Thiobacillus ferrooxidans, a facultative hydrogen oxidizer.

The type strain (ATCC 23270) and two other strains of Thiobacillus ferrooxidans were able to grow by hydrogen oxidation, a feature not recognized before. When cultivated on H2, a hydrogenase was induced and the strains were less extremely acidophilic than during growth on sulfidic ores. Cells of T. ferrooxidans grown on H2 and on ferrous iron showed 100% DNA homology. Hydrogen oxidation was not observed in eight other species of the genus Thiobacillus and in Leptospirillum ferrooxidans.     

Chemolithotrophic Bacteria in Copper Ores Leached at High Sulfuric Acid Concentration

Extensive bacterial growth was observed when copper sulfide ores were leached with 0.6 N sulfuric acid. The bacterial population developed in this condition was examined by characterization of the spacer regions between the 16S and 23S rRNA genetic loci obtained after PCR amplification of the DNA extracted from the leached ore. The spacers observed had the sizes found in strains of "Leptospirillum ferrooxidans" and Thiobacillus thiooxidans, except for a larger one, approximately 560 bp long, that was not observed in any of the strains examined, including those of Thiobacillus ferrooxidans. The bacteria with this last spacer were selected after culturing in mineral and elemental sulfur media containing 0.7 N sulfuric acid. The spacer and the 16S ribosomal DNA of this isolate were sequenced and compared with those in species commonly found in bioleaching processes. Though the nucleotide sequence of the spacer showed an extensive heterologous region with T. thiooxidans, the sequence of its 16S rDNA gene indicated a close relationship (99.85%) with this species. These results indicate that a population comprised of bacterial strains closely related to T. thiooxidans and of another strain, possibly related to "L. ferrooxidans," can develop during leaching at high sulfuric acid concentration. Iron oxidation in this condition is attributable to "L. ferrooxidans" and not T. ferrooxidans, based on the presence of spacers with the "L. ferrooxidans" size range and the absence of spacers characteristic of T. ferrooxidans.     

氧化亚铁硫杆菌分离复壮及固定化的研究

用稀释涂布平板法从已退化的氧化亚铁硫杆菌（Thiobacillus ferrooxidans）菌液中分离出氧化活性较高、生命力强的氧化亚铁硫杆菌T1。以H2软性填料作为氧化亚铁硫杆菌的固定化载体,构建了固定床生物反应器。考察了固定床生物反应器氧化Fe2+的情况：Fe2+最大氧化速率达7.67g/(L·h)。并对固定床生物反应器运行过程中在载体表面形成的沉淀物进行了研究,通过X衍射证明此沉淀物为黄钾铁矾［Kfe3(SO4)2(OH)6］。     

Monitoring of bacteria in acid mine environments by reverse sample genome probing

A variety of microorganisms can exist in acid mine drainage (AMD) environments, although their contribution to AMD problems is unclear. Environmental strains of Thiobacillus ferrooxidans and Thiobacillus acidophilus were purified by repeated plating and single-colony isolation on iron salts and tetrathionate media, respectively. Thiobacillus thiooxidans was enriched on sulfur-containing media. For the isolation of Leptospirillum ferrooxidans, iron salts and pyrite media were inoculated with environmental samples. However, L. ferrooxidans was never recovered on solid media. Denatured chromosomal DNAs from type and (or) isolated strains of T. ferrooxidans, T. acidophilus, T. thiooxidans, and L. ferrooxidans were spotted on a master filter for their detection in a variety of samples by reverse sample genome probing (RSGP). Analysis of enrichments of environmental samples by RSGP indicated that ferrous sulfate medium enriched T. ferrooxidans strains, whereas all thiobacilli grew in sulfur medium, T. thiooxidans strains being dominant. Enrichment in glucose medium followed by transfer to tetrathionate medium resulted in the selection of T. acidophilus strains. DNA was also extracted directly (without enrichment) from cells recovered from AMD water or sediments, and was analyzed by RSGP to describe the communities present. Strains showing homology with T. ferrooxidans and T. acidophilus were found to be major community components. Strains showing homology with T. thiooxidans were a minor community component, whereas strains showing homology with L. ferrooxidans were not detected.     

