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One of the key compounds engaged in the carbon dioxide fixation cycle (Calvin-Benson-Bassham cycle) is Ribulose 1, 5-Bisphosphate Carboxylase/ Oxygenase (RuBisCo). These are known to act as a carbon sink thus leading to decrease of the carbon level in the atmosphere. This unique property of RuBisCo can therefore help in diminishing an Earth-wide global warming problem, a noteworthy risk in the present world. In the present study, presence of RuBisCo in Sulfobacillus acidophilus strain NY-1 was studied. This strain was isolated from Neyveli lignite mines and their growth parameters such as pH and temperature wereoptimized. The ideal pH and temperature for S. acidophilus was at pH 1.7 and at 45oC respectively. The correlation of growth of S. acidophilus with light, carbon dioxide and aeration was investigated by enumerating the number of cells/mL using a hemocytometer. The cell count was highest in light condition whereas no growth was observed in dark condition. At the 60th hour of incubation, a cell density of 1.60×106 cells/mL was observed. Similarly, in the presence of carbon dioxide the maximum cell count was 2.72×106 at the 40th hour of incubation with aeration. The presence of RuBisCo in S. acidophilus was affirmed by ion exchange chromatography technique.

The semi-pilot scale bioleaching of electroplating sludge by the moderately thermophilic acidophilic consortium was carried out for the first time. During the microbial cultivation, Leptospirillum ferriphilum CS13, Acidithiobacillus caldus S2, and Sulfobacillus acidophilus CS5 could grow rapidly in a 300 L aeration packed reactor, in which the total suspended cell concentration could fluctuate around 3 × 108 cells/mL and the community structure remained relatively stable. During the bioleaching process, the microbial stock solution could effectively leach heavy metals from electroplating sludge in a stirred reactor within a few hours. Meanwhile, the effects of pH, temperature, the quantity of active culture, and liquid-solid ratio on the bioleaching behavior were also investigated. The optimal conditions for electroplating sludge bioleaching were pH 1.5, temperature 45 °C, bacterial liquid ratio 40%, liquid-solid ratio 4:1 L kg−1, and leaching time 5 h. The total removal rate of various heavy metals in electroplating sludge was over 99%. The bioleaching residue was successfully passed the TCLP test, and the total contents of heavy metals in the residue were also well below the regulatory criteria. In addition, the XRD analysis of the bioleaching residue was also confirmed that the moderately thermophilic consortium bioleaching provided a cleaner process than chemical leaching on the removal of the residual fraction metals, which was feasible and attractive for industrial treatment of electroplating sludge.

A mixotrophic and acidophilic bacterial strain BGR 140T was isolated from mine tailings in the Harz Mountains near Goslar, Germany. Cells of BGR 140T were Gram-stain-positive, endospore-forming, motile and rod-shaped. BGR 140T grew aerobically at 25–55 °C (optimum 45 °C) and at pH 1.5–5.0 (optimum pH 3.0). The results of analysis of the 16S rRNA gene sequences indicated that BGR 140T was phylogenetically related to different members of the genus Sulfobacillus , and the sequence identities to Sulfobacillus acidophilus DSM 10332T, Sulfobacillus thermotolerans DSM 17362T, and Sulfobacillus benefaciens DSM 19468T were 94.8, 91.8 and 91.6 %, respectively. Its cell wall peptidoglycan is A1γ, composed of meso-diaminopimelic acid. The respiratory quinone is DMK-6. The major polar lipids were determined to be glycolipid, phospholipid and phosphatidylglycerol. The predominant fatty acid is 11-cycloheptanoyl-undecanoate. The genomic DNA G+C content is 58.2 mol%. On the basis of the results of phenotypic and genomic analyses, it is concluded that strain BGR 140T represents a novel species of the genus Sulfobacillus , for which the name Sulfobacillus harzensis sp. nov. is proposed because of its origin. Its type strain is BGR 140T (=DSM 109850T=JCM 39070T).

This study aims to use neutral pH optimum arginase as the catalyst for high-efficiency l-ornithine production. Sulfobacillus acidophilus arginase was firstly cloned and overexpressed in Escherichia coli. The purified enzyme was obtained, and the molecular mass determination showed that this arginase was a hexamer. S. acidophilus arginase possessed similarities with the other arginases such as the conserved sequences, purification behavior, and the necessity for Mn2+ as a cofactor. The maximum enzyme activity was obtained at pH 7.5 and 70 °C. Thermostability and pH stability analysis showed that the arginase was stable at 30–60 °C and pH 7.0–8.5, respectively. The kinetic parameters suggested that S. acidophilus arginase could efficiently hydrolyze l-arginine. Bioconversion with this neutral pH optimum arginase had the advantages of avoiding producing by-product, high molar yield, and high-level production of l-ornithine. When the bioconversion was performed with a fed-batch strategy and a coupled-enzyme system involving S. acidophilus arginase and Jack bean urease, the final production of 2.87 mol/L was obtained with only 1.72 mmol/L l-arginine residue, and the molar yield was 99.9%. The highest production record suggests that S. acidophilus arginase has a great prospect in industrial l-ornithine production.

