Thermoacidophilic microbial community oxidizing the gold-bearing flotation concentrate of a pyrite-arsenopyrite ore

An aboriginal community of thermophilic acidophilic chemolithotrophic microorganisms (ACM) was isolated from a sample of pyrite gold-bearing flotation concentrate at 45–47°C and pH 1.8–2.0. Compared to an experimental thermoacidophilic microbial consortium formed in the course of cultivation in parallel bioreactors, it had lower rates of iron leaching and oxidation, while its rate of sulfur oxidation was higher. A new thermophilic acidophilic microbial community was obtained by mutual enrichment with the microorganisms from the experimental and aboriginal communities during the oxidation of sulfide ore flotation concentrate at 47°C. The dominant bacteria of this new ACM community were Acidithiobacillus caldus (the most active sulfur oxidize) and Sulfobacillus thermotolerans (active oxidizer of both iron and sulfur), while iron-oxidizing archaea of the family Ferroplasmaceae and heterotrophic bacteria Alicyclobacillus tolerans were the minor components. The new ACM community showed promise for leaching/oxidation of sulfides from flotation concentrate at high pulp density (S : L = 1 : 4).     

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.     

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.     

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.     

Leaching of pyrite-arsenopyrite concentrate in bioreactors during continuous cultivation of a thermoacidophilic microbial community

A community of thermoacidophilic chemolithotrophic microorganisms was shown to exhibit enhanced efficiency of leaching and biooxidation of the gold-bearing pyrite-arsenopyrite flotation concentrate in continuous mode of cultivation. Under the optimal values of growth parameters, the degree of oxidation of sulfide arsenic, iron, sulfur, and antimony in the line of three laboratory reactors (D = 0.004 h^?1) was 99.55, 98.87, 99.65, and 97.08%, respectively, while gold recovery from the solid biooxidation residue was 97.4%.     

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.     

Characteristics of the restriction profile of chromosomal DNA in strains of Acidithiobacillus ferroxidans,adapted to various 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 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 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 degree 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 chromosomal DNA structure in A. ferrooxidans strains and the physical and chemical peculiarities of the isolation substrate and habitat is discussed.     

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.     

Effect of temperature on the rate of oxidation of pyrrhotite-rich sulfide ore flotation concentrate and the structure of the acidophilic chemolithotrophic microbial community

Oxidation of flotation concentrate of a pyrrhotite-rich sulfide ore by acidophilic chemolithoautotrophic microbial communities at 35, 40, and 45°C was investigated. According to the physicochemical parameters of the liquid phase of the pulp, as well as the results of analysis of the solid residue after biooxidation and cyanidation, the community developed at 40°C exhibited the highest rate of oxidation. The degree of gold recovery at 35, 40, and 45°C was 89.34, 94.59, and 83.25%, respectively. At 40°C, the highest number of microbial cells (6.01 × 10⁹ cells/mL) was observed. While temperature had very little effect on the species composition of microbial communities (except for the absence of Leptospirillum ferriphilum at 35°C), the shares of individual species in the communities varied with temperature. Relatively high numbers of Sulfobacillus thermosulfidooxidans, the organism oxidizing iron and elemental sulfur at higher rates than other acidophilic chemolithotrophic species, were observed at 40°C.     

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.     

Effect of the aeration mode and yeast extract on the oxidation of high-pyrrhotite sulfide ore flotation concentrate and on the composition of the acidophilic chemolithotrophic microbial community

Optimal aeration conditions were determined and the effect of yeast extract on biooxidation of high-pyrrhotite sulfide ore flotation concentrate in the course of continuous cultivation of an acidophilic chemolithotrophic microbial community was studied in a line of four sequential laboratory reactors; the aeration rate was 3 L/(L min), yeast extract concentration was 0.02%. The gold recovery level was 96.45% at 2.23% elemental sulfur content in the solid residue. The dominant strains identified in the community responsible for biooxidation were Acidithiobacillus caldus OL13-1, At. caldus OL13-3 = At. caldus OL12-3, and an ‘Acidiferrobacter’ strain. Strains Sulfobacillus thermosulfidooxidans OL13-2 = S. thermosulfidooxidans OL12-1 and Ferroplasma acidiphilum OL13-4 = F. acidiphilum OL12-4 were isolated in pure culture and identified.     

Changes in the species composition of a thermotolerant community of acidophilic chemolithotrophic microorganisms upon switching to the oxidation of a new energy substrate

Construction and analysis of the 16S rDNA clone libraries was used to investigate the species composition of two thermotolerant communities of acidophilic chemolithotrophic microorganisms (ACM) isolated from the pulp of laboratory reactors used for oxidation of different gold-containing ore concentrates. The first community was formed during oxidation of the pyrite-arsenopyrite ore concentrate from the Kyuchus deposit. The clones of the bacterial component of this community belonged to the genera Sulfobacillus (32 clones) and Leptospirillum (33 clones). The Sulfobacillus clones belonged to three groups: Sb. thermosulfidooxidans, Sb. benefaciens, and Sb. thermotolerans. All Leptospirillum clones were closely related to L. ferriphilum. All clones of the archaeal component belonged to Ferroplasma acidiphilum. The microorganisms of this community were used as inoculum for biooxidation of a different mineral concentrate, the pyrrhotite-containing pyrite-arsenopyrite ore concentrate from the Olympiadinskoe deposit, and the structure of the community formed in the process was investigated. The clones of the bacterial component of the second community also belonged to the genera Sulfobacillus (14 clones) and Leptospirillum (48 clones). The Sulfobacillus clones belonged to the species Sb. thermosulfidooxidans (13 clones) and Sb. thermotolerans (1 clone). All Leptospirillum clones were closely related to L. ferriphilum. All clones of the archaeal component belonged to Ferroplasma acidiphilum. During the adaptation of the community to a new oxidized mineral substrate, both the composition and the ratio of the constituent microbial species changed.     

