Isolation and Characterization of Acidithiobacillus ferrooxidans Strain D3-2 Active in Copper Bioleaching from a Copper Mine in Chile

Acidithiobacillus ferrooxidans strain D3-2, which has a high copper bioleaching activity, was isolated from a low-grade sulfide ore dump in Chile. The amounts of Cu²⁺ solubilized from 1% chalcopyrite (CuFeS₂) concentrate medium (pH 2.5) by A. ferrooxidans strains D3-2, D3-6, and ATCC 23270 and 33020 were 1360, 1080, 650, and 600 mg·l ^?1·30 d^?1. The iron oxidase activities of D3-2, D3-6, and ATCC 23270 were 11.7, 13.2, and 27.9 μl O₂ uptake·mg protein^?1·min^?1. In contrast, the sulfite oxidase activities of strains D3-2, D3-6, and ATCC 23270 were 5.8, 2.9, and 1.0 μl O₂ uptake·mg protein^?1·min^?1. Both of cell growth and Cu-bioleaching activity of strains D3-6 and ATCC 23270, but not, of D3-2, in the chalcopyrite concentrate medium were completely inhibited in the presence of 5 mM sodium bisulfite. The sulfite oxidase of strain D3-2 was much more resistant to sulfite ion than that of strain ATCC 23270. Since sulfite ion is a highly toxic intermediate produced during sulfur oxidation that strongly inhibits iron oxidase activity, these results confirm that strain D3-2, with a unique sulfite resistant-sulfite oxidase, was able to solubilize more copper from chalcopyrite than strain ATCC 23270, with a sulfite-sensitive sulfite oxidase.     

Sulfite oxidation catalyzed by aa(3)-type cytochrome c oxidase in Acidithiobacillus ferrooxidans

Sulfite is produced as a toxic intermediate during Acidithiobacillus ferrooxidans sulfur oxidation. A. ferrooxidans D3-2, which posseses the highest copper bioleaching activity, is more resistant to sulfite than other A. ferrooxidans strains, including ATCC 23270. When sulfite oxidase was purified homogeneously from strain D3-2, the oxidized and reduced forms of the purified sulfite oxidase absorption spectra corresponded to those of A. ferrooxidans aa(3)-type cytochrome c oxidase. The confirmed molecular weights of the α-subunit (52.5 kDa), the β-subunit (25 kDa), and the γ-subunit (20 kDa) of the purified sulfite oxidase and the N-terminal amino acid sequences of the γ-subunit of sulfite oxidase (AAKKG) corresponded to those of A. ferrooxidans ATCC 23270 cytochrome c oxidase. The sulfite oxidase activities of the iron- and sulfur-grown A. ferrooxidans D3-2 were much higher than those cytochrome c oxidases purified from A. ferrooxidans strains ATCC 23270, MON-1 and AP19-3. The activities of sulfite oxidase purified from iron- and sulfur-grown strain D3-2 were completely inhibited by an antibody raised against a purified A. ferrooxidans MON-1 aa(3)-type cytochrome c oxidase. This is the first report to indicate that aa(3)-type cytochrome c oxidase catalyzed sulfite oxidation in A. ferrooxidans.     

Sulfite Oxidation Catalyzed by aa 3-Type Cytochrome c Oxidase in Acidithiobacillus ferrooxidans

Sulfite is produced as a toxic intermediate during Acidithiobacillus ferrooxidans sulfur oxidation. A. ferrooxidans D3-2, which posseses the highest copper bioleaching activity, is more resistant to sulfite than other A. ferrooxidans strains, including ATCC 23270. When sulfite oxidase was purified homogeneously from strain D3-2, the oxidized and reduced forms of the purified sulfite oxidase absorption spectra corresponded to those of A. ferrooxidans aa ₃-type cytochrome c oxidase. The confirmed molecular weights of the α-subunit (52.5 kDa), the β-subunit (25 kDa), and the γ-subunit (20 kDa) of the purified sulfite oxidase and the N-terminal amino acid sequences of the γ-subunit of sulfite oxidase (AAKKG) corresponded to those of A. ferrooxidans ATCC 23270 cytochrome c oxidase. The sulfite oxidase activities of the iron- and sulfur-grown A. ferrooxidans D3-2 were much higher than those cytochrome c oxidases purified from A. ferrooxidans strains ATCC 23270, MON-1 and AP19-3. The activities of sulfite oxidase purified from iron- and sulfur-grown strain D3-2 were completely inhibited by an antibody raised against a purified A. ferrooxidans MON-1 aa ₃-type cytochrome c oxidase. This is the first report to indicate that aa ₃-type cytochrome c oxidase catalyzed sulfite oxidation in A. ferrooxidans.     

