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

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

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

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

The Effect of the Facultative Chemolithotrophic Bacterium Thiobacillus acidophilus on the Leaching of Low-Grade Cu-Ni Sulfide Ore by Thiobacillus ferrooxidans

The purpose of this investigation was to determine the effect of Thiobacillus acidophilus on the leaching of a low-grade Cu-Ni sulfide ore by Thiobacillus ferrooxidans. A sample of low-grade Cu-Ni sulfide ore containing 0.36% Cu, 0.48% Ni, and 7.87% Fe was pulverized and initially leached for a 21-day period using two different pure cultures of T. ferrooxidans, an environmental strain (F2) and a strain from the American Type Culture Collection (ATCC 23270). Samples of the ore slurries were drawn and the pH was monitored over the course of the leaching period. The concentrations of Cu and Ni leached by each strain were determined and compared. No significant differences were observed in the concentrations of Cu and Ni leached by the two pure cultures of T. ferrooxidans. Subsequently, the ore was leached with mixed cultures of T. ferrooxidans and T. acidophilus to determine the effect of the latter on the concentrations of Cu and Ni leached from the ore. The environmental strain F2 of T. ferrooxidans was used in combination with both a type strain (ATCC 27807) and an environmental strain (64) of T. acidophilus. After 21 days, the mixed cultures of T. ferrooxidans and T. acidophilus leached significantly greater amounts of copper than the pure strain alone, but no such difference was observed for the leaching of nickel.     

Expression of Heterogenous Arsenic Resistance Genes in the Obligately Autotrophic Biomining Bacterium Thiobacillus ferrooxidans

Two arsenic-resistant plasmids were constructed and introduced into Thiobacillus ferrooxidans strains by conjugation. The plasmids with the replicon of wide-host-range plasmid RSF1010 were stable in T. ferrooxidans. The arsenic resistance genes originating from the heterotroph were expressed in this obligately autotrophic bacterium, but the promoter derived from T. ferrooxidans showed no special function in its original host.     

Controlled microbiological in-situ stope leaching of a sulphidic ore

A mixed culture of Thiobacillus ferrooxidans, T. thiooxidans, and Leptospirillum ferrooxidans was used for inoculation of a sulphidic ore body for a bacterial in-situ stope-leaching experiment in the Ilba mine in Romania. The ore body was inoculated with 10⁷ cells/g ore. Measurements at six main sites of the ore body indicated that microbial leaching was started by the inoculation. After about 8 weeks, sufficient microbial activity was measurable only in the upper third of the ore body. Due to the angle of incidence of the ore (75°), the leach liquor percolated only through the upper part leaving two-thirds humidified unsatisfactorily. The leach results, metal mobilization, indicated that by inoculation with the indigenous microorganisms efficient leaching was achieved. Metal output after 18 months of operation amounted to 10% of Cu and 78% of Zn. In the winter months energy for aeration and circulation was not available and this was reflected by reduced values for microbial activity, temperature, and daily metal output. The biological metal mobilisation after 18 months of operation was as active as at the beginning. Cu was mobilised predominantly by microbial leaching whereas Zn was leached mainly by chemical reactions. Both mechanisms contributed equally to iron output. Correspondence to: W. Sand     

Bacterial leaching of a sulfide ore by Thiobacillus ferrooxidans and Thiobacillus thiooxidans: I. Shake flask studies

Bacterial leaching of a sulfide ore containing pyrite, chalcopyrite, and sphalerite was studied in shake flask experiments using Thiobacillus ferrooxidans and Thiobacillus thiooxidans strains isolated from mine sites. The Fe(2+)grown T. ferrooxidans isolates solubilized sphalerite preferentially over chalcopyrite leaching 7-10% Cu, 68-76% Zn, and 10-22% Fe from the ore in 18 days. The sulfur grown T. thiooxidans isolates leached Zn much more slowly and very little Fe, with a Cu-Zn extraction ratio twice the value obtained with T. ferrooxidans. The ore adapted T. ferrooxidans started solubilizing Cu and Zn without a lag period. The ore-adapted T. thiooxidans extracted Cu as well as T. ferrooxidans, but the extraction of Zn or Fe was still much slower in the low-phosphate medium, while in the high-phosphate medium it approached the value obtained with T. ferrooxidans. A high Cu-Zn extraction ratio of 0.34 was obtained with T. thiooxidans in the low phosphate medium. In the mixed-culture experiments with T. ferrooxidans and T. thiooxidans, the culture behaved as T. thiooxidans in the low-phosphate medium with a higher Cu-Zn extraction ratio and as T. ferrooxidans in the high-phosphate medium with a lower Cu-Zn extraction ratio. It is concluded that T. ferrooxidans and T. thiooxidans solubilize sulfide minerals by different mechanisms.     

