T. A. Pivovarova

[Sulfobacillus sibiricus sp. nov., a new moderately thermophilic bacterium].

In the course of pilot industrial testing of a biohydrometallurgical technology for processing gold-arsenic concentrate obtained from the Nezhdaninskoe ore deposit (East Siberia, Sakha (Yakutiya)), a new gram-positive rod-shaped spore-forming moderately thermophilic bacterium (designated as strain N1) oxidizing Fe2+, S0, and sulfide minerals in the presence of yeast extract (0.02%) was isolated from a dense pulp. Physiologically, strain N1 differs from previously described species of the genus Sulfobacillus in having a somewhat higher optimal growth temperature (55°C). Unlike the type strain of S. thermosulfidooxidans, strain N1 could grow on a medium with 1 mM thiosulfate or sodium tetrathionate as a source of energy only within several passages and failed to grow in the absence of an inorganic energy source on media with sucrose, fructose, glucose, reduced glutathione, alanine, cysteine, sorbitol, sodium acetate, or pyruvate. The G+C content of the DNA of strain N1 was 48.2 mol %. The strain showed 42% homology after DNA–DNA hybridization with the type strain of S. thermosulfidooxidans and 10% homology with the type strain of S. acidophilus. The isolate differed from previously studied strains of S. thermosulfidooxidans in the structure of its chromosomal DNA (determined by the method of pulsed-field gel electrophoresis), which remained stable as growth conditions were changed. According to the results of the 16S rRNA gene analysis, the new strain forms a single cluster with the bacteria of the species Sulfobacillus thermosulfidooxidans (sequence similarity of 97.9–98.6%). Based on these genetic and physiological features, strain N1 is described as a new species Sulfobacillus sibiricus sp. nov.

[Phenotypic features of Ferroplasma acidiphilum strains Yt and Y-2].

Earlier, we described a new family of mesophilic, strictly autotrophic Fe2+-oxidizing archaebacteria, Ferroplasmaceae, which belongs to the order Thermoplasmales and includes the genus Ferroplasma and the species F. acidiphilum (strain YT) [1]. The present work is concerned with a comparative study of phenotypic characteristics of the type strain YТ and a new strain, F. acidiphilum Y-2, isolated from dense pulps during oxidation of gold-containing arsenopyrite/pyrite concentrates from the Bakyrchikskoe (Kazakhstan) and Olimpiadinskoe (Krasnoyarsk krai) ore deposits, respectively. The G+C content of DNA from strains YT and Y-2 comprised 35.1 and 35.2 mol %, respectively; the level of DNA–DNA homology between the strains was 84%. Restriction profiles of chromosomal DNA from both strains exhibited a similarity coefficient of 0.87. Genotypic characteristics of these strains indicate their affiliation to the same species. The cells of both strains are polymorphic and lack cell walls. Strains of F. acidiphilum oxidized ferrous iron and pyrite as the sole source of energy and fixed carbon dioxide as the sole carbon source. The strains required yeast extract as a growth factor. Optimum pH for cell growth ranged from 1.7 to 1.8; the temperature optima for the growth of strains YT and Y-2 were 34–36 and 40–42°С, respectively. Comparative analysis of the total lipids revealed their close similarity in the strains; two glycophospholipids comprised 90% of the total lipids: lipid I, β-D-glucopyranosylcaldarchaetidylglycerol (about 55%), and lipid II, trihexosylcaldarchaetidylglycerol (26%), whose isopranyl chains contained no cyclopentane rings. The carbohydrate fraction of lipid I hydrolysate contained only D-glucose, whereas hydrolysate of lipid II contained both D-glucose and D-galactose in a molar ratio of 2 : 1. Thus, it was established that the intraspecies phylogenetic divergence within F. acidiphilum is manifested in the two strains by different temperature optima against a background of similarity in other phenotypic properties.

Diversity of the communities of acidophilic chemolithotrophic microorganisms in natural and technogenic ecosystems

The main representatives of acidophilic chemolithotrophs oxidizing sulfide minerals, ferrous iron, elemental sulfur, and reduced sulfur compounds and forming microbial communities in the natural and technogenic ecosystems with low pH values and high concentrations of heavy metal ions are listed. The species and strain diversity of the communities and environmental factors affecting their composition (temperature, pH value, energy substrate, mineralogical composition of sulfide ore concentrates, the presence of organic substances, and level of aeration) are analyzed. Involvement of mobile genetic elements (IS elements and plasmids) in the structural changes of the chromosomal DNA in the course of switching microbial metabolism to the oxidation of new energy substrates or under increased concentrations of metal ions is shown to be a probable mechanism responsible for the intraspecific genetic heterogeneity of the populations. Importance of determination of the dominant strains of different microbial species in the communities and of their physiological peculiarities for stabilization, optimization, and enhancement of efficiency of biotechnological processes for sulfide mineral oxidation is stressed.

