Mineral and iron oxidation at low temperatures by pure and mixed cultures of acidophilic microorganisms

An enrichment culture from a boreal sulfide mine environment containing a low-grade polymetallic ore was tested in column bioreactors for simulation of low temperature heap leaching. PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing revealed the enrichment culture contained an Acidithiobacillus ferrooxidans strain with high 16S rRNA gene similarity to the psychrotolerant strain SS3 and a mesophilic Leptospirillum ferrooxidans strain. As the mixed culture contained a strain that was within a clade with SS3, we used the SS3 pure culture to compare leaching rates with the At. ferrooxidans type strain in stirred tank reactors for mineral sulfide dissolution at various temperatures. The psychrotolerant strain SS3 catalyzed pyrite, pyrite/arsenopyrite, and chalcopyrite concentrate leaching. The rates were lower at 5 degrees C than at 30 degrees C, despite that all the available iron was in the oxidized form in the presence of At. ferrooxidans SS3. This suggests that although efficient At. ferrooxidans SS3 mediated biological oxidation of ferrous iron occurred, chemical oxidation of the sulfide minerals by ferric iron was rate limiting. In the column reactors, the leaching rates were much less affected by low temperatures than in the stirred tank reactors. A factor for the relatively high rates of mineral oxidation at 7 degrees C is that ferric iron remained in the soluble phase whereas, at 21 degrees C the ferric iron precipitated. Temperature gradient analysis of ferrous iron oxidation by this enrichment culture demonstrated two temperature optima for ferrous iron oxidation and that the mixed culture was capable of ferrous iron oxidation at 5 degrees C.     

Relative contributions of biological and chemical reactions to the overall rate of pyrite oxidation at temperatures between 30 degrees C and 70 degrees C

The stoichiometry and kinetics of the spontaneous, chemical reaction between pyrite and ferric iron was studied at 30, 45, and 70 degrees C in shake flasks at pH 1.5 by monitoring the ferrous iron, total iron, elemental sulfur, and sulfate concentration profiles in time. It was found that the sulfur moiety of pyrite was oxidized completely to sulfate. Elemental sulfur was not produced in detectable amounts. The iron moiety of pyrite was released as ferrous iron. All observed initial reaction rates could be fitted into an empirical equation. This equation includes the concentrations of ferric iron and pyrite, and a constant which is dependent on the temperature and the nature of the main anion present. It was observed that ferrous iron formed during the reaction slowed down the oxidation of pyrite by ferric iron. The extent of this effect decreased with increasing temperature. With the aid of the empirical equation, the contribution of the chemical oxidation of pyrite by ferric iron to the overall oxidation in a hypothetical plug-flow reactor, in which biologically mediated oxdidation of pyrite and ferrous iron by oxygen also takes place, can be assessed. At 30, 45, and 70 degrees C, respectively, 2, 8-17, and 43% of the pyrite was oxidized chemically by ferric iron. Therefore, it is expected that only in reactors operating at high temperatures with extremely thermophilic bacteria, will chemical oxidation cause a significant deviation from the apparent first order overall kinetics of biological pyrite oxidation.     

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.     

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.     

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.     

Characterization of arsenic resistant and arsenopyrite oxidizing Acidithiobacillus ferrooxidans from Hutti gold leachate and effluents

Four arsenic resistant ferrous oxidizers were isolated from Hutti Gold Mine Ltd. (HGML) samples. Characterization of these isolates was done using conventional microbiological, biochemical and molecular methods. The ferrous oxidation rates with these isolates were 16, 48, 34 and 34 mg L(-1)h(-1) and 15, 47, 34 and 32 mg L(-1)h(-1) in absence and presence of 20 mM of arsenite (As3+) respectively. Except isolate HGM 8, other three isolates showed 2.9-6.3% inhibition due to the presence of 20 mM arsenite. Isolate HGM 8 was able to grow in presence of 14.7 g L(-1) of arsenite, with 25.77 mg L(-1)h(-1) ferrous oxidation rate. All the four isolates were able to oxidize iron and arsenopyrite from 20 g L(-1) and 40 g L(-1) refractory gold ore and 20 g L(-1) refractory gold concentrate. Once the growth was established pH adjustment was not needed inspite of ferrous oxidation, which could be due to concurrent oxidation of pyrite. Isolate HGM 8 showed the final cell count of as high as 1.12 x 10(8) cells mL(-1) in 40 g L(-1) refractory gold ore. The isolates were grouped into one haplotypes by amplified ribosomal DNA restriction analysis (ARDRA). The phylogenetic position of HGM 8 was determined by 16S rDNA sequencing. It was identified as Acidithiobacillus ferrooxidans and strain name was given as SRHGM 1.     