The magnesite and bauxite mines are a major source for some minerals and metals. The tailings of these magnesite and bauxite mines contains massive amount of Mn. (3221.2 +/- 6.51 & 7102.8.4 +/- 10.24), Cd (40.95 +/- 2.12 & 892.86 +/- 8.48), Zn (951.08 +/- 4.31 &724.12 +/- 3.18), and Pb (425.8 +/- 5.21 & 812.13 +/- 9.19 mg Kg(-1)) with alkaline and acid pH respectively. In the microbial diversity analysis, the results 8 (Thiobacillus thiooxidans, Leptospirillum ferrooxidans, Acetobacter methanolicus, T. intermedius, Bacillus cereus, Sulfobacillus acidophilus, Methylobacterium sp, and Thiobacillus ferooxidans) and 12 (Serratia marcescens, Metalogenium symbioticum 1, Metallogenium symbioticum 2, Bacillus alcalophilus, Aminobacter sp, Naumaniella neustonica, Staphylococcus aureus 1, Methylbacillus sp, Pandoraea sputorum, Acenetobacter sp, S. aureus 2, Pseudomonas aeruginosa) bacteria species were isolated from bauxite and magnesite mine tailings. The fungus such as Rhizopus arrhizus, Mucor sp, Aspergillus niger and Penicillium sp were obtained in both acid and alkaline environment. Among this microbial diversity, P. sputorum and B. cereus (identified through 16S rDNA sequencing) were shown better resistance to these four metals for up to 250 mg L-1. The bacterial diversity indexes, concludes that there was least diverse among the three sites. The evenness/equitability index also conform lower level of variation among these sites. (C) 2019 Elsevier Ltd. All rights reserved. info:eu-repo/semantics/publishedVersion

Land application of sewage sludge has received significant attention in recent years but the presence of elevated heavy metals in the sludge limits its land application. The purposes of this study were to investigate the effects of sulfur dosage and inoculum size on the thermophilic bioleaching of heavy metals from sewage sludge in a pilot-scale bioreactor. The microbial communities in this thermophilic bioleaching process were also identified using real-time polymerase chain reaction (real-time PCR). The results showed that the oxidation of sulfur and metal solubilization decreased with the increasing sulfur dosage. When the sulfur dosage was greater than 2% (w/v), the sulfur oxidation and metal solubilization rates decreased, indicating that the thermophilic bioleaching was hindered by high levels of substrate. However, it was found that the efficiency of metal solubilization and solid degradation was increased with the increase of inoculum size in the range from 5% to 20%. At the end of bioleaching, the efficiency of Mn, Zn, Ni, Cu and Cr from the sewage sludge reached 73–100%, 51–60%, 38–52%, 17–43% and 1–38%, respectively, while SS and VSS were degraded by 33–48% and 47–67%, respectively. Based on the analysis of real-time PCR, Sulfobacillus acidophilus was observed to be the predominant species (13–67% of total bacteria), whereas the populations of Sulfobacillus thermosulfidooxidans and Acidithiobacillus caldus were accounted relatively low (

Electrochemically active bacteria (EAB) use extracellular electron transfer (EET) to exchange electron with extracellular acceptors. Previous studies regarding the measurement of EAB were based on either extracellular reduction or oxidation. In this work, we developed a simple electrochemiluminescence (ECL) assay for the identification and detection of EAB. The results of this proposed method revealed that EET of EAB influenced the content of dissolved oxygen and the formation of Ru(bpy)32+• thus leading to qualitative changes of the ECL signal. EAB with the ability of extracellular reduction (such as Shewanella oneidensis MR-1) gave enhanced signal on ECL emission while those displaying the ability of extracellular oxidation (i.e., Sulfobacillus acidophilus) showed the opposite effect on ECL emission, but non-EAB (i.e., Escherichia coli) did not. These changes in ECL intensity were also proportional to the cell density that could be quantitatively detected in the concentration range of (1.1 ± 1) × 105–212 ± 2 CFU/mL (i.e. Shewanella oneidensis MR-1). Moreover, the measurement of the ability of EAB using this approach was in agreement with measurements using the dissimilatory Fe(III) reduction method. Compared to previous reports, this method displayed a continual and steady ECL signal that allowed accurate measurements of EAB. Most important, only a low cell density was needed in this Ru(bpy)32+ - based ECL method, which is beneficial for cell detection.