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.     

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%).     

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.     

分子生态网络揭示冶金微生物对能源底物的响应

通过分析不同铁硫比的能源底物驯化下冶金微生物群落的演替过程,基于分子生态网络揭示冶金微生物对能源底物的响应特征。对富铁少硫、富硫少铁条件下不同驯化批次的微生物样本进行高通量测序,分析物种组成,比较冶金微生物群落的α多样性和β多样性,并构建分子生态网络,探究驯化过程微生物之间的相互作用关系。鉴定关键物种,分析冶金微生物群落对能源底物的响应。研究发现在基于不同能源底物驯化过程中,富铁少硫群落的优势物种为嗜酸氧化亚铁硫杆菌Acidithiobacillus ferrooxidans和铁氧化嗜酸硫杆菌A. ferriphilus;而富硫少铁群落经过3个批次的驯化,氧化硫硫杆菌A. thiooxidans占比高达90%。α、β多样性分析结果表明,富硫少铁能源底物驯化过程使冶金微生物群落多样性降低,且随着驯化批次的增加群落组成发生显著变化。分子生态网络分析显示关键物种均为低丰度稀有物种,富硫少铁条件下菌种间具有更紧密的互作共生关系,群落更加稳定。通过该驯化实验,明确了不同能源底物对冶金微生物群落的影响。采用富硫少铁能源底物驯化冶金微生物,使冶金微生物群落更加稳定,有助于优势物种高效发挥铁、硫氧化功能,促进硫化矿物的氧化溶解,为生物冶金工业育种微生物群落的定向驯化提供理论基础。     

Effect of cultivation conditions on the growth and activities of sulfur metabolism enzymes and carboxylases of Sulfobacillus thermosulfidooxidans subsp. asporogenes strain 41

The moderately thermophilic acidophilic bacterium Sulfobacillus thermosulfidooxidans subsp. asporogenes strain 41 is capable of utilizing sulfides of gold-arsenic concentrate and elemental sulfur as a source of energy. The growth in the presence of S0 under auto- or mixotrophic conditions was less stable compared with the media containing iron monoxide. The enzymes involved in oxidation of sulfur inorganic compounds--thiosulfate-oxidizing enzyme, tetrathionate hydrolase, rhodonase, adenylyl sulfate reductase, sulfite oxidase, and sulfur oxygenase--were discovered in the cells of Sulfobacillus grown in the mineral medium containing 0.02% yeast extract and either sulfur or iron monoxide and thiosulfate. Cell-free extracts of the cultures grown in the medium with sulfur under auto- or mixotrophic conditions displayed activity of the key enzyme of the Calvin cycle--ribulose bisphosphate carboxylase--and several other enzymes involved in heterotrophic fixation of carbonic acid. Activities of carboxylases depended on the composition of cultivation media.     

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.     

A dynamic mathematical model for microbial removal of pyritic sulfur from coal

A dynamic mathematical model has been developed to describe microbial desulfurization of coal by Thiobacillus ferrooxidans. The model considers adsorption and desorption of cells on coal particles and microbial oxidation of pyritic sulfur on particle surfaces. The influence of certain parameters, such as microbial growth rate constants, adsorption-descrption constants, pulp density, coal particle size, initial cell and solid phase substrate concentration on the maximum rate of pyritic sulfur removal, have been elucidated. The maximum rate of pyritic sulfur removal was strongly dependent upon the number of attached cells per coal particle. At sufficiently high initial cell concentrations, the surfaces of coal particles are nearly saturated by the cells and the maximum leaching rate is limited either by total external surface area of coal particles or by the concentration of pyritic sulfur in the coal phase. The maximum volumetric rate of pyritic sulfur removal (mg S/h cm(3) mixture) increases with the pulp density of coal and reaches a saturation level at high pulp densities (e.g. 45%). The maximum rate also increases with decreasing particle diameter in a hyperbolic form. Increases in adsorption coefficient or decreases in the desorption coefficient also result in considerable improvements in this rate. The model can be applied to other systems consisting of suspended solid substrate particles in liquid medium with microbial oxidation occurring on the particle surfaces (e.g., bacterial ore leaching). The results obtained from this model are in good agreement with published experimental data on microbial desulfurization of coal and bacterial ore leaching.     

Characterization of sulfide-oxidizing microbial mats developed inside a full-scale anaerobic digester employing biological desulfurization

The microbial mats responsible for biological desulfurization from biogas in a full-scale anaerobic digester were characterized in terms of their structure, as well as their chemical and microbial properties. Filament-shaped elemental sulfur 100–500 μm in length was shown to cover the mats, which cover the entire headspace of the digester. This is the first report on filamentous sulfur production in a non-marine environment. The results of the analysis of the mats suggest that the key players in the sulfide oxidation and sulfur production in the bio-desulfurization in the headspace of the digester were likely to be two sulfide-oxidizing bacteria (SOB) species related to Halothiobacillus neapolitanus and Sulfurimonas denitrificans, and that the microbial community, cell density, activity for sulfide oxidation varied according to the environmental conditions at the various locations of the mats. Since the water and nutrients necessary for the SOB were provided by the digested sludge droplets deposited on the mats, and our results show that a higher rate of sulfide oxidation occurred with more frequent digested sludge deposition, the habitat of the SOB needs to be made in the lower part of the headspace near the liquid level of the digested sludge to maintain optimal conditions.