Inhibitory effects of particulate materials in growing cultures of Thiobacillus ferrooxidans

Particulate materials (flowers of sulfur, fluorapatite, glass beads, and pyrite) inhibited the growth of Thiobacillus ferrooxidans on ferrous iron at pH 1.5. The degree of inhabitation varied with the type of the particulate and culture conditions. The inhibition caused by pyrite involved a soluble toxic component. The inhibition caused by sulfur particles and glass beads was alleviated by incubation under static culture conditions (i.e., no shaking) but oxidation rates declined because of lack of aeration an increase in the pH above 2 also relieved the inhibition. The nature of inhibition. The nature of inhibition by different particles varied from declined iron oxidation rates to prolonged lag periods depending on the concentration of the test material. Thus, the results indicate that the bacterial activity at the liquid-solid interface is different from that in the bulk liquid phase and is influenced by the proton concentration and by the physiochemical properties of the solids.     

Solubilization and speciation of iron during pyrite oxidation by Thiobacillus ferrooxidans

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

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

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

Heterotrophic bacteria from cultures of autotrophic Thiobacillus ferrooxidans: relationships as studied by means of deoxyribonucleic acid homology.

From several presumably pure cultures of Thiobacillus ferrooxidans, we isolated a pair of stable phenotypes. One was a strict autotroph utilizing sulfur or ferrous iron as the energy source and unable to utilize glucose; the other phenotype was an acidophilic obligate heterotroph capable of utilizing glucose but not sulfur or ferrous iron. The acidophilic obligate heterotroph not only was encountered in cultures of T. ferrooxidans, but also was isolated with glucose-mineral salts medium, pH 2.0, directly from coal refuse. By means of deoxyribonucleic acid homology, we have demonstrated that the acidophilic heterotrophs are of a different genotype from T. ferrooxidans, not closely related to this species; we have shown also that the acidophilic obligate heterotrophs, regardless of their source of isolation, are related to each other. Therefore, cultures of T. ferrooxidans reported capable of utilizing organic compounds should be carefully examined for contamination. The acidophilic heterotrophs isolated by us are different from T. acidophilis, which is also associated with T. ferrooxidans but is facultative, utilizing both glucose and elemental sulfur as energy sources. Since they are so common and tenacious in T. ferrooxidans cultures, the heterotrophs must be associated with T. ferrooxidans in the natural habitat.     

Identification and characterization of four strains of Acidithiobacillus ferrooxidans isolated from different sites in China

Four strains of Acidithiobacillus ferrooxidans (A. ferrooxidans), AF1, AF2, AF3 and AFc, were isolated from samples with different geological sources using a 9K medium. These four isolates were identified as A. ferrooxidans by phenotypic and 16S rDNA sequence analyses. All four isolates were able to use ferrous ion (Fe(2+)), elemental sulfur (S0) or pyrite as a sole energy source, but they showed differences in pH optima and range of activity, optimum temperature of activity, resistance to chloride (KCl) and heavy metal ions, and oxidation rates of Fe(2+), S0 and pyrite. AF3 was the most active strain when using Fe(2+) as the energy source, while AFc grew best using pyrite as the energy source. AF2 appeared to differ from the other three strains in substrate utilization, as it oxidizes S0 and pyrite more effectively than Fe(2+). RAPD analysis of genomic DNA from these isolates showed that banding profiles of their genomic DNA exhibited some differences, and the genomic banding profile of AF2 was significantly different from that of others. To obtain an insight into the molecular biology of the process of the energy production of these strains, several genes involved in the iron respiratory chain were cloned and sequenced, including Fe(2+) oxidase (iro), rusticyanin (rus) and subunit III of aa3-type cytochrome oxidase (cox C) genes. The results revealed that the iro gene can be cloned from all of the four strains and the nucleotide sequences were shown to be completely identical in each. However, rus and coxC genes could be amplified only from AF1, AF3 and AFc, not from AF2. These results suggested that the phenotypic differences of the four strains of A. ferrooxidans from different sites correlated with their genetic polymorphism, which may result from the different environments in which they lived, and that the strain AF2 was phenotypically and genetically significantly different from the other three strains.     

