Pyrite oxidation by Thiobacillus ferrooxidans with special reference to the sulphur moiety of the mineral

Available cultures of Thiobacillus ferrooxidans were found to be contaminated with bacteria very similar to Thiobacillus acidophilus. The experiments described were performed with a homogeneous culture of Thiobacillus ferrooxidans.Pyrite (FeS₂) was oxidized by Thiobacillus ferrooxidans grown on iron (Fe²⁺), elemental sulphur (S^o) or FeS₂.Evidence for the direct utilization of the sulphur moiety of pyrite by Thiobacillus ferrooxidans was derived from the following observations: a. Known inhibitors of Fe²⁺ and S^o oxidation, NaN₃ and NEM, respectively, partially abolished FeS₂ oxidation. b. A b-type cytochrome was detectable in FeS₂-and S^o-grown cells but not in Fe²⁺-grown cells. c. FeS₂ and S^o reduced b-type cytochromes in whole cells grown on S^o. d. CO₂ fixation at pH 4.0 per mole of oxygen consumed was the highest with S^o, lowest with Fe²⁺ and medium with FeS₂ as substrate. e. Bacterial Fe²⁺ oxidation was found to be negligible at pH 5.0 whereas both FeS₂ and S^o oxidation was still appreciable above this pH. f. Separation of pyrite and bacteria by means of a dialysis bag caused a pronounced drop of the oxidation rate which was similar to the reduction of pyrite oxidation by NEM; indirect oxidation of the sulphur moiety by Fe³⁺ was not affected by separation of pyrite and bacteria.Bacterial oxidation and utilization of the sulphur moiety of pyrite were relatively more important with increasing pH.     

An ultraviolet spectrophotometric method for the determination of pyrite and ferrous ion oxidation by Thiobacillus ferrooxidans

Summary An ultraviolet spectrophotometric method was used to monitor the formation of soluble ferric iron in acid culture solutions of Thiobacillus ferrooxidans. This methodology was demonstrated to be applicable for determining both pyrite and ferrous ion oxidation. Kinetic parameters of Fe²⁺ oxidation determined with the use of this method were in close agreement with those previously obtained by measurement of oxygen uptake rates.     

Role of a Ferric Ion-Reducing System in Sulfur Oxidation of Thiobacillus ferrooxidans

The properties of a ferric ion-reducing system which catalyzes the reduction of ferric ion with elemental sulfur was investigated with a pure strain of Thiobacillus ferrooxidans. In anaerobic conditions, washed intact cells of the strain reduced 6 mol of Fe³⁺ with 1 mol of elemental sulfur to give 6 mol of Fe²⁺, 1 mol of sulfate, and a small amount of sulfite. In aerobic conditions, the 6 mol of Fe²⁺ produced was immediately reoxidized by the iron oxidase of the cell, with a consumption of 1.5 mol of oxygen. As a result, Fe²⁺ production was never observed under aerobic conditions. However, in the presence of 5 mM cyanide, which completely inhibits the iron oxidase of the cell, an amount of Fe²⁺ production comparable to that formed under anaerobic conditions was observed under aerobic conditions. The ferric ion-reducing system had a pH optimum between 2.0 and 3.8, and the activity was completely destroyed by 10 min of incubation at 60°C. A short treatment of the strain with 0.5% phenol completely destroyed the ferric ion-reducing system of the cell. However, this treatment did not affect the iron oxidase of the cell. Since a concomitant complete loss of the activity of sulfur oxidation by molecular oxygen was observed in 0.5% phenol-treated cells, it was concluded that the ferric ion-reducing system plays an important role in the sulfur oxidation activity of this strain, and a new sulfur-oxidizing route is proposed for T. ferrooxidans.     

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.     

Incubation factor in the inhibition of Fe2+ oxidation inThiobacillus ferrooxidans by uranyl ions

Kinetic analysis of Fe²⁺ oxidation by a nongrowing suspension ofThiobacillus ferooxidans in the presence of UO₂ ²⁺ demonstrated both qualitative and quantitative changes in the pattern of UO₂ ²⁺ inhibition during several hours of incubation. After 14 h the sensitivity of Fe²⁺ oxidation to uranyl ions rose to the level of a growing culture. Dedicated to Dr. D. Halama on the occasion of his life jubilee.     

