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.     

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.     

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.     

Bioleaching of zinc sulfide concentrate by Thiobacillus ferrooxidans

The kinetics of the bioleaching of ZnS concentrate by Thiobacillus ferrooxidans was studied in a well-mixed batch reactor. Experimental studies were made at 30 degrees C and pH 2.2 on adsorption of the bacteria to the mineral, ferric iron leaching, and bacterial leaching. The adsorption rate of the bacteria was fairly rapid in comparison with the bioleaching rate, indicating that the bacterial adsorption is at equilibrium during the leaching process. The adsorption equilibrium data were correlated by the Langmuir isotherm, which is a useful means for predicting the number of bacteria adsorbed on the mineral surface. The rate of chemical leaching varied with the concentration of ferric iron, and the first-order reaction rate constant was determined. Bioleaching in an iron-containing medium was found to take place by both direct bacterial attack on the sulfide mineral and indirect attack via ferric iron. In this case, the ferric iron was formed from the reaction product (ferrous iron) through the biological oxidation reaction. To develop rate expressions for the kinetics of bacterial growth and zinc leaching, the two bacterial actions were considered. The key parameters appearing in the rate equations, the growth yield and specific growth rate of adsorbed bacteria, were evaluated by curve fitting using the experimental data. This kinetic model allowed us to predict the liquid-phase concentrations of the leached zinc and free cells during the batch bioleaching process.     

Modeling continuous bioleach reactors

The results of recent research have shown that the bioleaching of sulfide minerals occurs via a two‐step mechanism. In this mechanism, the sulfide mineral is chemically oxidized by the ferric‐iron in the bioleaching liquor. The ferrous‐iron produced is subsequently oxidized to ferric‐iron by the microorganism. Further research has shown that the rates of both the ferric leaching and ferrous‐iron oxidation are governed by the ferric/ferrous‐iron ratio (i.e., the redox potential). During the steady‐state operation of a bioleach reactor, the rate of iron turnover between the chemical ferric leaching of the mineral and the bacterial oxidation of the ferrous‐iron will define the rate and the redox potential at which the system will operate. The balance between the two rates will in turn depend on the species used, the microbial concentration, the residence time employed, the nature of the sulfide mineral being leached, and its active surface area. The model described proposes that the residence time and microbial species present determine the microbial growth rate, which in turn determines the redox potential in the bioleach liquor. The redox potential of the solution, in turn, determines the degree of leaching of the mineral; that is, conversion in the bioleach reactor. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 671–677, 1999.     

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.     

Inhibitory effect of iron‐oxidizing bacteria on ferrous‐promoted chalcopyrite leaching

It is generally accepted that iron‐oxidizing bacteria, Thiobacillus ferrooxidans, enhance chalcopyrite leaching. However, this article details a case of the bacteria suppressing chalcopyrite leaching. Bacterial leaching experiments were performed with sulfuric acid solutions containing 0 or 0.04 mol/dm³ ferrous sulfate. Without ferrous sulfate, the bacteria enhance copper extraction and oxidation of ferrous ions released from chalcopyrite. However, the bacteria suppressed chalcopyrite leaching when ferrous sulfate was added. This is mainly due to the bacterial consumption of ferrous ions which act as a promoter for chalcopyrite oxidation with dissolved oxygen. Coprecipitation of copper ions with jarosite formed by the bacterial ferrous oxidation also causes the bacterial suppression of copper extraction. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 478–483, 1999.     

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.     

Control of the redox potential by oxygen limitation improves bacterial leaching of chalcopyrite

Shake flask and stirred tank bioleaching experiments showed that the dissolution of chalcopyrite is inhibited by ferric ion concentrations as low as 200 mg L(-1) and redox potentials >420 mV (vs. Ag/AgCl). Chemical leaching of chalcopyrite (4% suspension, surface area 2.3 m2 g(-1)) was enhanced fourfold in the presence of 0.1 M ferrous sulphate compared with 0.1 M ferric sulphate. A computer-controlled reactor was designed to function as a "potentiostat"-bioreactor by arresting the air supply to the reactor when the redox potential in solution was greater than a designated setpoint. Leaching at a low, constant redox potential (380 mV vs. Ag/AgCl) achieved final copper recoveries of 52%-61%, which was twice that achieved with a continuous supply of oxygen (<30% extraction). The bacterial populations were observed to continue growing under oxygen limitation but in a controlled manner that was found to improve chalcopyrite dissolution. As the control mechanism is easily established and is likely to decrease production cost, the use of this technology may find application in industry.     

