Activity of sulphite oxidase,thiosulphate oxidase and rhodanese in Thiobacillus ferrooxidans during covellite and chalcopyrite leaching

Summary The activities of enzymes involved in inorganic sulphur metabolism in crude, cell-free extracts of Thiobacillus ferrooxidans, strain F26-77 were determined during bacterial leaching of covellite and chalcopyrite. Increase in the specific activity of sulphide oxidase, thiosulphate oxidase and rhodanese during the leaching of both minerals was demonstrated. Simultaneously a decrease in the extraction rate of copper from the leached materials was observed.Offprint requests to: T. Wilczok     

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: 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.     

Inhibition of Sulfur Use by Sulfite Ion in Thiobacillus ferrooxidans

In Thiobacillus ferrooxidans AP19-3, elemental sulfur is oxidized by the cooperation of three enzymes, namely, hydrogen sulfide: ferric ion oxidoreductase (SFORase), sulfite: ferric ion oxidoreductase, and iron oxidase. Sulfite ions are one of the products when elemental sulfur is oxidized by SFORase. Under the conditions in which sulfite ions are accumulated in the cells, use of sulfur as an energy source by this strain was strongly inhibited. So the mechanism of inhibition by sulfite ions in T. ferrooxidans AP19-3 was studied. The activities of SFORase and iron oxidase were completely inhibited by 0.8 mm and 1.5 mm NaHSO₃, respectively. ¹⁴CO₂ uptake into washed intact cells was also completely inhibited by 1mm NaHSO₃ when ferrous ion or elemental sulfur was used as an energy source. However, the activities of ribulose-1,5-bisphosphate carboxylase, phosphoribulokinase, and ribosephosphate isomerase measured with a cell-free extract were not inhibited by NaHSO₃ at 1 mm, indicating that sulfite ions didn’t inhibit key enzymes of the Calvin cycle. Since the activity of CO₂ uptake into washed intact cells was absolutely dependent on Fe^{2 +} - or S0-oxidation, mechanism of inhibition of sulfur use by sulfite ions is proposed as follows: sulfite ions inhibit SFORase and iron oxidase, as a result T. ferrooxidans AP19-3 can not obtain a carbon source for CO₂ fixation and stops cell growth on sulfur-salts medium.     

Inhibition of Iron-oxidizing Activity by Bisulfite Ion in Thiobacillus ferrooxidans

Growth of Thiobacillus ferrooxidans on iron- and sulfur-salts media and iron oxidizing activity of this bacterium were strongly inhibited by bisulfite ion. The mechanism of inhibition by bisulfite ion of iron-oxidizing activity was studied with the plasma membrane of T. ferrooxidans AP19-3. The c-type cytochrome in the plasma membrane was reduced by ferrous ion and the cytochrome reduced by Fe²⁺ was oxidized by cytochrome c oxidase in the plasma membrane. In contrast, c-type cytochrome was reduced by bisulfite ion, but it was not oxidized by cytochrome c oxidase in the membrane. Cytochrome c-oxidizing activity was also inhibited by the ion when mammalian cytochrome c was used as an electron donor, suggesting that cytochrome c oxidase, one of the component of iron oxidase, is the site of inhibition by bisulfite ion.     

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.     

Role of cell attachment in leaching of chalcopyrite mineral by Thiobacillus ferrooxidans

Summary Experiments on the leaching of copper from chalcopyrite mineral by the bacterium Thiobacillus ferrooxidans show that, in the presence of adequate amounts of sulphide, iron-grown bacteria preferentially oxidise sulphur in the ore (through direct attachment) rather than ferrous sulphate in solution. At 20% pulp density, the leaching initially takes place by a predominantly direct mechanism. The cell density in the liquid phase increases, but the Fe²⁺ is not oxidised. However, in the later stages when less solid substrate is available and the cell density becomes very high, the bacteria start oxidising Fe²⁺ in the liquid phase, thus contributing to the indirect mechanism of leaching. Contrary to expectations, the rate of leaching increased with increasing particle size in spite of the decreasing specific surface area. This has been found to be due to increasing attachment efficiency with increase in particle size. Offprint requests to: R. Kumar     

Biodesulphurization of coal: rate enhancement by sulphur-grown cells

Pyritic sulphur was removed from coal by growing Thiobacillus ferrooxidans in a 250 ml batch bioreactor. Thiobacillus ferrooxidansgrown on sulphur and which was added 5 days after initial inoculation, enhanced the iron solubilization rate by 35% as compared to control (without addition of sulphur-grown cells). About 93% pyritic sulphur was removed in presence of sulphur-grown cells as compared to 77% in the control.     

