Growth and Maintenance of Thiobacillus ferrooxidans Cells

A rapid and sensitive spectrophotometric procedure was developed for monitoring the growth of Thiobacillus ferrooxidans in liquid culture. Values determined for the optical densities at 500 nm of washed T. ferrooxidans cell suspensions were directly proportional to both total cell number and total cell protein concentration and provided an accurate measurement of culture growth rate. The utility of this procedure was demonstrated by conducting physiological studies on the influence of CO₂ and FeSO₄ availability on the growth of T. ferrooxidans. In addition, we describe a procedure for the long-term maintenance of cells T. ferrooxidans that ensures culture purity and genetic stability.     

Sensitivity of Iron-Oxidizing Bacteria, Thiobacillus ferrooxidans and Leptospirillum ferrooxidans, to Bisulfite Ion

When grown on iron-salt medium supplemented with the bisulfite ion, Leptospirillum ferrooxidans was much more sensitive to the ion than was Thiobacillus ferrooxidans. The causes of the sensitivity of L. ferrooxidans to the bisulfite ion were studied. The bisulfite ion completely inhibited the iron-oxidizing activities of L. ferrooxidans and T. ferrooxidans at 0.02 and 0.2 mM, respectively. A trapping reagent for the bisulfite ion, formaldehyde, completely reversed the inhibition. The treatment of intact cells with 1.0 mM bisulfite ion for 1 h and washing the bisulfite ion from the cells had no harmful effects on the iron-oxidizing activity of T. ferrooxidans. However, the treatment of L. ferrooxidans with 0.1 mM bisulfite ion for 1 h completely destroyed the iron-oxidizing activity. T. ferrooxidans had sulfite:ferric ion oxidoreductase activity. In contrast, a quite low level of sulfite:ferric ion oxidoreductase activity was found in L. ferrooxidans, suggesting that it is much more difficult for L. ferrooxidans to oxidize the bisulfite ion to the less harmful sulfate than it is for T. ferrooxidans. These results suggest that the sensitivity of L. ferrooxidans to the bisulfite ion is due to a lack of an active sulfite:ferric ion oxidoreductase and the sensitivity of its iron oxidase to bisulfite ion.     

Purification and characterization of the hydrogenase from Thiobacillus ferrooxidans

Hydrogenase of Thiobacillus ferrooxidans ATCC 19859 was purified from cells grown lithoautotrophically with 80% hydrogen, 8.6% carbon dioxide, and 11.4% air. Hydrogenase was located in the 140,000 ×g supernatant in cell-free extracts. The enzyme was purified 7.3-fold after chromatography on Procion Red and Q-Sepharose with a yield of 19%, resulting in an 85% pure preparation with a specific activity of 6.0 U (mg protein)^–1. With native PAGE, a mol. mass of 100 and 200 kDa was determined. With SDS-PAGE, two subunits of 64 (HoxG) and of 34 kDa (HoxK) were observed. Hydrogenase reacted with methylene blue and other artificial electron acceptors, but not with NAD. The optimum of enzyme activity was at pH 9 and at 49° C. Hydrogenase contained 0.72 mol nickel and 6.02 mol iron per mol enzyme. The relationship of the T. ferrooxidans hydrogenase to other proteins was examined. A 9.5-kb EcoRI fragment of T. ferrooxidans ATCC 19859 hybridized with a 2.2-kb XhoI fragment from Alcaligenes eutrophus encoding the membrane-bound hydrogenase. Antibodies against this enzyme did not react with the T. ferrooxidans hydrogenase in Western blot analysis. The N-terminal amino acid sequence (40 amino acids) of HoxK was 46% identical to that of the hydrogen sensor HupU of Bradyrhizobium japonicum and 39% identical to that of the HupS subunit of the Desulfovibrio baculatus hydrogenase. The N-terminal sequence of 20 amino acids of HoxG of T. ferrooxidans was 83.3% identical to that of the 60-kDa subunit. HupL, of the hydrogenase of Anabaena sp. Sequences of ten internal peptides of HoxG were 50–100% identical to the respective sequences of HupL of the Anabaena sp. hydrogenase. Received: 17 November 1995 / Accepted: 2 February 1996     

