Surface Chemistry of Thiobacillus ferrooxidans Relevant to Adhesion on Mineral Surfaces

Thiobacillus ferrooxidans cells grown on sulfur, pyrite, and chalcopyrite exhibit greater hydrophobicity than ferrous ion-grown cells. The isoelectric points of sulfur-, pyrite-, and chalcopyrite-grown cells were observed to be at a pH higher than that for ferrous ion-grown cells. Microbe-mineral interactions result in change in the surface chemistry of the organism as well as that of the minerals with which it has interacted. Sulfur, pyrite, and chalcopyrite after interaction with T. ferrooxidans exhibited a significant shift in their isoelectric points from the initial values exhibited by uninteracted minerals. With antibodies raised against sulfur-grown T. ferrooxidans, pyrite- and chalcopyrite-grown cells showed immunoreactivity, whereas ferrous ion-grown cells failed to do so. Fourier transform infrared spectroscopy of sulfur-grown cells suggested that a proteinaceous new cell surface appendage synthesized in mineral-grown cells brings about adhesion to the solid mineral substrates. Such an appendage was found to be absent in ferrous ion-grown cells as it is not required during growth in liquid substrates.     

Selective Adhesion of Thiobacillus ferrooxidans to Pyrite

Bacterial adhesion to mineral surfaces plays an important role not only in bacterial survival in natural ecosystems, but also in mining industry applications. Selective adhesion was investigated with Thiobacillus ferrooxidans by using four minerals, pyrite, quartz, chalcopyrite, and galena. Escherichia coli was used as a control bacterium. Contact angles were used as indicators of hydrophobicity, which was an important factor in the interaction between minerals and bacteria. The contact angle of E. coli in a 0.5% sodium chloride solution was 31°, and the contact angle of T. ferrooxidans in a pH 2.0 sulfuric acid solution was 23°. E. coli tended to adhere to more hydrophobic minerals by hydrophobic interaction, while T. ferrooxidans selectively adhered to iron-containing minerals, such as pyrite and chalcopyrite. Ferrous ion inhibited the selective adhesion of T. ferrooxidans to pyrite competitively, while ferric ion scarcely inhibited such adhesion. When selective adhesion was quenched by ferrous ion completely, adhesion of T. ferrooxidans was controlled by hydrophilic interactions. Adhesion of E. coli to pyrite exhibited a liner relationship on langmuir isotherm plots, but adhesion of T. ferrooxidans did not. T. ferrooxidans recognized the reduced iron in minerals and selectively adhered to pyrite and chalcopyrite by a strong interaction other than the physical interaction.     

Bioleaching of chalcopyrite by pure and mixed cultures of Acidithiobacillus spp. and Leptospirillum ferriphilum

Bioleaching is an economical method for the recovery of metals that requires low investment and operation costs. Furthermore, it is generally more environmentally friendly than many physicochemical metal extraction processes. The bioleaching of chalcopyrite in shake flasks was investigated with pure and mixed cultures of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Acidithiobacillus caldus, and Leptospirillum ferriphilum. The mixed cultures containing both iron- and sulfur-oxidizing bacteria were more efficient than the pure culture alone. The presence of sulfur-oxidizing bacteria positively increased the dissolution rate and the percentage recovery of copper from chalcopyrite. Mixed cultures consisting of moderately thermophilic L. ferriphilum and A. caldus leached chalcopyrite more effectively than mesophilic A. ferrooxidans pure and mixed cultures. The decrease of the chalcopyrite dissolution rate in leaching systems containing A. ferrooxidans after 12–16 days coincided with the formation of jarosite precipitation as a passivation layer on the mineral surface during bioleaching. Low pH significantly reduces jarosite formation in pure and mixed cultures of L. ferriphilum and A. caldus.     

Dependence of effectiveness of leaching of metallic sulphides on enzymes involved in inorganic sulphur metabolism in Thiobacillus ferrooxidans

Summary The leaching activity of five batches of Thiobacillus ferrooxidans, strain F26-77, cultivated under various conditions, towards elemental sulphur, ferrous ions, pyrite, covellite, chalcopyrite and sphalerite was studied. The activities of sulphite oxidase, thiosulphate oxidase and rhodanese were determined in crude, cell-free bacterial extracts. The effectiveness of leaching was directly correlated with the enzymic activity of the cultures. The results suggest that the activities of the enzymes metabolizing sulphur and its inorganic compounds in Thiobacillus ferrooxidans, or bacterial leaching activity on sulphur and sulphides, rather than the rate of oxidation of ferrous ions, should be taken as the criterion of usefulness for the leaching of sulphide minerals.     