The cysteine desulfurase (SufS) gene of Sulfobacillus acidophilus TPY, a Gram-positive bacterium isolated from deep-sea hydrothermal vent, was cloned and over-expressed in E. coli BL21. The recombinant SufS protein was purified by one-step affinity chromatography. The TPY SufS contained a well conserved motif RXGHHCA as found in that of other microorganisms, suggesting that it belonged to group II of cysteine desulfurase family. The recombinant TPY SufS could catalyze the conversion of l-cysteine to l-alanine and produce persulfide, and the enzyme activity was 95 μ/μL of sulfur ion per minute. The growth of E. coli BL21 was promoted by over-expressing TPY SufS in vivo or by directly adding recombinant TPY SufS in the medium (4.3-4.5 × 108 cells/mL vs. 3.2-3.5 × 108 cells/mL). Furthermore, the highest cell density of E. coli BL21 when the TPY SufS was over-expressed was about 3.5 times that of the control groups in the presence of sodium thiosulfate. These results indicate that the SUF system as the only assembly system of iron-sulfur clusters not only has significant roles in survival of S. acidophilus TPY, but also might be important for combating with high content of sulfide.

EstS1, a newly identified thermostable esterase from Sulfobacillus acidophilus DSM10332, was heterologously expressed in Escherichia coli and shown to enzymatically degrade phthalate esters (PAEs) to their corresponding monoalkyl PAEs. The optimal pH and temperature of the esterase were found to be 8.0 and 70°C, respectively. The half-life of EstS1 at 60°C was 15 h, indicating that the enzyme had good thermostability. The specificity constant ( k cat / K m ) of the enzyme for p -nitrophenyl butyrate was as high as 6,770 mM −1 s −1 . The potential value of EstS1 was demonstrated by its ability to effectively hydrolyze 35 to 82% of PAEs (10 mM) within 2 min at 37°C, with all substrates being completely degraded within 24 h. At 60°C, the time required for complete hydrolysis of most PAEs was reduced by half. To our knowledge, this enzyme is a new esterase identified from thermophiles that is able to degrade various PAEs at high temperatures.

An enriched and adapted moderately thermophilic culture showed good performance during bioleaching of chalcopyrite under uncontrolled conditions. The copper extractions were up to 85.0%, 77.3% and 56.3% at pulp densities of 10%, 15% and 20% (w/v) within 22 days, respectively. The real-time quantitative PCR was employed to monitor planktonic and attached community dynamics during bioleaching of chalcopyrite by the moderately thermophilic microbial culture. Only three species, including Acidithiobacillus caldus , Sulfobacillus acidophilus and Ferroplasma thermophilum , were detected during the whole bioleaching process. The results show that pulp density had significant effects on planktonic and attached microbial community dynamics. The succession of attached cells was different from community dynamics of their planktonic counterparts. F. thermophilum and A. caldus preferred to attach to mineral surface, especially at pulp densities of 15% and 20%.

Copper sulphide ore bioleaching processes are currently under full development in Chile and worldwide, for treatment of: secondary sulphide in heaps [1], low-grade ores in dumps, and concentrates in reactors at high temperatures [2]. Abundant knowledge is available in the literature and great experience has been gathered about the behavior of mesophilic microorganisms and their application in bioleaching of sulfides in heaps and dumps [, as well as, about the behavior of extreme thermophilic microorganisms in reactors [. However, there is little information about the behavior of some moderately thermophilic microorganisms in the dissolution of chalcopyrite and pyrite at 45oC [. This is the case ofS. acidophilus, a gram-positive rod shaped that often forms endospores and grow at temperature optima, generally between 45 and 55°C. It has been characterized as a chemolithoheterotrophic bacteria that obtains energy from the oxidation of ferrous iron or from reduced sulfur compounds and that utilizes yeast extracts as a carbon source [.S. acidophiluswas isolated by Golovacheva and Karavaiko [ and described by Norris et al. [. Limited literature exists about its participation in bioleaching processes. In this work, we present experimental results obtained from the bioleaching of chalcopyrite and pyrite withS. acidophilusat 45°C, and compare them with those obtained withAt. ferrooxidansat 30°C.

