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Pyrite oxidation and copper sulfide ore leaching by halotolerant, thermotolerant bacteria
Hydrometallurgy 104 (2010) 432–436
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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
Pyrite oxidation and copper sulfide ore leaching by halotolerant, thermotolerant bacteria P.R. Norris a,⁎, C.S. Davis-Belmar a, J.Le C. Nicolle a, L.A. Calvo-Bado a, V. Angelatou b a b
Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom Institute of Geology and Mineral Exploration, Olympic Village, Acharnai 13677, Athens, Greece
a r t i c l e
i n f o
Available online 17 June 2010 Keywords: Halotolerant acidophiles Pyrite oxidation Copper sulfide ore leaching
a b s t r a c t Pyrite oxidation was observed in a mixed culture of salt-tolerant, thermotolerant, acidophilic bacteria from warm, acidic, coastal sediments of the island of Milos (Greece). Analysis of 16S rRNA gene sequences cloned from DNA extracted from the mixed culture indicated two species which were related to Thiobacillus prosperus. One of the sequences was found previously in warm, sediment samples from the island of Vulcano (Italy). Iron solubilization from pyrite by the Milos culture at 47 °C was most rapid in the presence of NaCl at 30 g l−1. A novel species was isolated from the mixed culture and grew in pure culture on pyrite with 50 g l−1 NaCl, but iron solubilization was most rapid at just below 50 °C with 20 g l−1 NaCl. Establishment of activity of the halotolerant, thermotolerant bacteria in copper sulfide ore leaching columns was more difficult than with related bacteria growing at lower temperatures. © 2010 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
Thiobacillus prosperus (Davis-Belmar et al., 2008; Huber and Stetter, 1989; Nicolle et al., 2009) and similar mesophilic bacteria (Norris and Simmons, 2004) have been isolated from close to shallow water hydrothermal vents at Vulcano, Italy. They oxidize ferrous iron and sulfur in the presence of salt (NaCl) concentrations at least twice that of seawater, which indicates potential application in biomining where only saline water is available. One of these bacteria, strain V8, was the dominant ferrous iron-oxidizing strain in pyrite oxidation by a mixed culture (Norris and Simmons, 2004). However, these bacteria were not represented among rRNA genes amplified and cloned (88 clones) from sediment samples from which the bacteria were isolated: a clone bank comprised only sequences of sulfur-oxidizing Acidithiobacillus species (Simmons and Norris, 2002). A third of cloned rRNA genes with origin at a nearby 45 °C sample site did indicate another species related to T. prosperus, a species therefore potentially more thermotolerant than the previously studied strains. The identification of T. prosperus-like rRNA gene sequences in samples from a similar environment of the island of Milos (Greece) is now described, together with the activity of T. prosperus-related, thermotolerant cultures from this environment in pyrite oxidation and copper sulfide ore leaching.
2.1. Sampling and enrichment cultures
⁎ Corresponding author. E-mail address: [email protected] (P.R. Norris). 0304-386X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.hydromet.2010.03.025
Sediment samples were from a shallow, acidic pool and geothermal sediments by the shore of Baia di Levante, Vulcano, as described previously (Simmons and Norris, 2002) and from acidic, geothermal sediments of Palaeochori Bay of the island of Milos in the Aegean Sea. The latter samples were from shallow, acidic pools, open to the sea, at the base of sulfur-rich cliffs. The sediment, about 60 °C a few centimetres below its surface, was covered with water at 35 to 40 °C. A water/sediment sample was used to establish a pyrite enrichment culture at 50 °C in a medium which contained (g l−1) MgSO4.7H2O (0.5), (NH4)2SO4 (0.4), K2HPO4 (0.2), NaCl (25) and a pyrite concentrate (10; minus 75 μm particle diameter). The effects of NaCl and the temperature on bacterial growth on pyrite (10 g l−1) were tested with cultures (100 ml) grown in shaken flasks. Isolation of a thermotolerant strains from the enrichment culture was through single colony isolation using ferrous iron-containing Phytagelsolidified medium (Davis-Belmar et al., 2008). Iron in the solution was measured by atomic absorption spectrophotometry. 2.2. 16S rRNA gene analysis 16S rRNA genes were amplified by PCR with universal bacterial primers (f27: 5′-AGAGTTTGATCMTGGCTCAG-3′; r1492: 5′- TACGGYTACCTTGTTACGACTT-3′) or a forward primer designed to be specific for T. prosperus-like sequences (5′-TAGCCCGGAAATCCGGAT-3′, used with
