Microbial desulfurization of coal and oxidation of pure pyrite byThiobacillus ferrooxidans andAcidianus brierleyi

Summary Thiobacillus ferrooxidans andAcidianus brierleyi were capable of oxidizing pure pyrite as well as oxidizing sulfur in coal. First order reactions were assumed in the kinetic analysis performed. For oxidation of pure pyrite the rate constant was higher forA. brierleyi than forT. ferrooxidans. For sulfur removal from coal the values of the rate constants were comparable for the two microorganisms.     

Microbial acceleration of aerobic pyrite oxidation at circumneutral pH

Pyrite (FeS₂) is the most abundant sulfide mineral on Earth and represents a significant reservoir of reduced iron and sulfur both today and in the geologic past. In modern environments, oxidative transformations of pyrite and other metal sulfides play a key role in terrestrial element partitioning with broad impacts to contaminant mobility and the formation of acid mine drainage systems. Although the role of aerobic micro‐organisms in pyrite oxidation under acidic‐pH conditions is well known, to date there is very little known about the capacity for aerobic micro‐organisms to oxidize pyrite at circumneutral pH. Here, we describe two enrichment cultures, obtained from pyrite‐bearing subsurface sediments, that were capable of sustained cell growth linked to pyrite oxidation and sulfate generation at neutral pH. The cultures were dominated by two Rhizobiales species (Bradyrhizobium sp. and Mesorhizobium sp.) and a Ralstonia species. Shotgun metagenomic sequencing and genome reconstruction indicated the presence of Fe and S oxidation pathways in these organisms, and the presence of a complete Calvin–Benson–Bassham CO₂ fixation system in the Bradyrhizobium sp. Oxidation of pyrite resulted in thin (30–50 nm) coatings of amorphous Fe(III) oxide on the pyrite surface, with no other secondary Fe or S phases detected by electron microscopy or X‐ray absorption spectroscopy. Rates of microbial pyrite oxidation were approximately one order of magnitude higher than abiotic rates. These results demonstrate the ability of aerobic microbial activity to accelerate pyrite oxidation and expand the potential contribution of micro‐organisms to continental sulfide mineral weathering around the time of the Great Oxidation Event to include neutral‐pH environments. In addition, our findings have direct implications for the geochemistry of modern sedimentary environments, including stimulation of the early stages of acid mine drainage formation and mobilization of pyrite‐associated metals.     

Sulfur colloids as temporary energy reservoirs for Thiobacillus ferrooxidans during pyrite oxidation

Thiobacillus ferrooxidans was cultivated on 100-nm-thick synthetic pyrite (FeS₂) films. The steps of biooxidation were studied with high-resolution transmission electron microscopy. The crystallized sulfide was transformed into colloidal sulfur (4–70 nm, depending on the age of the cell and the degree of substrate oxidation; 70nm initially and 4nm after oxidation of the pyrite substrate), which was taken up and distributed over an organic capsule around the bacteria. This colloidal sulfur acted as intermediate energy storage and was transferred by contact to daughter cells not directly attached to the sulfide substrate.     

Competitive adsorption of binary mixture of <Emphasis Type="Italic">Leptospirillum ferriphilum</Emphasis> and <Emphasis Type="Italic">Acidithiobacillus caldus</Emphasis> onto pyrite

Leptospirillum ferriphilum and Acidithiobacillus caldus are two important acidophilic microorganisms involved in iron and sulfur oxidation during bioleaching. Cell adsorption to mineral surfaces is important for the direct leaching or contact leaching of minerals. In this study, we report the competitive adsorption of binary mixtures of L. ferriphilum LF-104 and A. caldus MTH-04 onto pyrite surfaces. The Langmuir adsorption parameter (C_Am) indicated that these two bacteria underwent competitive adsorption to pyrite. Real-time quantitive PCR was used to quantify the relative amounts of L. ferriphilum and A. caldus adsorbed onto the surfaces of pyrite following exposure to a mixture of these two organisms. The adsorption of L. ferriphilum was not affected by A. caldus. However, adsorption of A. caldus was greatly affected by the presence of L. ferriphilum. Zeta-potential measurements and FT-IR spectroscopic studies showed that L. ferriphilum had a higher electrostatic attraction towards pyrite when compared to A. caldus. Based on the above results, we propose a competitive adsorption model to explain the mechanism by which L. ferriphilum and A. caldus compete in their adsorption to pyrite, although L. ferriphilum dominated in the competitive adsorption process. This work provides a better understanding of the adsorption behavior of mixed microbial populations onto mineral surfaces.     

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.     