Sulfobacillus acidophilus TPY, isolated from a hydrothermal vent in the Pacific Ocean, is a moderately thermoacidophilic Gram-positive bacterium that can oxidize ferrous iron or sulfur compounds to obtain energy. In this study, comparative transcriptomic analyses of S. acidophilus TPY were performed under different redox conditions. Based on these results, pathways involved in sulfur metabolism were proposed. Additional evidence was obtained by analyzing mRNA abundance of selected genes involved in the sulfur metabolism of sulfur oxygenase reductase (SOR)-overexpressed S. acidophilus TPY recombinant under different redox conditions. Comparative transcriptomic analyses of S. acidophilus TPY cultured in the presence of ferrous sulfate (FeSO4) or elemental sulfur (S0) were employed to detect differentially transcribed genes and operons involved in sulfur metabolism. The mRNA abundances of genes involved in sulfur metabolism decreased in cultures containing elemental sulfur, as opposed to cultures in which FeSO4 was present where an increase in the expression of sulfur metabolism genes, particularly sulfite reductase (SiR) involved in the dissimilatory sulfate reduction, was observed. SOR, whose mRNA abundance increased in S0 culture, may play an important role in the initial sulfur oxidation. In order to confirm the pathways, SOR overexpression in S. acidophilus TPY and subsequent mRNA abundance analysis of sulfur metabolism-related genes were carried out. Conjugation-based transformation of pTrc99A derived plasmid from heterotrophic E. coli to facultative autotrophic S. acidophilus TPY was developed in this study. Transconjugation between E. coli and S. acidophilus was performed on modified solid 2:2 medium at pH 4.8 and 37°C for 72 h. The SOR-overexpressed recombinant S. acidophilus TPY-SOR had a SO42−-accumulation increase, higher oxidation/ reduction potentials (ORPs) and lower pH compared with the wild type strain in the late growth stage of S0 culture condition. The transcript level of sor gene in the recombinant strain increased in both S0 and FeSO4 culture conditions, which influenced the transcription of other genes in the proposed sulfur metabolism pathways. Overall, these results expand our understanding of sulfur metabolism within the Sulfobacillus genus and provide a successful gene-manipulation method.

Barbiturases have scarce structural information available and do not fit in the conventional group of proteins. It is contemplated that they play a role in catabolism of s-triazine herbicide compounds. Structure as well as interaction data information of barbiturase with s-triazine compounds is missing. Sequence data is a goldmine of biological information and acts as raw material for structure and docking analysis. De novo structure prediction of the Sulfobacillus acidophilus DSM 10332 barbiturase has been attempted in this data article. Molecular docking analysis was carried out with atrazine, simazine and hexazinone belonging to s-triazine class of herbicides. The analysis revealed key residues necessary for these interactions. The generated data could be used by environmental scientists working on the enzyme assisted herbicide degradation.

The reductase component (MhpP) of the Sulfobacillus acidophilus TPY multicomponent phenol hydroxylase exhibits only 40 % similarity to Pseudomonas sp. strain CF600 phenol hydroxylase reductase. Amino acid sequence alignment analysis revealed that four cysteine residues (Cys-X 4 -Cys-X 2 -Cys-X 29-35 -Cys) are conserved in the N terminus of MhpP for [2Fe-2S] cluster binding, and two other motifs (RXYS and GXXS/T) are conserved in the C terminus for binding the isoalloxazine and phosphate groups of flavin adenine dinucleotide (FAD). Two motifs (S/T-R and yXCGp) responsible for binding to reduce nicotinamide adenine dinucleotide phosphate (NADPH) are also conserved in MhpP, although some residues differ. To confirm the function of this reductase, MhpP was heterologously expressed in Escherichia coli BL21(DE3) and purified. UV-visible spectroscopy and electron paramagnetic resonance spectroscopy revealed that MhpP contains a [2Fe-2S] cluster. MhpP mutants in which the four cysteine residues were substituted via site-directed mutagenesis lost the ability to bind the [2Fe-2S] cluster, resulting in a decrease in enzyme-specific oxidation of NADPH. Thin-layer chromatography revealed that MhpP contains FAD. Substrate specificity analyses confirmed that MhpP uses NADPH rather than NADH as an electron donor. MhpP oxidizes NADPH using cytochrome c, potassium ferricyanide, or nitro blue tetrazolium as an electron acceptor, with a specific activity of 1.7 ± 0.36, 0.78 ± 0.13, and 0.16 ± 0.06 U/mg, respectively. Thus, S. acidophilus TPY MhpP is a novel NADPH-dependent reductase component of phenol hydroxylase that utilizes FAD and a [2Fe-2S] cluster as cofactors.

Sulfobacillus acidophilus Norris et al. 1996 is a member of the genus Sulfobacillus which comprises five species of the order Clostridiales. Sulfobacillus species are of interest for comparison to other sulfur and iron oxidizers and also have biomining applications. This is the first completed genome sequence of a type strain of the genus Sulfobacillus, and the second published genome of a member of the species S. acidophilus. The genome, which consists of one chromosome and one plasmid with a total size of 3,557,831 bp harbors 3,626 protein-coding and 69 RNA genes, and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Arsenic resistance genes were isolated from the moderately thermophilic, Gram-positive iron and sulfur-oxidizing bacterium, Sulfobacillus thermosulfidooxidans . Only arsR and arsB genes were present and attempts to identify an arsC using degenerate PCR primers or Trx- or Grx-dependent arsC genes as probes in Southern hybridization experiments were unsuccessful. Although enhanced resistance to arsenite was not detected when the ars genes were cloned in Escherichia coli , the adjacent kumamolisin-As and arsRB genes were induced by arsenite. RT-PCR experiments suggested that transcription of the cloned kumamolisin-As-like and arsRB genes is linked in E. coli , but not in S. thermosulfidooxidans . The gene order kumamolisin-As precursor, arsR and arsB is maintained among three strains of S. thermosulfidooxidans isolated from three continents. Southern hybridization using a S. thermosulfidooxidans arsB gene fragment as a probe gave a positive hybridization signal using Sulfobacillus acidophilus but not with Sulfobacillus thermotolerans genomic DNA. Comparison of partial sequence data of the arsB and 16 S rRNA genes suggested that the two types of genes have undergone a similar evolutionary history and therefore that the arsB genes were present in the ancestral Sulfobacillus before its divergence into species.