Rate Equations and Kinetic Parameters of the Reactions Involved in Pyrite Oxidation by Thiobacillus ferrooxidans

Rate equations and kinetic parameters were obtained for various reactions involved in the bacterial oxidation of pyrite. The rate constants were 3.5 μM Fe²⁺ per min per FeS₂ percent pulp density for the spontaneous pyrite dissolution, 10 μM Fe²⁺ per min per mM Fe³⁺ for the indirect leaching with Fe³⁺, 90 μM O₂ per min per mg of wet cells per ml for the Thiobacillus ferrooxidans oxidation of washed pyrite, and 250 μM O₂ per min per mg of wet cells per ml for the T. ferrooxidans oxidation of unwashed pyrite. The K_m values for pyrite concentration were similar and were 1.9, 2.5, and 2.75% pulp density for indirect leaching, washed pyrite oxidation by T. ferrooxidans, and unwashed pyrite oxidation by T. ferrooxidans, respectively. The last reaction was competitively inhibited by increasing concentrations of cells, with a K_i value of 0.13 mg of wet cells per ml. T. ferrooxidans cells also increased the rate of Fe²⁺ production from Fe³⁺ plus pyrite.     

Bovine serum albumin functionalized blue emitting Ti3C2 MXene quantum dots as a sensitive fluorescence probe for Fe3+ ion detection and its toxicity analysis

In the present work, an improved class of protein functionalized fluorescent 2D Ti₃C₂ MXene quantum dots (MXene QDs) was prepared using a hydrothermal method. Exfoliated 2D Ti₃C₂ sheets were used as the starting precursor and transport protein bovine serum albumin (BSA) was used to functionalize the MXene QDs. BSA-functionalized MXene QDs exhibited excellent photophysical property and stability at various physiological parameters. High-resolution transmission electron microscopy analysis showed that the BSA@MXene QDs were quasispherical in shape with a size of ~2 nm. The fluorescence intensity of BSA@MXene QDs was selectively quenched in the presence of Fe³⁺ ions. The mechanism of fluorescence quenching was further substantiated using time-resolved fluorescence and Stern–Volmer analysis. The sensing assay showed a linear response within the concentration range 0–150 μM of Fe³⁺ ions with excellent limit of detection. BSA@MXene QDs probe showed good selectivity toward ferric ions even in the presence of other potential interferences. The practical applicability of BSA@MXene QDs was further tested in real samples for Fe³⁺ ion quantification and the sensor had good recovery rates. The cytotoxicity studies of the BSA@MXene QDs toward the human glioblastoma cells revealed that BSA@MXene QDs are biocompatible at lower doses and showed significant cytotoxicity at higher dosages.     

Synthesis of an Iron-Oxidizing System during Growth of Thiobacillus ferrooxidans on Sulfur-Basal Salts Medium

It was found that the de novo synthesis of not only sulfur:ferric ion oxidoreductase (ferric ion-reducing system) but also iron oxidase was absolutely required when Thiobacillus ferrooxidans AP19-3 was grown on sulfur-salts medium. The results strongly suggest that iron oxidase is involved in sulfur oxidation. This bacterium could not grow on sulfur-salts medium under anaerobic conditions with Fe³⁺ as a terminal electron acceptor, suggesting that energy conservation by electron transfer between elemental sulfur and Fe³⁺ is not available for this bacterium.     

Application of an M13 bacteriophage displaying tyrosine on the surface for detection of Fe3+ and Fe2+ ions

Ferric and ferrous ion plays critical roles in bioprocesses,their influences in many fields have not been fully explored due to the lack of methods for quantification of ferric and ferrous ions in biological system or complex matrix.In this study,an M13 bacteriophage(phage) was engineered for use as a sensor for ferric and ferrous ions via the display of a tyrosine residue on the P8 coat protein.The interaction between the specific phenol group of tyrosine and Fe~(3+)./ Fe~(2+).was used as the sensor.Transmission electron microscopy showed aggregation of the tyrosine-displaying phages after incubation with Fe~(3+) and Fe~(2+).The aggregated phages infected the host bacterium inefficiently.This phenomenon could be utilized for detection of ferric and ferrous ions.For ferric ions,a calibration curve ranging from 200 nmol/L to 8 μmol/L with a detection limit of 58 nmol/L was acquired.For ferrous ions,a calibration curve ranging from 800 nmol/L to 8μmol/L with a detection limit of 641.7 nmol/L was acquired.The assay was specific for Fe~((3+)) and Fe~((2+)) when tested against Ni~(2+),Pb~(2+),Zn~(2+),Mn~(2+),Co~(2+),Ca~(2+),Cu~(2+),Cr~(3+),Ba~(2+),and K~+.The tyrosine displaying phage to Fe~(3+) and Fe~(2+) interaction would have plenty of room in application to biomatenals and bionanotechnology.     