Development of a Laboratory-Scale Leaching Plant for Metal Extraction from Fly Ash by Thiobacillus Strains

Semicontinuous biohydrometallurgical processing of fly ash from municipal waste incineration was performed in a laboratory-scale leaching plant (LSLP) by using a mixed culture of Thiobacillus thiooxidans and Thiobacillus ferrooxidans. The LSLP consisted of three serially connected reaction vessels, reservoirs for a fly ash suspension and a bacterial stock culture, and a vacuum filter unit. The LSLP was operated with an ash concentration of 50 g liter⁻¹, and the mean residence time was 6 days (2 days in each reaction vessel). The leaching efficiencies (expressed as percentages of the amounts applied) obtained for the economically most interesting metal, Zn, were up to 81%, and the leaching efficiencies for Al were up to 52%. Highly toxic Cd was completely solubilized (100%), and the leaching efficiencies for Cu, Ni, and Cr were 89, 64, and 12%, respectively. The role of T. ferrooxidans in metal mobilization was examined in a series of shake flask experiments. The release of copper present in the fly ash as chalcocite (Cu₂S) or cuprite (Cu₂O) was dependent on the metabolic activity of T. ferrooxidans, whereas other metals, such as Al, Cd, Cr, Ni, and Zn, were solubilized by biotically formed sulfuric acid. Chemical leaching with 5 N H₂SO₄ resulted in significantly increased solubilization only for Zn. The LSLP developed in this study is a promising first step toward a pilot plant with a high capacity to detoxify fly ash for reuse for construction purposes and economical recovery of valuable metals.     

Effects of Cinnabar on Pyrite Oxidation by Thiobacillus ferrooxidans and Cinnabar Mobilization by a Mercury-Resistant Strain

The effect of cinnabar on pyrite oxidation by mercury-sensitive and mercury-resistant strains of Thiobacillus ferrooxidans was investigated by using percolation columns. Mercury-resistant strains oxidized pyrite in pyrite-cinnabar mixtures (1 and 10%, wt/wt), whereas a mercury-sensitive strain did not. Elemental mercury was produced by the mercury-resistant strains growing in the pyrite-cinnabar mixtures in percolation columns and in flasks containing cinnabar only. Manometric experiments showed that cinnabar had little effect on oxygen uptake of mercury-sensitive or mercury-resistant cells growing on ferrous sulfate, pyrite, or pyrite-ferrous sulfate mixtures. In addition, shake flask leaching experiments showed that cinnabar had little effect on pyrite oxidation at 1% (wt/wt) but inhibited growth of mercury-sensitive and mercury-resistant strains at 10%. Mercury-resistant strains were unable to grow on cinnabar as an energy source.     

Leaching of Pyrites of Various Reactivities by Thiobacillus ferrooxidans

Wide variations were found in the rate of chemical and microbiological leaching of iron from pyritic materials from various sources. Thiobacillus ferrooxidans accelerated leaching of iron from all of the pyritic materials tested in shake flask suspensions at loadings of 0.4% (wt/vol) pulp density. The most chemically reactive pyrites exhibited the fastest bioleaching rates. However, at 2.0% pulp density, a delay in onset of bioleaching occurred with two of the pyrites derived from coal sources. T. ferrooxidans was unable to oxidize the most chemically reactive pyrite at 2.0% pulp density. No inhibition of pyrite oxidation by T. ferrooxidans occurred with mineral pyrite at 2.0% pulp density. Experiments with the most chemically reactive pyrite indicated that the leachates from the material were not inhibitory to iron oxidation by T. ferrooxidans.     

Attachment of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum cultured under varying conditions to pyrite, chalcopyrite, low-grade ore and quartz in a packed column reactor

The attachment of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum spp. grown on ferrous medium or adapted to a pyrite mineral concentrate to four mineral substrata, namely, chalcopyrite and pyrite concentrates, a low-grade chalcopyrite ore (0.5 wt%) and quartzite, was investigated. The quartzite represented a typical gangue mineral and served as a control. The attachment studies were carried out in a novel particle-coated column reactor. The saturated reactor containing glass beads, which were coated with fine mineral concentrates, provided a quantifiable surface area of mineral concentrate and maintained good fluid flow. A. ferrooxidans and Leptospirillum spp. had similar attachment characteristics. Enhanced attachment efficiency occurred with bacteria grown on sulphide minerals relative to those grown on ferrous sulphate in an ore-free environment. Selective attachment to sulphide minerals relative to gangue materials occurred, with mineral adapted cultures attaching to the minerals more efficiently than ferrous grown cultures. Mineral-adapted cultures showed highest levels of attachment to pyrite (74% and 79% attachment for A. ferrooxidans and L. ferriphilum, respectively). This was followed by attachment of mineral-adapted cultures to chalcopyrite (63% and 58% for A. ferrooxidans and L. ferriphilum, respectively). A. ferrooxidans and L. ferriphilum exhibited lower levels of attachment to low-grade ore and quartz relative to the sulphide minerals.     

