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Existence of aa3-type ubiquinol oxidase as a terminal oxidase in sulfite oxidation of Acidithiobacillus thiooxidans.

It was found that Acidithiobacillus thiooxidans has sulfite:ubiquinone oxidoreductase and ubiquinol oxidase activities in the cells. Ubiquinol oxidase was purified from plasma membranes of strain NB1-3 in a nearly homogeneous state. A purified enzyme showed absorption peaks at 419 and 595 nm in the oxidized form and at 442 and 605 nm in the reduced form. Pyridine ferrohaemochrome prepared from the enzyme showed an alpha-peak characteristic of haem a at 587 nm, indicating that the enzyme contains haem a as a component. The CO difference spectrum of ubiquinol oxidase showed two peaks at 428 nm and 595 nm, and a trough at 446 nm, suggesting the existence of an aa(3)-type cytochrome in the enzyme. Ubiquinol oxidase was composed of three subunits with apparent molecular masses of 57 kDa, 34 kDa, and 23 kDa. The optimum pH and temperature for ubiquinol oxidation were pH 6.0 and 30 degrees C. The activity was completely inhibited by sodium cyanide at 1.0 mM. In contrast, the activity was inhibited weakly by antimycin A(1) and myxothiazol, which are inhibitors of mitochondrial bc(1) complex. Quinone analog 2-heptyl-4-hydoroxyquinoline N-oxide (HOQNO) strongly inhibited ubiquinol oxidase activity. Nickel and tungstate (0.1 mM), which are used as a bacteriostatic agent for A. thiooxidans-dependent concrete corrosion, inhibited ubiquinol oxidase activity 100 and 70% respectively.

Acidithiobacillus thiooxidans↗

Leaching characteristics of heavy metals from sewage sludge by Acidithiobacillus thiooxidans MET.

An acidophilic, sulfur-oxidizing Acidithiobacillus thiooxidans MET bacterium was isolated from anaerobically digested, dewatered sewage sludge. This bacterium showed sulfur-oxidizing ability at both acidic and neutral conditions, and allowed metal leaching even at a high (130 g L(-1)) sludge solids concentration. We found that low metal leaching efficiency at high solids concentration was mainly due to an increase in buffering capacity resulting in retardation of pH reduction. Therefore, metal leaching was mainly influenced not by sludge solids concentration, but by the pH (or sulfate concentration per unit sludge mass) of the sludge solutions. The relationship between the pH of the sludge solution and the efficiency of metal leaching was obtained by quantitatively investigating the effect of pH reduction or the amount of sulfate produced per unit sludge mass on leaching of each metal. Furthermore, the relationship between total metal content in the sludge and metal leached to the solution was obtained for each metal. Such a relationship allowed estimation of leachable metal at various amounts of total metal content in sludge.

Gammaproteobacteria↗

Growth inhibition by tungsten in the sulfur-oxidizing bacterium Acidithiobacillus thiooxidans.

Growth of five strains of sulfur-oxidizing bacteria Acidithiobacillus thiooxidans, including strain NB1-3, was inhibited completely by 50 microM of sodium tungstate (Na(2)WO(4)). When the cells of NB1-3 were incubated in 0.1 M beta-alanine-SO(4)(2-) buffer (pH 3.0) with 100 microM Na(2)WO(4) for 1 h, the amount of tungsten bound to the cells was 33 microg/mg protein. Approximately 10 times more tungsten was bound to the cells at pH 3.0 than at pH 7.0. The tungsten binding to NB1-3 cells was inhibited by oxyanions such as sodium molybdenum and ammonium vanadate. The activities of enzymes involved in elemental sulfur oxidation of NB1-3 cells such as sulfur oxidase, sulfur dioxygenase, and sulfite oxidase were strongly inhibited by Na(2)WO(4). These results indicate that tungsten binds to NB1-3 cells and inhibits the sulfur oxidation enzyme system of the cells, and as a result, inhibits cell growth. When portland cement bars supplemented with 0.075% metal nickel and with 0.075% metal nickel and 0.075% calcium tungstate were exposed to the atmosphere of a sewage treatment plant containing 28 ppm of H(2)S for 2 years, the weight loss of the portland cement bar with metal nickel and calcium tungstate was much lower than the cement bar containing 0.075% metal nickel.