SULFATE REQUIREMENT FOR IRON OXIDATION BY THIOBACILLUS FERROOXIDANS

Lazaroff, Norman (British Columbia Research Council, Vancouver, B.C., Canada). Sulfate requirement for iron oxidation by Thiobacillus ferrooxidans. J. Bacteriol. 85:78-83. 1963.-The growth of Thiobacillus ferrooxidans is initially inhibited in media containing ferrous chloride in place of ferrous sulfate. This inhibition of growth is due to the requirement of a high relative proportion of sulfate ions to chloride (or other anions) for iron oxidation. Adaptation takes place, producing strains which are able to oxidize iron in media containing an initially unfavorable anionic composition. Adaptation is possibly due to the selection of spontaneous mutants capable of oxidizing iron in high chloride, low sulfate media. Such cells are found at a frequency of 10(-5) of the population of unadapted cultures.     

Mobilization of Thiobacillus ferrooxidans plasmids among Escherichia coli strains

Nonconjugative Thiobacillus ferrooxidans plasmids were mobilized at high frequencies among Escherichia coli strains by the IncP plasmid RP4 and at low frequencies by the IncN plasmid R46, but not by the IncW plasmid pSa. The mobilization region of a nonconjugative T. ferrooxidans plasmid was located on a 5.3-kilobase T. ferrooxidans DNA fragment.     

Mobilization of Thiobacillus ferrooxidans plasmids among Escherichia coli strains.

Nonconjugative Thiobacillus ferrooxidans plasmids were mobilized at high frequencies among Escherichia coli strains by the IncP plasmid RP4 and at low frequencies by the IncN plasmid R46, but not by the IncW plasmid pSa. The mobilization region of a nonconjugative T. ferrooxidans plasmid was located on a 5.3-kilobase T. ferrooxidans DNA fragment.     

Thermal characterization of different isolates of Thiobacillus ferrooxidans

Abstract Ten different isolates of Thiobacillus ferrooxidans were characterized with respect to temperature in the range 2–35°C. Four of the ten strains oxidized ferrous iron exponentially over the entire range of incubation temperatures, including the lowest temperature tested (2°C), and were therefore characterized as psychrotrophic. Jarosite production was substantially reduced at temperatures less than 10°C and was not observed at 2°C. Energy of activation values were in the range 75.2–96.6 kJ/mol°C and indicated that iron oxidation at low temperatures was governed by both a chemical and a physical control.     

Preliminary proteomic analysis of Thiobacillus ferrooxidans growing on elemental sulphur and Fe2+ separately

Thiobacillus ferrooxidans is one of the most important bacterium used in bioleaching, and can utilize Fe2+ or sulphide as energy source. Growth curves for Thiobacillus ferrooxidans have been tested, which show lag, logarithmic, stationary and aging phases as seen in other bacteria. The logarithmic phases were from 10 to 32 hours for Thiobacillus ferrooxidans cultivated with Fe2+ and from 4 to 12 days for Thiobacillus ferrooxidans cultivated with elemental sulphur. Differences of protein patterns of Thiobacillus ferrooxidans growing on elemental sulphur and Fe2+ separately were investigated after cultivation at 30 degrees C by the analysis of two-dimensional gel electrophoresis (2-DE), matrix-assisted laser desorption/ ionization (MALDI)-Mass spectrometry and ESI-MS/MS. From the 17 identified protein spots, 11 spots were found more abundant when growing on elemental sulphur. By contrast 6 protein spots were found decreased at elemental cultivation condition. Among the proteins identified, cytochrome C have been previously identified as necessary elements of electron-transferring pathway for Thiobacillus ferrooxidans to oxidize Fe2+; ATP synthase alpha chain and beta are expressed increased when Thiobacillus ferrooxidans cultivated with Fe2+ as energy source. ATP synthase Beta chain is the catalytic subunit, and ATP synthase alpha chain is a regulatory subunit. The function of ATPase produces ATP from ADP in the presence of a proton gradient across the membrane.     

Mercuric reductase enzyme from a mercury-volatilizing strain of Thiobacillus ferrooxidans.