Thiobacillus cuprinus sp. nov., a Novel Facultatively Organotrophic Metal-Mobilizing Bacterium

Five strains of mesophilic, facultatively organotrophic, ore-leaching eubacteria were isolated from solfatara fields in Iceland and a uranium mine in the Federal Republic of Germany. The new organisms are aerobic gram-negative rods. They can use sulfidic ores or elemental sulfur as sole energy source, indicating that they belong to the genus Thiobacillus. Alternatively, they grow on organic substrates such as yeast extract, peptone, and pyruvate. In contrast to the other leaching bacteria known so far, the new isolates are unable to oxidize ferrous iron. They consist of extreme and moderate acidophiles growing optimally at pH 3 and 4, respectively. The extreme acidophiles showed leaching characteristics similar to those shown by Thiobacillus ferrooxidans, while the moderate acidophiles exhibited a pronounced preference for copper leaching on some chalcopyrite ores. The G+C content of the DNA is between 66 and 69 mol%, depending on the isolate. In DNA-DNA hybridization experiments, the new strains showed homologies among each other of >70%, indicating that they belong to the same species. No significant DNA homology to Thiobacillus reference strains was detectable. Therefore, the new isolates represent a new species of Thiobacillus, which we name Thiobacillus cuprinus. Isolate Hö5 is designated as the type strain (DSM 5495).     

Energy Transduction by Anaerobic Ferric Iron Respiration in Thiobacillus ferrooxidans

Formate-grown cells of the obligately chemolithoautotrophic acidophile Thiobacillus ferrooxidans were capable of formate- and elemental sulfur-dependent reduction of ferric iron under anaerobic conditions. Under aerobic conditions, both oxygen and ferric iron could be simultaneously used as electron acceptors. To investigate whether anaerobic ferric iron respiration by T. ferrooxidans is an energy-transducing process, uptake of amino acids was studied. Glycine uptake by starved cells did not occur in the absence of an electron donor, neither under aerobic conditions nor under anaerobic conditions. Uptake of glycine could be driven by formate- and ferrous iron-dependent oxygen uptake. Under anaerobic conditions, ferric iron respiration with the electron donors formate and elemental sulfur could energize glycine uptake. Glycine uptake was inhibited by the uncoupler 2,4-dinitrophenol. The results indicate that anaerobic ferric iron respiration can contribute to the energy budget of T. ferrooxidans.     

Microbial leaching of arsenic from low‐sulfide gold mine material

Drainages from high‐sulfide tailings near abandoned lode deposits in Alaska, U.S.A., and Yukon, Canada, were found to be acidic, to contain large numbers of Thiobacillus ferrooxidans, and to have high concentrations of dissolved arsenic. Drainages from active placer gold mines are not acidic, but T. ferrooxidans and concentrations of dissolved arsenic exceeding 10 μg/L are found in some streams affected by placer mine drainage. Placer mine material containing low amounts of sulfides (326 (μg/g) and moderately high amounts of arsenic (700 μg/g) was leached with growing cultures of T. ferrooxidans, T. ferrooxidans‐spent filtrate, and acid ferric sulfate. The results showed that while more arsenic was released from this material by growing cultures of T. ferrooxidans than by abiotic controls, acid ferric sulfate released much more arsenic than did either growing cultures of T. ferrooxidans or spent culture filtrate containing oxidized iron. Cation exchange chromatography showed that oxidized iron from T. ferrooxidans culture filtrate is chemically less reactive than the iron in aqueous solutions of ferric sulfate salt. These results indicate that arsenic release from both high‐ and low‐sulfide mine wastes is enhanced biologically, but that rates and amounts of arsenic release are primarily controlled by iron species.     