Increase in Fe2+-producing activity during growth of Acidithiobacillus ferrooxidans ATCC23270 on sulfur

When Acidithiobacillus ferrooxidans ATCC23270 cells, grown for many generations on sulfur were grown in sulfur medium with and without Fe(3+), the bacterium markedly increased not only in iron oxidase activity but also in Fe(2+)-producing sulfide:ferric ion oxidoreductase (SFORase) activity during the early log phase, and retained part of these activities during the late log phase. The activity of SFORase, which catalyzes the production of Fe(2+) from Fe(3+) and sulfur, of sulfur-grown cells was approximately 10-20 fold higher than that of iron-grown cells. aa(3) type cytochrome c oxidase, an important component of iron oxidase in A. ferrooxidans, was partially purified from sulfur-grown cells. A. ferrooxidans ATCC23270 cells grown for many generations on sulfur had the ability to grow on iron as rapidly as that did iron-grown cells. These results suggest that both iron oxidase and Fe(2+)-producing SFORase have a role in the energy generation of A. ferrooxidans ATCC23270 from sulfur.     

Reduction of Hg2+ with reduced mammalian cytochrome c by cytochrome c oxidase purified from a mercury-resistant acidithiobacillus ferrooxidans strain, MON-1

Acidithiobacillus ferrooxidans AP19-3, ATCC 23270, and MON-1 are mercury-sensitive, moderately mercury-resistant, and highly mercury-resistant strains respectively. It is known that 2,3,5,6-tetramethyl-p-phenylendiamine (TMPD) and reduced cytochrome c are used as electron donors specific for cytochrome c oxidase. Resting cells of strain MON-1 had TMPD oxidase activity and volatilized metal mercury with TMPD as an electron donor. Cytochrome c oxidase purified from strain MON-1 reduced mercuric ions to metalic mercury with reduced mammalian cytochrome c as well as TMPD. These mercury volatilization activities with reduced cytochrome c and TMPD were completely inhibited by 1 mM NaCN. These results indicate that cytochrome c oxidase is involved in mercury reduction in A. ferrooxidans cells. The cytochrome c oxidase activities of strains AP19-3 and ATCC 23270 were completely inhibited by 1 muM and 5 muM of mercuric chloride respectively. In contrast, the activity of strain MON-1 was inhibited 33% by 5 muM, and 70% by 10 muM of mercuric chloride, suggesting that the levels of mercury resistance in A. ferrooxidans strains correspond well with the levels of mercury resistance of cytochrome c oxidase.     

Thiobacillus ferrooxidans, a facultative hydrogen oxidizer.

The type strain (ATCC 23270) and two other strains of Thiobacillus ferrooxidans were able to grow by hydrogen oxidation, a feature not recognized before. When cultivated on H2, a hydrogenase was induced and the strains were less extremely acidophilic than during growth on sulfidic ores. Cells of T. ferrooxidans grown on H2 and on ferrous iron showed 100% DNA homology. Hydrogen oxidation was not observed in eight other species of the genus Thiobacillus and in Leptospirillum ferrooxidans.     

Thiobacillus ferrooxidans, a facultative hydrogen oxidizer

The type strain (ATCC 23270) and two other strains of Thiobacillus ferrooxidans were able to grow by hydrogen oxidation, a feature not recognized before. When cultivated on H2, a hydrogenase was induced and the strains were less extremely acidophilic than during growth on sulfidic ores. Cells of T. ferrooxidans grown on H2 and on ferrous iron showed 100% DNA homology. Hydrogen oxidation was not observed in eight other species of the genus Thiobacillus and in Leptospirillum ferrooxidans.     

