Mechanism of pyrite dissolution in the presence of Thiobacillus ferrooxidans.

In spite of the environmental and commercial interests in the bacterial leaching of pyrite, two central questions have not been answered after more than 35 years of research: does Thiobacillus ferrooxidans enhance the rate of leaching above that achieved by ferric sulfate solutions under the same conditions, and if so, how do the bacteria affect such an enhancement? Experimental conditions of previous studies were such that the concentrations of ferric and ferrous ions changed substantially throughout the course of the experiments. This has made it difficult to interpret the data obtained from these previous works. The aim of this work was to answer these two questions by employing an experimental apparatus designed to maintain the concentrations in solution at a constant value. This was achieved by using the constant redox potential apparatus described previously (P. I. Harvey, and F. K. Crundwell, Appl. Environ. Microbiol. 63:2586-2592, 1997; T. A. Fowler, and F. K. Crundwell, Appl. Environ. Microbiol. 64:3570-3575, 1998). Experiments were conducted in both the presence and absence of T. ferrooxidans, maintaining the same conditions in solution. The rate of dissolution of pyrite with bacteria was higher than that without bacteria at the same concentrations of ferrous and ferric ions in solution. Analysis of the dependence of the rate of leaching on the concentration of ferric ions and on the pH, together with results obtained from electrochemical measurements, provided clear evidence that the higher rate of leaching with bacteria is due to the bacteria increasing the pH at the surface of the pyrite.     

Leaching of Zinc Sulfide by Thiobacillus ferrooxidans: Experiments with a Controlled Redox Potential Indicate No Direct Bacterial Mechanism

The role of Thiobacillus ferrooxidans in bacterial leaching of mineral sulfides is controversial. Much of the controversy is due to the fact that the solution conditions, especially the concentrations of ferric and ferrous ions, change during experiments. The role of the bacteria would be more easily discernible if the concentrations of ferric and ferrous ions were maintained at set values throughout the experimental period. In this paper we report results obtained by using the constant redox potential apparatus described previously (P. I. Harvey and F. K. Crundwell, Appl. Environ. Microbiol. 63:2586–2592, 1997). This apparatus is designed to control the redox potential in the leaching compartment of an electrolytic cell by reduction or oxidation of dissolved iron. By controlling the redox potential the apparatus maintains the concentrations of ferrous and ferric ions at their initial values. Experiments were conducted in the presence of T. ferrooxidans and under sterile conditions. Analysis of the conversion of zinc sulfide in the absence of the bacteria and analysis of the conversion of zinc sulfate in the presence of the bacteria produced the same results. This indicates that the only role of the bacteria under the conditions used is regeneration of ferric ions in solution. In this work we found no evidence that there is a direct mechanism for bacterial leaching.     

Leaching of Zinc Sulfide by Thiobacillus ferrooxidans: Bacterial Oxidation of the Sulfur Product Layer Increases the Rate of Zinc Sulfide Dissolution at High Concentrations of Ferrous Ions

This paper reports the results of leaching experiments conducted with and without Thiobacillus ferrooxidans at the same conditions in solution. The extent of leaching of ZnS with bacteria is significantly higher than that without bacteria at high concentrations of ferrous ions. A porous layer of elemental sulfur is present on the surfaces of the chemically leached particles, while no sulfur is present on the surfaces of the bacterially leached particles. The analysis of the data using the shrinking-core model shows that the chemical leaching of ZnS is limited by the diffusion of ferrous ions through the sulfur product layer at high concentrations of ferrous ions. The analysis of the data shows that diffusion through the product layer does not limit the rate of dissolution when bacteria are present. This suggests that the action of T. ferrooxidans in oxidizing the sulfur formed on the particle surface is to remove the barrier to diffusion by ferrous ions.     