The effects of Fe(II) and Fe(III) concentration and initial pH on microbial leaching of low-grade sphalerite ore in a column reactor

In this study the effects of initial concentration of Fe(II) and Fe(III) ions as well as initial pH on the bioleaching of a low-grade sphalerite ore in a leaching column over a period of 120 days with and without bacteria were investigated. Four different modifications of medium were used as column feed solutions to investigate the effects of initial concentration of Fe(II) and Fe(III) ions on zinc extraction. The experiments were carried out using a bench-scale, column leaching reactor, which was inoculated with mesophilic iron oxidizing bacteria, Acidithiobacillus ferrooxidans, initially isolated from the Sarcheshmeh chalcopyrite concentrate (Kerman, Iran). The effluent solutions were periodically analyzed for Zn, total Fe, Fe(II) and Fe(III) concentrations as well as pH values. Bacterial population was measured in the solution (free cells). Maximum zinc recovery in the column was achieved about 76% using medium free of initial ferrous ion and 11.4 g/L of ferric ion (medium 2) at pH 1.5. The extent of leaching of sphalerite ore with bacteria was significantly higher than that without bacteria (control) in the presence of ferrous ions. Fe(III) had a strong influence in zinc extraction, and did not adversely affect the growth of the bacteria population.     

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures was examined, using on-line off-gas analyses to measure the oxygen and carbon dioxide consumption rates continuously. A cell suspension from continuous cultures at steady state was used as the inoculum. It was observed that a dynamic phase occurred in the initial phase of the experiment. In this phase the bacterial ferrous iron oxidation and growth were uncoupled. After about 16 h the bacteria were adapted and achieved a pseudo-steady state, in which the specific growth rate and oxygen consumption rate were coupled and their relationship was described by the Pirt equation. In pseudo-steady state, the growth and oxidation kinetics were accurately described by the rate equation for competitive product inhibition. Bacterial substrate consumption is regarded as the primary process, which is described by the equation for competitive product inhibition. Subsequently the kinetic equation for the specific growth rate, μ, is derived by applying the Pirt equation for bacterial substrate consumption and growth. The maximum specific growth rate, μ _max, measured in the batch culture agrees with the dilution rate at which washout occurs in continuous cultures. The maximum oxygen consumption rate, q _O2,max, of the cell suspension in the batch culture was determined by respiration measurements in a biological oxygen monitor at excess ferrous iron, and showed changes of up to 20% during the course of the experiment. The kinetic constants determined in the batch culture slightly differ from those in continuous cultures, such that, at equal ferric to ferrous iron concentration ratios, biomass-specific rates are up to 1.3 times higher in continuous cultures. Received: 8 February 1999 / Accepted: 17 February 1999     

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.     

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.     

Diferric transferrin reduction by K562 cells. A critical study

This paper critically examines the redox activity of K562 cells (chronic myelogenous leukemia cells) and normal peripheral blood lymphocytes (PBL). Ferricyanide reduction, diferric transferrin reduction, and ferric ion reduction were measured spectrophotometrically by following the time-dependent changes of absorbance difference characteristic for ferricyanide disappearance and for the formation of ferrous ion:chelator complexes. Bathophenanthroline disulfonate (BPS) and ferrozine (FZ) were used to detect the appearance of ferrous ions in the reaction mixtures when diferric transferrin or ferric reduction was studied. Special attention was devoted to the analysis of time-dependent absorbance changes in the presence and absence of cells under different assay conditions. It was observed and concluded that: (i) FZ was far less sensitive and more sluggish than BPS for detecting ferrous ions at concentrations commonly used for BPS; (ii) FZ, at concentrations of at least 10-times the commonly used BPS concentrations, seemed to verify the results obtained with BPS; (iii) ferricyanide reduction, diferric transferrin reduction and ferric ion reduction by both K562 cells and peripheral blood lymphocytes did not differ significantly; and (iv) earlier values published for the redox activities of different cells might be overestimated, partly because of the observation published in 1988 that diferric transferrin might have loosely bound extra iron which is easily reduced. It is suggested that the specific diferric transferrin reduction by cells might be considered as a consequence of (i) changing the steady-state equilibrium in the diferric transferrin-containing solution by addition of ferrous ion chelators which effectively raised the redox potential of the iron bound in holotransferrin, and (ii) changing the steady-state equilibrium by addition of cells which would introduce, via their large and mostly negatively charged plasma membrane surface, a new phase which would favor release and reduction of the iron in diferric transferrin by a ferric ion oxidoreductase. The reduction of ferricyanide is also much slower than activities reported for other cells which may indicate reduced plasma membrane redox activity in these cells.     