Acidithiobacillus thiooxidans↗

Purification and properties of thiosulfate dehydrogenase from Acidithiobacillus thiooxidans JCM7814.

A key enzyme of the thiosulfate oxidation pathway in Acidithiobacillus thiooxidans JCM7814 was investigated. As a result of assaying the enzymatic activities of thiosulfate dehydrogenase, rhodanese, and thiosulfate reductase at 5.5 of intracellular pH, the activity of thiosulfate dehydrogenase was measured as the key enzyme. The thiosulfate dehydrogenase of A. thiooxidans JCM7814 was purified using three chromatographies. The purified sample was electrophoretically homogeneous. The molecular mass of the enzyme was 27.9 kDa and it was a monomer. This enzyme had cytochrome c. The optimum pH and temperature of this enzyme were 3.5 and 35 degrees C. The enzyme was stable in the pH range from 5 to 7, and it was stable up to 45 degrees C. The isoelectric point of the enzyme was 8.9. This enzyme reacted with thiosulfate as a substrate. The Km was 0.81 mM.

Acidithiobacillus thiooxidans↗

Influence of initial pH on bioleaching of heavy metals from contaminated soil employing indigenous Acidithiobacillus thiooxidans.

Bioleaching of heavy metals from contaminated soil was carried out employing indigenous sulfur oxidizing bacterium Acidithiobacillus thiooxidans. Experiments were carried out to assess the influence of initial pH of the system on bioleaching of chromium, zinc, copper, lead and cadmium from metal contaminated soil. pH at the end of four weeks of bioleaching at different initial pH of 3-7 was between 0.9 and 1.3, ORP between 567 and 617mV and sulfate production was in the range of 6090-8418mgl(-1). Chromium, zinc, copper, lead and cadmium solubilization ranged from "59% to 98%" at different initial pH. A. thiooxidans was not affected by the increasing pH of the bioleaching system towards neutral and it was able to utilize elemental sulfur. The results of the present study are encouraging to develop the bioleaching process for decontamination of heavy metal contaminated soil.

Acidithiobacillus thiooxidans↗

The effects of metabolites from the indigenous Acidithiobacillus thiooxidans and temperature on the bioleaching of cadmium from soil.

The effect of metabolites from the indigenous Acidithiobacillus thiooxidans and temperature on the bioleaching of cadmium from soil was investigated in the present study. Bioleaching was found to be more effective than chemical leaching of cadmium. The metabolite, mainly sulfuric acid, which was shown to be growth-associated in the exponential phase, plays a major role in bioleaching. The maximum amount of cadmium leached was obtained after 8 days of precultivation when cells were directly involved in the leaching process. It indicates that cells in the exponential growth phase exhibit higher activity toward bioleaching. In contrast, the maximum amount of cadmium leached and the maximum initial rate for bioleaching were reached after 16 days of precultivation when only metabolites were involved in the bioleaching process. It implies that higher sulfuric acid concentration results in higher leaching efficiency. In addition, higher temperature leads to higher leaching efficiency. The optimal operation condition for bioleaching was determined to be a two-stage process: The first stage involves the precultivation of the indigenous A. thiooxidans at 30 degrees C for 8 days followed by 20 minutes of centrifugation to discard cells. The second stage involves the bioleaching with the subsequent supernatant at 50 degrees C.

Acidithiobacillus thiooxidans↗

Biochemical and molecular characterization of the NAD(+)-dependent isocitrate dehydrogenase from the chemolithotroph Acidithiobacillus thiooxidans.

An isocitrate dehydrogenase (ICDH) with an unique coenzyme specificity from Acidithiobacillus thiooxidans was purified and characterized, and its gene was cloned. The native enzyme was homodimeric with a subunit of M(r) 45000 and showed a 78-fold preference for NAD(+) over NADP(+). The cloned ICDH gene (icd) was expressed in an icd-deficient strain of Escherichia coli EB106; the activity was found in the cell extract. The gene encodes a 429-amino acid polypeptide and is located between open reading frames encoding a putative aconitase gene (upstream of icd) and a putative succinyl-CoA synthase beta-subunit gene (downstream of icd). A. thiooxidans ICDH showed high sequence similarity to bacterial NADP(+)-dependent ICDH rather than eukaryotic NAD(+)-dependent ICDH, but the NAD(+)-preference of the enzyme was suggested due to residues conserved in the coenzyme binding site of the NAD(+)-dependent decarboxylating dehydrogenase.