Patterns of pyrite oxidation by different microorganisms

The bacterial-chemical oxidation of natural pyrites with different physical, chemical, and electrophysical characteristics by bacteria Acidithiobacillus ferrooxidans, Sulfobacillus thermotolerans, and the archaeon Ferroplasma acidiphilum were studied. The electrophysical characteristics of three natural pyrites differed in the K _thermoEMF value (pyrites 3, 4, hole conduction (p-type conductivity); pyrite 5, mixed type conductivity (n-p)) and in the logarithm of electric resistance. Chemical oxidation of pyrites 3 and 5 resulted in no changes of K _thermoEMF. When pyrite 4 was oxidized chemically, the K _thermoEMF values remained in the same range as in the initial sample, but the ratio of grains with different K _thermoEMF values in the sample was changed: the number of grains with a higher K _thermoEMF value increased. The same changes were also observed in the course of bacterio-chemical oxidation of pyrite 4. Of the three pyrites studied, an increase in the logarithm of resistance was observed only for chemical oxidation of pyrite 4 at 28°C. At higher experimental temperatures, the logarithm of resistance increased accordingly; more active bacterial-chemical oxidation resulted in a more pronounced increase in the logarithm of resistance than chemical oxidation. On bacterial-chemical oxidation of pyrites 3 and 5 by A. ferrooxidans and S. thermotolerans strains, iron was leached more actively than sulfur. Preferred bacterial-chemical oxidation of certain fractions from the pyrite samples was shown, namely of the pyrite 3 fraction with higher K _thermoEMF values by the F. acidiphilum strain and of a fraction from the pyrite 5 sample with medium K _thermoEMF values by the A. ferrooxidans and S. thermotolerans strains. The comparative assessment of bacterial-chemical pyrite oxidation by three types of microorganisms showed the direction of changes in the K _thermoEMF values to be the same in the case of bacteria Acidithiobacillus ferrooxidans and Sulfobacillus thermotolerans and different in the case of the archaeon Ferroplasma acidiphilum.     

Intermediary sulfur compounds in pyrite oxidation: implications for bioleaching and biodepyritization of coal

Accumulation of elemental sulfur during pyrite oxidation lowers the efficiency of coal desulfurization and bioleaching. In the case of pyrite bioleaching by Leptospirillum ferrooxidans, an iron(II)-ion-oxidizing organism without sulfur-oxidizing capacity, from the pyritic sulfur moiety about 10% elemental sulfur, 2% pentathionate, and 1% tetrathionate accumulated by a recently described cyclic pyrite oxidation mechanism. In the case of pure cultures of Thiobacillus ferrooxidans and mixed cultures of L. ferrooxidans and T. thiooxidans, pyrite was nearly completely oxidized to sulfate because of the capacity of these cultures to oxidize both iron(II) ions and sulfur compounds. Pyrite oxidation in acidic solutions, mediated chemically by iron(III) ion, resulted in an accumulation of similar amounts of sulfur compounds as obtained with L. ferrooxidans. Changes of pH to values below 2 or in the iron ion concentration are not decisive for diverting the flux of sulfur compounds. The literature on pyrite bioleaching is in agreement with the findings indicating that the chemistry of direct and indirect pyrite leaching is identical. Received: 20 April 1998 / Received revision: 27 August 1998 / Accepted: 3 September 1998     

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.     

Influence of the sulfur species reactivity on biofilm conformation during pyrite colonization by Acidithiobacillus thiooxidans

Massive pyrite (FeS₂) electrodes were potentiostatically modified by means of variable oxidation pulse to induce formation of diverse surface sulfur species (S _n ^2?, S⁰). The evolution of reactivity of the resulting surfaces considers transition from passive (e.g., Fe_1?x S₂) to active sulfur species (e.g., Fe_1?x S_2?y , S⁰). Selected modified pyrite surfaces were incubated with cells of sulfur-oxidizing Acidithiobacillus thiooxidans for 24 h in a specific culture medium (pH 2). Abiotic control experiments were also performed to compare chemical and biological oxidation. After incubation, the attached cells density and their exopolysaccharides were analyzed by confocal laser scanning microscopy (CLMS) and atomic force microscopy (AFM) on bio-oxidized surfaces; additionally, S _n ^2?/S⁰ speciation was carried out on bio-oxidized and abiotic pyrite surfaces using Raman spectroscopy. Our results indicate an important correlation between the evolution of S _n ^2?/S⁰ surface species ratio and biofilm formation. Hence, pyrite surfaces with mainly passive-sulfur species were less colonized by A. thiooxidans as compared to surfaces with active sulfur species. These results provide knowledge that may contribute to establishing interfacial conditions that enhance or delay metal sulfide (MS) dissolution, as a function of the biofilm formed by sulfur-oxidizing bacteria.     

