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

Restriction profiles of chromosomal DNA were studied in different Acidithiobacillus ferrooxidans 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.     

Plasmid profiles of Acidithiobacillus ferrooxidans strains adapted to different oxidation substrates

Plasmid profiles were studied in five Acidithiobacillus ferrooxidans strains of various origin cultivated on medium with Fe2+, as well as adapted to such oxidation substrates as S0, FeS2, and sulfide concentrate. The method used revealed plasmids in all A. ferrooxidans strains grown on medium with Fe2+. One plasmid was found in strain TFL-2, two plasmids, in strains TFO, TFBk, and TFV-1, and three plasmids were detected in strain TFN-d. The adaptation of strain TFN-d to sulfide concentrate and the adaptation of strain TFV-1 to S0, FeS2, or sulfide concentrate resulted in a change in the number of plasmids occurring in cells. In cells of strain TFN-d adapted to sulfide concentrate, the number of plasmids decreased from three to two. The number of plasmids in cells of strain TFV-1 adapted to different substrates varied from three to six depending on the energy source present in the medium: three plasmids were found after growth on FeS2, four after growth on S0, and six after growth on sulfide concentrate. The possible role of plasmids in the adaptation of A. ferrooxidans to new energy substrates and in the regulation of the intensity of their oxidation is discussed.     

Phenotypic characteristics of Thiobacillus ferrooxidans strains

Phenotypic polymorphism of strains of Thiobacillus ferrooxidans isolated from various ecological niches was studied. The strains differed both in rates of growth and oxidation of Fe2+, S0, FeS2, and sulfide minerals contained in concentrate. Each strain, irrespective of its original environment, required a period of adaptation to a new substrate. Strains TFN-d, TFBk, TFO, and TFL-2, isolated from ores and concentrates rich in oxidized substrates, showed an equal adaptation pace (five culture transfers) but differed in their adaptation efficiency. Strain TFV-1, isolated from base ore and showing the lowest rates of growth and oxidation of all the substrates, required five culture transfers to adapt to S0 and FeS2 and seven culture transfers to adapt to the concentrate. It is concluded that the phenotypic properties of the strains correlate with their genotypic polymorphism and the environmental conditions under which their microevolution took place.     

Identification of IS elements in <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> strains grown in a medium with ferrous iron or adapted to elemental sulfur

IS elements were identified in the genomes of five Acidithiobacillus ferrooxidans strains isolated from various media. IST2 elements were revealed in all the strains grown in a medium with ferrous iron, ISAfe1 elements were detected in four strains (TFBk, TFL-2, TFV-1 and TFO). Three strains (TFV-1, TFN-d and TFO) were found to contain IS elements, ~600 bp long. These were named preliminary as ISAfe600. Partial sequencing of the 5′- and 3′-terminal nucleotide stretches of an ISAfe1 element in TFBk and TFL-2 strains and complete sequencing of the ISAfe1 element in the TFBk strain has revealed nucleotide substitutions as compared to the prototype, i.e., the ISAfe1 element of an ATCC 19859 strain. Partial sequencing of the 5′- and 3′-terminal nucleotide stretches of the IST2 elements in TFO, TFBk and TFL-2 strains has shown numerous nucleotide substitutions when compared to the IST2 element of an ATCC 19859 strain. Complete sequencing of the IST2 element in the TFBk strain has revealed: the divergence between the IST2 elements in the TFBk strain and the prototype was 21.2%. Southern hybridization of EcoRI fragments of the chromosomal DNA from five A. ferrooxidans strains grown in a medium with ferrous iron using an internal region of ISAfe1, a full-length ISAfe1 or a full-length IST2 as probes has shown them to differ in the number of copies of IS elements and their localization on the chromosomes. Adaptation to elemental sulfur in A. ferrooxidans strains caused changes in the number, intensity and localization of hybridization bands. The authors discuss the role of IS elements in the adaptation of A. ferrooxidans to the new energy substrate. The nucleotide sequence data reported in this paper were deposited in GenBank under accession numbers: AY823401, the ISAfe1 from A. ferrooxidans TFBk; AY825254, the IST2 from TFBk; DQ002894, the 5′-terminal nucleotide sequence of ISAfe1 from TFL-2; DQ002895, the 3′-terminal nucleotide sequence of ISAfe1 from TFL-2; DQ005952, the 5′-terminal nucleotide sequence of IST2 from TFV-1; DQ005953, the 3′-terminal nucleotide sequence of IST2 from TFV-1.     