Cell-free mercury volatilization activity (mercuric reductase) was obtained from a mercury-volatilizing Thiobacillus ferrooxidans strain, and the properties of intact-cell and cell-free activities were compared with those determined by plasmid R100 in Escherichia coli. Intact cells of T. ferrooxidans volatilized mercury at pH 2.5, whereas cells of E. coli did not. Cell-free enzyme preparations from both bacteria functioned best at or above neutral pH and not at all at pH 2.5. The T. ferrooxidans mercuric reductase was a soluble enzyme that was dependent upon added NAD(P)H. The enzyme activity was stable at 80 degrees C, required an added thiol compound, and was stimulated by EDTA. Antisera against purified mercuric reductases from transposon Tn501 and plasmid R831 (which inactivated mercuric reductases from a wide range of enteric and pseudomonad strains) did not inactivate the enzyme from T. ferrooxidans.     

Identification of Thiobacillus ferrooxidans strains based on restriction fragment length polymorphism analysis of 16S rDNA.

The 16S rDNA sequences from ten strains of Thiobacillus ferrooxidans were amplified by PCR. The products were compared by performing restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases Alu I, Hap II, Hha I, and Hae III. The RFLP patterns revealed that T. ferrooxidans could be distinguished from other iron- or sulphur-oxidizing bacteria such as T. thiooxidans NB1-3, T. caldus GO-1, Leptospirillum ferrooxidans and the marine iron-oxidizing bacterium strain KU2-11. The RFLP patterns obtained with Alu I, Hap II, and Hae III were the same for nine strains of T. ferrooxidans except for strain ATCC 13661. The RFLP patterns for strains NASF-1 and ATCC 13661 with Hha I were distinct from those for other T. ferrooxidans strains. The 16S rDNA sequence of T. ferrooxidans NASF-1 possessed an additional restriction site for Hha I. These results show that iron-oxidizing bacteria isolated from natural environments were rapidly identified as T. ferrooxidans by the method combining RFLP analysis with physiological analysis.     

氧化亚铁硫杆菌氧化亚硫酸盐的动力学研究

实验用Ms培养基,利用去除铁离子的氧化亚铁硫杆菌(Thiobacillus ferrooxidans)进行了细菌亚硫酸盐的生长代谢研究。实验结果表明氧化亚铁硫杆菌对亚硫酸根具有一定的氧化能力。用Origin 7.0对实验数据进行拟合处理,表明了氧化亚铁硫杆菌催化氧化亚硫酸盐的动力学方程符合Hill方程。氧化亚铁硫杆菌催化氧化亚硫酸盐是一个底物抑制的细胞反应,其KS值随pH值和底物浓度的改变而变化。pH值对反应有很大的影响,pH值越接近中性KS就越小,反应速率就越大。     

Production of rhodanese by bacteria present in bio-oxidation plants used to recover gold from arsenopyrite concentrates

Considerably larger quantities of cyanide are required to solubilize gold following the bio-oxidation of gold-bearing ores compared with oxidation by physical-chemical processes. A possible cause of this excessive cyanide consumption is the presence of the enzyme rhodanese. Rhodanese activities were determined for the bacteria most commonly encountered in bio-oxidation tanks. Activities of between 6.4 and 8.2 micromol SCN min(-1) mg protein(-1) were obtained for crude enzyme extracts of Thiobacillus ferrooxidans, Thiobacillus thiooxidans and Thiobacillus caldus, but no rhodanese activity was detected in Leptospirillum ferrooxidans. Rhodanese activities 2-2.5-fold higher were found in the total mixed cell mass from a bio-oxidation plant. T. ferrooxidans synthesized rhodanese irrespective of whether it was grown on iron or sulphur. With a PCR-based detection technique, only L. ferrooxidans and T. caldus cells were detected in the bio-oxidation tanks. As no rhodanese activity was associated with L. ferrooxidans, it was concluded that T. caldus was responsible for all of the rhodanese activity. Production of rhodanese by T. caldus in batch culture was growth phase-dependent and highest during early stationary phase. Although the sulphur-oxidizing bacteria were clearly able to convert cyanide to thiocyanate, it is unlikely that this rhodanese activity is responsible for the excessive cyanide wastage at the high pH values associated with the gold solubilization process.     