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primer r1492). Cloned sequences (TOPO TA Cloning Kit, Invitrogen) were aligned before analysis with PHYLIP programmes (Felsenstein, 2006). 2.3. Ore leaching columns Laboratory ore leaching columns were operated and monitored essentially as described previously (Davis-Belmar et al., 2008), but with addition of ferric sulfate to the irrigation solution to give an iron concentration of 1 g l−1. This solution at pH 1.7 also contained 25 g NaCl l−1. Each column contained 0.7 kg of copper ore as fragments with a mean weight of 1.3 g. The ore contained 0.6% w/v copper which was mainly present in chalcopyrite, chalcocite and some covellite. The solution flow was 150 ml day−1, corresponding to a surface application rate of approximately 4 l m2 h−1 and there was no solution recycling. Aeration from the base of the columns was at 20 ml min−1. A column at 36 °C was inoculated with a halotolerant, mesophilic mixed culture as described previously (Davis-Belmar et al., 2008); the culture contained T. prosperus-like bacteria and salt-tolerant, sulfur-oxidizing Acidithiobacillus species. A column at 47 °C was inoculated with a mixed culture of two halotolerant, thermotolerant strains which are related to T. prosperus (and which are noted in Section 3.3) and Acidithiobacillus caldus. Adjustments to operating conditions are indicated with the results.
Fig. 1. A phylogenetic distance tree of Thiobacillus prosperus-related 16S rRNA gene sequences which were cloned from environmental DNA samples or enrichment cultures. The sequences from T. prosperus, from the Acidithiobacillus ferrooxidans type strain (as an out-group sequence) and from an Alkalilimnicola species (see text) are also included. Bootstrap values greater than 70 from 100 replicates are shown. The scale bar indicates 0.1 substitutions per site.
3. Results and discussion 3.1. T. prosperus-like bacteria in acidic samples from Milos and Vulcano Using universal bacterial primers, 16 S rRNA genes were amplified from DNA extracted from three water/sediment samples from the base of sulfur-rich cliffs of Milos. Cloned sequences with different RFLP patterns were analyzed. 29% (of 52), 12% (of 49) and 3% (of 73) of clones from the three samples were derived from rRNA genes with sequence identity to a sequence designated as clone type V3 when it was found previously in environmental DNA from a Vulcano sample (Simmons and Norris, 2002). No other T. prosperus-like sequences were obtained from two of the Milos samples while 44% (of 73 clones) of the third clone bank represented a novel T. prosperus-like sequence, designated clone M14. The majority of cloned sequences from sample two were similar to that of Sulfobacillus thermosulfidooxidans, while the other clone banks mainly comprised sequences related to those of heterotrophic, marine bacteria. A small clone bank (20 clones) was constructed using primers designed to be specific for the Acidithiobacillus genus and comprised only A. caldus sequences. Primers designed to be specific for the T. prosperus group were also used with DNA from the first two samples. The first of these specific clone banks comprised sequences of clone V3 (96% of clones), clone V6 which represents T. prosperus (2%), and clone M14 (2%). The second group specific clone bank comprised clones V3 (98%) and V6 (2%). Strain V8, which was previously found to dominate 35 °C pyrite enrichment cultures (Norris and Simmons, 2004), was not represented in the clone banks, but PCR with strain V8 16 S rRNA gene-specific primers amplified strain V8 rRNA sequences from all three samples (not shown). Universal primers were used in PCR amplification of 16 S rRNA genes from a 45 °C sediment sample from Vulcano which had been stored frozen since a previous analysis (Simmons and Norris, 2002). As before, clone type V3 sequences comprised over 30% of the clones, while a T. prosperus-like sequence not recorded previously, and now designated clone type V12, represented 12% of the clones. The phylogenetic relationships of the cloned T. prosperus-like sequences, including a novel clone type from the Milos pyrite enrichment culture (clone M7, see Section 3.2.), are shown (Fig. 1). The sequences are phylogenetically closer to sequences from many alkaliphiles (Alkalilimnicola sp. is shown as an example) than to those