Amino Acid Sequence↗

[Expression of phosphofructokinase gene from Escherichia coli K-12 in obligately autotrophic bacterium Acidithiobacillus thiooxidans].

A plasmid pSDK-1 containing the Escherichia coli phosphofructokinase-1 (EC 2.7.1. 11) gene (pfkA) was constructed and transferred into Acidithiobacillus thiooxidans Tt-Z2 by conjugation. The transfer frequency of plasmid from E. coli to Tt-Z2 was 2.6 x 10(-6). More than 68% of Tt-Z2 cells carried the recombinant plasmids after being cultured for 50 generations without selective pressure, which showed that pSDK-1 was maintained consistently in Tt-Z2. The pfkA gene from E. coli could be expressed in this obligately autotrophic bacterium but the enzyme activity (14 U/g was lower than that in E. coli (K-12: 86 U/g; DF1010 carrying plasmid pSDK-1: 97 U/g). In th presence of glucose, the Tt-Z2 transconjugant consumed glucose leading to a better growth yield.

Acidithiobacillus thiooxidans↗

Effects of inhibitors and NaCl on the oxidation of reduced inorganic sulfur compounds by a marine acidophilic, sulfur-oxidizing bacterium, Acidithiobacillus thiooxidans strain SH.

The effect of NaCl and the pathways of the oxidation of reduced inorganic sulfur compounds were studied using resting cells and cell-free extracts of Acidithiobacillus thiooxidans strain SH. This isolate specifically requires NaCl for growth. The oxidation of sulfur and sulfite by resting cells was strongly inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide. Carbonylcyanide m-chlorophenyl-hydrazone and monensin were also relatively strong inhibitors. Thiosulfate-oxidizing activity was not inhibited by these uncouplers. Valinomycin did not inhibit the oxidation of sulfur compounds. NaCl stimulated the sulfur- and sulfite-oxidizing activities in resting cells but not in cell-free extracts. The tetrathionate-oxidizing activity in resting cells was slightly stimulated by NaCl, whereas it did not influence the thiosulfate-oxidizing activity. Sulfide oxidation was biphasic, suggesting the formation of intermediate sulfur. The initial phase of sulfide oxidation was not affected by NaCl, whereas the subsequent oxidation of sulfur in the second phase was Na+-dependent. A model is proposed for the role of NaCl in the metabolism of reduced sulfur compounds in A. thiooxidans strain SH.

Acidithiobacillus thiooxidans↗

Reduction of vanadium(V) with Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans.

Biotechnological leaching has been proposed as a suitable method for extraction of vanadium from spent catalysts and oil ash. In the biological leaching process, the vanadium(V) can be reduced to vanadium(IV), which is a less toxic and more soluble form of the vanadium. The present investigation showed that Acidithiobacillus ferrooxidans efficiently reduced vanadium(V) in the form of vanadium pentaoxide, to vanadyl(IV) ions, and tolerated high concentrations of vanadium(IV) and vanadium(V). A. ferrooxidans was compared with Acidithiobacillus thiooxidans, which has previously been utilized for vanadium leaching and reduction. Vanadium pentaoxide and sodium vanadate were used as model compounds. The results of this study indicate possibilities to develop an economical and technically feasible process for biotechnological vanadium recovery.

Acidithiobacillus↗

Removal of hydrogen sulfide by sulfate-resistant Acidithiobacillus thiooxidans AZ11.