Ferrous iron-dependent volatilization of mercury by the plasma membrane of Thiobacillus ferrooxidans

Of 100 strains of iron-oxidizing bacteria isolated, Thiobacillus ferrooxidans SUG 2-2 was the most resistant to mercury toxicity and could grow in an Fe(2+) medium (pH 2.5) supplemented with 6 microM Hg(2+). In contrast, T. ferrooxidans AP19-3, a mercury-sensitive T. ferrooxidans strain, could not grow with 0.7 microM Hg(2+). When incubated for 3 h in a salt solution (pH 2.5) with 0.7 microM Hg(2+), resting cells of resistant and sensitive strains volatilized approximately 20 and 1.7%, respectively, of the total mercury added. The amount of mercury volatilized by resistant cells, but not by sensitive cells, increased to 62% when Fe(2+) was added. The optimum pH and temperature for mercury volatilization activity were 2.3 and 30 degrees C, respectively. Sodium cyanide, sodium molybdate, sodium tungstate, and silver nitrate strongly inhibited the Fe(2+)-dependent mercury volatilization activity of T. ferrooxidans. When incubated in a salt solution (pH 3.8) with 0.7 microM Hg(2+) and 1 mM Fe(2+), plasma membranes prepared from resistant cells volatilized 48% of the total mercury added after 5 days of incubation. However, the membrane did not have mercury reductase activity with NADPH as an electron donor. Fe(2+)-dependent mercury volatilization activity was not observed with plasma membranes pretreated with 2 mM sodium cyanide. Rusticyanin from resistant cells activated iron oxidation activity of the plasma membrane and activated the Fe(2+)-dependent mercury volatilization activity of the plasma membrane.     

Optimal conditions for bio-oxidation of ferrous ions to ferric ions using Thiobacillus ferrooxidans

A chemo-biochemical process using Thiobacillus ferrooxidans for desulphurization of gaseous fuels and emissions containing hydrogen sulphide (H2S) has been developed. In the first stage, H2S present in fuel gas and emissions is selectively oxidized to elemental sulphur using ferric sulphate. The ferrous sulphate produced in the first stage of the process is oxidized to ferric sulphate using Thiobacillus ferrooxidans for recycle and reuse in the process. The effects of process variables, temperature, pH, total dissolved solids (TDS), elemental sulphur, ferric and magnesium ions on bio-oxidation of ferrous ions to ferric ions were investigated using flask culture experiments. The bio-oxidation of ferrous ions to ferric ions could be achieved efficiently in the temperature range of 20(+/-1)-44(+/-1) degrees C. A pH range of 1.8(+/-0.02)-2.2(+/-0.02) was optimum for the growth of culture and effective bio-oxidation of ferrous ions to ferric ions. The effect of TDS on bio-oxidation of ferrous ions indicated that a preacclimatized culture in a growth medium containing high dissolved solid was required to achieve effective bio-oxidation of ferrous ions. Elemental sulphur ranging from 1000 to 100,000 mg/l did not have any effect on efficiency of ferrous ion oxidation. The efficiency of bio-oxidation of ferrous ions to ferric ions was not affected in the presence of ferric ions up to a concentration of 500 mg/l while 3 mg/l of magnesium ion was optimal for achieving effective bio-oxidation.     

Activation of bovine plasminogen by Streptococcus uberis

Abstract Thiosulfate and tetrathionate oxidation activity of Thiobacillus ferrooxidans were found to be absent in iron-growth cell as well as in the cells grown anaerobically on elemental sulfur. While the thiosulfate oxidase activity was absent in the cell-free extract of the above cells, the activity of rhodanese was present irrespective of the culture condition of T. ferrooxidans . It is thus conceivable that rhodanese is not involved in thiosulfate metabolism. During growth in presence of ferrous sulfate plus elemental sulfur, the thiosulfate/tetrathionate oxidation activity was absent till the oxidation of ferrous iron was complete and the cells harvested only in the latter period acquired the thiosulfate/tetrathionate oxidation activity. Thus it becomes evident that the inhibition of thiosulfate and tetrathionate oxidation is solely due to presence of ferrous iron.     

Cultural and phylogenetic analysis of mixed microbial populations found in natural and commercial bioleaching environments.