from the most studied pyrite-oxidizing acidophile, Acidithiobacillus ferrooxidans. 3.2. Pyrite oxidation in thermotolerant, halotolerant enrichment cultures A pyrite enrichment culture of a Milos sample was maintained through many serial sub-cultures at 47 °C in medium with NaCl (25 g l−1). One sequence in a clone bank (29 clones) of 16 S rRNA genes amplified from DNA extracted from the tenth serial sub-culture represented clone type V3. The other clones represented a novel T. prosperus-like sequence, designated clone type M7, which was not seen in the analysis of the environmental samples. The effect of temperature on the dissolution of pyrite during growth of the thermotolerant enrichment culture was compared to the effect on an enrichment culture which had been originally established with samples from Vulcano (Simmons and Norris, 2002) and had been maintained at 30 °C. The original Vulcano culture oxidized the pyrite extensively at 40 °C but did not survive incubation at 45 °C (Fig. 2A). Analysis of this mesophilic culture revealed T. prosperus-like 16S rRNA gene sequences corresponding to the strains V6 and V8, while no V3, M7 or M14 clone-type sequences were found. Dissolution of pyrite by the 47 °C enrichment culture was not inhibited at 50 °C (Fig. 2B). It grew at 47 °C with NaCl at 50 g l−1, while most iron was solubilized and remained in solution with NaCl at 30 g l−1 (Fig. 2C). 3.3. Isolation of thermotolerant strains and pyrite oxidation in pure culture Pure cultures of thermotolerant strains from the Milos enrichment culture were obtained from colonies on solid medium. Two strains were isolated with 16S rRNA gene sequences that corresponded to the cloned V3 and M7 sequences (Fig. 1). Both strains oxidized ferrous iron, sulfur and tetrathionate with some differences in their capacities for autotrophic growth (data not shown). The solubilization of iron from pyrite during growth of strain M7 is shown (Fig. 3). Iron in solution did not increase significantly in a sterile control at 50 °C over the same period (not shown). The effect of temperature on the growth of strain M7 was measured with an inoculum grown at 47 °C. The maximum rate of solubilization occurred
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Fig. 2. Solubilization of iron from pyrite by salt-tolerant enrichment cultures which were established with samples of water/sediment from the islands of Vulcano (A) and Milos (B and C). The medium contained NaCl at 25 g l−1 (A and B) or at the indicated concentrations (C). The incubation temperatures were as indicated (A and B) or 47 °C (C).
at a temperature of about 50 °C (Fig. 3A) and (at 44 °C) with a NaCl concentration of about 20 g l−1 (Fig. 3B). 3.4. Copper sulfide ore leaching There was sufficient ore in columns in relation to the concentration of ferric iron in the irrigation solution, and to the flow rate, to maintain almost all of the iron in the effluent as ferrous iron in the absence of ironoxidizing bacteria, as shown with un-inoculated ore at 47 °C (Fig. 4C). Occasional concentrations of ferrous iron higher than total iron at some
sampling times may have reflected ferrous iron measurement immediately after collection of a sample over 10 min whereas total iron was measured in the effluent collected between ferrous iron assays, usually over one or two days. The acidity of the feed solution and the oxidation of sulfides by added ferric iron resulted in an initial, relatively rapid release of copper, which was probably from the chalcocite fraction of the ore. This early release of copper was not affected by inoculation with bacteria but was enhanced at 47 °C versus 36 °C (Fig. 4A). The mesophilic, halotolerant T. prosperus-like bacteria maintained ferric iron in solution through their oxidation of the ferrous iron which
Fig. 3. Solubilization of iron from pyrite (10 g l−1) during autotrophic growth of strain M7 at (A) different temperatures in the presence of NaCl (25 g l−1) and (B) at different NaCl concentrations at 44 °C.