Toxic H2S gas is an important industrial pollutant that is applied to biofiltration. Here, we examined the effects of factors such as inlet concentration and space velocity on the removal efficiency of a bacterial strain capable of tolerating high sulfate concentrations and low pH conditions. We examined three strains of Acidithiobacillus thiooxidans known to have sulfur-oxidizing activity, and identified strain AZ11 as having the highest tolerance for sulfate. A. thiooxidans AZ11 could grow at pH 0.2 in the presence of 74 g l(-1) sulfate, the final oxidation product of elemental sulfur, in the culture broth. Under these conditions, the specific sulfur oxidation rate was 2.9 g-S g-DCW (dry cell weight)(-1) d(-1). The maximum specific sulfur oxidation rate of A. thiooxidans AZ11 was 21.2 g-S g-DCW(-1) d(-1), which was observed in the presence of 4.2 g-SO4(2-) l(-1) and pH 1.5, in the culture medium. To test the effects of various factors on biofiltration by this strain, A. thiooxidans AZ11 was inoculated into a porous ceramic biofilter. First, a maximum inlet loading of 670 g-S m(-3) h(-1) was applied with a constant space velocity (SV) of 200 h(-1) (residence time, 18 s) and the inlet concentration of H2S was experimentally increased from 200 ppmv to 2200 ppmv. Under these conditions, less than 0.1 ppmv H2S was detected at the biofilter outlet. When the inlet H2S was maintained at a constant concentration of 200 ppmv and the SV was increased from 200 h(-1) to 400 h(-1) (residence time, 9 s), an H2S removal of 99.9% was obtained. However, H2S removal efficiencies decreased to 98% and 94% when the SV was set to 500 h(-1) (residence time, 7.2 s) and 600 h(-1) (residence time, 6 s), respectively. The critical elimination capacity guaranteeing 96% removal of the inlet H2S was determined to be 160 g-S m(-3) h(-1) at a space velocity of 600 h(-1). Collectively, these findings show for the first time that a sulfur oxidizing bacterium has a high sulfate tolerance and a high sulfur oxidizing activity below pH 1.

Acidithiobacillus thiooxidans↗

Conversion of an obligate autotrophic bacteria to heterotrophic growth: expression of a heterogeneous phosphofructokinase gene in the chemolithotroph Acidithiobacillus thiooxidans.

A plasmid pSDK-1 containing the Escherichia coli phosphofructokinase-1 gene (pfkA) was constructed, and transferred into Acidithiobacillus thiooxidans Tt-7 by conjugation. The pfkA gene from E. coli could be expressed in this obligately autotrophic bacterium but the enzyme activity (18 U g-1) was lower than that in E. coli (K12: 86 U g-1; DF1010 carrying plasmid pSDK-1: 97 U g-1). In the presence of glucose, the Tt-7 transconjugant consumed glucose leading to a better growth yield.

Conjugation, Genetic↗

Simultaneous removal of H2S and NH3 in biofilter inoculated with Acidithiobacillus thiooxidans TAS.

H2S and NH3 gases are toxic, corrosive and malodorous air pollutants. Although there are numerous well-established physicochemical techniques presently available for the treatment of these gases, the growing demand for a more economical and improved process has prompted investigations into biological alternatives. In biological treatment methods, H2S is oxidized to SO4(2-) by sulfur-oxidizing bacteria, and then NH3 is removed by chemical neutralization with SO4(2-) to (NH4)2SO4. Since the accumulated (NH4)2SO4 can inhibit microbial activity, it is important to utilize an effective sulfur-oxidizing bacterium that has tolerance to high concentrations of (NH4)2SO4 for the simultaneous removal of H2S and NH3. In this study, a sulfur-oxidizing bacterium with tolerance to high concentrations of (NH4)2SO4 was isolated from activated sludge and identified as Acidithiobacillus thiooxidans TAS. A. thiooxidans TAS could display its sulfur-oxidizing activity in a medium supplemented with 60 g.l(-1) (NH4)2SO4, even though its growth and sulfur-oxidizing activity were completely inhibited in 80 g.l(-1) (NH4)2SO4. When H2S alone was supplied to a ceramic biofilter inoculated with A. thiooxidans TAS, an almost 100% H2S removal efficiency was maintained until the inlet H2S concentration was increased up to 900 microl.l(-1) and the space velocity up to 500 h(-1), at which the amount of H2S eliminated was 810 g-S.m(-3).h(-1). However, when NH3 (50-500 microl.l(-1)) was simultaneously supplied to the biofilter with H2S, the maximum amount of H2S eliminated decreased to 650 g-S.m(-3).h(-1). The inhibition of H2S removal by low NH3 concentrations (50-200 microl.l(-1)) was similar to that by high NH3 concentrations (300-500 microl.l(-1)). The critical inlet H2S load that resulted in over 99% removal was determined as 400 g-S.m(-3).h(-1) in the presence of NH3.

