n.o n.o. 204
223 57 230
240 136 68
n.o., no ferrous iron oxidation.
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Fig. 3. Leaching of 2% (w/v) pyrite by Sulfobacillus L-15 under controlled pH in a stirred bioreactor (phase 1, pH 1.5; phase 2, pH 1.0; phase 3, pH 0.8). Key: n, total soluble iron; w, soluble ferrous iron; E, soluble ferric iron; , redox potentials (Eh).
with changes in ferrous/ferric ratios (and redox potentials) which indicated that abiotic, ferric iron-catalysed oxidation of the mineral was occurring, but not bacterial regeneration of ferric iron. The rate of pyrite leaching in the At. ferrooxidans culture was also less than when the culture was maintained at pH 1.5 (47 mg, compared with 81 mg iron solubilised per day). During this second leaching phase, viable At. ferrooxidans cells were detected only on day 14, and were fewer than those counted at the end of phase 1 of the experiment (Fig. 5). Most, if not all, of the bacteria counted under the microscope (total counts) were therefore nonviable during phase 2. In contrast, pyrite leaching continued uninterrupted in the Sulfobacillus L-15 culture, and similar ferrous/ferric ratios were maintained, when the pH was lowered from pH 1.5 to 1.0 (Fig. 4). Numbers of total and viable Sulfobacillus L-15 both increased and remained similar to each other during leaching phase 2 (Fig. 5). The third phase of the fixed pH pyrite leaching experiment was different for the two cultures, in view of the differential patterns that had emerged during phase 2. In the case of Sulfobacillus L-15, the culture pH was lowered further to 0.8. This caused a temporary halt in pyrite leaching, a decline in the ferric/ferrous iron ratio and a corresponding decrease in redox potential. However, after 10 days had elapsed, the
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Fig. 4. Leaching of 2% (w/v) pyrite by At. ferrooxidans under controlled pH in a stirred bioreactor (phase 1, pH 1.5; phase 2, pH 1.0; phase 3, pH 1.5). Key: n, total soluble iron; w, soluble ferrous iron; E, soluble ferric iron; , redox potentials (Eh).
Fig. 5. Cell counts of Sulfobacillus L-15 and At. ferrooxidans during the leaching of 2% (w/v) pyrite under controlled pH in stirred bioreactor cultures. Total numbers of cells were estimated microscopically using a Thoma cell counting chamber, and numbers of viable bacteria using overlaid solid media. Phase 1, pH 1.5; phase 2, pH 1.2; phase 3, pH 0.8 (Sulfobacillus L-15) or pH 1.5 (At. ferrooxidans).
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culture appeared to recover and the subsequent rate of pyrite oxidation (238 mg iron solubilised per day) appeared to be greater than rates recorded at pH 1.5 or 1.0, though this was calculated from fewer data points. While numbers of both total and viable Sulfobacillus L-15 were less than those counted at pH 1.0, the ratio of viable/total cells was similar to that found during the earlier phases of the experiment (Fig. 5). In the case of the At. ferrooxidans culture, it was decided, in phase 3, to raise the pH to 1.5 in an attempt to restart microbial leaching. However, this resulted in no additional leaching of pyrite or isolation of viable bacteria (Figs. 3 and 5).