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resulted from iron reduction by the sulfide minerals in the ore (Fig. 4B). This indicated the potential of such bacteria to enhance copper extraction by regeneration of the ferric iron, as shown previously (Davis-Belmar et al., 2008). Replacement of aeration by a flow of nitrogen gas for three days, depriving the bacteria of the electron acceptor for ferrous iron oxidation, resulted in a peak of ferrous iron in solution (Fig. 4B). In contrast, to the rapid establishment of ferrous iron oxidation at 36 °C (Fig. 4B), ferrous iron remained in the effluent from the column inoculated with the halotolerant, thermotolerant bacteria and A. caldus (Fig. 4D). A previous attempt also failed to establish these bacteria during 70 days of operation, whereas complete ferrous iron oxidation occurred at 47 °C with this ore in the absence of salt when it was inoculated with moderately thermophilic species of Acidimicrobium, Acidimicrobium-like bacteria and Sulfobacillus (data not shown). Reinoculation of the 47 °C column after 25 days (indicated on Fig. 4D) failed to establish any apparent bacterial activity. A second reinoculation after 72 days was made concurrent with CO2 enrichment of the gas flow (20 ml min−1 of 5% CO2 v/v in air) and a halving of the irrigation rate (indicated on Fig. 4D; the flow rate to the un-inoculated ore at 47 °C was also halved). After this third inoculation, most of the iron in the effluent was oxidized, indicating bacterial activity. A supplement of tetrathionate (1 mM) was added to the feed solution when the bacterial activity appeared to decline. A readily available source of reduced sulfur was previously shown to stimulate growthassociated ferrous iron oxidation by T. prosperus (Nicolle et al., 2009) and by the thermotolerant strains with clone type M7 and V3 16S rRNA gene sequences (data not shown). The alterations to several parameters in the attempt to establish the thermotolerant bacteria in the column preclude any conclusions with regard to the influence of individual factors, but the activity of these bacteria was clearly more difficult to establish than that of the mesophiles. In addition, their activity was less than that of the mesophiles, either per unit biomass or because less growth was established at the higher temperature. Approximately half of the iron in the effluent remained oxidized after withdrawal of the tetrathionate and CO2 supplements (Fig. 4D). At 47 °C, there was more deposition of iron from the feed solution in the inoculated column, with approximately 25% less iron in the final effluent from the inoculated column in comparison to that from the un-inoculated column. The pH of the effluents was similar and close to that of the feed solution, after rising slightly in the initial days of leaching. Finally, the copper extraction from the 36 °C, 47 °C (uninoculated) and 47 °C (inoculated) columns was still increasing and had reached 44, 49 and 54% of the total copper (Fig. 4D). Effluent from the columns at 47 °C (10 ml) was used to inoculate medium at 47 °C in shaken flasks (100 ml growth medium with 20 g NaCl l−1, 10 g pyrite l−1 and 100 mg yeast extract l−1). Growth and pyrite oxidation was obtained only with effluent from the inoculated column. The activity of T. prosperus-related halotolerant, thermotolerant, acidophilic bacteria in pyrite oxidation has been demonstrated but it has not been established whether they have the robustness required for application in biomining. A greater sensitivity to copper than that shown by potentially useful mineral sulfide-oxidizing bacteria (data not shown) is one their characteristics that requires further investigation. Acknowledgements We thank Billiton PLC, The States of Guernsey and the University of Warwick for supporting this work.
Fig. 4. Leaching of a copper sulfide ore in the presence of 25 g NaCl l−1. Total and ferrous iron concentrations are shown for effluents from (B) an ore column at 36 °C inoculated with mesophilic, T. prosperus-like bacteria (see Section 2.3), (C) an un-inoculated ore column at 47 °C and (D) an ore column at 47 °C inoculated with thermotolerant T. prosperus-like bacteria (see Section 2.3). The total copper leached from the ore columns is shown (A).
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References Davis-Belmar, C., Nicolle, J.Le C., Norris, P.R., 2008. Ferrous iron oxidation and leaching of copper ore with halotolerant bacteria in ore columns. Hydrometallurgy 94, 144–147. Felsenstein, J., 2006. PHYLIP (Phylogeny Inference Package). Department of Genome Sciences, University of Washington, Seattle. version 3.6a3. Huber, H., Stetter, K.O., 1989. Thiobacillus prosperus sp. nov., represents a new group of halotolerant metal-mobilizing bacteria isolated from a marine geothermal field. Arch. Microbiol. 151, 479–485.