Acidithiobacillus thiooxidans↗

The sulfane sulfur of persulfides is the actual substrate of the sulfur-oxidizing enzymes from Acidithiobacillus and Acidiphilium spp.

To identify the actual substrate of the glutathione-dependent sulfur dioxygenase (EC 1.13.11.18) elemental sulfur oxidation of the meso-acidophilic Acidithiobacillus thiooxidans strains DSM 504 and K6, Acidithiobacillus ferrooxidans strain R1 and Acidiphilium acidophilum DSM 700 was analysed. Extraordinarily high specific sulfur dioxygenase activities up to 460 nmol x min(-1) (mg protein)(-1) were found in crude extracts. All cell-free systems oxidized elemental sulfur only via glutathione persulfide (GSSH), a non-enzymic reaction product from glutathione (GSH) and elemental sulfur. Thus, GSH plays a catalytic role in elemental sulfur activation, but is not consumed during enzymic sulfane sulfur oxidation. Sulfite is the first product of sulfur dioxygenase activity; it further reacted non-enzymically to sulfate, thiosulfate or glutathione S-sulfonate (GSSO(-3)). Free sulfide was not oxidized by the sulfur dioxygenase. Persulfide as sulfur donor could not be replaced by other sulfane-sulfur-containing compounds (thiosulfate, polythionates, bisorganyl-polysulfanes or monoarylthiosulfonates). The oxidation of H(2)S by the dioxygenase required GSSG, i.e. the disulfide of GSH, which reacted non-enzymically with sulfide to give GSSH prior to enzymic oxidation. On the basis of these results and previous findings a biochemical model for elemental sulfur and sulfide oxidation in Acidithiobacillus and Acidiphilium spp. is proposed.

Acetobacteraceae↗

Succession of sulfur-oxidizing bacteria in the microbial community on corroding concrete in sewer systems.

Microbially induced concrete corrosion (MICC) in sewer systems has been a serious problem for a long time. A better understanding of the succession of microbial community members responsible for the production of sulfuric acid is essential for the efficient control of MICC. In this study, the succession of sulfur-oxidizing bacteria (SOB) in the bacterial community on corroding concrete in a sewer system in situ was investigated over 1 year by culture-independent 16S rRNA gene-based molecular techniques. Results revealed that at least six phylotypes of SOB species were involved in the MICC process, and the predominant SOB species shifted in the following order: Thiothrix sp., Thiobacillus plumbophilus, Thiomonas intermedia, Halothiobacillus neapolitanus, Acidiphilium acidophilum, and Acidithiobacillus thiooxidans. A. thiooxidans, a hyperacidophilic SOB, was the most dominant (accounting for 70% of EUB338-mixed probe-hybridized cells) in the heavily corroded concrete after 1 year. This succession of SOB species could be dependent on the pH of the concrete surface as well as on trophic properties (e.g., autotrophic or mixotrophic) and on the ability of the SOB to utilize different sulfur compounds (e.g., H2S, S0, and S2O3(2-)). In addition, diverse heterotrophic bacterial species (e.g., halo-tolerant, neutrophilic, and acidophilic bacteria) were associated with these SOB. The microbial succession of these microorganisms was involved in the colonization of the concrete and the production of sulfuric acid. Furthermore, the vertical distribution of microbial community members revealed that A. thiooxidans was the most dominant throughout the heavily corroded concrete (gypsum) layer and that A. thiooxidans was most abundant at the highest surface (1.5-mm) layer and decreased logarithmically with depth because of oxygen and H2S transport limitations. This suggested that the production of sulfuric acid by A. thiooxidans occurred mainly on the concrete surface and the sulfuric acid produced penetrated through the corroded concrete layer and reacted with the sound concrete below.

Acidithiobacillus thiooxidans↗

Effects of bacterial activities on the release of heavy metals from contaminated dredged sediments.