A genomic island provides <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> ATCC 53993 additional copper resistance: a possible competitive advantage

There is great interest in understanding how extremophilic biomining bacteria adapt to exceptionally high copper concentrations in their environment. Acidithiobacillus ferrooxidans ATCC 53993 genome possesses the same copper resistance determinants as strain ATCC 23270. However, the former strain contains in its genome a 160-kb genomic island (GI), which is absent in ATCC 23270. This GI contains, amongst other genes, several genes coding for an additional putative copper ATPase and a Cus system. A. ferrooxidans ATCC 53993 showed a much higher resistance to CuSO₄ (>100 mM) than that of strain ATCC 23270 (<25 mM). When a similar number of bacteria from each strain were mixed and allowed to grow in the absence of copper, their respective final numbers remained approximately equal. However, in the presence of copper, there was a clear overgrowth of strain ATCC 53993 compared to ATCC 23270. This behavior is most likely explained by the presence of the additional copper-resistance genes in the GI of strain ATCC 53993. As determined by qRT-PCR, it was demonstrated that these genes are upregulated when A. ferrooxidans ATCC 53993 is grown in the presence of copper and were shown to be functional when expressed in copper-sensitive Escherichia coli mutants. Thus, the reason for resistance to copper of two strains of the same acidophilic microorganism could be determined by slight differences in their genomes, which may not only lead to changes in their capacities to adapt to their environment, but may also help to select the more fit microorganisms for industrial biomining operations.     

Volatilization of mercury by an iron oxidation enzyme system in a highly mercury-resistant Acidithiobacillus ferrooxidans strain MON-1

A highly mercury-resistant strain Acidithiobacillus ferrooxidans MON-1, was isolated from a culture of a moderately mercury-resistant strain, A. ferrooxidans SUG 2-2 (previously described as Thiobacillus ferrooxidans SUG 2-2), by successive cultivation and isolation of the latter strain in a Fe2+ medium with increased amounts of Hg2+ from 6 microM to 20 microM. The original stain SUG 2-2 grew in a Fe2+ medium containing 6 microM Hg2+ with a lag time of 22 days, but could not grow in a Fe2+ medium containing 10 microM Hg2+. In contrast, strain MON-1 could grow in a Fe2+ medium containing 20 microM Hg2+ with a lag time of 2 days and the ability of strain MON-1 to grow rapidly in a Fe2+ medium containing 20 microM Hg2+ was maintained stably after the strain was cultured many times in a Fe2+ medium without Hg2+. A similar level of NADPH-dependent mercury reductase activity was observed in cell extracts from strains SUG 2-2 and MON-1. By contrast, the amounts of mercury volatilized for 3 h from the reaction mixture containing 7 microM Hg2+ using a Fe(2+)-dependent mercury volatilization enzyme system were 5.6 nmol for SUG 2-2 and 67.5 nmol for MON-1, respectively, indicating that a marked increase of Fe(2+)-dependent mercury volatilization activity conferred on strain MON-1 the ability to grow rapidly in a Fe2+ medium containing 20 microM Hg2+. Iron oxidizing activities, 2,3,5,6-tetramethyl-p-phenylenediamine (TMPD) oxidizing activities and cytochrome c oxidase activities of strains SUG 2-2 and MON-1 were 26.3 and 41.9 microl O2 uptake/mg/min, 15.6 and 25.0 microl O2 uptake/mg/min, and 2.1 and 6.1 mU/mg, respectively. These results indicate that among components of the iron oxidation enzyme system, especially cytochrome c oxidase activity, increased by the acquisition of further mercury resistance in strain MON-1. Mercury volatilized by the Fe(2+)-dependent mercury volatilization enzyme system of strain MON-1 was strongly inhibited by 1.0 mM sodium cyanide, but was not by 50 nM rotenone, 5 microM 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO), 0.5 microM antimycin A, or 0.5 microM myxothiazol, indicating that cytochrome c oxidase plays a crucial role in mercury volatilization of strain MON-1 in the presence of Fe2+.     