Nicolle, J.Le C., Bathe, S., Norris, P.R., 2009. Ferrous iron oxidation and rusticyanin in halotolerant, acidophilic ‘Thiobacillus prosperus’. Microbiology 155, 1302–1309. Norris, P.R., Simmons, S., 2004. S., Pyrite oxidation by halotolerant, acidophilic bacteria. In: Tsezos, M., Hatzikioseyian, A., Remoundaki, E. (Eds.), Biohydrometallurgy: A Sustainable Technology in Evolution Part II. National Technical University of Athens, Athens, pp. 1347–1351. Simmons, S., Norris, P.R., 2002. Acidophiles of saline water at thermal vents of Vulcano, Italy. Extremophiles 6, 201–207.
Contents lists available at ScienceDirect
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
Pyrite oxidation and copper sulfide ore leaching by halotolerant, thermotolerant bacteria P.R. Norris a,⁎, C.S. Davis-Belmar a, J.Le C. Nicolle a, L.A. Calvo-Bado a, V. Angelatou b a b
Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom Institute of Geology and Mineral Exploration, Olympic Village, Acharnai 13677, Athens, Greece
a r t i c l e
i n f o
Available online 17 June 2010 Keywords: Halotolerant acidophiles Pyrite oxidation Copper sulfide ore leaching
a b s t r a c t Pyrite oxidation was observed in a mixed culture of salt-tolerant, thermotolerant, acidophilic bacteria from warm, acidic, coastal sediments of the island of Milos (Greece). Analysis of 16S rRNA gene sequences cloned from DNA extracted from the mixed culture indicated two species which were related to Thiobacillus prosperus. One of the sequences was found previously in warm, sediment samples from the island of Vulcano (Italy). Iron solubilization from pyrite by the Milos culture at 47 °C was most rapid in the presence of NaCl at 30 g l−1. A novel species was isolated from the mixed culture and grew in pure culture on pyrite with 50 g l−1 NaCl, but iron solubilization was most rapid at just below 50 °C with 20 g l−1 NaCl. Establishment of activity of the halotolerant, thermotolerant bacteria in copper sulfide ore leaching columns was more difficult than with related bacteria growing at lower temperatures. © 2010 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
Thiobacillus prosperus (Davis-Belmar et al., 2008; Huber and Stetter, 1989; Nicolle et al., 2009) and similar mesophilic bacteria (Norris and Simmons, 2004) have been isolated from close to shallow water hydrothermal vents at Vulcano, Italy. They oxidize ferrous iron and sulfur in the presence of salt (NaCl) concentrations at least twice that of seawater, which indicates potential application in biomining where only saline water is available. One of these bacteria, strain V8, was the dominant ferrous iron-oxidizing strain in pyrite oxidation by a mixed culture (Norris and Simmons, 2004). However, these bacteria were not represented among rRNA genes amplified and cloned (88 clones) from sediment samples from which the bacteria were isolated: a clone bank comprised only sequences of sulfur-oxidizing Acidithiobacillus species (Simmons and Norris, 2002). A third of cloned rRNA genes with origin at a nearby 45 °C sample site did indicate another species related to T. prosperus, a species therefore potentially more thermotolerant than the previously studied strains. The identification of T. prosperus-like rRNA gene sequences in samples from a similar environment of the island of Milos (Greece) is now described, together with the activity of T. prosperus-related, thermotolerant cultures from this environment in pyrite oxidation and copper sulfide ore leaching.