Species composition of the association of acidophilic chemolithotrophic microorganisms participating in the oxidation of gold-arsenic ore concentrate

The species composition of the microbial association involved in industrial tank biooxidation of the concentrate of refractory pyrrhotite-containing pyrite-arsenopyrite gold-arsenic ore of the Olympiadinskoe deposit at 39°C was studied by cultural and molecular biological techniques. Pure microbial cultures were isolated, their physiological characteristics were investigated, and their taxonomic position was determined by 16S rRNA gene sequencing. The library of 16S rRNA gene clones obtained from the total DNA isolated from the biomass of the pulp of industrial reactors was analyzed. The diversity of microorganisms revealed by cultural techniques in the association of acidophilic chemolithotrophs (Acidithiobacillus ferrooxidans, Leptospirillum ferriphilum, Sulfobacillus thermosulfidooxidans, Ferroplasma acidiphilum, Alicyclobacillus tolerans, and Acidiphilium cryptum) was higher than the diversity of the 16S rDNA clone library (At. ferrooxidans, L. ferriphilum, and F. acidiphilum). The combination of microbiological and molecular biological techniques for the investigation of the biodiversity in natural and anthropogenic microbial communities promotes detection of new phylogenetic microbial groups in these communities.

Strain diversity of Thiobacillus ferrooxidans and its significance in biohydrometallurgy

Structural changes in chromosomal DNA of Thiobacillus ferrooxidans strains that occur under the influence of varied growth conditions were studied by pulsed-field gel electrophoresis. Strain diversity of T. ferrooxidans was manifested in different growth rates and oxidation rates of inorganic substrates under extreme conditions, in different resistance to metal ions and low pH values, and also in polymorphism of the chromosomal DNA fragments generated by the macrorestriction endonucleases. Adaptation of some strains to growth on media containing new substrates was accompanied by changes in the number and size of restriction fragments. Thus, new 177 and 164 kb DNA fragments were revealed after the substitution of the oxidation substrate from Fe2+ to FeS2 or from Fe2+ to So, respectively, whereas 115 and 77 kb DNA fragments disappeared. The switching from Fe2+ to So resulted in the change in the number of 27 kb DNA fragments. Another type of chromosomal DNA variability was found in the strains adapted to high concentrations of metal ions. A comparison of XbaI-restriction patterns in parent strains and in strains with acquired enhanced resistance to zinc (from 40 to 70 g/l) or arsenic (from 1.5 to 4.0 g/l) revealed amplification of 98 and 28 kb fragments, respectively. When both strains were subcultured on medium with Fe2+ without the inducing factors, amplification of DNA fragments was no longer detectable. However, the strain adapted tp 50 g/l of Fe2+/Fe3+ had a mutation in the structure of chromosomal DNA. The data obtained on the natural and experimental genomic variability of T. ferrooxidans strains provide biotechnologists with practical recommendations for selection aimed at the intensification of bioleaching processes and testify about possibilities of strain monitoring in natural and technological conditions. Strains with the labile genome have an advantage in biohydrometallurgy.

Strain Polymorphism of the Plasmid Profiles in Acidithiobacillus ferrooxidans

Plasmid profiles were studied in 27 Acidithiobacillus ferrooxidans strains isolated from different geographic zones and substrates differing in composition of the main sulfide minerals, and also in experimentally obtained strains with acquired enhanced resistance to the ions of heavy metals (Fe, Ni, Cu, Zn, As). In 16 out of 20 strains isolated from different substrates, one to four 2- to 20-kb and larger plasmids were revealed. Plasmids were found in all five strains isolated from gold-containing pyrite–arsenopyrite ores and concentrates, in nine of 11 strains isolated from the ores and concentrates containing nonferrous metals, and in two of four strains isolated from the oxidation substrates of simple composition (mine waters, pyritized coals, active sludge). Changes in the plasmid profiles in some A. ferrooxidans strains (TFZ, TFI-Fe, TFV-1-Cu) with experimentally enhanced resistance to Zn2+, Fe3+, and Cu2+, respectively, were noted as compared with the initial strains. After 30 passages on a S0-containing medium, strain TFBk showed changes in the copy number of plasmids. The role of plasmids in the processes of oxidation of energy substrates and in the acquired enhanced resistance to heavy metal ions is discussed.

Polymorphism of Sulfobacillus thermosulfidooxidans strains dominating in processes of high-temperature oxidation of gold-arsenic concentrate

The composition was studied of the microbial association involved in tank biooxidation of the concentrate of a refractory pyrrhotite-containing pyrite-arsenopyrite gold-arsenic ore from the Olympiadinskoe deposit at 50°C. The two Sulfobacillus thermosulfidooxidans strains predominant in the association were phylogenetically different from the strains used as inocula. The isolates were found to differ significantly both from each other and from the strains that dominated in the processes of biooxidation of a similar concentrate by traditional tank technology at 39°C or at 39°C with treatment of the concentrate with ferric iron prior to biooxidation. These results indicate the strain and species diversity of sulfobacilli in microbial associations involved in biooxidation of the concentrates under different technological modes.