The removal of pyritic sulphur from coal by Leptospirillum-like bacteria

Summary The microbial oxidation of pyritic sulphur was studied in a 4.5-l airlift fermentor at pH 1.5 and 100 g/l pulp density. By microbial leaching with Leptospirillum-like bacteria 85% of the pyritic sulphur was removed within 40 days; 30% of the removed pyrite was oxidized to elemental sulphur, the rest being transformed to soluble sulphate. Accumulation of elemental sulphur could be avoided by using a mixed culture of Leptospirillum-like bacteria and Thiobacillus ferrooxidans. Apart from oxidation of elemental sulphur neither the pure nor the mixed culture showed a significant difference as to removal of pyrite.     

Ferrous iron oxidation and uranium extraction by Thiobacillus ferrooxidans

The microbiological oxidation of ferrous iron in batch and continuous systems has been investigated in relation to uranium extraction from a low-grade ore by Thiobacillus ferrooxidans. The influence of the parameters, agitation, and aeration on oxygen saturation concentration, rate of oxygen mass transfer, and rate of ferrous iron oxidation was demonstrated. The kinetic values, V_max and K were determined using an adapted Monod equation for different dilution rates and initial concentrations of ferrous iron. The power requirements for initial leaching conditions were also calculated. Uranium extraction as high as 68% has been realized during nine days of treatment. Regrinding the leach residue and its subsequent leaching yielded 87% uranium solubilization.     

Microbiological leaching of a chalcopyrite concentrate and the influence of hydrostatic pressure on the activity of Thiobacillus ferrooxidans

Summary The effect of hydrostatic pressure on the activity of Thiobacillus ferrooxidans grown on chalcopyrite concentrate has been investigated. It was found that bacterial activity, measured by conventional respirometry, was little affected by subjecting these microorganisms to a pressure of 100 lbs/in² (690 kPa). The total cooper concentration was as high as 18 g/l in 28 days of leaching at atmospheric pressure.     

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.     

Continuous microbial leaching of a pyritic concentrate by Leptospirillum-like bacteria

Summary Continuous leaching of a pyritic flotation concentrate by mixed cultures of acidophilic bacteria was studied in a laboratory scale airlift reactor. Enrichment cultures adapted to the flotation concentrate contained Thiobacillus ferrooxidans and Thiobacillus thiooxidans. During the late stationary growth phase of these thiobacilli growth of Leptospirillum-like bacteria was observed, too. In discontinuous cultivation no significant influence of Leptospirillum-like bacteria on leaching rates could be detected. During continuous leaching at pH 1.5 Leptospirillum-like bacteria displaced Thiobacillus ferrooxidans. The iron leaching rate achieved by Leptospirillum-rich cultures was found to be up to 3.9 times higher than that by Leptospirillum-free cultures.     

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.     

Kinetics of uranous ion and ferrous iron oxidation byThiobacillus ferrooxidans

Kinetic constants for the oxidation of uranous and ferrous ions byThiobacillus ferrooxidans were estimated. The kinetics indicate a direct biological mechanism for uranium oxidation. The complex interrelations of ferric, uranyl and uranous ion inhibition are considered.     