Competitive Inhibition of Ferrous Iron Oxidation by Thiobacillus ferrooxidans by Increasing Concentrations of Cells

The oxidation of ferrous iron (Fe²⁺) to ferric iron (Fe³⁺) with dioxygen (O₂) by various strains of Thiobacillus ferrooxidans was studied by measuring the rate of O₂ consumption at various Fe²⁺ concentrations and cell concentrations. The apparent K_m values for Fe²⁺ remained constant at different cell concentrations of laboratory strains ATCC 13661 and ATCC 19859 but increased with increasing cell concentrations of mine isolates SM-4 and SM-5. The latter results are explained by the competitive inhibition of the Fe²⁺-binding site of a cell by other cells in the reaction mixture. Possible mechanisms involving cell surface properties are discussed.     

Sulfur Transformation in Microbially Mediated Pyrite Oxidation by Acidithiobacillus ferrooxidans: Insights From X-ray Photoelectron Spectroscopy-Based Quantitative Depth Profiling

The oxidation of pyrite and other sulfides is responsible for the generation of acid mine drainage and acid rock drainage, which leads to further contamination of soil and water. In these processes, microbial oxidation usually prevails over chemical oxidation. To determine the mechanism of microbial oxidation of pyrite, the interaction of Acidithiobacillus ferrooxidans with pyrite was comprehensively studied, and the sulfur transformation in the interaction was disclosed using X-ray photoelectron spectroscopy (XPS) depth profiling. Abundant bacterial cells attach to pyrite surface and form biofilms, which greatly enhances surface corrosion and results in two types of etching pits: bacteria-driven rod-shaped and chemically driven hexagonal etching pits. The details of XPS depth profiles on a reacted pyrite surface reveal that the surface sulfur was first oxidized into elemental sulfur. Thereafter, elemental sulfur was further oxidized to intermediate species S₂O₃^2?, SO₃^2?, and ultimately to SO₄^2?. The oxidation sequence of sulfur is S₂^2?/S^2?→S_n^2?, S⁰→SO₃^2?, and S₂O₃^2?→SO₄^2?. Meanwhile, the remnant ferrous iron in the surface layer was released into solution and subsequently oxidized into Fe³⁺ by A. ferrooxidans and dissolved oxygen, which in turn enhanced the oxidation of sulfur. Fe³⁺, sulfate, and other ions (e.g., K⁺, Na⁺, NH₄⁺) in the solution precipitated as jarosite, hydroniumjarosite, and ammoniojarosite. On the basis of results, a three-staged model is proposed to interpret the kinetics of microbial oxidation of pyrite.     

Kinetics of Iron Oxidation by Thiobacillus ferrooxidans

A statistical relationship between the rate of ferric ion production by a strain of Thiobacillus ferrooxidans and various levels of cell concentration, Fe²⁺ concentration, Na⁺ concentration, and temperature was studied by a direct colorimetric method at 304 nm. The relationship was linear (90 to 93%), cross-product (3 to 4%), and quadratic (1 to 2%). The levels of cell concentration and Fe²⁺ concentration and their respective interactions with one another and the other factors had the most significant effects on the regression models. The solution of the quadratic response surface for optimum oxidation was a saddle point, and the predicted critical levels of temperature, cell concentration, Fe²⁺ concentration, and Na⁺ concentration ranged between −6 and 2°C, 0.43 and 0.62 mg/ml, 72 and 233 mM, and 29.6 mM, respectively.     

Uptake and degradation of EDTA by Escherichia coli

It was found that Escherichia coli exhibited a growth by utilization of Fe(III)EDTA as a sole nitrogen source. No significant growth was detected when Fe(III)EDTA was replaced by EDTA complexes with other metal ions such as Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, and Zn²⁺. When EDTA uptake was measured in the presence of various ions, it was remarkable only when Fe³⁺ was present. The cell extract of E. coli exhibited a significant degradation of EDTA only in the presence of Fe³⁺. It is likely that the capability of E. coli for the growth by utilization of Fe(III)EDTA results from the Fe³⁺-dependent uptake and degradation of EDTA.     