A range of autotrophic and heterotrophic enrichment cultures were established to determine the cultural bacterial diversity present in samples obtained from the acidic runoff of a chalcocite overburden heap and from laboratory-scale (1- to 4-liter) batch and continuous bioreactors which were being used for the commercial assessment of the bioleachability of zinc sulfide ore concentrates. Strains identified as Thiobacillus ferrooxidans, Thiobacillus thiooxidans, "Leptospirillum ferrooxidans," and Acidiphilium cryptum were isolated from both the natural site and the batch bioreactor, but only "L. ferrooxidans," a moderately thermophilic strain of T. thiooxidans, and a moderately thermophilic iron-oxidizing bacterium could be recovered from the continuous bioreactor running under steady-state conditions. Sequence analysis of the 16S rRNA genes of 33 representative strains revealed that all of the strains were closely related to strains which have been sequenced previously and also confirmed the phylogenetic diversity of bacteria present in bioleaching environments.     

Existence of a new type of sulfite oxidase which utilizes ferric ions as an electron acceptor in Thiobacillus ferrooxidans

A new type of sulfite oxidase which utilizes ferric ion (Fe3+) as an electron acceptor was found in iron-grown Thiobacillus ferrooxidans. It was localized in the plasma membrane of the bacterium and had a pH optimum at 6.0. Under aerobic conditions, 1 mol of sulfite was oxidized by the enzyme to produce 1 mol of sulfate. Under anaerobic conditions in the presence of Fe3+, sulfite was oxidized by the enzyme as rapidly as it was under aerobic conditions. In the presence of o-phenanthroline or a chelator for Fe2+, the production of Fe2+ was observed during sulfite oxidation by this enzyme under not only anaerobic conditions but also aerobic conditions. No Fe2+ production was observed in the absence of o-phenanthroline, suggesting that the Fe2+ produced was rapidly reoxidized by molecular oxygen. Neither cytochrome c nor ferricyanide, both of which are electron acceptors for other sulfite oxidases, served as an electron acceptor for the sulfite oxidase of T. ferrooxidans. The enzyme was strongly inhibited by chelating agents for Fe3+. The physiological role of sulfite oxidase in sulfur oxidation of T. ferrooxidans is discussed.     

Existence of a new type of sulfite oxidase which utilizes ferric ions as an electron acceptor in Thiobacillus ferrooxidans.

A new type of sulfite oxidase which utilizes ferric ion (Fe3+) as an electron acceptor was found in iron-grown Thiobacillus ferrooxidans. It was localized in the plasma membrane of the bacterium and had a pH optimum at 6.0. Under aerobic conditions, 1 mol of sulfite was oxidized by the enzyme to produce 1 mol of sulfate. Under anaerobic conditions in the presence of Fe3+, sulfite was oxidized by the enzyme as rapidly as it was under aerobic conditions. In the presence of o-phenanthroline or a chelator for Fe2+, the production of Fe2+ was observed during sulfite oxidation by this enzyme under not only anaerobic conditions but also aerobic conditions. No Fe2+ production was observed in the absence of o-phenanthroline, suggesting that the Fe2+ produced was rapidly reoxidized by molecular oxygen. Neither cytochrome c nor ferricyanide, both of which are electron acceptors for other sulfite oxidases, served as an electron acceptor for the sulfite oxidase of T. ferrooxidans. The enzyme was strongly inhibited by chelating agents for Fe3+. The physiological role of sulfite oxidase in sulfur oxidation of T. ferrooxidans is discussed.     

Electrotransformation of Thiobacillus ferrooxidans with plasmids containing a mer determinant.

The mer operon from a strain of Thiobacillus ferrooxidans (C. Inoue, K. Sugawara, and T. Kusano, Mol. Microbiol. 5:2707-2718, 1991) consists of the regulatory gene merR and an operator-promoter region followed by merC and merA structural genes and differs from other known gram-negative mer operons. We have constructed four potential shuttle plasmids composed of a T. ferrooxidans-borne cryptic plasmid, a pUC18 plasmid, and the above-mentioned mer determinant as a selectable marker. Mercury ion-sensitive T. ferrooxidans strains were electroporated with constructed plasmids, and one strain, Y4-3 (of 30 independent strains tested), was found to have a transformation efficiency of 120 to 200 mercury-resistant colonies per microgram of plasmid DNA. This recipient strain was confirmed to be T. ferrooxidans by physiological, morphological, and chemotaxonomical data. The transformants carried a plasmid with no physical rearrangements through 25 passages under no selective pressure. Cell extracts showed mercury ion-dependent NADPH oxidation activity.