Growth of Thiobacillus ferrooxidans: a Novel Experimental Design for Batch Growth and Bacterial Leaching Studies

The concentrations of ferrous and ferric ions change dramatically during the course of the batch experiments usually performed to study the kinetics of the bacterial oxidation of ferrous ions and sulfide minerals. This change in concentration of the iron species during the course of the experiment often makes it difficult to interpret the results of these experiments, as is evidenced by the lack of consensus concerning the mechanism of bacterial leaching. If the concentrations of ferrous and ferric ions were constant throughout the course of the batch experiment, then the role of the bacteria could be easily established, because the rate of the chemical leaching should be the same at a given redox potential in the presence and in the absence of bacteria. In this paper we report an experiment designed to obtain kinetic data under these conditions. The redox potential is used as a measure of the concentrations of ferrous and ferric ions, and the redox potential of the leaching solution is controlled throughout the experiment by electrolysis. The effects of ferrous, ferric, and arsenite ions on the rate of growth of Thiobacillus ferrooxidans on ferrous ions in this redox-controlled reactor are presented. In addition, the growth of this bacterium on ferrous ions in batch culture was also determined, and it is shown that the parameters obtained from the batch culture and the redox-controlled batch culture are the same. An analysis of the results from the batch culture indicates that the initial number of bacteria that are adapted to the solution depends on the concentrations of ferrous and arsenite ions.     

Coal Depyritization by the Thermophilic Archaeon Metallosphaera sedula

The kinetics of pyrite oxidation by Metallosphaera sedula were investigated with mineral pyrite and two coals with moderate (Pittsburgh no. 8) and high (New Brunswick, Canada) pyritic sulfur content. M. sedula oxidized mineral pyrite at a greater rate than did another thermophile, Acidianus brierleyi, or a mesophile, Thiobacillus ferrooxidans. Maximum rates of coal depyritization were also greater with M. sedula, although the magnitude of biological stimulation above abiotic rates was notably less than with mineral pyrite. Coal depyritization appears to be limited by the oxidation of pyrite with ferric ions and not by the rate of biotic oxidation of ferrous iron, as evidenced by the maintenance of a high ratio of ferric to ferrous iron in solution by M. sedula. Significant precipitation of hydronium jarosite at elevated temperature occurred only with New Brunswick coal.     

Solubilization of Minerals by Bacteria: Electrophoretic Mobility of Thiobacillus ferrooxidans in the Presence of Iron, Pyrite, and Sulfur

Thiobacillus ferroxidans is an obligate acidophile that respires aerobically on pyrite, elemental sulfur, or soluble ferrous ions. The electrophoretic mobility of the bacterium was determined by laser Doppler velocimetry under physiological conditions. When grown on pyrite or ferrous ions, washed cells were negatively charged at pH 2.0. The density of the negative charge depended on whether the conjugate base was sulfate, perchlorate, chloride, or nitrate. The addition of ferric ions shifted the net charge on the surface asymptotically to a positive value. When grown on elemental sulfur, washed cells were close to their isoelectric point at pH 2.0. Both pyrite and colloidal sulfur were negatively charged under the same conditions. The electrical double layer around the bacterial cells under physiological conditions exerted minimal electrostatic repulsion in possible interactions between the cell and either of its charged insoluble substrates. When Thiobacillus ferrooxidans was mixed with either pyrite or colloidal sulfur at pH 2.0, the mobility spectra of the free components disappeared with time to be replaced with a new colloidal particle whose electrophoretic properties were intermediate between those of the starting components. This new particle had the charge and size properties anticipated for a complex between the bacterium and its insoluble substrates. The utility of such measurements for the study of the interactions of chemolithotrophic bacteria with their insoluble substrates is discussed.     

Dependence of effectiveness of leaching of metallic sulphides on enzymes involved in inorganic sulphur metabolism in Thiobacillus ferrooxidans

Summary The leaching activity of five batches of Thiobacillus ferrooxidans, strain F26-77, cultivated under various conditions, towards elemental sulphur, ferrous ions, pyrite, covellite, chalcopyrite and sphalerite was studied. The activities of sulphite oxidase, thiosulphate oxidase and rhodanese were determined in crude, cell-free bacterial extracts. The effectiveness of leaching was directly correlated with the enzymic activity of the cultures. The results suggest that the activities of the enzymes metabolizing sulphur and its inorganic compounds in Thiobacillus ferrooxidans, or bacterial leaching activity on sulphur and sulphides, rather than the rate of oxidation of ferrous ions, should be taken as the criterion of usefulness for the leaching of sulphide minerals.     