Effect of oxyanions of sulfur onThiobacillus ferrooxidans: Ferrous ion oxidation,oxygen uptake,and cytochrome reduction

Ferrous ion oxidation byThiobacillus ferrooxidans was completely inhibited by 10 mM each of thiosulfate, sulfite, metabisulfite, bisulfite, and tetrathionate. The inhibition was enhanced in a low pH medium (pH 1.5 versus pH 2.5). Oxygen uptake measurements with Fe²⁺ as the electron donor confirmed the toxicity of thiosulfate, but also indicated its dependency on the concentration of Fe²⁺. Cytochrome spectra of intact cells ofT. ferrooxidans showed that metabisulfite, and thiosulfate to a lesser extent, directly reduced electron transport components, in contrast to no direct reduction of cytochromes by tetrathionate and sulfite.     

Heme-mediated binding of α-casein to ferritin: evidence for preferential α-casein binding to ferrous iron

Bovine milk α-casein was identified as a ferritin-binding protein, and ferritin is known to be a heme-binding protein. In this study, we found that the binding of α-casein to bovine spleen ferritin in vitro was blocked by hemin, but not by iron-free hemin (protoporphyrin IX) or zinc-protoporphyrin IX, suggesting that the presence of iron in heme play a key role in this interaction. Indeed, the binding of α-casein to ferritin and biotinylated hemin was inhibited by adding excess ferrous ammonium sulfate (FAS). To further elucidate the binding mechanism of α-casein to biotinylated hemin, Ferrozine and nitrilotriacetic acid (NTA) were used as ferrous and ferric iron chelators, respectively. FAS-mediated inhibition of α-casein to biotinylated hemin was neutralized with Ferrozine, but not NTA, while FAS- as well as ferric chloride-mediated inhibition in their interaction was neutralized by NTA. The following ions also inhibited α-casein-biotinylated hemin binding in order of potency of inhibition: FAS (Fe²⁺) ≪ ferric chloride (Fe³⁺) < copper sulfate (Cu²⁺) < zinc sulfate (Zn²⁺) < manganese chloride (Mn²⁺) < calcium chloride (Ca²⁺) < magnesium sulfate (Mg²⁺). These results suggests that the binding of α-casein to ferritin is heme-mediated through direct binding of α-casein to iron in the heme on the surface of ferritin molecule, and that α-casein preferentially binds Fe²⁺ compared with any other metal ions, including Fe³⁺.     

Protein Scission by Metal Ion–Ascorbate System

About 14 proteins were tested for specific oxidative scission catalyzed by metal ions in the presence of ascorbate and oxidizing agents (O₂ or hydrogen peroxide). Only four of them were degraded by Fe³⁺/Fe²⁺- ascorbate, twelve – by Cu²⁺/Cu⁺-ascorbate and two proteins (α- and β-caseins) were degraded by Pd²⁺ ions. The rate and the intensity of degradation are very different for various proteins. For the most of tested proteins only a small fraction of molecules was degraded. None of them was degraded completely. Two possible reasons of protein stability against oxidative degradation may be proposed as follows: either there is no metal binding site in a protein molecule, or metal binding ligands of protein undergo a rapid oxidative modification and the metal ion is released from the binding site. Human growth hormone was cut specifically at two sites by Cu²⁺/Cu⁺-ascorbate system. At least one of amino acid residues of this protein was modified by formation of reactive carbonyl.     

Fast Kinetics of Fe2+ Oxidation in Packed-Bed Reactors

Thiobacillus ferrooxidans was used in fixed-film bioreactors to oxidize ferrous sulfate to ferric sulfate. Glass beads, ion-exchange resin, and activated-carbon particles were tested as support matrix materials. Activated carbon was tested in both a packed-bed bioreactor and a fluidized-bed bioreactor; the other matrix materials were used in packed-bed reactors. Activated carbon displayed the most suitable characteristics for use as a support matrix of T. ferrooxidans fixed-film formation. The reactors were operated within a pH range of 1.35 to 1.5, which effectively reduced the amount of ferric iron precipitation and eliminated diffusion control of mass transfer due to precipitation. The activated-carbon packed-bed reactor displayed the most favorable biomass holdup and kinetic performance related to ferrous sulfate oxidation. The fastest kinetic performance achieved with the activated-carbon packed-bed bioreactor was 78 g of Fe²⁺ oxidized per liter per h (1,400 mmol of Fe²⁺ oxidized per liter per h) at a true dilution rate of 40/h, which represents a hydraulic retention time of 1.5 min.     