2.1. Sampling and enrichment cultures
⁎ Corresponding author. E-mail address: [email protected] (P.R. Norris). 0304-386X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.hydromet.2010.03.025
Sediment samples were from a shallow, acidic pool and geothermal sediments by the shore of Baia di Levante, Vulcano, as described previously (Simmons and Norris, 2002) and from acidic, geothermal sediments of Palaeochori Bay of the island of Milos in the Aegean Sea. The latter samples were from shallow, acidic pools, open to the sea, at the base of sulfur-rich cliffs. The sediment, about 60 °C a few centimetres below its surface, was covered with water at 35 to 40 °C. A water/sediment sample was used to establish a pyrite enrichment culture at 50 °C in a medium which contained (g l−1) MgSO4.7H2O (0.5), (NH4)2SO4 (0.4), K2HPO4 (0.2), NaCl (25) and a pyrite concentrate (10; minus 75 μm particle diameter). The effects of NaCl and the temperature on bacterial growth on pyrite (10 g l−1) were tested with cultures (100 ml) grown in shaken flasks. Isolation of a thermotolerant strains from the enrichment culture was through single colony isolation using ferrous iron-containing Phytagelsolidified medium (Davis-Belmar et al., 2008). Iron in the solution was measured by atomic absorption spectrophotometry. 2.2. 16S rRNA gene analysis 16S rRNA genes were amplified by PCR with universal bacterial primers (f27: 5′-AGAGTTTGATCMTGGCTCAG-3′; r1492: 5′- TACGGYTACCTTGTTACGACTT-3′) or a forward primer designed to be specific for T. prosperus-like sequences (5′-TAGCCCGGAAATCCGGAT-3′, used with
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primer r1492). Cloned sequences (TOPO TA Cloning Kit, Invitrogen) were aligned before analysis with PHYLIP programmes (Felsenstein, 2006). 2.3. Ore leaching columns Laboratory ore leaching columns were operated and monitored essentially as described previously (Davis-Belmar et al., 2008), but with addition of ferric sulfate to the irrigation solution to give an iron concentration of 1 g l−1. This solution at pH 1.7 also contained 25 g NaCl l−1. Each column contained 0.7 kg of copper ore as fragments with a mean weight of 1.3 g. The ore contained 0.6% w/v copper which was mainly present in chalcopyrite, chalcocite and some covellite. The solution flow was 150 ml day−1, corresponding to a surface application rate of approximately 4 l m2 h−1 and there was no solution recycling. Aeration from the base of the columns was at 20 ml min−1. A column at 36 °C was inoculated with a halotolerant, mesophilic mixed culture as described previously (Davis-Belmar et al., 2008); the culture contained T. prosperus-like bacteria and salt-tolerant, sulfur-oxidizing Acidithiobacillus species. A column at 47 °C was inoculated with a mixed culture of two halotolerant, thermotolerant strains which are related to T. prosperus (and which are noted in Section 3.3) and Acidithiobacillus caldus. Adjustments to operating conditions are indicated with the results.
Fig. 1. A phylogenetic distance tree of Thiobacillus prosperus-related 16S rRNA gene sequences which were cloned from environmental DNA samples or enrichment cultures. The sequences from T. prosperus, from the Acidithiobacillus ferrooxidans type strain (as an out-group sequence) and from an Alkalilimnicola species (see text) are also included. Bootstrap values greater than 70 from 100 replicates are shown. The scale bar indicates 0.1 substitutions per site.
3. Results and discussion 3.1. T. prosperus-like bacteria in acidic samples from Milos and Vulcano Using universal bacterial primers, 16 S rRNA genes were amplified from DNA extracted from three water/sediment samples from the base of sulfur-rich cliffs of Milos. Cloned sequences with different RFLP patterns were analyzed. 29% (of 52), 12% (of 49) and 3% (of 73) of clones from the three samples were derived from rRNA genes with sequence identity to a sequence designated as clone type V3 when it was found previously in environmental DNA from a Vulcano sample (Simmons and Norris, 2002). No other T. prosperus-like sequences were obtained from two of the Milos samples while 44% (of 73 clones) of the third clone bank represented a novel T. prosperus-like sequence, designated clone M14. The majority of cloned sequences from sample two were similar to that of Sulfobacillus thermosulfidooxidans, while the other clone banks mainly comprised sequences related to those of heterotrophic, marine bacteria. A small clone bank (20 clones) was constructed using primers designed to be specific for the Acidithiobacillus genus and comprised only A. caldus sequences. Primers designed to be specific for the T. prosperus group were also used with DNA from the first two samples. The first of these specific clone banks comprised sequences of clone V3 (96% of clones), clone V6 which represents T. prosperus (2%), and clone M14 (2%). The second group specific clone bank comprised clones V3 (98%) and V6 (2%). Strain V8, which was previously