Optimal conditions for bio-oxidation of ferrous ions to ferric ions using Thiobacillus ferrooxidans

A chemo-biochemical process using Thiobacillus ferrooxidans for desulphurization of gaseous fuels and emissions containing hydrogen sulphide (H2S) has been developed. In the first stage, H2S present in fuel gas and emissions is selectively oxidized to elemental sulphur using ferric sulphate. The ferrous sulphate produced in the first stage of the process is oxidized to ferric sulphate using Thiobacillus ferrooxidans for recycle and reuse in the process. The effects of process variables, temperature, pH, total dissolved solids (TDS), elemental sulphur, ferric and magnesium ions on bio-oxidation of ferrous ions to ferric ions were investigated using flask culture experiments. The bio-oxidation of ferrous ions to ferric ions could be achieved efficiently in the temperature range of 20(+/-1)-44(+/-1) degrees C. A pH range of 1.8(+/-0.02)-2.2(+/-0.02) was optimum for the growth of culture and effective bio-oxidation of ferrous ions to ferric ions. The effect of TDS on bio-oxidation of ferrous ions indicated that a preacclimatized culture in a growth medium containing high dissolved solid was required to achieve effective bio-oxidation of ferrous ions. Elemental sulphur ranging from 1000 to 100,000 mg/l did not have any effect on efficiency of ferrous ion oxidation. The efficiency of bio-oxidation of ferrous ions to ferric ions was not affected in the presence of ferric ions up to a concentration of 500 mg/l while 3 mg/l of magnesium ion was optimal for achieving effective bio-oxidation.     

Growth of free and attached Thiobacillus ferrooxidans in ore suspension

The growth of Thiobacillus ferrooxidans in a copper-containing ore suspension incubated in shake flasks was studied by determining the number of colony-forming units both in solution and attached to ore particles. The amounts of iron and copper released from the ore under experimental conditions were also determined. The total ferrous iron either released from the minerals or generated by reduction of the ferric iron in the minerals could account for the observed growth of bacteria in solution. Only a small fraction of the total colony-forming units-about 500 per mg ore-was found to be associated with the ore particles throughout the experiments. However, the rapid development of these colonies when ore particles were plated suggested that they were produced by a number of bacteria associated with each ore particle. Accordingly, when the amount of bacteria attached to ore particles was determined by monitoring the formation of ferric iron in the plates, the percentage of the total activity associated with attached bacteria was found to be between 1 and 10%.     

Estimations on the degradability of ores and bacterial leaching activity using short-time microcalorimetric tests

Abstract: This work is based on calorimetric measurements on batch cultures of ferrous iron-oxidizing thiobacilli and leaching cultures degrading solid substrates in the titration vessel under aerobic conditions. It has been demonstrated that heat-flow levels are a direct measure for oxidation rates of known reactions under non-limiting conditions. The total loss of heat-energy per unit of ferrous iron oxidized is identical under similar incubation conditions with different Thiobacillus ferrooxidans strains and different cell-densities in culture suspension. Under limiting conditions a reduced heat-loss occurred. This may have been due to either a more effective use of the substrate or to a shift to different redox reactions. Oxygen was proved to be a limiting factor in dense bacterial cultures with high substrate concentrations in the titration vessel. The experimental duration decreased significantly with the use of increasing cell densities. Therefore cell densities in degradation experiments with solid substrates were adjusted to between 5X10⁹ and 1x10¹⁰ cells/ml in short-term experiments. T. ferrooxidans is the most important species for short-term leaching experiments. Calorimetric measurements allow the best strains for a degradation of unknown substrates to be found. The calculated heat energy is a measure of the amount of converted substrate. High heat-flow only occurs if catabolic reactions take place and maintenance reactions and phenomena like adhesion of cells to surface do not cause significant loss of heat.     

