A. E. Zhuravleva

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.

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.

Regulation of metabolic pathways in sulfobacilli under different aeration regimes

A comparative study of the activities of the enzymes of carbon metabolism from the cells of moderately thermophilic chemolithotrophic bacteria Sulfobacillus sibiricus (strains N1 and SSO) and Sulfobacillus thermosulfidooxidans subsp. asporogenes (strain 41) was carried out grown in a high layer of medium without forced aeration and cells grown with intense aeration. Limited air access to the growing S. sibiricus N1 cells resulted in switching from the pentose phosphate pathway of glucose metabolism to the Entner-Doudoroff pathway while the Embden-Meyerhof-Parnas pathway persisted. Irrespective of the level of the aeration, in the cells of S. sibiricus SSO and S. thermosulfidooxidans subsp. asporogenes 41, degradation of the glucose occurred via the Entner-Doudoroff and pentose phosphate metabolic pathways, respectively, as well as via the Embden-Meyerhof-Parnas pathway. Prolonged growth of S. sibiricus, strains N1 and SSO, in a high layer of the medium without forced aeration led to the repression of synthesis of most of the tricarboxylic acid cycle (TCA cycle) enzymes, in particular dehydrogenases, as well as of some carboxylases including RuBisCO. The traits of carbon metabolism in various strains of Sulfobacillus under conditions of oxygen deficiency are discussed.

Phenotypic properties of Sulfobacillus thermotolerans: Comparative aspects

The phenotypic characteristics of the species Sulfobacillus thermotolerans Kr1T, as dependent on the cultivation conditions, are described in detail. High growth rates (0.22–0.30 h−1) and high oxidative activity were recorded under optimum mixotrophic conditions at 40 °C on medium with inorganic (Fe(II), S0, or pyrite-arsenopyrite concentrate) and organic (glucose and/or yeast extract) substrates. In cells grown under optimum conditions on medium with iron, hemes a, b, and, most probably, c were present, indicating the presence of the corresponding cytochromes. Peculiar extended structures in the form of cylindrical cords, never observed previously, were revealed; a mucous matrix, likely of polysaccharide nature, occurred around the cells. In the cells of sulfobacilli grown litho-, organo-, and mixotrophically at 40 °C, the enzymes of the three main pathways of carbon utilization and some enzymes of the TCA cycle were revealed. The enzyme activity was maximum under mixotrophic growth conditions. The growth rate in the regions of limiting temperatures (55 °C and 12–14 °C) decreased two-and tenfold, respectively; no activity of 6-phosphogluconate dehydrogenase, one of the key enzymes of the oxidative pentose phosphate pathway, could be revealed; and a decrease in the activity of almost all enzymes of glucose metabolism and of the TCA cycle was observed. The rate of 14CO2 fixation by cells under auto-, mixo-, and heterotrophic conditions constituted 31.8, 23.3, and 10.3 nmol/(h mg protein), respectively. The activities of RuBP carboxylase (it peaked during lithotrophic growth) and of carboxylases of heterotrophic carbon dioxide fixation were recorded. The physiological and biochemical peculiarities of the thermotolerant bacillus are compared versus moderately thermophilic sulfobacilli.

The dependence of intracellular ATP level on the nutrition mode of the acidophilic bacteria Sulfobacillus thermotolerans and Alicyclobacillus tolerans

The dynamics of the ATP pool in the aerobic spore-forming acidothermophilic mixotrophic bacteria Sulfobacillus thermotolerans Kr1T and Alicyclobacillus tolerans K1T were studied in the course of their chemolithoheterotrophic, chemoorganoheterotrophic, and chemolithoautotrophic growth. It was established that, during mixotrophic growth, the maximum ATP concentrations in the cells of S. thermotolerans Kr1 and A. tolerans K1 were 3.8 and 0.6 nmol/mg protein, respectively. The ATP concentrations in sulfobacilli and alicyclobacilli during organotrophic growth were 2.2 and 3.1 nmol/mg protein, respectively. In the cells of the obligately heterotrophic bacterium Alicyclobacillus cycloheptanicus 4006T, the maximum ATP concentration was several times higher and reached 12.3 nmol/mg protein. During lithotrophic growth, the maximum values of the ATP concentration in the cells of S. thermotolerans Kr1 and A. tolerans K1 were 0.3 and <0.1 nmol/mg protein, respectively; in the cells of the autotrophic bacterium Acidithiobacillus ferrooxidans TFBk, the ATP content was about 60–300 times higher (17.0 nmol/mg protein). It is concluded that low ATP content is among the possible causes of growth cessation of S. thermotolerans Kr1 and A. tolerans K1 under auto-and heterotrophic conditions after several culture transfers.