found to dominate 35 °C pyrite enrichment cultures (Norris and Simmons, 2004), was not represented in the clone banks, but PCR with strain V8 16 S rRNA gene-specific primers amplified strain V8 rRNA sequences from all three samples (not shown). Universal primers were used in PCR amplification of 16 S rRNA genes from a 45 °C sediment sample from Vulcano which had been stored frozen since a previous analysis (Simmons and Norris, 2002). As before, clone type V3 sequences comprised over 30% of the clones, while a T. prosperus-like sequence not recorded previously, and now designated clone type V12, represented 12% of the clones. The phylogenetic relationships of the cloned T. prosperus-like sequences, including a novel clone type from the Milos pyrite enrichment culture (clone M7, see Section 3.2.), are shown (Fig. 1). The sequences are phylogenetically closer to sequences from many alkaliphiles (Alkalilimnicola sp. is shown as an example) than to those
from the most studied pyrite-oxidizing acidophile, Acidithiobacillus ferrooxidans. 3.2. Pyrite oxidation in thermotolerant, halotolerant enrichment cultures A pyrite enrichment culture of a Milos sample was maintained through many serial sub-cultures at 47 °C in medium with NaCl (25 g l−1). One sequence in a clone bank (29 clones) of 16 S rRNA genes amplified from DNA extracted from the tenth serial sub-culture represented clone type V3. The other clones represented a novel T. prosperus-like sequence, designated clone type M7, which was not seen in the analysis of the environmental samples. The effect of temperature on the dissolution of pyrite during growth of the thermotolerant enrichment culture was compared to the effect on an enrichment culture which had been originally established with samples from Vulcano (Simmons and Norris, 2002) and had been maintained at 30 °C. The original Vulcano culture oxidized the pyrite extensively at 40 °C but did not survive incubation at 45 °C (Fig. 2A). Analysis of this mesophilic culture revealed T. prosperus-like 16S rRNA gene sequences corresponding to the strains V6 and V8, while no V3, M7 or M14 clone-type sequences were found. Dissolution of pyrite by the 47 °C enrichment culture was not inhibited at 50 °C (Fig. 2B). It grew at 47 °C with NaCl at 50 g l−1, while most iron was solubilized and remained in solution with NaCl at 30 g l−1 (Fig. 2C). 3.3. Isolation of thermotolerant strains and pyrite oxidation in pure culture Pure cultures of thermotolerant strains from the Milos enrichment culture were obtained from colonies on solid medium. Two strains were isolated with 16S rRNA gene sequences that corresponded to the cloned V3 and M7 sequences (Fig. 1). Both strains oxidized ferrous iron, sulfur and tetrathionate with some differences in their capacities for autotrophic growth (data not shown). The solubilization of iron from pyrite during growth of strain M7 is shown (Fig. 3). Iron in solution did not increase significantly in a sterile control at 50 °C over the same period (not shown). The effect of temperature on the growth of strain M7 was measured with an inoculum grown at 47 °C. The maximum rate of solubilization occurred
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Fig. 2. Solubilization of iron from pyrite by salt-tolerant enrichment cultures which were established with samples of water/sediment from the islands of Vulcano (A) and Milos (B and C). The medium contained NaCl at 25 g l−1 (A and B) or at the indicated concentrations (C). The incubation temperatures were as indicated (A and B) or 47 °C (C).
at a temperature of about 50 °C (Fig. 3A) and (at 44 °C) with a NaCl concentration of about 20 g l−1 (Fig. 3B). 3.4. Copper sulfide ore leaching There was sufficient ore in columns in relation to the concentration of ferric iron in the irrigation solution, and to the flow rate, to maintain almost all of the iron in the effluent as ferrous iron in the absence of ironoxidizing bacteria, as shown with un-inoculated ore at 47 °C (Fig. 4C). Occasional concentrations of ferrous iron higher than total iron at some
sampling times may have reflected ferrous iron measurement immediately after collection of a sample over 10 min whereas total iron was measured in the effluent collected between ferrous iron assays, usually over one or two days. The acidity of the feed solution and the oxidation of sulfides by added ferric iron resulted in an initial, relatively rapid release of copper, which was probably from the chalcocite fraction of the ore. This early release of copper was not affected by inoculation with bacteria but was enhanced at 47 °C versus 36 °C (Fig. 4A). The mesophilic, halotolerant T. prosperus-like bacteria maintained ferric iron in solution through their oxidation of the ferrous iron which
Fig. 3. Solubilization of iron from pyrite (10 g l−1) during autotrophic growth of strain M7 at (A) different temperatures in the presence of NaCl (25 g l−1) and (B) at different NaCl concentrations at 44 °C.