4. Discussion It is now recognised that a wide range of acidophilic bacteria and archaea can accelerate the oxidative dissolution of sulfidic minerals due to their abilities to oxidise reduced iron and/or sulfur. The two known Sulfobacillus species that catalyse pyrite oxidation (Sb. thermosulfidooxidans and Sb. acidophilus) are both ‘‘moderate thermophiles’’. In contrast, while the two Montserrat Sulfobacillus isolates (L-15 and Riv-14) described in this paper have a number of physiological characteristics that are in common with these classified species (e.g. in being Gram-positive, spore-forming iron-oxidisers), they differ from them in being mesophilic (neither strain Riv-14 nor L-15 can grow at 40 C), and in their tolerance of extreme acidity (Sb. thermosulfidooxidans and Sb. acidophilus do not grow below pH 1.5; Norris and Johnson, 1998). Sulfobacillus strains L-15 and Riv-14 displayed different responses to the addition of yeast extract to mineral medium. The greatly enhanced leaching displayed by strain Riv-14 in yeast extract-amended cultures was similar to the response of the moderately thermophilic Sulfobacillus spp., while the negligible effect of yeast extract on pyrite leaching by strain L-15 suggests that this bacterium has a superior capacity for autotrophic growth. Commercial mineral leaching processes operate without addition of organic substrates, and this would tend to select chemoautotrophic bacteria and archaea rather than heterotrophic acidophiles that require extraneous fixed carbon for optimum leaching activity. However, commensal interactions, whereby heterotrophs scavenge organic compounds (lysates and
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exudates) originating from CO2-fixing acidophiles is known to occur (Johnson, 1998). Shake flask leaching studies, using media set at different pH values, showed that only Sulfobacillus L15 (of the three iron oxidisers that were tested) was able to oxidise pyrite at pH 1.2. In general, L. ferrooxidans tends to be slightly more tolerant of low pH than At. ferrooxidans (Norris and Johnson, 1998), though strains of both of these bacteria vary in this respect. The rate of pyrite oxidation displayed by Sulfobacillus L-15 at pH 1.2 was only slightly less than at pH 1.5 (the pH optimum of this iron oxidiser is 1.6; Yahya et al., 1999). This, in turn, was similar to the maximum rate displayed by At. ferrooxidans in cultures adjusted to the initial pH of 2.5. The different abilities of Sulfobacillus L-15 and At. ferrooxidans to leach pyrite at extremely low pH values was even more clearly illustrated in batch cultures grown in pH-statted bioreactors. Whereas Sulfobacillus L-15 leached pyrite when the culture was maintained at pH 1.5, 1.0, and (after a period of adaptation) 0.8, At. ferrooxidans was active only at pH 1.5. Comparison of total and plate counts of these bacteria showed that Sulfobacillus L-15 remained viable throughout the experiment. In contrast, lowering the pH of the leach liquor to 1.0 caused rapid mortality of At. ferrooxidans, and the bacterial population did not recover when the pH was raised subsequently back to 1.5. While it is possible that the plating technique might have enumerated spores of Sulfobacillus L-15 rather than active vegetative cells, endospores were rarely seen when the culture was viewed under the microscope, at any phase of the experiment, and it was concluded that plate counts recorded numbers of viable vegetative cells. During pyrite leaching, Sulfobacillus L-15 maintained a redox potential of between 760 and 800 mV when the bioreactor was set at pH 1.5 and 1.0, though this fell to < 650 mV when the culture pH was adjusted to 0.8. The redox potential in pyrite leaching systems is determined by the ratio of ferric to ferrous iron in solution; the E0 of the ferrous/ferric couple is 770 mV at pH 2.0. The ferrous/ferric iron ratios and measured redox potentials showed good agreement in both bioreactor cultures. Concentrations of soluble ferrous and ferric iron in the Sulfobacillus L-15 culture increased during leaching but remained very similar to each other up to the time that the culture pH was low-
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ered to 0.8. In contrast, the redox potential in the At. ferrooxidans bioreactor increased rapidly to 890 mV in phase 1 (pH 1.5) of the experiment, and > 99% of the soluble iron was present as ferric iron, which is typical of pyrite leaching by this iron oxidiser. However, a major disadvantage of high Eh values in leachate liquors, so far as commercial leaching operations are concerned, is that elevated concentrations of ferric iron promote the formation of secondary ferric iron-containing minerals, such as jarosites. In this regard, it would be advantageous, particularly with some ores and concentrates (e.g. chalcopyrite), to utilise microorganisms such as Sulfobacillus L-15 that are able to leach sulfide minerals effectively while maintaining relatively low redox potentials and correspondingly low concentrations of soluble ferric iron. Formation of jarosites is also precluded at extremely low pH (these are basic ferric sulphates, e.g. natrojarosite: NaFe3(SO4)2(OH)6) where Sulfobacillus L-15, unlike other mesophilic iron-oxidising bacteria, is active. These physiological characteristics, together with its capacity for chemolithotrophic growth on sulfide minerals (and sulfur), suggest that this novel mesophilic, Grampositive bacterium could have considerable potential for bioprocessing of ores and concentrates. Acknowledgements Adibah Yahya is grateful to the University Technology Malaysia for providing a research studentship. References Atkinson, T., Cairns, S., Cowan, D.A., Danson, M.J., Hough, D.W., Johnson, D.B., Norris, P.R., Raven, N., Robson, R., Robinson, C., Sharp, R.J., 2000. A microbiological survey of Montserrat island hydrothermal biotopes. Extremophiles 4, 305 – 313. Boon, M., Brasser, H.J., Hansford, G.S., Heijnen, J.J., 1999. Comparison of the oxidation kinetics of different pyrites in the presence of Thiobacillus ferrooxidans or Leptospirillum ferrooxidans. Hydrometallurgy 53, 57 – 72. Brierley, C.L., 2001. Bacterial succession in bioheap leaching. Hydrometallurgy 59, 249 – 256. Brierley, J.A., Brierley, C.L., 2001. Present and future commercial applications in biohydrometallurgy. Hydrometallurgy 59, 233 – 240. Clark, D.A., Norris, P.R., 1996. Acidimicrobium ferrooxidans gen.