Mechanism of growth inhibition by tungsten in Acidithiobacillus ferrooxidans

Cell growth of three hundred iron-oxidizing bacteria isolated from natural environments was inhibited strongly by 0.05 mM, and completely by 0.2 mM of sodium tungstate (Na2WO4), respectively. Since no great difference in the level of tungsten inhibition was observed among the 300 strains tested, the mechanism of inhibition by Na2WO4 was studied with Acidithiobacillus ferrooxidans strain AP19-3. When resting cells of AP19-3 were incubated in 0.1 M beta-alanine-SO4(2-) buffer (pH 3.0) with 0.1 mM Na2WO4 for 1 h, the amount of tungsten bound to the cells was 12 microg/mg protein. The optimum pH for tungsten binding to the resting cells was 2 to approximately 3. Approximately 2 times more tungsten bound to the cells at pH 3.0 than at pH 6.0. The tungsten binding was specifically inhibited by sodium molybdenum. However, copper, nickel, cadmium, zinc, manganese, cobalt, and vanadate did not disturb tungsten binding to the resting cells. The iron-oxidizing activity of AP19-3 was inhibited 24, 62, and 77% by 1, 5, and 10 mM of Na2WO4, respectively. Among the components of iron oxidation enzyme system, iron:cytochrome c oxidoreductase activity was not inhibited by 10 mM of Na2WO4. In contrast, the activity of cytochrome c oxidase purified highly from the strain was inhibited 50 and 72%, respectively, by 0.05 and 0.1 mM of Na2WO4. The amounts of tungsten bound to plasma membrane, cytosol fraction, and a purified cytochrome c oxidase were 8, 0.5, and 191 microg/mg protein, respectively. From the results, the growth inhibition by Na2WO4 observed in A. ferrooxidans is explained as follows: tungsten binds to cytochrome c oxidase in plasma membranes and inhibits cytochrome c oxidase activity, and as a results, the generation of energy needed for cell growth from the oxidation of Fe2+ is stopped.     

Effect of sulfite exposure on zinc, iron, and copper levels in rat liver and kidney tissues

Sulfite is a potentially toxic molecule that might enter the body via ingestion, inhalation, or injection. For cellular detoxification, mammalians rely on sulfite oxidase to convert sulfite to sulfate. The purpose of this research was to determine the effect of sulfite on zinc, iron, and copper levels in rat liver and kidney tissues. Forty normal and sulfite oxidase-deficient male albino rats were divided into four groups that included untreated controls (group C), a sulfite-supplemented group that received 70 mg sodium metabisulfite per kilogram per day (group S), a sulfite oxidase-deficient group (group D), and a sulfite oxidase-deficient group that was also given 70 mg sodium metabisulfite per kilogram per day (group DS). The iron and zinc levels in the liver and kidney in groups S and DS were not affected by sulfite treatment compared to their respective controls (groups C and D). Sulfite exposure led to an increase of kidney copper content in the S group when compared to untreated controls. The kidney copper levels were significantly increased in the unexposed deficient rats, but it was not different than that of the deficient rats that were given oral sulfite treatment. These results suggest that kidney copper levels might be affected by exogenous or endogenous sulfite. An erratum to this article is available at .     

Sulfite oxidation by iron-grown cells of Thiobacillus ferrooxidans at pH 3 possibly involves free radicals, iron, and cytochrome oxidase

Thiobacillus ferrooxidans cells grown on ferrous iron oxidized sulfite to sulfate at pH 3, possibly by a free radical mechanism involving iron and cytochrome oxidase. A purely chemical system with low concentrations of Fe3+ simulated the T. ferrooxidans system. Metal chelators, ethylenediamine tetraacetic acid (EDTA), 4,5-dihydroxy-1-3-benzene disulfonic acid (Tiron), o-phenanthroline, and 2,2'-dipyridyl, inhibited both sulfite oxidation systems, but the T. ferrooxidans system was inhibited only after the initial brief oxygen consumption. EDTA and Tiron, strong chelators of Fe3+, inhibited the oxidation at lower concentrations than o-phenanthroline and 2,2'-dipyridyl, strong chelators of Fe2+. Inhibition of Fe3+-catalyzed sulfite oxidation by EDTA and Tiron was instant, but the inhibition by o-phenanthroline and dipyridyl was briefly delayed, presumably for the reduction of Fe3+ to Fe2+. Mannitol, a free radical scavenger, inhibited both systems to the same extent. Cyanide and azide inhibited only the T. ferrooxidans system, suggesting a role of cytochrome oxidase. It is proposed that sulfite is oxidized by a free radical mechanism initiated by Fe3+ on the cell surface of T. ferrooxidans. Cytochrome oxidase is possibly involved in the regeneration of Fe3+ from Fe2+ by the normal Fe2+-oxidizing system of T. ferrooxidans.     