Sixteen strains of acidophilic bacteria were screened for their abilities to adhere to pyrite ore, glass beads and ferric hydroxysulfates. These were three culture collection and two isolated strains of the iron- and sulfur-oxidizer, Acidithiobacillus ferrooxidans, two each of the sulfur-oxidizer Acidithiobacillus thiooxidans and the iron-oxidizer Leptospirillum ferrooxidans (the type strain and a mine isolate in either case), five heterotrophic acidophiles (four Acidiphilium and one Acidocella sp.) and two moderately thermophilic iron/sulfur-oxidizers (Sulfobacillus thermosulfidooxidans and Sulfobacillus acidophilus). Considerable variations were found between different species of acidophiles, and also between different strains of the same species, in how they attached to the three solid materials tested. Attachment to the solid substrata generally increased with time (over 100 min) though > 99% of one At. ferrooxidans isolate (strain OP14) were attached to pyrite after just 10 min exposure. Most acidophiles attached more readily to pyrite than to glass beads, and attachment to ferric hydroxysulfates was highly variable, though one At. ferrooxidans isolate (strain SJ2) and one heterotrophic acidophile (Acidocella sp. het-4) both attached strongly to ferric iron precipitates (jarosites and schwertmannite) that formed in cultures of At. ferrooxidans grown at pH > 2. The results of these experiments showed that even closely related strains of acidophilic bacteria can display very different propensities to attach to solid materials, an observation that may explain the somewhat disparate results reported on occasions by research groups that have examined single, or limited numbers of strains, of acidophiles (mostly At. ferrooxidans). The significance of differential attachment of mineral-oxidizing and other acidophiles to pyrite and other solids is discussed in the context of biohydrometallurgy.

Due to its toxicity and volatility, phenol must be cleared from the environment. Sulfobacillus acidophilus TPY, which was isolated from a hydrothermal vent in the Pacific Ocean as a moderately thermoacidophilic Gram-positive bacterium, was capable of aerobically degrading phenol. This bacterium could tolerate up to 1300mg/L phenol and degrade 100mg/L phenol in 40h completely at 45°C and pH 1.8 with a maximal degradation rate of 2.32mg/L/h at 38h. Genome-wide search revealed that one gene (TPY_3176) and 14 genes clustered together in two regions with locus tags of TPY_0628-0634 and TPY_0640-0646 was proposed to be involved in phenol degradation via the meta-pathway with both the 4-oxalocrotonate branch and the hydrolytic branch. Real-time PCR analysis of S. acidophilus TPY under phenol cultivation condition confirmed the transcription of proposed genes involved in the phenol degradation meta-pathway. Degradation of 3-methylphenol and 2-methylphenol confirmed that the hydrolytic branch was utilised by S. acidophilus TPY. Phylogenetic analysis revealed that S. acidophilus TPY was closely related to sulphate-reducing bacteria and some Gram-positive phenol-degrading bacteria. This was the first report demonstrating the ability of S. acidophilus to degrade phenol and characterising the putative genes involved in phenol metabolism in S. acidophilus TPY.

Heavy metals can be removed from the sludge using bioleaching technologies at thermophilic condition, thereby providing an option for biotreatment of wasted sludge generated from wastewater treatment. The purposes of this study were to establish a molecular biology technique, real-time PCR, for the detection and enumeration of the sulfur-oxidizing bacteria during the thermophilic sludge bioleaching. The 16S rRNA gene for real-time PCR quantification targeted the bioleaching bacteria: Sulfobacillus thermosulfidooxidans, Sulfobacillus acidophilus, and Acidithiobacillus caldus. The specificity and stringency for thermophilic sulfur-oxidizing bacteria were tested before the experiments of monitoring the bacterial community, bacterial number during the thermophilic sludge bioleaching and the future application on testing various environmental samples. The results showed that S. acidophilus was identified as the dominant sulfur-oxidizing bacteria, while A. caldus and S. thermosulfidooxidans occurred in relatively low numbers. The total number of the sulfur-oxidizing bacteria increased during the thermophilic bioleaching process. Meanwhile, the decrease of pH, production of sulfate, degradation of SS/VSS, and solubilization of heavy metal were found to correlate well with the population of thermophilic sulfur-oxidizing bacteria during the bioleaching process. The real-time PCR used in this study is a suitable method to monitor numbers of thermophilic sulfur-oxidizing bacteria during the bioleaching process.