Existence of a Hydrogen Sulfide:Ferric Ion Oxidoreductase in Iron-Oxidizing Bacteria

The existence of a hydrogen sulfide:ferric ion oxidoreductase, which catalyzes the oxidation of elemental sulfur with ferric ions as an electron acceptor to produce ferrous and sulfite ions, was assayed with washed intact cells and cell extracts of various kinds of iron-oxidizing bacteria, such as Thiobacillus ferrooxidans 13598, 13661, 14119, 19859, 21834, 23270, and 33020 from the American Type Culture Collection, Leptospirillum ferrooxidans 2705 and 2391 from the Deutsche Sammlung von Mikroorganismen, L. ferrooxidans BKM-6-1339 and P3A, and moderately thermophilic iron-oxidizing bacterial strains BC1, TH3, and Alv. It was found that hydrogen sulfide:ferric ion oxidoreductase activity comparable to that of T. ferrooxidans AP19-3 was present in all iron-oxidizing bacteria tested, suggesting a wide distribution of this enzyme in iron-oxidizing bacteria.     

Selective Adhesion of Thiobacillus ferrooxidans to Pyrite

Bacterial adhesion to mineral surfaces plays an important role not only in bacterial survival in natural ecosystems, but also in mining industry applications. Selective adhesion was investigated with Thiobacillus ferrooxidans by using four minerals, pyrite, quartz, chalcopyrite, and galena. Escherichia coli was used as a control bacterium. Contact angles were used as indicators of hydrophobicity, which was an important factor in the interaction between minerals and bacteria. The contact angle of E. coli in a 0.5% sodium chloride solution was 31°, and the contact angle of T. ferrooxidans in a pH 2.0 sulfuric acid solution was 23°. E. coli tended to adhere to more hydrophobic minerals by hydrophobic interaction, while T. ferrooxidans selectively adhered to iron-containing minerals, such as pyrite and chalcopyrite. Ferrous ion inhibited the selective adhesion of T. ferrooxidans to pyrite competitively, while ferric ion scarcely inhibited such adhesion. When selective adhesion was quenched by ferrous ion completely, adhesion of T. ferrooxidans was controlled by hydrophilic interactions. Adhesion of E. coli to pyrite exhibited a liner relationship on langmuir isotherm plots, but adhesion of T. ferrooxidans did not. T. ferrooxidans recognized the reduced iron in minerals and selectively adhered to pyrite and chalcopyrite by a strong interaction other than the physical interaction.     

Leaching of zinc sulfide by Thiobacillus ferrooxidans: bacterial oxidation of the sulfur product layer increases the rate of zinc sulfide dissolution at high concentrations of ferrous ions

This paper reports the results of leaching experiments conducted with and without Thiobacillus ferrooxidans at the same conditions in solution. The extent of leaching of ZnS with bacteria is significantly higher than that without bacteria at high concentrations of ferrous ions. A porous layer of elemental sulfur is present on the surfaces of the chemically leached particles, while no sulfur is present on the surfaces of the bacterially leached particles. The analysis of the data using the shrinking-core model shows that the chemical leaching of ZnS is limited by the diffusion of ferrous ions through the sulfur product layer at high concentrations of ferrous ions. The analysis of the data shows that diffusion through the product layer does not limit the rate of dissolution when bacteria are present. This suggests that the action of T. ferrooxidans in oxidizing the sulfur formed on the particle surface is to remove the barrier to diffusion by ferrous ions.     

The effect of ferric ion on the rate of ferrous oxidation by Thiobacillus ferrooxidans

 In this study, the effect of ferric ion and cell concentrations on the oxidation of ferrous ion by T. ferrooxidans was investigated. Ferric ions competitively inhibited ferrous ion oxidation by the bacteria. The inhibitory effect of ferric ion was, however, reduced by increasing cell concentration. The apparent ferric ion inhibition constant did not change with increasing cell concentration. The ferrous ion oxidation kinetics in the absence and presence of ferric ion changes from the standard Michaelis-Menten type at low cell concentrations to pseudo-first-order kinetics at high cell concentration. Received: 8 August 1995/Received revision: 31 October 1995/Accepted: 10 November 1995     