Plasmid Profiles of Acidithiobacillus ferrooxidans Strains Adapted to Different Oxidation Substrates

Plasmid profiles were studied in five Acidithiobacillus ferrooxidans strains of various origin cultivated on a medium with Fe²⁺, as well as adapted to such oxidation substrates as S⁰, FeS₂, and sulfide concentrate. The method used revealed plasmids in all A. ferrooxidans strains grown on a medium with Fe²⁺. One plasmid was found in strain TFL-2; two plasmids, in strains TFO, TFBk, and TFV-1; and three plasmids were detected in strain TFN-d. The adaptation of strain TFN-d to sulfide concentrate and the adaptation of strain TFV-1 to S⁰, FeS₂, or sulfide concentrate resulted in a change in the number of plasmids occurring in cells. In cells of strain TFN-d adapted to sulfide concentrate, the number of plasmids decreased from three to two. The number of plasmids in cells of strain TFV-1 adapted to different substrates varied from three to six depending on the energy source present in the medium: three plasmids were found after growth on FeS₂, four after growth on S⁰, and six after growth on sulfide concentrate. The possible role of plasmids in the adaptation of A. ferrooxidans to new energy substrates and in the regulation of the intensity of their oxidation is discussed.     

Phenotypic Characteristics of Thiobacillus ferrooxidansStrains

Phenotypicpolymorphism of Thiobacillus ferrooxidansstrains isolated from various ecological niches was studied. The strains differed both in rates of growth and oxidation of Fe²⁺, S⁰, FeS₂, and sulfide minerals contained in concentrate. Each strain, irrespective of its original environment, required a period of adaptation to a new substrate. Strains TFN-d, TFBk, TFO, and TFL-2, isolated from ores and concentrates rich in oxidizable substrates, showed an equal adaptation rate (five culture transfers) but differed in their adaptation efficiency. Strain TFV-1, isolated from low-grade ore and showing the lowest rates of growth and oxidation of all the substrates, required five culture transfers to adapt to S⁰and FeS₂and seven culture transfers to adapt to the concentrate. It is concluded that the phenotypic properties of the strains correlate with their genotypic polymorphism and the environmental conditions under which their microevolution took place.     

Dependence of the genotypic characteristics of Acidithiobacillus ferrooxidans on the physical, chemical, and electrophysical properties of pyrites

Comparison of Acidithiobacillus ferrooxidans strains TFV-1 and TFBk with respect to their capacity to oxidize pyrite 1, with hole-type (p-type) conductivity, or pyrite 2, with an electron-type (n-type) conductivity, showed that, at a pulp density of 1%, both before and after its adaptation to the pyrites, strain TFBk, isolated from a substrate with a more complex mineral composition, grew faster and oxidized the pyrites of both conductivity types more efficiently than strain TFV-1, which was isolated from a mineralogically simple ore. At a pulp density of 3-5%, the oxidation of pyrite 1 by strain TFV-1 and both of the pyrites by strain TFBk began only after an artificial increase in Eh to 600 mV. If the pulp density was increased gradually, strain TFBk could oxidize the pyrites at its higher values than strain TFV-1, with the rate of pyrite 2 oxidation being higher than that of pyrite 1. During chemical oxidation of both of the pyrites, an increase was observed in the absolute values of the coefficients of thermoelectromotive force (KTEMF); during bacterial-chemical oxidation, the KTEMF of pyrite 1 changed insignificantly, whereas the KTEMF of pyrite 2 decreased.     

Strain polymorphism of the plasmid profiles in <Emphasis Type="Italic">Sulfobacillus</Emphasis> species

Plasmids were discovered for the first time in strains belonging to different species of the genus Sulfobacillus: S. thermosulfidooxidans, S. sibiricus, S. thermotolerans, “S. olympiadicus”, and S. acidophilus. The plasmids were detected in the cells of four out of eight strains grown on a medium with ferrous iron. Adaptation to elementary sulfur was accompanied by changes in the plasmid profiles in two out of seven strains. Plasmids were detected in all the studied strains of sulfobacilli after adaptation to the pyrite-arsenopyrite ore concentrate from the Nezhdaninskoe deposit containing gold, silver, zinc, copper, and lead. No plasmids were found in S. thermotolerans Kr1^T after four transfers on a medium containing iron and 0.018 mM Ag⁺. After adaptation of the same strain to 765 mM Zn²⁺, only one plasmid was found in the cells, the largest among those detected earlier in this culture adapted to the Nezhdaninskoe ore concentrate. The strain S. thermotolerans Kr1^T, after four transfers on media with either 78 mM Cu²⁺ or 2 mM Pb²⁺, did not contain plasmids. The presence of plasmids in the cells of sulfobacilli did not influence their resistance to the ions of the studied metals.     