Characteristics of a moderately thermophilic and acidophilic iron-oxidizing Thiobacillus

Summary Thiobacillus TH1 is an acidophilic chemolithotrophic heterotroph growing at temperatures up to about 50°C on media containing ferrous iron or pyrite when supplemented with yeast extract or glutathione. Virtually no carbon dioxide fixation occurred during growth on iron with yeast extract. Its DNA contains 48 mol % guanine + cytosine. The organism effects the thermophilic leaching of metals from pyrite, chalcopyrite, CuS, and copper concentrates. Oxidation of soluble ferrous iron at pH 1.6 was competitively inhibited by ferric iron and had a K_m of 7.3 mM FeSO₄.     

Respiratory components in acidophilic bacteria that respire on iron

Abstract

Microorganisms capable of aerobic respiration on ferrous ions are spread throughout eubacterial and archaebacterial phyla. Phylogenetically distinct organisms were shown to express spectrally distinct redox‐active biomolecules during autotrophic growth on soluble iron. A new iron‐oxidizing eubacterium, designated as strain Funis, was investigated. Strain Funis was judged to be different from other known iron‐oxidizing bacteria on the bases of comparative lipid analyses, 16S rRNA sequence analyses, and cytochrome composition studies. When grown autotrophically on ferrous ions, Funis produced conspicuous levels of a novel acid‐stable, acid‐soluble yellow cytochrome with a distinctive absorbance peak at 579 nm in the reduced state.

Stopped‐flow spectrophotometric kinetic studies were conducted on respiratory chain components isolated from cell‐free extracts of Thiobacillus ferrooxidans. Experimental results were consistent with a model where the primary oxidant of ferrous ions is a highly aggregated c‐type cytochrome that then reduces the periplasmic rusticyanin. The Fe(II)‐dependent, cytochrome c‐catalyzed reduction of the rusticyanin possessed three kinetic properties in common with corresponding intact cells that respire on iron: the same anion specificity, a similar dependence of the rate on the concentration of ferrous ions, and similar rates at saturating concentrations of ferrous ions     

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.     

不同能源条件下中度嗜热嗜酸细菌多样性分析

采用黄铁矿、黄铜矿、硫酸亚铁和硫粉混合物作为主要能源物质在50°C条件下分别培养中度嗜热细菌混合物,研究其细菌多样性。提取细菌基因组总DNA,采用PCR结合限制性酶切片段多态性分析(RFLP)方法进行细菌16SrRNA基因的系统发育分析,比较不同能源条件下富集培养的混合细菌群落构成的差异。从3个培养物中共获得阳性克隆303个并进行RFLP分析,对29种不同酶切谱型的克隆插入序列进行测定和系统发育分析。大部分序列与已报道的浸矿微生物16SrRNA序列相似性较高(89.1%~99.7%),归属于硫化叶菌属的耐温氧化硫化杆菌(Sulfobacillus thermotolerans)和热氧化硫化杆菌(Sulfobacillus thermosulfidooxidans),嗜酸硫杆菌属的喜温硫杆菌(Acidithiobacillus caldus),钩端螺旋菌属的嗜铁钩端螺旋菌(Leptospirillumferriphilum)以及unculturedforest soil bacterium、uncultured proteobacterium。其中Acidithiobacillus caldus,Sulfobacillus thermotolerans,Leptospirillumferriphilum3种细菌为三类能源物质培养物中的优势细菌类群。L.ferriphilum在黄铁矿培养体系(53.8%)和硫酸亚铁和硫粉为能源的培养体系中(45.9%)中丰度最高;在以黄铜矿为能源物质的培养体系中,S.thermotolerans的比例大幅上升(70.1%)。关键词:中度嗜热细菌;生物多样性;生物浸矿;喜温硫杆菌(Acidithiobacillus caldus);耐温氧化硫化杆菌(Sulfobacillus thermotolerans);嗜铁钩端螺旋菌(Leptospirillumferriphilum)     

Iron and carbon metabolism by a mineral-oxidizing Alicyclobacillus-like bacterium

A novel iron-oxidizing, moderately thermophilic, acidophilic bacterium (strain “GSM”) was isolated from mineral spoil taken from a gold mine in Montana. Biomolecular analysis showed that it was most closely related to Alicyclobacillus tolerans, although the two bacteria differed in some key respects, including the absence (in strain GSM) of ϖ-alicyclic fatty acids and in their chromosomal base compositions. Isolate GSM was able to grow in oxygen-free media using ferric iron as terminal electron acceptor confirming that it was a facultative anaerobe, a trait not previously described in Alicyclobacillus spp.. The acidophile used both organic and inorganic sources of energy and carbon, although growth and iron oxidation by isolate GSM was uncoupled in media that contained both fructose and ferrous iron. Fructose utilization suppressed iron oxidation, and oxidation of ferrous iron occurred only when fructose was depleted. In contrast, fructose catabolism was suppressed when bacteria were harvested while actively oxidizing iron, suggesting that both ferrous iron- and fructose-oxidation are inducible in this acidophile. Isolate GSM accelerated the oxidative dissolution of pyrite in liquid media either free of, or amended with, organic carbon, although redox potentials were significantly different in these media. The potential of this isolate for commercial mineral processing is discussed.     