Reduction of Hg2+ with Reduced Mammalian Cytochrome c by Cytochrome c Oxidase Purified from a Mercury-Resistant Acidithiobacillus ferrooxidans Strain,MON-1

Acidithiobacillus ferrooxidans AP19-3, ATCC 23270, and MON-1 are mercury-sensitive, moderately mercury-resistant, and highly mercury-resistant strains respectively. It is known that 2,3,5,6-tetramethyl-p-phenylendiamine (TMPD) and reduced cytochrome c are used as electron donors specific for cytochrome c oxidase. Resting cells of strain MON-1 had TMPD oxidase activity and volatilized metal mercury with TMPD as an electron donor. Cytochrome c oxidase purified from strain MON-1 reduced mercuric ions to metalic mercury with reduced mammalian cytochrome c as well as TMPD. These mercury volatilization activities with reduced cytochrome c and TMPD were completely inhibited by 1 mM NaCN. These results indicate that cytochrome c oxidase is involved in mercury reduction in A. ferrooxidans cells. The cytochrome c oxidase activities of strains AP19-3 and ATCC 23270 were completely inhibited by 1 μM and 5 μM of mercuric chloride respectively. In contrast, the activity of strain MON-1 was inhibited 33% by 5 μM, and 70% by 10 μM of mercuric chloride, suggesting that the levels of mercury resistance in A. ferrooxidans strains correspond well with the levels of mercury resistance of cytochrome c oxidase.     

Identification of Thiobacillus ferrooxidans strains based on restriction fragment length polymorphism analysis of 16S rDNA.

The 16S rDNA sequences from ten strains of Thiobacillus ferrooxidans were amplified by PCR. The products were compared by performing restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases Alu I, Hap II, Hha I, and Hae III. The RFLP patterns revealed that T. ferrooxidans could be distinguished from other iron- or sulphur-oxidizing bacteria such as T. thiooxidans NB1-3, T. caldus GO-1, Leptospirillum ferrooxidans and the marine iron-oxidizing bacterium strain KU2-11. The RFLP patterns obtained with Alu I, Hap II, and Hae III were the same for nine strains of T. ferrooxidans except for strain ATCC 13661. The RFLP patterns for strains NASF-1 and ATCC 13661 with Hha I were distinct from those for other T. ferrooxidans strains. The 16S rDNA sequence of T. ferrooxidans NASF-1 possessed an additional restriction site for Hha I. These results show that iron-oxidizing bacteria isolated from natural environments were rapidly identified as T. ferrooxidans by the method combining RFLP analysis with physiological analysis.     

Effect of anions on selective solubilization of zinc and copper in bacterial leaching of sulfide ores

Bacterial leaching of sulfide ores using Thiobacillus ferrooxidans, Thiobacillus thiooxidans, or a combination of the two was studied at various concentrations of specific anions. Selective zinc and copper solubilization was obtained by inhibiting iron oxidation without affecting sulfur/sulfide oxidation. Phosphate reduced iron solubilization from a pyrite (FeS(2))-sphalerite (ZnS) mixture without significantly affecting zinc solubilization. Copper leaching from a chalcopyrite (CuFeS(2))-sphalerite mixture was stimulated by phosphate, whereas chloride accelerated zinc extraction. In a complex sulfide ore containing pyrite, chalcopyrite, and sphalerite, both phosphate and chloride reduced iron solubilization and increased copper extraction, whereas only chloride stimulated zinc extraction. Maximum leaching obtained was 100% zinc and 50% copper. Time-course studies of copper and zinc solubilization suggest the possibility of selective metal recovery following treatment with specific anions.     

The bc(1) complex of the iron-grown acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans functions in the reverse but not in the forward direction. Is there a second bc(1) complex?

Acidithiobacillus ferrooxidans is an acidophilic chemolithotrophic bacterium that can grow in the presence of either a weak reductant, Fe(2+), or reducing sulfur compounds that provide more energy for growth than Fe(2+). Here we first review the latest findings about the uphill electron transfer pathway established in iron-grown A. ferrooxidans, which has been found to involve a bc(1) complex. We then provide evidence that this bc(1) complex cannot function in the forward direction (exergonic reaction), even with an appropriate substrate. A search for the sequence of the three redox subunits of the A. ferrooxidans bc(1) complex (strain ATCC 19859) in the complete genome sequence of the A. ferrooxidans ATCC 23270 strain showed the existence of two different bc(1) complexes in A. ferrooxidans. Cytochrome b and Rieske protein sequence comparisons allowed us to point out some sequence particularities of these proteins in A. ferrooxidans. Lastly, we discuss the possible reasons for the existence of two different "classical" bc(1) complexes and put forward some suggestions as to what role these putative complexes may play in this acidophilic chemolithotrophic bacterium.