Degradation of massive pyrite: Physical,chemical, and bacterial effects

Massive pyrite was shown to produce soluble iron, hydrogen, and sulfate ions on exposure to air and water. The rate of this process was directly proportional to the surface area of the mineral; it was unaffected by a drop in the pH and the presence of the ferrous and sulfate ions formed. Cupic ion had no effect but ferric ion accelerated pyrite degradation until all the ferric ion was consumed, in accordance with FeS₂ + 2Fe³⁺ —>‐3Fe²⁺ + 2S°. Thiobacillus ferrooxidans increased pyrite degradation considerably; the presence of Thiobacillus thiooxidans had no influence on pyrite degradation.     

Evaluation of a Fluorescent Lectin-Based Staining Technique for Some Acidophilic Mining Bacteria

A fluorescence-labeled wheat germ agglutinin staining technique (R. K. Sizemore et al., Appl. Environ. Microbiol. 56:2245–2247, 1990) was modified and found to be effective for staining gram-positive, acidophilic mining bacteria. Bacteria identified by others as being gram positive through 16S rRNA sequence analyses, yet clustering near the divergence of that group, stained weakly. Gram-negative bacteria did not stain. Background staining of environmental samples was negligible, and pyrite and soil particles in the samples did not interfere with the staining procedure.     

Ferric Iron Reduction by Acidophilic Heterotrophic Bacteria

Fifty mesophilic and five moderately thermophilic strains of acidophilic heterotrophic bacteria were tested for the ability to reduce ferric iron in liquid and solid media under aerobic conditions; about 40% of the mesophiles (but none of the moderate thermophiles) displayed at least some capacity to reduce iron. Both rates and extents of ferric iron reduction were highly strain dependent. No acidophilic heterotroph reduced nitrate or sulfate, and (limited) reduction of manganese(IV) was noted in only one strain (Acidiphilium facilis), an acidophile which did not reduce iron. Insoluble forms of ferric iron, both amorphous and crystalline, were reduced, as well as soluble iron. There was evidence that, in at least some acidophilic heterotrophs, iron reduction was enzymically mediated and that ferric iron could act as a terminal electron acceptor. In anaerobically incubated cultures, bacterial biomass increased with increasing concentrations of ferric but not ferrous iron. Mixed cultures of Thiobacillus ferrooxidans or Leptospirillum ferrooxidans and an acidophilic heterotroph (SJH) produced sequences of iron cycling in ferrous iron-glucose media.     

Solubilization and speciation of iron during pyrite oxidation by Thiobacillus ferrooxidans

Various species of soluble iron in pyrite‐grown cultures of Thiobacillus ferrooxidans were determined by colorimetry, atomic absorption spectrometry, and ultraviolet spectroscopy. All the cultures were incubated for six weeks before iron analysis. The effects of the following factors were investigated: particle size, initial pH, shaking (aeration), concentration of pyrite, and concentration of yeast extract. Shaking, but not initial pH nor particle size, influenced the relative proportion of different iron species. Polynomial regressions could be used to describe the functional relationship between the different iron species and concentration of pyrite; fewer relationships were evident with respect to concentration of yeast extract. The variance‐covariance matrices indicated a linear dependence among the different iron species. Canonical correlations indicated perfect correlations between group variables of iron, copper, and zinc, with the exception of an absence of significant correlation with the hydroxy complex of iron (FeOH²⁺).

The dissolved ferrous iron (dissociated and weakly chelated) always remained less than 7% of the total iron in solution. The total ferrous iron, which included complexed species, amounted to 7–34% of the total iron in solution. The concentrations of dissociated ferrous and ferric iron and their weak chelates (the dissolved iron) remained mostly constant, irrespective of the concentration of the total iron in solution. Most of the total iron was complexed as ferric species and the amount correlated with culture conditions. The hydroxy complex (FeOH²⁺), which was indicative of the relative amount of hydrolyzable ferric iron upon dilution in CO₂‐free water, usually ranged between 60 and 80% of the total iron. The amount of the total iron in uninoculated controls was less than 12% of that solu‐bilized in the presence of iron‐oxidizing thiobacilli.