Analysis of Community Composition during Moderately Thermophilic Bioleaching of Pyrite,Arsenical Pyrite,and Chalcopyrite.

An analysis of the community composition of three previously undefined mixed cultures of moderately thermophilic bioleaching bacteria grown at 45°C on pyrite, arsenical pyrite, and chalcopyrite has been carried out. The bacterial species present were identified by comparative sequence analysis of the 16S rRNA gene isolated from the bioleaching vessels and analyzed by denaturing gradient gel electrophoresis, cloning, and sequencing. The mixed cultures leached all three minerals, as shown by the increase in iron released from the mineral concentrates. The species identified from the mixed cultures during bioleaching of pyrite, arsenical pyrite, and chalcopyrite were clones closely related to Acidithiobacillus caldus C-SH12, Sulfobacillus thermosulfidooxidans AT-1, Sulfobacillus montserratensis L15, and an uncultured thermal soil bacterium YNP. It was also found that the same mixed culture maintained for over a year on chalcopyrite mineral selected approximately the same consortia of bacteria as the original mixed culture grown on chalcopyrite.     

Investigation of energy gene expressions and community structures of free and attached acidophilic bacteria in chalcopyrite bioleaching

In order to better understand the bioleaching mechanism, expression of genes involved in energy conservation and community structure of free and attached acidophilic bacteria in chalcopyrite bioleaching were investigated. Using quantitative real-time PCR, we studied the expression of genes involved in energy conservation in free and attached Acidithiobacillus ferrooxidans during bioleaching of chalcopyrite. Sulfur oxidation genes of attached A. ferrooxidans were up-regulated while ferrous iron oxidation genes were down-regulated compared with free A. ferrooxidans in the solution. The up-regulation may be induced by elemental sulfur on the mineral surface. This conclusion was supported by the results of HPLC analysis. Sulfur-oxidizing Acidithiobacillus thiooxidans and ferrous-oxidizing Leptospirillum ferrooxidans were the members of the mixed culture in chalcopyrite bioleaching. Study of the community structure of free and attached bacteria showed that A. thiooxidans dominated the attached bacteria while L. ferrooxidans dominated the free bacteria. With respect to available energy sources during bioleaching of chalcopyrite, sulfur-oxidizers tend to be on the mineral surfaces whereas ferrous iron-oxidizers tend to be suspended in the aqueous phase. Taken together, these results indicate that the main role of attached acidophilic bacteria was to oxidize elemental sulfur and dissolution of chalcopyrite involved chiefly an indirect bioleaching mechanism.     

Isolation of a strain of <Emphasis Type="Italic">Acidithiobacillus caldus</Emphasis> and its role in bioleaching of chalcopyrite

A moderately thermophilic and acidophilic sulfur-oxidizing bacterium named S₂, was isolated from coal heap drainage. The bacterium was motile, Gram-negative, rod-shaped, measured 0.4 to 0.6 by 1 to 2 μm, and grew optimally at 42–45°C and an initial pH of 2.5. The strain S₂ grew autotrophically by using elemental sulfur, sodium thiosulfate and potassium tetrathionate as energy sources. The strain did not use organic matter and inorganic minerals including ferrous sulfate, pyrite and chalcopyrite as energy sources. The morphological, biochemical, physiological characterization and analysis based on 16S rRNA gene sequence indicated that the strain S₂ is most closely related to Acidithiobacillus caldus (>99% similarity in gene sequence). The combination of the strain S₂ with Leptospirillum ferriphilum or Acidithiobacillus ferrooxidans in chalcopyrite bioleaching improved the copper-leaching efficiency. Scanning electron microscope (SEM) analysis revealed that the chalcopyrite surface in a mixed culture of Leptospirillum ferriphilum and Acidithiobacillus caldus was heavily etched. The energy dispersive X-ray (EDX) analysis indicated that Acidithiobacillus caldus has the potential role to enhance the recovery of copper from chalcopyrite by oxidizing the sulfur formed during the bioleaching progress.     

