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.     

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.     

Evaluation of Leptospirillum ferrooxidans for Leaching

The importance of Leptospirillum ferrooxidans for leach processes has been evaluated by studying the lithotrophic flora of three mine biotopes and a heap leaching operation, by percolation experiments with inoculated, sterilized ore, and by morphological, physiological, and genetic investigations of pure and mixed cultures of L. ferrooxidans, Thiobacillus ferrooxidans, and Thiobacillus thiooxidans. In biotopes of 20°C or above, Leptospirillum-like bacteria are as abundant as T. ferrooxidans. Leptospirilli represent at least one-half of the ferrous-iron-oxidizing population. Percolation experiments confirmed this result. Leptospirilli were as numerous as T. ferrooxidans. At reduced temperatures, the generation times of leptospirilli increase more so than those of T. ferrooxidans. At 14°C, Leptospirillum grows slowly and T. ferrooxidans dominates the population. Physiological investigations indicate that L. ferrooxidans is a strict chemolithoautotroph, metabolizing only ferrous iron and pyrite. Even an addition of 0.05% (wt/vol) yeast extract inhibited its growth. The maximum ferrous-iron-oxidizing activity of L. ferrooxidans amounts to about 40% of the activity of T. ferrooxidans. After growth on sulfidic ore, both species exhibit reduced iron-oxidizing activities, L. ferrooxidans exhibiting one-third and T. ferrooxidans exhibiting one-seventh of their maximum activities. Surprisingly, the absolute values are similar. For indirect leaching, L. ferrooxidans is as important as T. ferrooxidans. This was confirmed by the results of percolation experiments. L. ferrooxidans together with T. thiooxidans mobilized metals at least as well as T. ferrooxidans did. The best results were obtained with a mixed culture of all three species.     

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.     

Growth and Maintenance of Thiobacillus ferrooxidans Cells

A rapid and sensitive spectrophotometric procedure was developed for monitoring the growth of Thiobacillus ferrooxidans in liquid culture. Values determined for the optical densities at 500 nm of washed T. ferrooxidans cell suspensions were directly proportional to both total cell number and total cell protein concentration and provided an accurate measurement of culture growth rate. The utility of this procedure was demonstrated by conducting physiological studies on the influence of CO₂ and FeSO₄ availability on the growth of T. ferrooxidans. In addition, we describe a procedure for the long-term maintenance of cells T. ferrooxidans that ensures culture purity and genetic stability.     

Construction of arsenic-resistant Thiobacillus ferrooxidans recombinant plasmids and the expression of autotrophic plasmid genes in a heterotrophic cell-free system

Recombinant plasmids between Thiobacillus ferrooxidans and Escherichia coli which contain arsenic and antibiotic resistance genes and unique restriction sites have been constructed. The expression of autotrophic T. ferrooxidans genes in a heterotrophic E. coli cell-free system was demonstrated.     

The effect of different Fe2+ concentrations in culture media on the recycling of ground tyre rubber by Acidithiobacillus ferrooxidans YT-1

Waste rubber has posed challenging environmental and disposal problems across the world. This study focused on the microbial reclaiming of ground tyre rubber (GTR) by Acidithiobacillus ferrooxidans YT-1 cultured in media with variable Fe²⁺ concentrations. The Acidithiobacillus ferrooxidans YT-1 strain with the ability of oxidizing sulfur and reclaiming waste rubber was isolated and identified. Toxicity tests of different rubber and additives in tyre rubber compounds to microorganisms was quantitatively investigated. After desulfurization, there were many small colonies on the surface of the desulfurizated GTR (DGTR), due to surface degradation by A. ferrooxidans YT-1. The amount of small colonies increased and sulfur content decreased with the increase of Fe²⁺ concentrations in the media, implying that Fe²⁺ concentration had a great influence on the degradation ability of A. ferrooxidans YT-1. A medium with a high Fe²⁺ concentration was good for growth of A. ferrooxidans YT-1. Compared with styrene butadiene rubber (SBR)/GTR blends, the tensile strength and elongation at the break of the SBR/DGTR blends were significantly improved. The scanning electron microscope (SEM) photographs of the fracture surface further indicated a good coherency between DGTR and the SBR matrix. These results revealed that A. ferrooxidans YT-1 cultured in a medium with a high Fe²⁺ concentration could improve the reclaiming efficiency of waste rubber.     

Existence of a Hydrogen Sulfide:Ferric Ion Oxidoreductase in Iron-Oxidizing Bacteria

The existence of a hydrogen sulfide:ferric ion oxidoreductase, which catalyzes the oxidation of elemental sulfur with ferric ions as an electron acceptor to produce ferrous and sulfite ions, was assayed with washed intact cells and cell extracts of various kinds of iron-oxidizing bacteria, such as Thiobacillus ferrooxidans 13598, 13661, 14119, 19859, 21834, 23270, and 33020 from the American Type Culture Collection, Leptospirillum ferrooxidans 2705 and 2391 from the Deutsche Sammlung von Mikroorganismen, L. ferrooxidans BKM-6-1339 and P3A, and moderately thermophilic iron-oxidizing bacterial strains BC1, TH3, and Alv. It was found that hydrogen sulfide:ferric ion oxidoreductase activity comparable to that of T. ferrooxidans AP19-3 was present in all iron-oxidizing bacteria tested, suggesting a wide distribution of this enzyme in iron-oxidizing bacteria.     