T. ferrooxidans was enumerated by a most‐probable‐number technique after three and six weeks of growth on pyrite. The counts after three weeks indicated an increase in the number of free and loosely attached bacteria, followed by a decline of about one order of magnitude in bacterial numbers after six weeks. The technique for bacterial enumeration was deemed unsatisfactory because it could not account for cells attached on pyrite.     

A mathematical model for the bacterial oxidation of a sulfide ore concentrate

The effect of dilution rate and feed solids concentration on the bacterial leaching of a pyrite/arsenopyrite ore concentrate was studied. A mathematical model was developed for the process based on the steady-state data collected over the range of dilution rates (20 to 110 h) and feed solids concentrations (6 to 18% w/v) studied. A modified Monod model with inhibition by arsenic was used to model bacterial ferrous ion oxidation rates. The model assumes that (i) pyrite and arsenopyrite leaching occurs solely by the action of ferric iron produced from the bacterial oxidation of ferrous iron and (ii) bacterial growth rates are proportional to ferrous ion oxidation rate. The equilibrium among the various ionic species present in the leach solution that are likely to have a significant effect on the bioleach process were included in the model. (c) 1994 John Wiley & Sons, Inc.     

Enhancement of Copper Dissolution from a Sulfide Ore by Using Thiobacillus thiooxidans

Copper dissolution from a sulfide ore (with covellite as the main copper phase) was investigated in cultures of Thiobacillus ferrooxidans or Thiobacillus thiooxidans and in abiotic controls. In unsupplemented media, T. ferrooxidans was more efficient than T. thiooxidans. In the presence of ferric iron, the dissolution of covellite was not significantly different in cultures inoculated with T. ferrooxidans or T. thiooxidans. However, the most extraction was found in T. thiooxidans cultures supplemented with ferrous sulfate. The first results were explained by the mechanism proposed by Schippers and Sand (Appl Envir Microbiol 65:319-321, 1999), which involves polysulfides and sulfur as intermediates. This mechanism was extended to explain the behavior of T. thiooxidans culture supplemented with ferrous iron.     

Bioregeneration of Leaching Solutions during Two-Step Processing of Copper-Zinc Concentrate

A comparative study of the oxidation of ferrous iron ions by various cultures of acidophilic chemolithotrophic microorganisms in solutions obtained after ferric leaching of copper-zinc concentrate at 80°C has been carried out. It was shown that the use of a moderately thermophilic culture for bioregeneration of leaching solutions was preferable. At the same time, the oxidation rate of Fe²⁺ ions reached 0.88 g/(L h), or 21.1 g/(L day). We propose that the activity of the moderately thermophilic culture was due to the presence of the mixotrophic bacteria Sulfobacillus spp., which used organic products of the microbial lysis for their growth. These products were formed during high-temperature ferric leaching of the copper-zinc concentrate with the biosolution.     

Attachment of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum cultured under varying conditions to pyrite, chalcopyrite, low-grade ore and quartz in a packed column reactor

The attachment of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum spp. grown on ferrous medium or adapted to a pyrite mineral concentrate to four mineral substrata, namely, chalcopyrite and pyrite concentrates, a low-grade chalcopyrite ore (0.5 wt%) and quartzite, was investigated. The quartzite represented a typical gangue mineral and served as a control. The attachment studies were carried out in a novel particle-coated column reactor. The saturated reactor containing glass beads, which were coated with fine mineral concentrates, provided a quantifiable surface area of mineral concentrate and maintained good fluid flow. A. ferrooxidans and Leptospirillum spp. had similar attachment characteristics. Enhanced attachment efficiency occurred with bacteria grown on sulphide minerals relative to those grown on ferrous sulphate in an ore-free environment. Selective attachment to sulphide minerals relative to gangue materials occurred, with mineral adapted cultures attaching to the minerals more efficiently than ferrous grown cultures. Mineral-adapted cultures showed highest levels of attachment to pyrite (74% and 79% attachment for A. ferrooxidans and L. ferriphilum, respectively). This was followed by attachment of mineral-adapted cultures to chalcopyrite (63% and 58% for A. ferrooxidans and L. ferriphilum, respectively). A. ferrooxidans and L. ferriphilum exhibited lower levels of attachment to low-grade ore and quartz relative to the sulphide minerals.