Phylogeny of anoxygenic filamentous phototrophic bacteria (AFPB) of the family Oscillochloridaceae (Oscillochloris trichoides DG6T and the recently isolated strains Oscillochloris sp. R and C6) was studied based on comparative analyses of the genes coding for 16S rRNA (rrs), ribulose-1,5-bisphosphate carboxylase/oxygenase (cbbL), and nitrogenase (nifH). The sequences of the genes studied proved to be identical in the three strains, which is in agreement with data obtained earlier that showed a lack of differentiating phenotypic distinctions between these strains; therefore, it is proposed that the new strains should be identified as representatives of the species O. trichoides. Using an earlier designed system of oligonucleotide primers and a specially designed additional primer, fragments of the cbbL genes of the “red-like” form I RuBisCO were amplified and sequenced for all of the O. trichoides strains. Analysis of the cbbL genes suggested a separate position of the bacteria studied in the phylogenetic tree, where O. trichoides strains formed an independent branch, which, apart from this species, also included the only studied species of gram-positive facultatively chemoautotrophic bacteria, Sulfobacillus acidophilus. In the phylogenetic tree inferred from the analysis of nifH genes, the bacteria under study also formed a new separate branch, deviating near the root, which indicated a lack of relatedness between them and other phototrophic bacteria. The data obtained support the conclusion that AFPB has an ancient origin and their allocation as one of the main evolutionary lineages of eubacteria, which was made based on the analysis of ribosomal genes.

A SybrGreen real-time PCR assay was developed to detect and quantify both total and selected 16S rDNA species of bacteria and archaea involved in the bioleaching of metals from sulfide ores. A set of specific and universal primers based on 16S rDNA sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of Acidianus brierleyi, Sulfolobus sp., Sulfobacillus thermosulfidooxidans, Sulfobacillus acidophilus, Acidithiobacillus caldus, and Leptospirillum ferrooxidans. An artificial sequence based on 16S rDNA was constructed to quantify total 16S rDNA in mixed DNA samples. The real-time PCR assay was further validated using a mixture of 16S rDNA amplicons derived from the six different species, each added at a known amount. Finally, the real-time PCR assay was used to monitor the change of 16S rDNA copies of four bioleaching strains inoculated into chalcopyrite airlift column reactors operated at different temperatures. The growth dynamics of these strains correlated well with the expected effects of temperature in the chalcopyrite-leaching environment. The suitability of this method for monitoring microbial populations in industrial bioleaching environments is discussed.

A systematic study of the bioleaching of chalcopyrite (CuFeS 2 ) was conducted using axenic cultures of 11 species of acidophilic Bacteria and Archaea to obtain a direct comparison of the microbial chalcopyrite leaching capabilities of the different cultures and to determine the factors that affect Cu release. The characteristics of chalcopyrite leaching by the moderate thermophile Sulfobacillus thermosulfidooxidans , the mesophile Acidithiobacillus ferrooxidans , and the thermophile Acidianus brierleyi were used to elucidate the leaching process. Moderately thermophilic cultures of Sulfobacillus acidophilus, Acidimicrobium ferrooxidans , and Acidithiobacillus caldus were used to study the effects of different metabolic capabilities and relate those to leaching efficiency. The greatest rate of Cu solubilization from chalcopyrite was achieved at high temperatures (up to 70°C) at redox potentials below +550 mV (Ag/AgCl). The enhanced Cu solubilization observed at high temperatures resulted from accelerated ...

The bioleaching of chalcopyrite in an acidic sulphate nutrient medium was investigated using Sulfobacillus thermosulfidooxidans, a moderately thermophilic iron- and sulphur oxidising bacterium. Copper release to solution was initially rapid but this slowed significantly after about SO hours. The decrease in chalcopyrite dissolution rate coincided with significant precipitation of jarosite on the mineral surface. Cultures of the moderately thermophilic acidophilic bacteria Acidimicrobium ferrooxidans, Sulfobacillus acidophilus and Sulfobacillus thermosulfidooxidans were grown in anaerobic media containing chalcopyrite passivated by jarosite. The moderate thermophiles used the ferric ion in the jarositic surface precipitate as a terminal electron acceptor in place of oxygen in the anoxic environment. Despite extensive bioreduction of the iron-hydroxy precipitates, it was found that the jarosite was not completely removed and that subsequent biooxidation of the treated concentrate achieved no significant increases in copper release compared with concentrate that had not been subjected to prior biooxidation or bioreduction.