nov., sp. nov.: mixed-culture ferrous iron oxidation with Sulfobacillus species. Microbiology 141, 785 – 790. Crane, A.G., Holden, P.J., 1999. Leaching of harbour sediments by estuarine iron-oxidising bacteria. In: Amils, R., Ballester, A. (Eds.), Biohydrometallurgy and the Environment Toward the Mining of the 21st Century, Process Microbiology 9A. Elsevier, Amsterdam, pp. 347 – 356. Dopson, M., Lindstrom, E.B., 1999. Potential role of Thiobacillus caldus in arsenopyrite leaching. Appl. Environ. Microbiol. 65, 36 – 40. Fowler, T.A., Holmes, P.R., Crundwell, F.K., 2001. On the kinetics and mechanism of the dissolution of pyrite in the presence of Thiobacillus ferrooxidans. Hydrometallurgy 59, 257 – 270. Ghauri, M.A., 1991. A study of the diversity of acidophilic bacteria. PhD Thesis, University of Wales. Goebel, B.M., Stackebrandt, E., 1994. Cultural and phylogenetic analysis of mixed microbial populations found in natural and commercial bioleaching environments. Appl. Environ. Microbiol. 60, 1614 – 1621. Johnson, D.B., 1995. Selective solid media for isolating and enumerating acidophilic bacteria. J. Microbiol. Methods, 205 – 218. Johnson, D.B., 1998. Biodiversity and ecology of acidophilic microorganisms. FEMS Microbiol. Ecol. 27, 307 – 317. Johnson, D.B., 2001. Genus Leptospirillum. In: Boone, D.R., Castenholz, R.W., Garrity, G.M. (Eds.), Bergey’s Manual of Systematic Bacteriology (2nd edn.), vol. 1. Springer, New York, pp. 453 – 456. Lovley, D.R., Phillips, E.J.P., 1987. Rapid assay for microbially reducible ferric iron in aquatic sediments. Appl. Environ. Microbiol. 53, 1536 – 1540. Norris, P.R., Johnson, D.B., 1998. Acidophilic microorganisms. In: Horikoshi, K., Grant, W.D. (Eds.), Extremophiles: Microbial Life in Extreme Environments. Wiley, New York, pp. 133 – 154. Norris, P.R., Clark, D.A., Owen, J.P., Waterhouse, S., 1996. Characteristics of Sulfobacillus acidophilus so. nov. and other moderately thermophilic mineral sulphide-oxidizing bacteria. Microbiology 141, 775 – 783. Norris, P.R., Burton, N.P., Foulis, N.A.M., 2000. Acidophiles in bioreactor mineral processing. Extremophiles 4, 71 – 76. Rawlings, D.E. (Ed.), 1997. Biomining: Theory, Microbes and Industrial Processes. Springer-Verlag/Landes Bioscience, Georgetown, TX. Rawlings, D.E., Tributsch, H., Hansford, G.S., 1999. Reasons why ‘‘Leptospirillum-like’’ species rather than Thiobacillus ferrooxidans are the dominant iron-oxidizing bacteria in many commercial processes for the biooxidation of pyrite and related ores. Microbiology 145, 5 – 13. Schippers, A., Sand, W., 1999. Bacterial leaching of metal sulfides proceeds by two indirect mechanisms via thiosulfate or via polysulfides and sulfur. Appl. Environ. Microbiol. 65, 319 – 321. Yahya, A., Roberto, F.F., Johnson, D.B., 1999. Novel mineral-oxidising bacteria from Montserrat (W.I.): physiological and phylogenetic characteristics. In: Amils, R., Ballester, A. (Eds.), Biohydrometallurgy and the Environment Toward the Mining of the 21st Century Process Microbiology 9A. Elsevier, Amsterdam, pp. 72 – 740.