Reduction of ferric iron by acidophilic heterotrophic bacteria: evidence for constitutive and inducible enzyme systems in Acidiphilium spp

AIMS: To compare the abilities of two obligately acidophilic heterotrophic bacteria, Acidiphilium acidophilum and Acidiphilium SJH, to reduce ferric iron to ferrous when grown under different culture conditions. METHODS AND RESULTS: Bacteria were grown in batch culture, under different aeration status, and in the presence of either ferrous or ferric iron. The specific rates of ferric iron reduction by fermenter-grown Acidiphilium SJH were unaffected by dissolved oxygen (DO) concentrations, while iron reduction by A. acidophilum was highly dependent on DO concentrations in the growth media. The ionic form of iron present (ferrous or ferric) had a minimal effect on the abilities of harvested cells to reduce ferric iron. Whole cell protein profiles of Acidiphilium SJH were very similar, regardless of the DO status of the growth medium, while additional proteins were present in A. acidophilum grown microaerobically compared with aerobically-grown cells. CONCLUSIONS: The dissimilatory reduction of ferric iron is constitutive in Acidiphilium SJH while it is inducible in A. acidophilum. SIGNIFICANCE AND IMPACT OF THE STUDY: Ferric iron reduction by Acidiphilium spp. may occur in oxygen-containing as well as anoxic acidic environments. This will detract from the effectiveness of bioremediation systems where removal of iron from polluted waters is mediated via oxidation and precipitation of the metal.     

Sulfur chemistry,biofilm, and the (in)direct attack mechanism — a critical evaluation of bacterial leaching

It has been shown (a) that bacterial leaching of metal sulfides apparently requires the attachment of leach bacteria to metal sulfides, (b) that exopolymerbound iron compounds are responsible for or at least considerably increase the rate of the biological attack over the chemical rate, (c) that the primary attacking agent in leaching environments is the ferric iron hexahydrate ion, (c) that thiosulfate is the first intermediate sulfur compound, giving rise to a variety of other compounds including polythionate-containing periplasmic granula, and (d) that we have no idea about the actual concentrations of protons, ferrous/ferric and/or other cations, and sulfur compounds in the reaction space between the bacterium and the sulfide surface.     

Comparison of ferric iron generation by different species of acidophilic bacteria immobilized in packed-bed reactors

Flooded packed-bed bioreactors, prepared by immobilizing four different species of acidophilic iron-oxidizing bacteria on porous glass beads, were compared for their ferric iron-generating capacities when operated in batch and continuous flow modes over a period of up to 9 months, using a ferrous iron-rich synthetic liquor and acid mine drainage (AMD) water. The bacteria used were strains of Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans, a Ferrimicrobium-like isolate (TSTR) and a novel Betaproteobacterium (isolate PSTR), which were all isolated from relatively low-temperature mine waters. Three of the bacteria used were chemoautotrophs, while the Ferrimicrobium isolate was an obligate heterotroph. Greater biomass yields achievable with the Ferrimicrobium isolate resulted in greater iron oxidation efficiency in the newly commissioned bioreactor containing this bacterium, though long-term batch testing with organic carbon-free solution resulted in similar maximum iron oxidation rates in all four bioreactors. Two of the bioreactors (those containing immobilized L. ferrooxidans and Ferrimicrobium TSTR) were able to generate significantly lower concentrations of ferrous iron than the others when operated in batch mode. In contrast, when operated as continuous flow systems, the bioreactor containing immobilized PSTR was superior to the other three when challenged with either synthetic or actual AMD at high flow rates. The least effective bacterium overall was At. ferrooxidans, which has previously been the only iron-oxidizer used in the majority of reports describing ferric iron-generating bioreactors. The results of these experiments showed that different species of iron-oxidizing acidophiles have varying capacities to oxidize ferrous iron when immobilized in packed-bed bioreactors, and that novel isolates may be superior to well-known species.