Biooxidation of a gold-containing sulfide concentrate in relation to changes in physical and chemical conditions

The growth of a microbial community and the oxidation of iron- and sulfur-containing substrates in batch culture during the leaching/oxidation of the flotation concentrate of refractory gold-arsenic sulfide ore were optimized with respect to the following medium parameters: temperature, pH, and requirement in organic substances. It was revealed that the optimum mode is (i) to maintain the pH at 1.6–1.7 and the temperature at 34–35 and 38°C and (ii) to add Corg in the form of yeast extract (0.02%). Mutually beneficial or competitive relationships among groups of microorganisms of the community were established, depending on the cultivation conditions.

Oxidation of gold-antimony ores by a thermoacidophilic microbial consortium

Antimony leaching from sulfide ore samples by an experimental consortium of thermoacidophilic microorganisms, including Sulfobacillus, Leptospirillum, and Ferroplasma strains was studied. The ores differed significantly in the content of the major metal sulfides (%): SbS, 0.84 to 29.95; FeS, 0.47 to 2.5, and AsS, 0.01 to 0.4. Independent of the SbS concentration in the experimental sample, after adaptation to a specific ore and pulp compaction, the microorganisms grew actively and leached/oxidized all gold-antimony ores at 39 ± 1°C. The lower was the content of iron and arsenic sulfides, the higher was antimony leaching. For the first time the investigations conducted with the use of X-ray microanalysis made it possible to conclude that, in a natural high-antimony ore, Sb inhibits growth of only a part of the cell population and that Ca, Fe, and Sb may compete for the binding centers of the cell.

Functional diversity of an aboriginal microbial community oxidizing the ore with high antimony content at 46–47°C

A procedure for rapid (7–10 days) obtaining of enrichment cultures of aboriginal thermoacidophilic microbial communities from ores with high antimony content (Sb 26%) was developed. This technique allows for rapid alkalization of the medium due to the abundance of calcites, as well as the low antioxidant status of the initial cells. The ore concentration in the medium was gradually increased to 10 g/l. In the course of this process, selection of enrichment cultures containing microbial strains preferentially oxidizing ore, S0, or Fe2+ is carried out. A combination of three enrichment cultures allowed us to rapidly (in six days) adapt the aboriginal strains to high-density pulp (16%) in the reactor at 46°C, as well as to carry out a three-stage semi-continuous cultivation in the reactors at D = 0.0042 h−1 and to isolate from each reactor the pure cultures of predominant bacteria involved in the process of bioleaching/oxidation of the mixture of antimonite-containing ores and sulfide flotation concentrates. It was demonstrated that, in the microbial community of reactor I, strain Sb-K exhibiting high rates of growth and initial substrate oxidation was predominant. In reactor II, strain Sb-F prevailed, showing a high substrate specificity with respect to Fe2+. A sulfur-oxidizing strain involved in active oxidation of reduced inorganic sulfur compounds (RISCs) was predominant in reactor III. Nevertheless, together, all three strains showed synergism and were able to oxidize S0, Fe2+, and sulfide minerals (including antimonite Sb2S3 in the presence of 0.02% yeast extract) in reactors. The strains differed from each other in their DNA restriction profiles, growth rates, and the rates of inorganic substrate oxidation under mixotrophic conditions. The phenotypic properties of all the studied isolates have a certain similarity to those of sulfobacilli.

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%).

Specific characteristics of the strains isolated from a thermoacidophilic microbial community oxidizing antimony sulfide ore

Investigation of the phenotypic properties of three mixotrophic bacteria, strains Sb-K, Sb-F, and Sb-S, isolated from an aboriginal thermoacidophilic microbial community participating in biooxidation of ore with high antimony content (26%) and ore concentrates from the Olympiadinskoe deposit under semicontinuous cultivation conditions at 46 ± 1°C, revealed the differentiating characteristics of these strains. The isolated cultures grew lithotrophically through different numbers of transfers: strains Sb-F and Sb-K grew through seven and eight transfers, respectively, and strain Sb-S grew through two or three transfers. Strains Sb-K and Sb-S utilized a wide range of organic substrates for active organotrophic growth during nine or ten transfers, while strain Sb-F was less tolerant to organic compounds. Strain Sb-K grew on a medium with the ore and sulfide ore concentrates in the pH range of 1.0–3.0. Growth of strains Sb-F and Sb-S occurred in the pH ranges of 1.0–2.5 and 1.5–5.5 on media with Fe2+ and S0, respectively.. The optimal initial pH values of the media, corresponding to the maximum specific growth rates, were 1.6–1.7, 1.9, and 2.0–3.0 for strains Sb-K, Sb-F, and Sb-S, respectively. All three strains were able to grow within a broad temperature range, 20–65°C, with an optimum at 46°C (Sb-K), 40–46°C (Sb-F), and 48–50°C (Sb-S). According to the results of DNA-DNA hybridization and phylogenetic analysis, as well as their phenotypic characteristics, the isolates can be classified as novel strains of species of the genus Sulfobacillus. Strains Sb-K, Sb-F, and Sb-S, isolated as predominant cultures on the media with sulfide compounds, iron, or sulfur, respectively, were affiliated to the species S. thermotolerans, S. sibiricus, and S. thermosulfidooxidans.