Kinetic study of ferrous sulphate oxidation of Acidithiobacillus ferrooxidans in the presence of heavy metal ions

Acidophilic microorganisms such as Acidithiobacillus ferrooxidans have the capability to carry out processes of bioleaching, biosorption and bioprecipitation of heavy metal ions, which have important environmental applications. At. ferrooxidans derives the energy for their metabolism from ferrous iron oxidation, process, which can be affected by the presence of heavy metals in the medium. Moreover, organic matter produces an inhibitory effect over the ferrous iron oxidation of At. ferrooxidans. In this work, heterotrophic bacterium Acidiphilium sp. was added when the medium is supplemented with organic matter to reduce this negative effect. The purpose of this work is the kinetic study of ferrous sulphate oxidation by At. ferrooxidans in the presence of different concentrations of several heavy metal ions (Cr(III), Cu(II), Cd(II), Zn(II) and Ni(II)) and compare this kinetic behaviour with a mixed culture with Acidiphilium sp.The obtained results show a non-competitive inhibition of heavy metals over bacterial oxidation of ferrous sulphate. In accordance with this kind of inhibition, a kinetic equation has been proposed to predict the behaviour of At. ferrooxidans in the presence of heavy metals in the range of concentrations studied.     

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.     

Specific Dot-Immunobinding Assay for Detection and Enumeration of Thiobacillus ferrooxidans

A specific and very sensitive dot-immunobinding assay for the detection and enumeration of the bioleaching microorganism Thiobacillus ferrooxidans was developed. Nitrocellulose spotted with samples was incubated with polyclonal antisera against whole T. ferrooxidans cells and then in ¹²⁵I-labeled protein A or ¹²⁵I-labeled goat antirabbit immunoglobulin G; incubation was followed by autoradiography. Since a minimum of 10³ cells per dot could be detected, the method offers the possibility of simultaneous processing of numerous samples in a short time to monitor the levels of T. ferrooxidans in bioleaching operations.     

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.     

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.     

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.     

Amplification of ribulose bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU) gene fragments from Thiobacillus ferrooxidans and a moderate thermophile using polymerase chain reaction

Southern blot analysis of DNA from an iron-oxidising moderate thermophile NMW-6 and from Thiobacillus ferrooxidans strain TF1–35 demonstrated sequences homologous to the RuBisCO LSU gene of Synechococcus. DNA fragments (457 bp) encoding part of the RuBisCO LSU gene (amino acids 73–200) were amplified from the genomic DNA of Thiobacillus ferrooxidans and the moderate thermophile NMW-6 using the polymerase chain reaction (PCR) technique (Saiki et al. (1985) Science 233, 1350–1354). A comparison with the LSU sequences from T. ferrooxidans, Alcaligenes eutrophus, Chromatium vinosum, Synechococcus and Spinacea oleracea, which all have RuBisCOs with a hexadecameric structure, showed that the RuBisCO LSU gene sequence from NMW-6 appeared to be most closely related to that of the hydrogen bacterium A. eutrophus which showed 71.9% homology at the amino acid level. Despite its physiological similarity, T. ferrooxidans showed only 64.1% homology to the amino acid sequence from NMW-6 and had the lowest DNA homology (60.9%) of the hexadecameric type RuBisCOs. In the region sequenced, T. ferrooxidans and the RuBisCOs of the phototrophs C. vinosum, Synechococcus and S. oleracea, had 17 residues that were completely conserved which were substituted in both NMW-6 and A. eutrophus, 11 of these being identical substitutions. Comparison of the nucleotide and derived amino acid sequences of the RuBisCO LSU fragment from T. ferrooxidans with other RuBisCO sequences indicated a closer relationship to the hexadecameric type LSU genes of photosynthetic origin than to that of A. eutrophus. The T. ferrooxidans amino acid sequence showed 93.8%, 78.9% and 77.3% homology, respectively, to the C. vinosum, Synechococcus and S. oleracea (spinach) sequences but only 56.2% to A. eutrophus. The DNA sequence from Rhodospirillum rubrum, which has the atypical large subunit dimer RuBisCO structure with no small subunit, showed 39.2% and 42.7% homology, respectively, with the sequences of NMW-6 and T. ferrooxidans, and 25.0% and 29.7% amino acid homology, indicating that the DNA homology was substantially random in nature. PCR fragments (126 bp) that overlaped the last 15 codons of the fragments above were also amplified and sequenced. They showed incomplete homology with the larger fragments, supporting evidence obtained from Southern hybridizations that T. ferrooxidans and the moderate thermophile NMW-6 have multiple copies of RuBisCO LSU genes.     

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

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

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.