Evaluation of Leptospirillum ferrooxidans for Leaching

The importance of Leptospirillum ferrooxidans for leach processes has been evaluated by studying the lithotrophic flora of three mine biotopes and a heap leaching operation, by percolation experiments with inoculated, sterilized ore, and by morphological, physiological, and genetic investigations of pure and mixed cultures of L. ferrooxidans, Thiobacillus ferrooxidans, and Thiobacillus thiooxidans. In biotopes of 20°C or above, Leptospirillum-like bacteria are as abundant as T. ferrooxidans. Leptospirilli represent at least one-half of the ferrous-iron-oxidizing population. Percolation experiments confirmed this result. Leptospirilli were as numerous as T. ferrooxidans. At reduced temperatures, the generation times of leptospirilli increase more so than those of T. ferrooxidans. At 14°C, Leptospirillum grows slowly and T. ferrooxidans dominates the population. Physiological investigations indicate that L. ferrooxidans is a strict chemolithoautotroph, metabolizing only ferrous iron and pyrite. Even an addition of 0.05% (wt/vol) yeast extract inhibited its growth. The maximum ferrous-iron-oxidizing activity of L. ferrooxidans amounts to about 40% of the activity of T. ferrooxidans. After growth on sulfidic ore, both species exhibit reduced iron-oxidizing activities, L. ferrooxidans exhibiting one-third and T. ferrooxidans exhibiting one-seventh of their maximum activities. Surprisingly, the absolute values are similar. For indirect leaching, L. ferrooxidans is as important as T. ferrooxidans. This was confirmed by the results of percolation experiments. L. ferrooxidans together with T. thiooxidans mobilized metals at least as well as T. ferrooxidans did. The best results were obtained with a mixed culture of all three species.     

Reduction of dichromate by Thiobacillus ferrooxidans

Chromium(VI) was reduced by Thiobacillus ferrooxidans grown with elemental sulphur as the sole energy source. Chromium(VI) reduction (as high as 2000 M), was due to the presence of sulphite and thiosulphate, among others with high reducing power which was generated during the sulphur oxidation by the bacteria. Therefore, Thiobacillus ferrooxidans could be used to treat chromium(VI)-containing industrial effluents.     

Pyrite oxidation in acid sulphate soils: The role of miroorganisms

Summary A study has been made of microbial processes in the oxidation of pyrite in aicd sulphate soil material. Such soils are formed during aeration of marine muds rich in pyrite (FeS₂). Bacteria of the type ofThiobacillus ferrooxidans are mainly responsible for the oxidation of pyrite, causing a pronounced acidification of the soil. However, becauseThiobacillus ferrooxidans functions optimally at pH values bellow 4.0, its activity cannot explain the initial pH drop from approximately neutral to about 4. This was shown to be a non-biological process, in which bacteria play an insignificant part. AlthoughThiobacillus thioparus andThiobacillus thiooxidans were isolated from the acidifying soil, they did not stimulate oxidation of FeS₂, but utilized reduced sulphur compounds, which are formed during the non-biological oxidation of FeS₂.Ethylene-oxide-sterilized and dry-sterilized soil inoculated with pure cultures of mixtures of various thiobacilli or with freshly sampled acid sulphate soil soil did not acidify faster than sterile blanks.Thiobacillus thiooxians. Thiobacillus thioparus. Thiobacillus intermedius andThiobacillus perometabolis increased from about 10⁴ to 10⁵ cells/ml in media with FeS₂ as energy source. However, FeS₂ oxidation in the inoculated media was not faster than in sterile blanks.Attempts to isolate microorganisms other thanThiobacillus ferrooxidans, like metallogenium orLeptospirillum ferrooxidans, which might also be involved in the oxidation of FeS₂ were not successful.Addition of CaCO₃ to the soil prevented acidification but did not stop non-biological oxidation of FeS₂.     

Bulk and surface characterization of crystalline and plastic sulphur oxidized by two Thiobacillus species

This work studies the surface interaction between Thiobacillus ferrooxidans and Thiobacillus thiooxidans with crystalline and plastic elemental sulphur. The interaction mechanisms were analysed by fractal geometry which describes textural modifications of the substrate caused by bacterial action. The results demonstrated that the bacteria are able to produce two different effects depending on the substrates. Only surface smoothing (decrease on fractal dimension values) was detected on crystalline sulphur (this effect being stronger with T. ferrooxidans than with T. thiooxidans), but, perforation of the bulk was also observed in plastic sulphur