Enzyme-Linked Immunofiltration Assay To Estimate Attachment of Thiobacilli to Pyrite

An enzyme-linked immunofiltration assay (ELIFA) has been developed in order to estimate directly and specifically Thiobacillus ferrooxidans attachment on sulfide minerals. This method derives from the enzyme-linked immunosorbent assay but is performed on filtration membranes which allow the retention of mineral particles for a subsequent immunoenzymatic reaction in microtiter plates. The polyclonal antiserum used in this study was raised against T. ferrooxidans DSM 583 and recognized cell surface antigens present on bacteria belonging to the genus Thiobacillus. This antiserum and the ELIFA allowed the direct quantification of attached bacteria with high sensitivity (10⁴ bacteria were detected per well of the microtiter plate). The mean value of bacterial attachment has been estimated to be about 10⁵ bacteria mg⁻¹ of pyrite at a particle size of 56 to 65 μm. The geometric coverage ratio of pyrite by T. ferrooxidans ranged from 0.25 to 2.25%. This suggests an attachment of T. ferrooxidans on the pyrite surface to well-defined limited sites with specific electrochemical or surface properties. ELIFA was shown to be compatible with the measurement of variable levels of adhesion. Therefore, this method may be used to establish adhesion isotherms of T. ferrooxidans on various sulfide minerals exhibiting different physicochemical properties in order to understand the mechanisms of bacterial interaction with mineral surfaces.     

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.     

A Novel Mineral Flotation Process Using Thiobacillus ferrooxidans

Oxidative leaching of metals by Thiobacillus ferrooxidans has proven useful in mineral processing. Here, we report on a new use for T. ferrooxidans, in which bacterial adhesion is used to remove pyrite from mixtures of sulfide minerals during flotation. Under control conditions, the floatabilities of five sulfide minerals tested (pyrite, chalcocite, molybdenite, millerite, and galena) ranged from 90 to 99%. Upon addition of T. ferrooxidans, the floatability of pyrite was significantly suppressed to less than 20%. In contrast, addition of the bacterium had little effect on the floatabilities of the other minerals, even when they were present in relatively large quantities: their floatabilities remained in the range of 81 to 98%. T. ferrooxidans thus appears to selectively suppress pyrite floatability. As a consequence, 77 to 95% of pyrite was removed from mineral mixtures while 72 to 100% of nonpyrite sulfide minerals was recovered. The suppression of pyrite floatability was caused by bacterial adhesion to pyrite surfaces. When normalized to the mineral surface area, the number of cells adhering to pyrite was significantly larger than the number adhering to other minerals. These results suggest that flotation with T. ferrooxidans may provide a novel approach to mineral processing in which the biological functions involved in cell adhesion play a key role in the separation of minerals.     

Selection of a community of acidochemolithotrophic microorganisms with a high oxidation rate of pyrrhotite-containing sulphide ore flotation concentrate

A community of acidochemolithotrophic microorganisms with a high oxidation rate of pyrrhotite-containing sulphide ore flotation concentrate was selected. The Acidithiobacillus caldus OP-1 and Ferroplasma acidiphilum OP-2 cultures were identified to be dominating members. The presence of the Acidithiobacillus ferrooxidans OP-3, Leptospirillum ferriphilum OP-4, and Sulfobacillus thermosulfidooxidans OP-5 cultures in the community’s composition was also mentioned. The analysis results of solid residues of the process showed a greater elemental sulfur oxidation level and gold recovery when the initial pH value in tank I was maintained at a level of 1.8–2.0 (90.5%) rather than 1.6–1.8 (86.3%).     

Evidence of cell surface iron speciation of acidophilic iron-oxidizing microorganisms in indirect bioleaching process

While indirect model has been widely accepted in bioleaching, but the evidence of cell surface iron speciation has not been reported. In the present work the iron speciation on the cell surfaces of four typically acidophilic iron-oxidizing microorganism (mesophilic Acidithiobacillus ferrooxidans ATCC 23270, moderately thermophilic Leptospirillum ferriphilum YSK and Sulfobacillus thermosulfidooxidans St, and extremely thermophilic Acidianus manzaensis YN25) grown on different energy substrates (chalcopyrite, pyrite, ferrous sulfate and elemental sulfur (S⁰)) were studied in situ firstly by using synchrotron-based micro- X-ray fluorescence analysis and X-ray absorption near-edge structure spectroscopy. Results showed that the cells grown on iron-containing substrates had apparently higher surface iron content than the cells grown on S⁰. Both ferrous iron and ferric iron were detected on the cell surface of all tested AIOMs, and the Fe(II)/Fe(III) ratios of the same microorganism were affected by different energy substrates. The iron distribution and bonding state of single cell of A. manzaensis were then studied in situ by scanning transmission soft X-ray microscopy based on dual-energy contrast analysis and stack analysis. Results showed that the iron species distributed evenly on the cell surface and bonded with amino, carboxyl and hydroxyl groups.     