Leaching of Zinc Sulfide by Thiobacillus ferrooxidans: Bacterial Oxidation of the Sulfur Product Layer Increases the Rate of Zinc Sulfide Dissolution at High Concentrations of Ferrous Ions

This paper reports the results of leaching experiments conducted with and without Thiobacillus ferrooxidans at the same conditions in solution. The extent of leaching of ZnS with bacteria is significantly higher than that without bacteria at high concentrations of ferrous ions. A porous layer of elemental sulfur is present on the surfaces of the chemically leached particles, while no sulfur is present on the surfaces of the bacterially leached particles. The analysis of the data using the shrinking-core model shows that the chemical leaching of ZnS is limited by the diffusion of ferrous ions through the sulfur product layer at high concentrations of ferrous ions. The analysis of the data shows that diffusion through the product layer does not limit the rate of dissolution when bacteria are present. This suggests that the action of T. ferrooxidans in oxidizing the sulfur formed on the particle surface is to remove the barrier to diffusion by ferrous ions.     

Leptospirillum-Like Bacteria and Evaluation of Their Role in Pyrite Oxidation

Two strains of Leptospirillum-like bacteria isolated from dumps of Alaverdi and Akhtala sulfide ore deposits in Armenia were studied. The optimum and maximum temperatures for the growth of both strains were 37 and 40°C, respectively. The pH optimum was 2.0–2.3. Bacterial growth and ferrous iron oxidation were inhibited by yeast extract. The pyrite-leaching activity of the Leptospirillum-like bacteria under mesophilic conditions was close to that of Acidithiobacillus ferrooxidans and exceeded by 2.0–2.7 times the activity of these moderately thermophilic bacteria at 37°C. The leaching of pyrite by Leptospirillum-like bacteria increased in the presence of sulfur-oxidizing bacteria, particularly, in their association with a thermotolerant sulfur-oxidizing bacterium.     

Sensitivity of Iron-Oxidizing Bacteria, Thiobacillus ferrooxidans and Leptospirillum ferrooxidans, to Bisulfite Ion

When grown on iron-salt medium supplemented with the bisulfite ion, Leptospirillum ferrooxidans was much more sensitive to the ion than was Thiobacillus ferrooxidans. The causes of the sensitivity of L. ferrooxidans to the bisulfite ion were studied. The bisulfite ion completely inhibited the iron-oxidizing activities of L. ferrooxidans and T. ferrooxidans at 0.02 and 0.2 mM, respectively. A trapping reagent for the bisulfite ion, formaldehyde, completely reversed the inhibition. The treatment of intact cells with 1.0 mM bisulfite ion for 1 h and washing the bisulfite ion from the cells had no harmful effects on the iron-oxidizing activity of T. ferrooxidans. However, the treatment of L. ferrooxidans with 0.1 mM bisulfite ion for 1 h completely destroyed the iron-oxidizing activity. T. ferrooxidans had sulfite:ferric ion oxidoreductase activity. In contrast, a quite low level of sulfite:ferric ion oxidoreductase activity was found in L. ferrooxidans, suggesting that it is much more difficult for L. ferrooxidans to oxidize the bisulfite ion to the less harmful sulfate than it is for T. ferrooxidans. These results suggest that the sensitivity of L. ferrooxidans to the bisulfite ion is due to a lack of an active sulfite:ferric ion oxidoreductase and the sensitivity of its iron oxidase to bisulfite ion.     

A Combined Immunofluorescence-DNA-Fluorescence Staining Technique for Enumeration of Thiobacillus ferrooxidans in a Population of Acidophilic Bacteria

An antiserum raised against whole cells of Thiobacillus ferrooxidans was allowed to react with a variety of acidophilic and nonacidophilic bacteria in an enzyme-linked immunosorbent assay and an indirect immunofluorescence assay. Both experiments demonstrated that the antiserum was specific at the species level. This preparation was used to evaluate the role of T. ferrooxidans in the microbial desulfurization process. Leaching experiments were performed, and the numbers of T. ferrooxidans cells and other bacteria were estimated by using a combined immunofluorescence-DNA-fluorescence staining technique that was adapted for this purpose. Nonsterile coal samples inoculated with T. ferrooxidans yielded high concentrations of soluble iron after 16 days. After this period, however, T. ferrooxidans cells could no longer be detected by the immunofluorescence assay, whereas the DNA-fluorescence staining procedure demonstrated a large number of microorganisms on the coal particles. These results indicate that T. ferrooxidans is removed by competition with different acidophilic microorganisms that were originally present on the coal.     

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.     

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

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

Isolation, chemical structure, acute toxicity, and some physicochemical properties of territrem B'' from Aspergillus terreus.

We investigated the production and regeneration of Thiobacillus ferrooxidans spheroplasts. Regeneration frequencies of up to 90% reversion were obtained. Attempts to transform T. ferrooxidans spheroplasts with a recombinant T. ferrooxidans plasmid were unsuccessful.