Three kinds of samples (acid mine drainage, coal mine wastewater, and thermal spring) derived from different sites were collected in China. Thereafter, these samples were combined and then inoculated into a basal salts solution in which different substrates (ferrous sulfate, elemental sulfur, and chalcopyrite) served as energy sources. After that, the mixed cultures growing on different substrates were pooled equally, resulting in a final mixed culture. After being adapted to gradually increasing pulp densities of chalcopyrite concentrate by serial subculturing for more than 2 years, the final culture was able to efficiently leach the chalcopyrite at a pulp density of 20% (wt/vol). At that pulp density, the culture extracted 60.4% of copper from the chalcopyrite in 25 days. The bacterial and archaeal diversities during adaptation were analyzed by denaturing gradient gel electrophoresis and constructing clone libraries of the 16S rRNA gene. The results show that the culture consisted mainly of four species, including Leptospirillum ferriphilum , Acidithiobacillus caldus , Sulfobacillus acidophilus , and Ferroplasma thermophilum , before adapting to a pulp density of 4%. However, L. ferriphilum could not be detected when the pulp density was greater than 4%. Real-time quantitative PCR was employed to monitor the microbial dynamics during bioleaching at a pulp density of 20%. The results show that A. caldus was the predominant species in the initial stage, while S. acidophilus rather than A. caldus became the predominant species in the middle stage. F. thermophilum accounted for the greatest proportion in the final stage.

While many prokaryotic species are known to use hydrogen as an electron donor to support their growth, this trait has only previously been reported for two acidophilic bacteria, Hydrogenobaculum acidophilum (in the presence of reduced sulfur) and Acidithiobacillus (At.) ferrooxidans. To test the hypothesis that hydrogen may be utilized more widely by acidophilic bacteria, 38 strains of acidophilic bacteria, including representatives of 20 designated and four proposed species, were screened for their abilities to grow via the dissimilatory oxidation of hydrogen. Growth was demonstrated in several species of acidophiles that also use other inorganic electron donors (ferrous iron and sulfur) but in none of the obligately heterotrophic species tested. Strains of At. ferrooxidans, At. ferridurans and At. caldus, grew chemolithotrophically on hydrogen, though those of At. thiooxidans and At. ferrivorans did not. Growth was also observed with Sulfobacillus acidophilus, Sb. benefaciens and Sb. thermosulfidooxidans, though not with other iron-oxidizing Firmicutes. Similarly, Acidimicrobium ferrooxidans grew on hydrogen, closely related acidophilic actinobacteria did not. Growth yields of At. ferrooxidans and At. ferridurans grown aerobically on hydrogen (c. 10(10) cells mL(-1) ) were far greater than typically obtained using other electron donors. Several species also grew anaerobically by coupling hydrogen oxidation to the reduction of ferric iron.

Several isolates of Gram-positive, acidophilic, moderately thermophilic, ferrous-iron- and mineral-sulphide-oxidizing bacteria were examined to establish unequivocally the characteristics of Sulfobacillus-like bacteria. Two species were evident: Sulfobacillus thermosulfidooxidans with 48-50 mol% G + C and Sulfobacillus acidophilus sp. nov. with 55-57 mol% G + C. Both species grew autotrophically and mixotrophically on ferrous iron, on elemental sulphur in the presence of yeast extract, and heterotrophically on yeast extract. Autotrophic growth on sulphur was consistently obtained only with S. acidophilus.

A new species of ferrous-iron-oxidizing, moderately thermophilic, acidophilic bacteria, Acidimicrobium ferrooxidans, has been described. Two isolates of the species differed only in the tendency of one, previously known as strain TH3, to grow in filaments. The chromosomal DNA base composition is between 67 and 69 mol% G + C. The capacity of this species to fix CO2 from air was greater than that of iron-oxidizing thermoacidophiles of the genus Sulfobacillus, which required an enhanced CO2 concentration for optimum autotrophic growth. Under air, ferrous iron oxidation in mixed cultures of A. ferrooxidans with either Sulfobacillus thermosulfidooxidans or Sulfobacillus acidophilus was more extensive than in pure cultures of these three strains. The greater part of ferrous iron oxidation in mixed cultures probably resulted from activity of the Sulfobacillus species, which possess a greater tolerance of ferric iron, and which presumably grew mixotrophically utilizing organic compounds from A. ferrooxidans.

Sequence alignments revealed that the conserved motifs of SORSa which formed an independent branch between archaea and Gram-negative bacteria SORs according to the phylogenetic relationship were similar with the archaea and Gram-negative bacteria SORs. In order to investigate the active sites of SORSa, cysteines 31, 101 and 104 (C31, C101, C104), histidines 86 and 90 (H86 and H90) and glutamate 114 (E114) of SORSa were chosen as the target amino acid residues for site-specific mutagenesis. The wild type and six mutant SORs were expressed in E. coli BL21, purified and confirmed by SDS-PAGE and Western blotting analysis. Enzyme activity determination revealed that the active sites of SORSa were identical with the archaea and Gram-negative bacteria SORs reported. Replacement of any cysteine residues reduced SOR activity by 53-100%, while the mutants of H86A, H90A and E114A lost their enzyme activities largely, only remaining 20%, 19% and 32% activity of the wild type SOR respectively. This study will enrich our awareness for active sites of SOR in a Gram-positive bacterium.