Restriction Profiles of the Chromosomal DNA from Acidithiobacillus ferrooxidans Strains Adapted to Different Oxidation Substrates

Restriction profiles of chromosomal DNA were studied in different Acidithiobacillusferrooxidans strains grown on medium with Fe2+ and further adapted to another oxidation substrate (S0, FeS2, or sulfide ore concentrates). The restriction endonuclease XbaI digested the chromosomal DNA from different strains into different numbers of fragments of various sizes. Adaptation of two strains (TFBk and TFN-d) to new oxidation substrates resulted in structural changes in XbaI-restriction patterns of their chromosomal DNA. Such changes in the DNA restriction patterns occurred in strain TFBk after the adaptation to precyanidated gravitational pyrite-arsenopyrite concentrate (no. 1) from the Nezhdaninskoe deposit or to copper-containing ore from the Udokanskoe deposit and also in strain TFN-d adapted to untreated pyrite-arsenopyrite concentrate (no. 2) from the Nezhdaninskoe deposit. No changes in the number or size of the XbaI-restriction patterns of chromosomal DNA were revealed in either strain TFBk cultivated on media with pyrite from the Angren and Tulun deposits or in strains TFN-d and TFO grown on media with S0 and pyrite. Neither were changes observed in the XbaI-restriction patterns of the DNA from strain TFV-1, isolated from the copper ore of the Volkovskoe deposit, when Fe2+ was substituted with alternative substrates—S0, pyrite or concentrate no. 2 from the ore of the Nezhdaninskoe deposit. In strain TFO, no differences in the XbaI-restriction patterns of the chromosomal DNA were revealed between the culture grown on medium containing concentrate no. 2 or the concentrate of surface-lying ore from the Olimpiadinskoe deposit and the culture grown on medium with Fe2+. When strain TFO was cultivated on the ore concentrate from deeper horizons of the Olimpiadinskoe deposit, which are characterized by lower oxidation degrees and high antimony content, mutant TFO-2 differing from the parent strain in the chromosomal DNA structure was isolated. The correlation between the lability of the chromosomal DNA structure in A. ferrooxidans strains and the physical and chemical peculiarities of the isolation substrate and habitat is discussed.

Bioprocessing of Mining and Metallurgical Wastes Containing Non-Ferrous and Precious Metals

Mining and metallurgical treatments of sulphide ores are characterised by present significant losses of non-ferrous and precious metals as different types of waste. These elements are accumulated in heaps due to the lack of efficient technology for the recovery of the metals from metallurgical waste. The treatment of two types of industrial metallurgical waste (copper converter slag and old flotation pyrite tailings) containing non-ferrous and precious metals were examined in the laboratory. Leaching of the slag containing 2.74% Cu (as digenite, bornite, and free metal) and 2.49% Zn (as a ferrite ZnFe2O4 and silicate) by an Fe3+-containing solution was studied. The effect of various experimental parameters on the leaching dynamics of copper, zinc, and iron under batch conditions was investigated. The following experimental parameters were recommended: a pH of 1.5, a pulp density of 10% (w/v), a temperature of 70 °C, and an initial Fe3+ concentration of 15 g/L. Leaching under these conditions resulted in the solubilisation of 89.4% copper and 35.3% zinc within 2.5 hours. Percolation leaching of the pyrite tailings containing 0.29% Cu (as chalcopyrite), 0.26% Zn (as sphalerite), 0.00007% gold, and 0.00108% silver was also studied. Acidic percolation leaching and the resulting biooxidation lasting 134 days resulted in the solubilisation of 73.4% zinc and 50.8% copper. The recovery rates of gold and silver from the bioleaching residues by cyanidation were 57.2% and 50.7%, respectively. The data obtained in the present work may be used to estimate the operating parameters for the industrial-scale processing of non-ferrous and precious metals from mining and metallurgical waste.

Two-stage process of bacterial-chemical oxidation of refractory pyrite-arsenopyrite gold-bearing concentrate

A technology for tank biooxidation of refractory gold-bearing concentrate under variable temperature conditions has been improved: the temperature of the first of two stages was changed from 30°C to 34–36°C. Gold in this concentrate is mainly associated with sulfide minerals: arsenopyrite and pyrite, which underlies a low gold recovery (16.68%) as a result of cyanidation. To resolve the problem, an association of mesophilic acidophilic chemolithotrophic microorganisms and moderately thermophilic bacteria of the Sulfobacillus genus were used for the concentrate oxidation. The composition of the used microbial association was studied; it was shown that it depends upon temperature: at 42°C, the population of the mesophilic thiobacteria decreased, whereas that of thermophilic sulfobacilli enhanced as compared to 36°C. The accepted scheme of the process ensures a high extent of gold recovery (94.6%) within a short space of time for biooxidation (96 h).