The potential impact of indigenous bacterial processes on the release of heavy metals from dredged sediment deposits was investigated. Batch re-suspension experiments were conducted in order to investigate the release of Zn, Cd, Cu and Pb from a polluted anoxic sediment submitted to oxidative perturbations. The concentrations of heavy metals, sulphate and dissolved organic carbon (DOC) were periodically recorded, and cell counts were performed to follow the evolution of several bacterial species. The specific effects of microbial processes were quantified by performing re-suspension assays on sterilised samples. Moreover, the effect of an initial acidification of the system was studied. The results showed that metal release was mainly due to oxidation of sulphide minerals contained in the sediment. Sulphur-oxidising bacteria such as Acidithiobacillus thiooxidans were identified to play a major role in the process, by enhancing the oxidation kinetic. However, the acid production resulting from these reactions was almost totally buffered by the dissolution of the calcite present in the sediment. Copper was released to a lesser extent, and a strong association with organic matter was observed. Lead was not observed in solution, because of its low solubility at neutral conditions and of its re-adsorption on the solid phase. The initial acidification of the system resulted in an faster growth of the acidophilic A. thiooxidans. A subsequent pH drop originating from microbial processes was then observed during the first stages of the experiment. As a consequence, drastic increases in metal (Zn, Cd) release were observed.

Acidithiobacillus thiooxidans↗

Marine acidophilic sulfur-oxidizing bacterium requiring salts for the oxidation of reduced inorganic sulfur compounds.

An acidophilic sulfur-oxidizing bacterium was isolated from seawater, and designated as strain SH. Strain SH was a Gram-negative, rod-shaped and motile bacterium, which had an optimum temperature and pH value for growth of 30 degrees C and 4.0, respectively. The mol% guanine plus cytosine of the DNA was 46.0. Chemolithotrophic growth was observed with elemental sulfur and tetrathionate at pH 4.0, and was not observed with ferrous ion. The isolate was able to utilize carbon dioxide as a carbon source, and was unable to grow heterotrophically with yeast extract or glucose. The growth of strain SH was activated in medium supplemented with NaCl. However, LiCl and KCl did not sustain the growth of strain SH. The results indicate that strain SH was an acidophilic, halophilic, and obligately chemolithotrophic sulfur-oxidizing bacterium. Phylogenetic analysis based on 16S rDNA sequences indicated that strain SH had a close relationship to Acidithiobacillus thiooxidans. The oxidizing activities of sulfur and sulfite with resting cells were stimulated not only by the addition of NaCl, but also by KCl and LiCl. The oxidation of sulfite was inhibited by ionophores, carbonyl cyanide- m-chlorophenylhydrazone (CCCP), and monensin, and respiratory inhibitors, KCN and 2-heptyl-4-hydroxy-quinoline-N-oxode (HQNO).

Acidithiobacillus thiooxidans↗

Bioleaching of sulfidic tailing samples with a novel, vacuum-positive pressure driven bioreactor.

This study presents a design for a novel bioreactor that uses alternating vacuum and positive pressure cycles to transfer acidic leach solution in and out of contact with finely ground sulfidic mine tailings. These tailings constitute an environmental problem that needs experimental data to support the development of management and control strategies. A conventional stirred tank bioreactor was used as a reference system. Both bioreactors were inoculated with mixed cultures of acidophilic iron and sulfur oxidizers. The rate of the bioleaching of tailings was 0.50 +/- 0.14 g Fe/L . day in the stirred tank bioreactor and 0.17 +/- 0.05 g Fe/L . day in the novel bioreactor. Microbial populations were identified in the two-bioreactor systems by analysis of 16S rRNA genes involving amplification, denaturing gradient gel electrophoresis (DGGE), cloning, and sequencing. The inoculum contained sulfur-oxidizing Acidithiobacillus caldus and Acidithiobacillus thiooxidans, iron oxidizers from the genera Leptospirillum and Ferroplasma, and a chemoorganotrophic Alicyclobacillus sp. During bioleaching of the tailings, the microbial populations in both bioreactors were similar to the inoculum culture, except that At. thiooxidans outgrew At. caldus. Sequences consistent with a Sulfobacillus sp. were amplified from both bioreactor samples although this bacterium was initially below the level of detection in the inoculum. After prolonged operation, Ferroplasma acidiphilum and an uncultured bacterium related to the CFB group were also detected in the novel bioreactor, whereas Sulfobacillus sp. was no longer detected. The novel bioreactor has potential uses in other areas of environmental biotechnology that involves periodic contact of liquids with solid substrates.

Acidithiobacillus thiooxidans↗