Sulfobacillus acidophilus strain TPY is a moderately thermoacidophilic bacterium originally isolated from a hydrothermal vent in the Pacific Ocean. Ferrous iron and sulfur oxidation in acidic environments in strain TPY have been confirmed. Here we report the genome sequence and annotation of the strain TPY, which is the first complete genome of Sulfobacillus acidophilus .

The Calvin-Benson-Bassham (CBB) cycle has been extensively studied in proteobacteria, cyanobacteria, algae and plants, but hardly at all in Gram-positive bacteria. Some characteristics of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) and a cluster of potential CBB cycle genes in a Gram-positive bacterium are described in this study with two species of Sulfobacillus (Gram-positive, facultatively autotrophic, mineral sulfide-oxidizing acidophiles). In contrast to the Gram-negative, iron-oxidizing acidophile Acidithiobacillus ferrooxidans, Sulfobacillus thermosulfidooxidans grew poorly autotrophically unless the CO(2) concentration was enhanced over that in air. However, the RuBisCO of each organism showed similar affinities for CO(2) and for ribulose 1,5-bisphosphate, and similar apparent derepression of activity under CO(2) limitation. The red-type, form I RuBisCO of Sulfobacillus acidophilus was confirmed as closely related to that of the anoxygenic phototroph Oscillochloris trichoides. Eight genes potentially involved in the CBB cycle in S. acidophilus were clustered in the order cbbA, cbbP, cbbE, cbbL, cbbS, cbbX, cbbG and cbbT.

Five moderately thermophilic iron-oxidizing bacteria, including representative strains of the three classified species ( Sulfobacillus thermosulfidooxidans , Sulfobacillus acidophilus , and Acidimicrobium ferrooxidans ), were shown to be capable of reducing ferric iron to ferrous iron when they were grown under oxygen limitation conditions. Iron reduction was most readily observed when the isolates were grown as mixotrophs or heterotrophs with glycerol as an electron donor; in addition, some strains were able to couple the oxidation of tetrathionate to the reduction of ferric iron. Cycling of iron between the ferrous and ferric states was observed during batch culture growth in unshaken flasks incubated under aerobic conditions, although the patterns of oxidoreduction of iron varied in different species of iron-oxidizing moderate thermophiles and in strains of a single species ( S. acidophilus ). All three bacterial species were able to grow anaerobically with ferric iron as a sole electron acceptor; the growth yields correlated with the amount of ferric iron reduced when the isolates were grown in the absence of oxygen. One of the moderate thermophiles (identified as a strain of S. acidophilus ) was able to bring about the reductive dissolution of three ferric iron-containing minerals (ferric hydroxide, jarosite, and goethite) when it was grown under restricted aeration conditions with glycerol as a carbon and energy source. The significance of iron reduction by moderately thermophilic iron oxidizers in both environmental and applied contexts is discussed.

Currently more than 90% of the world’s copper is obtained through sulfide mineral processing. Among the copper sulfides, chalcopyrite is the most abundant and therefore economically relevant. However, primary copper sulfide bioleaching is restricted due to high ionic strength raffinate solutions and particularly chloride coming from the dissolution of ores. In this work we describe the chalcopyrite bioleaching capacity of Sulfobacillus thermosulfidooxidans strain Cutipay (DSM 27601) previously described at the genomic level (Travisany et al. (2012) Draft genome sequence of the Sulfobacillus thermosulfidooxidans Cutipay strain, an indigenous bacterium isolated from a naturally extreme mining environment in Northern Chile. J Bacteriol 194:6327–6328). Bioleaching assays with the mixotrophic strain Cutipay showed a strong increase in copper recovery from chalcopyrite concentrate at 50°C in the presence of chloride ion, a relevant inhibitory element present in copper bioleaching processes. Compared to the abiotic control and a test with Sulfobacillus acidophilus DSM 10332, strain Cutipay showed an increase of 42 and 69% in copper recovery, respectively, demonstrating its high potential for chalcopyrite bioleaching. Moreover, a genomic comparison highlights the presence of the 2-Haloacid dehalogenase predicted-protein related to a potential new mechanism of chloride resistance in acidophiles. This novel and industrially applicable strain is under patent application CL 2013–03335. Electronic supplementary material The online version of this article (doi:10.1186/s13568-014-0084-1) contains supplementary material, which is available to authorized users.