Interaction of chromosomal and plasmid DNA in Acidithiobacillus ferrooxidans strains adapted to different oxidation substrates

Restriction analysis of plasmids pTFK1 and pTFK2 of the Acidithiobacillus ferrooxidans strain TFBk was carried out, and the sizes of these plasmids were determined (13.5 and 30 kb, respectively). A macrorestriction map was built for plasmid pTFK1. DNA-DNA hybridization revealed that the plasmids contained homologous nucleotide sequences. Plasmid pTFK2 labeled with 32P was used as a probe for Southern hybridization with blots of XbaI-generated fragments of the chromosomal DNA of A. ferrooxidans strains grown on a medium containing Fe2+ or adapted to different oxidation substrates. Low-intensity hybridization signals were observed for many fragments of the chromosomal DNA of the strains studied. In the process of adaptation to new oxidation substrates, the localization of bands producing the low-intensity hybridization signals changed in a number of cases. Certain fragments of the chromosomal DNA of the strains adapted to different oxidation substrates produced strong hybridization signals with pTFK2. The data obtained are discussed in terms of the possible role of IST elements and plasmids in the adaptation of A. ferrooxidans to new energy substrates, microevolution, and strain polymorphism.     

Rates of sulfide mineral oxidation by acidophilic chemolithotrophic microbial communities from various sources

A correlation was observed between the rate of oxidation of pure sulfide minerals (pyrite, pyrrhotite, and arsenopyrite) by communities of acidophilic chemolithotrophic microorganisms (ACM) and the mineral substrate where these communities were formed. The ACM community formed during continuous oxidation of the pyrite-arsenopyrite ore concentrate (Kyuchus deposit) exhibited the highest rate of pyrite oxidation. The highest rate of pyrrhotite oxidation was observed for the ACM community developed during semicontinuous oxidation of the pyrrhotite-containing pyrite-arsenopyrite ore concentrate (Olympiadinskoe deposit), by the communities isolated from the pyrrhotite concentrate, and ore of the Shanuch deposit. In the case of arsenopyrite oxidation, the ACM community isolated during oxidation of the Olympiadinskoe ore concentrate grew without a lag phase. Other communities commenced arsenopyrite oxidation at various rates only after a two-day lag phase. The similarity of the mineralogical characteristics of pure sulfide minerals with those of the minerals in the substrates where the ACM communities developed may affect the rates of oxidation.     

Dependence of the Genotypic Characteristics of <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> on the Physical,Chemical, and Electrophysical Properties of Pyrites

This study focused on the effect of physical, chemical, and electrophysical properties of two pyrites, pyrite 1, which had electron-type (n-type) conductivity, and pyrite 2, with hole-type (p-type) conductivity, on the genotypic characteristics of Acidithiobacillus ferrooxidans strains TFV-1 and TFBk, which were isolated from different substrates. After the adaptation of the strains to the pyrites at a pulp density of 1%, pulsed-field electrophoresis revealed changes in the chromosomal DNA of strain TFV-1 adapted to pyrite 1, and strain TFBk adapted to either of the pyrite types. In pyrite-adapted strain TFBk, the plasmid composition was the same as after growth on a medium containing ferrous iron, whereas, in strain TFV-1, changes in plasmid sizes or both in plasmid sizes and plasmid number occurred. After an increase in the density of the pyrite 2 pulp from 1 to 10%, the plasmid number increased from three to four, and, after an increase in the density of the pyrite 1 pulp from 1 to 7%, the plasmid number increased from two to six.     

Genotypic and phenotypic polymorphism of environmental strains of the moderately thermophilic bacterium Sulfobacillus sibiricus

Five cultures of moderately thermophilic spore-forming acidophilic chemolithotrophic bacteria were isolated from the zones of spontaneous heating of pyrrhotine-containing pyrite-arsenopyrite gold-arsenic sulfide ores in an operating open pit (strains B1, B2, B3, OFO, and SSO). Analysis of the chromosomal DNA structure revealed differences between these cultures at the strain level (apart from B3 and SSO, which had identical restriction profiles). All the strains had a similar G + C DNA molar content (47.4-48.3%). The level of DNA reassociation was 85 to 95%. The similarity between the DNA of the type strain Sulfobacillus sibiricus N1 isolated from arsenopyrite ore concentrate and that of these strains (83-93%) indicates that they belong to the same species. The strains had similar values of pH and temperature optimal for growth on ferrous iron (1.6-2.0 and 45-55 degrees C, respectively). They were mixotrophs; Fe(II), S0, and sulfide minerals along with organic compounds were used as energy sources and electron donors. However, the kinetic parameters of growth and substrate oxidation varied from strain to strain. Genetic variety of the strains from diverse ecosystems and environments is possibly the result of the different rates of microevolution processes.     