Bioleaching of chalcopyrite concentrate using Leptospirillum ferriphilum, Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans in a continuous bubble column reactor

To estimate the bioleaching performance of chalcopyrite for various hydraulic residence times (HRTs), laboratory-scale bioleaching of chalcopyrite concentrate was carried out in a continuous bubble column reactor with three different HRTs of 120, 80 and 40 h, respectively. An extraction rate and ratio of 0.578 g Cu l⁻¹ h⁻¹ and 39.7%, respectively, were achieved for an HRT of 80 h at a solids concentration of 10% (w/v). Lower bioleaching performances than this were obtained for a longer HRT of 120 h and a shorter HRT of 40 h. In addition, there was obvious competition between Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans to oxidize ferrous iron, causing large compositional differences between the microbial communitys obtained for the different HRTs. Leptospirillum ferriphilum and Acidithiobacillus thiooxidans were found to be the dominant microbes for the longer HRT (120 h). Acidithiobacillus ferrooxidans became the dominant species when the HRT was decreased. The proportion of Acidithiobacillus thiooxidans was comparatively constant in the microbial community throughout the three process stages.     

Role of Thiobacillus ferrooxidans and sulphur (sulphide)-dependent ferric-ion-reducing activity in the oxidation of sulphide minerals

 Thiobacillus ferrooxidans oxidized the sulphide minerals e.g., pyrite, pyrrhotite and copper concentrate under anaerobic conditions in the presence of ferric ion as sole electron acceptor. Copper and iron were solubilized from sulphide ores by the sulphur (sulphide)-dependent ferric-ion oxidoreductase activity. Treatment of resting cells of T. ferrooxidans with 0.5% phenol for 30 min completely destroyed the iron- and copper-solubilizing activity. The above treatment destroyed the sulphur(sulphide)-dependent ferric-ion-reducing activity completely but did not affect the iron-oxidizing activity. The results suggest that sulphur(sulphide)-dependent ferric-ion-reducing activity actively participates in the oxidation of sulphide minerals under anaerobic conditions. The activity of sulphur(sulphide)-dependent ferric ion reduction in the solubilization of iron and copper from the sulphide ores were also observed under aerobic conditions in presence of sodium azide (0.1 μmol), which completely inhibits the iron-oxidizing activity. Received: 23 May 1995/Received revision: 10 October 1995/Accepted: 16 October 1995     

A Moderately Thermophilic Mixed Microbial Culture for Bioleaching of Chalcopyrite Concentrate at High Pulp Density

Three kinds of samples (acid mine drainage, coal mine wastewater, and thermal spring) derived from different sites were collected in China. Thereafter, these samples were combined and then inoculated into a basal salts solution in which different substrates (ferrous sulfate, elemental sulfur, and chalcopyrite) served as energy sources. After that, the mixed cultures growing on different substrates were pooled equally, resulting in a final mixed culture. After being adapted to gradually increasing pulp densities of chalcopyrite concentrate by serial subculturing for more than 2 years, the final culture was able to efficiently leach the chalcopyrite at a pulp density of 20% (wt/vol). At that pulp density, the culture extracted 60.4% of copper from the chalcopyrite in 25 days. The bacterial and archaeal diversities during adaptation were analyzed by denaturing gradient gel electrophoresis and constructing clone libraries of the 16S rRNA gene. The results show that the culture consisted mainly of four species, including Leptospirillum ferriphilum, Acidithiobacillus caldus, Sulfobacillus acidophilus, and Ferroplasma thermophilum, before adapting to a pulp density of 4%. However, L. ferriphilum could not be detected when the pulp density was greater than 4%. Real-time quantitative PCR was employed to monitor the microbial dynamics during bioleaching at a pulp density of 20%. The results show that A. caldus was the predominant species in the initial stage, while S. acidophilus rather than A. caldus became the predominant species in the middle stage. F. thermophilum accounted for the greatest proportion in the final stage.     