Contribution to the chemical and bacteriological oxidation of pyrite in soil

Summary 1. The rate of pyrite oxidation and the influence of microbes on this process were studied, since this oxidation proved to be one of the most important phenomena in the process of weathering and aging of newly reclaimed marine soils.2. Pyrite oxidation does not depend on the activity of microbes, but is significantly stimulated by the metabolism of the specific sulphur-oxidising bacteria. The most important species proved to beThiobacillus thiooxidans, the same organism that takes part in the oxidation of monosulphides, thiosulphates, etc.3. Factors depressing the solubility of iron (high pH level or high concentration of phosphate ions) retard the oxidation of pyrite.4. It is probable that the oxidative disintegration of pyrite starts with the oxidation of the ferrous ions on the surface of the crystals, releasing the sulphur. The latter is then oxidised primarily by the specific sulphur-oxidising microbes and transformed to sulphates.     

Bacterial dissolution of pyrite by Thiobacillus ferrooxidans

The kinetics of the dissolution of pure pyrite (FeS₂) particles by Thiobacillus ferrooxidans were studied both theoretically and experimentally. Adsorption and dissolution experiments were carried out at 30 °C and pH=2, by using a batch reactor. The adsorption process of T. ferrooxidans to pyrite surface was rapid in comparison with the bacterial dissolution process. The experimental results for the adsorption equilibrium were well correlated by the Langmuir type isotherm. The growth rate of adsorbed bacteria was found to be proportional to the product of the number of adsorbed cells and the fraction of solid surface unoccupied by cells. A new kinetic model for the bacterial dissolution was presented, and shown to correlate well with the experimental data for the rate of bacterial dissolution and for the time variation in the number of cells in the liquid phase. The specific growth rate of adsorbed bacteria was also evaluated.List of Symbols f weight fraction of iron in pyrite - K _A m³/cells equilibrium constant for cell adsorption - R _A cells/d m³-mixture growth rate of bacteria adsorbed on solid surface - R _L cells/d m³-mixture growth rate of free bacteria in the liquid phase - t d time - V m³ volume of solid-liquid mixture - W kg weight of pyrite - W ₀ kg initial weight of pyrite - X _A cells/kg-solid number of adsorbed cells on solid surface - X _Am cells/kg-solid maximum adsorption capacity - X _L cells/m³-liquid number of free cells existing in the liquid phase - X _T cells/m³-mixture total number of cells - X _TO cells/m³ initial total number of cells - Y _A cells/kg-FeS₂ growth yield of adsorbed bacteria - Y _L cells/kg-Fe²⁺ growth yield of free bacteria - [Fe] _T kg/m³-liquid concentration of total iron in the liquid phase - fraction of pyrite dissolved - _V fraction of adsorption sites unoccupied by cells - _A d^–1 specific growth rate of adsorbed bacteria - _L d^–1 specific growth rate of free bacteria - volume fraction of solid particles in solid-liquid mixture     

Autotrophic carbon dioxide fixation inAcidianus brierleyi

The autotrophic CO₂ fixation pathway inAcidianus brierleyi, a facultatively anaerobic thermoacidophilic archaebacterium, was investigated by measuring enzymatic activities from autotrophic, mixotrophic, and heterotrophic cultures. Contrary to the published report that the reductive tricarboxylic acid cycle operates inA. brierleyi, the enzymatic activity of ATP:citrate lyase, the key enzyme of the cycle, was not detected. Instead, activities of acetyl-CoA carboxylase and propionyl-CoA carboxylase, key enzymes of the 3-hydroxypropionate cycle, were detected only whenA. brierleyi was growing autotrophically. We conclude that a modified 3-hydroxypropionate pathway operates inA. brierleyi.Abbreviations TCA tricarboxylic acid - BV Benzyl viologen     

A direct observation of bacterial coverage and biofilm formation by Acidithiobacillus ferrooxidans on chalcopyrite and pyrite surfaces

To obtain a fundamental understanding of the population behaviour of Acidithiobacillus ferrooxidans at chalcopyrite and pyrite surfaces, the early stage attachment behaviour and biofilm formation by this bacterium on chalcopyrite (CuFeS₂) and pyrite (FeS₂) were studied by optical microscopy, Raman spectroscopy, time-of-flight secondary ion mass spectrometry (ToF-SIMS) and electron backscatter diffraction (EBSD). The results indicate there was no significant difference in selectivity of bacterial attachment between chalcopyrite and pyrite. However, the result of ToF-SIMS analysis suggests that the surface of the pyrite was covered more extensively by biofilm than that of the chalcopyrite, which may indicate more extracellular polymeric substances (EPS) formation by bacterial cells growing on pyrite. EBSD and optical image analysis indicated that selectivity of bacterial attachment to chalcopyrite was not significantly affected by crystal orientation. The results also suggest that the bacterial population in defective areas of chalcopyrite was significantly higher than on the polished surfaces.     