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resulted from iron reduction by the sulfide minerals in the ore (Fig. 4B). This indicated the potential of such bacteria to enhance copper extraction by regeneration of the ferric iron, as shown previously (Davis-Belmar et al., 2008). Replacement of aeration by a flow of nitrogen gas for three days, depriving the bacteria of the electron acceptor for ferrous iron oxidation, resulted in a peak of ferrous iron in solution (Fig. 4B). In contrast, to the rapid establishment of ferrous iron oxidation at 36 °C (Fig. 4B), ferrous iron remained in the effluent from the column inoculated with the halotolerant, thermotolerant bacteria and A. caldus (Fig. 4D). A previous attempt also failed to establish these bacteria during 70 days of operation, whereas complete ferrous iron oxidation occurred at 47 °C with this ore in the absence of salt when it was inoculated with moderately thermophilic species of Acidimicrobium, Acidimicrobium-like bacteria and Sulfobacillus (data not shown). Reinoculation of the 47 °C column after 25 days (indicated on Fig. 4D) failed to establish any apparent bacterial activity. A second reinoculation after 72 days was made concurrent with CO2 enrichment of the gas flow (20 ml min−1 of 5% CO2 v/v in air) and a halving of the irrigation rate (indicated on Fig. 4D; the flow rate to the un-inoculated ore at 47 °C was also halved). After this third inoculation, most of the iron in the effluent was oxidized, indicating bacterial activity. A supplement of tetrathionate (1 mM) was added to the feed solution when the bacterial activity appeared to decline. A readily available source of reduced sulfur was previously shown to stimulate growthassociated ferrous iron oxidation by T. prosperus (Nicolle et al., 2009) and by the thermotolerant strains with clone type M7 and V3 16S rRNA gene sequences (data not shown). The alterations to several parameters in the attempt to establish the thermotolerant bacteria in the column preclude any conclusions with regard to the influence of individual factors, but the activity of these bacteria was clearly more difficult to establish than that of the mesophiles. In addition, their activity was less than that of the mesophiles, either per unit biomass or because less growth was established at the higher temperature. Approximately half of the iron in the effluent remained oxidized after withdrawal of the tetrathionate and CO2 supplements (Fig. 4D). At 47 °C, there was more deposition of iron from the feed solution in the inoculated column, with approximately 25% less iron in the final effluent from the inoculated column in comparison to that from the un-inoculated column. The pH of the effluents was similar and close to that of the feed solution, after rising slightly in the initial days of leaching. Finally, the copper extraction from the 36 °C, 47 °C (uninoculated) and 47 °C (inoculated) columns was still increasing and had reached 44, 49 and 54% of the total copper (Fig. 4D). Effluent from the columns at 47 °C (10 ml) was used to inoculate medium at 47 °C in shaken flasks (100 ml growth medium with 20 g NaCl l−1, 10 g pyrite l−1 and 100 mg yeast extract l−1). Growth and pyrite oxidation was obtained only with effluent from the inoculated column. The activity of T. prosperus-related halotolerant, thermotolerant, acidophilic bacteria in pyrite oxidation has been demonstrated but it has not been established whether they have the robustness required for application in biomining. A greater sensitivity to copper than that shown by potentially useful mineral sulfide-oxidizing bacteria (data not shown) is one their characteristics that requires further investigation. Acknowledgements We thank Billiton PLC, The States of Guernsey and the University of Warwick for supporting this work.
Fig. 4. Leaching of a copper sulfide ore in the presence of 25 g NaCl l−1. Total and ferrous iron concentrations are shown for effluents from (B) an ore column at 36 °C inoculated with mesophilic, T. prosperus-like bacteria (see Section 2.3), (C) an un-inoculated ore column at 47 °C and (D) an ore column at 47 °C inoculated with thermotolerant T. prosperus-like bacteria (see Section 2.3). The total copper leached from the ore columns is shown (A).
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Nicolle, J.Le C., Bathe, S., Norris, P.R., 2009. Ferrous iron oxidation and rusticyanin in halotolerant, acidophilic ‘Thiobacillus prosperus’. Microbiology 155, 1302–1309. Norris, P.R., Simmons, S., 2004. S., Pyrite oxidation by halotolerant, acidophilic bacteria. In: Tsezos, M., Hatzikioseyian, A., Remoundaki, E. (Eds.), Biohydrometallurgy: A Sustainable Technology in Evolution Part II. National Technical University of Athens, Athens, pp. 1347–1351. Simmons, S., Norris, P.R., 2002. Acidophiles of saline water at thermal vents of Vulcano, Italy. Extremophiles 6, 201–207.