Patterns of growth and oxidation of natural pyrites by the representatives of acidophilic chemolithotrophic microorganisms

The patterns of the growth and oxidation of different types of natural pyrites were studied for the three microbial species adapted to these substrates and belonging to phylogenetically remote groups: gram-negative bacterium Acidithiobacillus ferrooxidans, gram-positive bacterium Sulfobacillus thermotolerans, and the archaeon Ferroplasma acidiphilum. For both A. ferrooxidans strains, TFV-1 and TFBk, pyrite 4 appeared to be the most difficult to oxidize and grow; pyrite 5 was oxidized by both strains at an average rate, and pyrite 3 was the most readily oxidized. On each of the three pyrites, growth and oxidation by TFBk were more active than by TFV-1. The effectiveness of the adaptation of S. thermotolerans Kr1^T was low compared to the A. ferrooxidans strains; however, the adapted strain Kr1^T showed the highest growth rate on pyrite 3 among all the cultures studied. No adaptation of strain Kr1^T to pyrite 5 was observed; the rates of growth and pyrite oxidation in the third transfer were lower than in the first transfer. The strain F. acidiphilum Y^T was not adapted to pyrites 3 and 5; the rates of growth and pyrite oxidation were the same in the first five transfers. The strains of three species of the microorganisms studied, A. ferrooxidans, S. thermotolerans, and F. acidiphilum, grew on pyrite 3 (holetype (p) conductivity) and oxidized it better than pyrite 5 (mixed-type (n-p) conductivity). The most readily oxidized were the pyrites with a density of 5.6–5.7 g/cm³ and high resistance values (ln R = 8.8). The pyrite oxidation rate did not depend on the type of conductivity. Changes in the chromosomal DNA structure were revealed in strain TFBk on adaptation to pyrites 3 and 4 and in the TFV-1 plasmid profile on adaptation to pyrite 3. Correlation between genetic variability and adaptive capabilities was shown for A. ferrooxidans. No changes in the chromosomal DNA structure were found in S. thermotolerans Kr1^T and F. acidiphilum Y^T on adaptation to pyrites 3 and 5. Plasmids were absent in the cells of these cultures.     

Genotypic and phenotypic polymorphism of environmental strains of the moderately thermophilic bacterium <Emphasis Type="Italic">Sulfobacillus sibiricus</Emphasis>

Five cultures of moderately thermophilic spore-forming acidophilic chemolithotrophic bacteria were isolated from the zones of spontaneous heating of pyrrhotite-containing pyrite-arsenopyrite gold-arsenic sulfide ores in an operating open pit (strains B1, B2, B3, OFO, and SSO). Analysis of the chromosomal DNA structure revealed the differences between these cultures at the strain level (apart from B3 and SSO, which had identical restriction profiles). All the strains had a similar G+C DNA molar content (47.4–48.3%). The level of DNA reassociation was 85 to 95%. The similarity between the DNA of the type strain Sulfobacillus sibiricus N1 isolated from arsenopyrite ore concentrate and that of these strains (83–93%) indicates that they belong to the same species. The strains had similar values of pH and temperature optimal for growth on ferrous iron (1.6–2.0 and 45–55°C, respectively). They were mixotrophs; Fe(II), S^o, and sulfide minerals along with organic compounds were used as energy sources and electron donors. However, the kinetic parameters of growth and substrate oxidation varied from strain to strain. Genetic variety of the strains from diverse ecosystems and environments is possibly the result of the different rates of microevolution processes.     

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.     

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.     

Polymorphism of <Emphasis Type="Italic">Sulfobacillus thermosulfidooxidans</Emphasis> 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.     

Dependence of the Phenotypic Characteristics of <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> on the Physical,Chemical, and Electrophysical Properties of Pyrites

Comparison of Acidithiobacillus ferrooxidans strains TFV-1 and TFBk with respect to their capacity to oxidize pyrite 1, with an electron-type (n-type) conductivity, or pyrite 2, with hole-type (p-type) conductivity, showed that, at a pulp density of 1%, both before and after its adaptation to the pyrites, strain TFBk, isolated from a substrate with a more complex mineral composition, grew faster and oxidized the pyrites of both conductivity types more efficiently than strain TFV-1, which was isolated from a mineralogically simple ore. At a pulp density of 3–5%, the oxidation of pyrite 2 by strain TFV-1 and both of the pyrites by strain TFBk began only after an artificial increase in Eh to 600 mV. If the pulp density was increased gradually, strain TFBk could oxidize the pyrites at its higher values than strain TFV-1, with the rate of pyrite 2 oxidation being higher than that of pyrite 1. During chemical oxidation of both of the pyrites, an increase was observed in the absolute values of the coefficients of thermoelectromotive force (K_TEMF); during bacterial-chemical oxidation, the K_TEMF of pyrite 1 changed insignificantly, whereas the K_TEMF of pyrite 2 decreased.     

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

In the course of pilot industrial testing of a biohydrometallurgical technology for processing goldarsenic 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 degrees C). Unlike the type strain of S. thermosulfidooxidans, strain N1 could grow on 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.     

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.