Morphology of bacterial leaching patterns by Thiobacillus ferrooxidans on synthetic pyrite

Thiobacillus ferrooxidans has been cultivated on synthetic pyrite (FeS₂) single crystals as the only energy source and the pyrite interface investigated with respect to characteristic morphological changes using scanning electron microscopy. Corrosion patterns of bacterial size were identified in different stages of development and correlated with bacterial activity. It appears that bacterial attack of the sulfide interface starts by secretion of organic substances around the contact area between the bacterial cell and the sulfide energy source. They might either be part of a pseudo capsule which shields the contact area or may form a sulfur absorbing and transporting organic film. Degradation of the sulfide occurs in the contact area below the bacterial cell leading to a corrosion pit which the bacterium may abandon after it has reached a depth of bacterial dimension. Electron spectroscopic (XPS) and X-ray fluorescence studies indicate a layer of organic substances covering the sulfide surface under bacterial leaching conditions, which is sufficiently thick for consideration in interfacial chemical mechanisms.     

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.     

Bioleaching of pyrite by acidophilic thermophile Acidianus brierleyi

The kinetics of bioleaching of pyrite (FeS(2)) by the acidophilic thermophilic bacterium Acidianus brierleyi was studied in a well-mixed batch reactor. Experiments were done at 65 degrees C and pH 1.5 on adsorption of A. brierleyi onto pyrite particles, liquid-phase oxidation of ferrous iron by A. brierleyi, and microbial leaching of pyrite. The adsorption of A. brierleyi was a fast process; equilibrium was attained within the first 30 min of exposure to pyrite. The adsorption equilibrium data were well correlated with the Langmuir isotherm. The oxidation of ferrous iron was markedly accelerated in the presence of A. brierleyi, and the growth yield on ferrous iron was determined. The bioleaching of pyrite by A. brierleyi was found to take place with a direct attack by adsorbed cells on the surface of pyrite, the chemical leaching of pyrite by ferric iron being insignificant. Rate data collected under a wide variety of operating variables were analyzed to determine kinetic and stoichiometric parameters for the microbial pyrite leaching. The specific growth rate on pyrite for A. brierleyi was about four times that for the mesophilic bacterium, Thiobacillus ferrooxidans, whereas the growth yields on pyrite for the two microbes were approximately equal to one another in magnitude. A comparison of A. brierleyi with T. ferrooxidans for pyrite leachability demonstrated the thermophile to be much more effective. (c) 1995 John Wiley & Sons, Inc.     

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.     

Evolution of biofilms during the colonization process of pyrite by Acidithiobacillus thiooxidans

We have applied epifluorescence principles, atomic force microscopy, and Raman studies to the analysis of the colonization process of pyrite (FeS₂) by sulfuroxidizing bacteria Acidithiobacillus thiooxidans after 1, 15, 24, and 72 h. For the stages examined, we present results comprising the evolution of biofilms, speciation of S_n²⁻/S⁰ species, adhesion forces of attached cells, production and secretion of extracellular polymeric substances (EPS), and its biochemical composition. After 1 h, highly dispersed attached cells in the surface of the mineral were observed. The results suggest initial non-covalent, weak interactions (e.g., van der Waal’s, hydrophobic interactions), mediating an irreversible binding mechanism to electrooxidized massive pyrite electrode (eMPE), wherein the initial production of EPS by individual cells is determinant. The mineral surface reached its maximum cell cover between 15 to 24 h. Longer biooxidation times resulted in the progressive biofilm reduction on the mineral surface. Quantification of attached cell adhesion forces indicated a strong initial mechanism (8.4 nN), whereas subsequent stages of mineral colonization indicated stability of biofilms and of the adhesion force to an average of 4.2 nN. A variable EPS (polysaccharides, lipids, and proteins) secretion at all stages was found; thus, different architectural conformation of the biofilms was observed during 120 h. The main EPS produced were lipopolysaccharides which may increase the hydrophobicity of A. thiooxidans biofilms. The highest amount of lipopolysaccharides occurred between 15–72 h. In contrast with abiotic surfaces, the progressive depletion of S_n²⁻/S⁰ was observed on biotic eMPE surfaces, indicating consumption of surface sulfur species. All observations indicated a dynamic biooxidation mechanism of pyrite by A. thiooxidans, where the biofilms stability and composition seems to occur independently from surface sulfur species depletion.