Isolation and characterization of an acidophilic, heterotrophic bacterium capable of oxidizing ferrous iron.

A heterotrophic bacterium, isolated from an acidic stream in a disused pyrite mine which contained copious growths of "acid streamers," displayed characteristics which differentiated it from previously described mesophilic acidophiles. The isolate was obligately acidophilic, with a pH range of 2.0 to 4.4 and an optimum pH of 3.0. The bacterium was unable to fix carbon dioxide but oxidized ferrous iron, although at a slower rate than either Thiobacillus ferrooxidans or Leptospirillum ferrooxidans. Elemental sulfur and manganese(II) were not oxidized. In liquid media, the isolate produced macroscopic streamerlike growths. Microscopic examination revealed that the bacterium formed long (greater than 100 microns) filaments which tended to disintegrate during later growth stages, producing single, motile cells and small filaments. The isolate did not appear to utilize the energy from ferrous iron oxidation. Both iron (ferrous or ferric) and an organic substrate were necessary to promote growth. The isolate displayed a lower tolerance to heavy metals than other iron-oxidizing acidophiles, and growth was inhibited by exposure to light. There was evidence of extracellular sheath production by the isolate. In this and some other respects, the isolate resembles members of the Sphaerotilus-Leptothrix group of filamentous bacteria. The guanine-plus-cytosine content of the isolate was 62 mol%, which is less than that recorded for Sphaerotilus-Leptothrix spp. and greater than those of L. ferrooxidans and most T. ferrooxidans isolates.     

Isolation and characterization of an acidophilic, heterotrophic bacterium capable of oxidizing ferrous iron.

A heterotrophic bacterium, isolated from an acidic stream in a disused pyrite mine which contained copious growths of "acid streamers," displayed characteristics which differentiated it from previously described mesophilic acidophiles. The isolate was obligately acidophilic, with a pH range of 2.0 to 4.4 and an optimum pH of 3.0. The bacterium was unable to fix carbon dioxide but oxidized ferrous iron, although at a slower rate than either Thiobacillus ferrooxidans or Leptospirillum ferrooxidans. Elemental sulfur and manganese(II) were not oxidized. In liquid media, the isolate produced macroscopic streamerlike growths. Microscopic examination revealed that the bacterium formed long (greater than 100 microns) filaments which tended to disintegrate during later growth stages, producing single, motile cells and small filaments. The isolate did not appear to utilize the energy from ferrous iron oxidation. Both iron (ferrous or ferric) and an organic substrate were necessary to promote growth. The isolate displayed a lower tolerance to heavy metals than other iron-oxidizing acidophiles, and growth was inhibited by exposure to light. There was evidence of extracellular sheath production by the isolate. In this and some other respects, the isolate resembles members of the Sphaerotilus-Leptothrix group of filamentous bacteria. The guanine-plus-cytosine content of the isolate was 62 mol%, which is less than that recorded for Sphaerotilus-Leptothrix spp. and greater than those of L. ferrooxidans and most T. ferrooxidans isolates.     

Characterization of a new metal-mobilizing Thiobacillus isolate

A new acidophilic, mineral sulphide oreoxidizing bacterium was isolated from a uranium mine near Salamanca, Spain. Cells were rod-shaped, motile and gram-negative. They were aerobes, could grow on pyrite and use sulphur or thiosulphate as sole energy source, suggesting this new isolate belongs to the genus Thiobacillus. It could grow neither with glucose nor with yeast extract as sole substrates. It could not grow on ferrous sulphate as the only energy source, although it grew in the same medium supplemented with glucose, yeast extract or thiosulphate. It was a mesophilic and extremely acidophilic Thiobacillus, with an optimal pH of 1.5 2. The G+C content of the DNA was 58%. The new isolate could grow in cultures on pyrite where electrophoretic pattern was clearly different from those of other thiobacilli, such as T. ferrooxidans.Abbreviations G+C Guanine + Cytosine     

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.