Iron Specifically Protects Corn Protoplasts from T-Toxin of Cochliobolus heterostrophus

Ferric ion reduced the damaging effects of T-toxin, a series of linear β-polyketols produced by the pathogenic fungus Cochliobolus heterostrophus, on leaf mesophyll protoplasts from susceptible T-cytoplasm corn. Of nine metals tested, only ferric and ferrous ions had this effect. Despite the presence of 12 available oxygen atoms in each T-toxin molecule, there was no evidence for the formation of an aqueous Fe²⁺- or Fe³⁺-T-toxin complex. The protective effect of iron was eliminated by a molar excess of EDTA. Iron had no effect on the sensitivity of T-cytoplasm mitochondria to T-toxin, even at a 1000-fold molar excess, nor did it protect roots of T-cytoplasm corn seedlings from inhibition by T-toxin. The mechanism by which iron specifically protects protoplasts from T-toxin is not understood, but time lapse experiments suggest that iron acts on some intracellular site to modify T-toxin sensitivity and not on a transport system at the cell surface.     

In vitro antioxidant activity of silymarin

Silymarin, a known standardized extract obtained from seeds of Silybum marianum is widely used in treatment of several diseases of varying origin. In the present paper, we clarified the antioxidant activity of silymarin by employing various in vitro antioxidant assay such as 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH·) scavenging, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, total antioxidant activity determination by ferric thiocyanate, total reducing ability determination by Fe³⁺ ? Fe²⁺ transformation method and Cuprac assay, superoxide anion radical scavenging by riboflavin/methionine/illuminate system, hydrogen peroxide scavenging and ferrous ions (Fe²⁺) chelating activities. Silymarin inhibited 82.7% lipid peroxidation of linoleic acid emulsion at 30 μg/mL concentration; butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol and trolox indicated inhibition of 83.3, 82.1, 68.1 and 81.3% on peroxidation of linoleic acid emulsion at the same concentration, respectively. In addition, silymarin had an effective DPPH· scavenging, ABTS^√+ scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, ferric ions (Fe³⁺) reducing power by Fe³⁺ ? Fe²⁺ transformation, cupric ions (Cu²⁺) reducing ability by Cuprac method, and ferrous ions (Fe²⁺) chelating activities. Also, BHA, BHT, α-tocopherol and trolox, were used as the reference antioxidant and radical scavenger compounds. Moreover, this study, which clarifies antioxidant mechanism of silymarin, brings new information on the antioxidant properties of silymarin. According to the present study, silymarin had effective in vitro antioxidant and radical scavenging activity. It could be used in the pharmacological and food industry because of its antioxidant properties.     

Evaluation of Bacterial Community Structure and Its Influence on Sulfide Oxidation in a Bio-Leaching Environment

The mechanism of sulfide oxidation by adhering bacteria (direct oxidation mechanism) and by ferric ion in the aqueous phase was studied by quantitative assessment of bacterial activity on the sulfide surface. To probe for the principal bacterial species on the surface and in the supernatant, a library of DNA genes encoding portions of bacterial 16S rRNA was constructed. The PCR-amplified DNA from the bacterial populations was cloned employing PROMEGA's pGEM-T Easy Vector system. The clone frequency indicated that iron-oxidizing bacteria were dominant in the liquid phase, while Acidithiobacillus ferroixdans, which is both sulfur and iron oxidizer was the most prevalent on the sulfide surface. Samples of crystalline pyrite were exposed to the bacterial consortium to evaluate surface alterations caused by bacteria. Chemical (abiotic) oxidation experiments with ferric ion as the oxidant were carried out in parallel with the biological oxidation tests. Changes in the surface topography were monitored by atomic force microscopy (AFM) while changes in surface chemistry were examined by Raman spectroscopy. Bacterial attachment resulted in a 53% increase in the specific surface area in comparison to a 13% increase caused by chemical (ferric ion) oxidation. The oxidation rate was assessed by evaluating the iron release. After corrections for surface area changes, the specific abiotic (oxidation by Fe^{3 +}) and biotic oxidation rates with adhering bacteria were nearly the same (2.6 × 10^{? 9} mol O₂/s/m² versus 3.3 × 10^{? 9} mol O₂/s/m²) at pH = 2 and a temperature of 25°C. The equality of rates implies that the availability of ferric ion as the oxidant is rate limiting.