The effect of the introduction of exogenous strain Acidithiobacillus thiooxidans A01 on functional gene expression, structure and function of indigenous consortium during pyrite bioleaching

Acidithiobacillus thiooxidans A01 was added to a consortium of bioleaching bacteria including Acidithiobacilluscaldus, Leptospirillumferriphilum, Acidithiobacillus ferrooxidans, Sulfobacillus thermosulfidooxidans, Acidiphilium spp., and Ferroplasma thermophilum cultured in modified 9 K medium containing 0.5% (w/v) pyrite, and 10.7% increase of bioleaching rate was observed. Changes in community structure and gene expression were monitored with real-time PCR and functional gene arrays (FGAs). Real-time PCR showed that addition of At. thiooxidans caused increased numbers of all consortium members except At. caldus, and At. caldus, L. ferriphilum, and F. thermophilum remained dominant in this community. FGAs results showed that after addition of At. thiooxidans, most genes involved in iron, sulfur, carbon, and nitrogen metabolisms, metal resistance, electron transport, and extracellular polymeric substances of L. ferriphilum, F. thermophilum, and Acidiphilium spp., were up-regulated while most of these genes were down-regulated at 70-78 h in At. caldus and up-regulated in At. ferrooxidans, then down-regulated at 82-86 h.     

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.     

Leaching of Pyrite by Acidophilic Heterotrophic Iron-Oxidizing Bacteria in Pure and Mixed Cultures

Seven strains of heterotrophic iron-oxidizing acidophilic bacteria were examined to determine their abilities to promote oxidative dissolution of pyrite (FeS₂) when they were grown in pure cultures and in mixed cultures with sulfur-oxidizing Thiobacillus spp. Only one of the isolates (strain T-24) oxidized pyrite when it was grown in pyrite-basal salts medium. However, when pyrite-containing cultures were supplemented with 0.02% (wt/vol) yeast extract, most of the isolates oxidized pyrite, and one (strain T-24) promoted rates of mineral dissolution similar to the rates observed with the iron-oxidizing autotroph Thiobacillus ferrooxidans. Pyrite oxidation by another isolate (strain T-21) occurred in cultures containing between 0.005 and 0.05% (wt/vol) yeast extract but was completely inhibited in cultures containing 0.5% yeast extract. Ferrous iron was also needed for mineral dissolution by the iron-oxidizing heterotrophs, indicating that these organisms oxidize pyrite via the “indirect” mechanism. Mixed cultures of three isolates (strains T-21, T-23, and T-24) and the sulfur-oxidizing autotroph Thiobacillus thiooxidans promoted pyrite dissolution; since neither strains T-21 and T-23 nor T. thiooxidans could oxidize this mineral in yeast extract-free media, this was a novel example of bacterial synergism. Mixed cultures of strains T-21 and T-23 and the sulfur-oxidizing mixotroph Thiobacillus acidophilus also oxidized pyrite but to a lesser extent than did mixed cultures containing T. thiooxidans. Pyrite leaching by strain T-23 grown in an organic compound-rich medium and incubated either shaken or unshaken was also assessed. The potential environmental significance of iron-oxidizing heterotrophs in accelerating pyrite oxidation is discussed.     

Growth of Leptospirillum ferriphilum in sulfur medium in co-culture with Acidithiobacillus caldus

Leptospirillum ferriphilum and Acidithiobacillus caldus are both thermotolerant acidophilic bacteria that frequently co-exist in natural and man-made environments, such as biomining sites. Both are aerobic chemolithotrophs; L. ferriphilum is known only to use ferrous iron as electron donor, while A. caldus can use zero-valent and reduced sulfur, and also hydrogen, as electron donors. It has recently been demonstrated that A. caldus reduces ferric iron to ferrous when grown aerobically on sulfur. Experiments were carried out which demonstrated that this allowed L. ferriphilum to be sustained for protracted periods in media containing very little soluble iron, implying that dynamic cycling of iron occurred in aerobic mixed cultures of these two bacteria. In contrast, numbers of viable L. ferriphilum rapidly declined in mixed cultures that did not contain sulfur. Data also indicated that growth of A. caldus was partially inhibited in the presence of L. ferriphilum. This was shown to be due to greater sensitivity of the sulfur-oxidizer to ferric than to ferrous iron, and to highly positive redox potentials, which are characteristic of cultures containing Leptospirillum spp. The implications of these results in the microbial ecology of extremely acidic environments and in commercial bioprocessing applications are discussed.

     

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.