氧化亚铁硫杆菌的形态及对Fe2+的氧化研究

在纯培养的条件下,对江西德兴铜矿酸性矿坑水中分离出的一株氧化亚铁硫杆菌(Thiobacillus ferrooxidans)的细胞形态、生长条件以及对Fe2 的氧化进行了初步研究。透射电子显微镜检查的结果表明,其成熟菌体大小均一,有较好的运动性;采用光学显微镜对微生物进行菌群观测和利用血小板计数器法对细菌计数的结果表明,在摇床转速为160r/min的条件下,T.f.菌在9K液体培养基中最适生长条件为温度30℃左右,最佳初始pH 2.0;用重铬酸钾滴定法测定铁的结果表明,在摇床转速为160r/min的条件下,pH值1.7,温度30℃时T.f.菌对Fe2 的氧化速率最大,约为0.58g/L·h。     

Iron Oxidation and Precipitation of Ferric Hydroxysulfates by Resting Thiobacillus ferrooxidans Cells

The oxidation of ferrous ions, in acid solution, by resting suspensions of Thiobacillus ferrooxidans produced sediments consisting of crystalline jarosites, amorphous ferric hydroxysulfates, or both. These products differed conspicuously in chemical composition and infrared spectra from precipitates formed by abiotic oxidation under similar conditions. The amorphous sediments, produced by bacterial oxidation, exhibited a distinctive fibroporous microstructure when examined by scanning electron microscopy. Infrared spectra indicated outer-sphere coordination of Fe(III) by sulfate ions, as well as inner-sphere coordination by water molecules and bridging hydroxo groups. In the presence of excess sulfate and appropriate monovalent cations, jarosites, instead of amorphous ferric hydroxysulfates, precipitated from bacterially oxidized iron solutions. It is proposed that the jarositic precipitates result from the conversion of outer-sphere (T_d) sulfate, present in a soluble polymeric Fe(III) complex, to inner-sphere (C_3v) bridging sulfate. The amorphous precipitates result from the further polymerization of hydroxo-linked iron octahedra and charge stabilized aggregation of the resulting iron complexes in solution. This view was supported by observations that bacterially oxidized iron solutions gave rise to either amorphous or jarositic sediments in response to ionic environments imposed after oxidation had been completed and the bacteria had been removed by filtration.     

Combined degradation of covellite by Thiobacillus thiooxidans and Thiobacillus ferrooxidans

Summary In the presence of iron, which is always associated with natural sulphide ores, the percentages of copper dissolution in the bioleaching of covellite were 34 and 45 % when Thiobacillus thiooxidans and Thiobacillus ferrooxidans were used together and when an indirect bioleaching with attached bacteria was performed respectively. In the latter, the percentage of copper dissolution was still higher than the percentages obtained with pure cultures (36 % with a T. thiooxidans culture and 40 % with a T. ferrooxidans culture).     

包埋氧化亚铁硫杆菌的海藻酸钙凝胶扩散特性研究

采用非稳态法测定了FeSO4在未包埋氧化亚铁硫杆菌的凝胶中的有效扩散系数,分析包埋细菌的氧化情况.结果表明,FeSO4在凝胶中的有效扩散系数De随着海藻酸钠浓度的升高而降低,当海藻酸钠浓度为2%时最优;凝胶剂CaCl2的浓度对扩散系数的影响较小.包埋的氧化亚铁硫杆菌在10h达到增殖平衡,而FeSO4在包埋细菌的凝胶内扩散系数明显减少.包埋的氧化亚铁硫杆菌在初始铁浓度为5g/L时,完全氧化所需时间最短但氧化速率变化较快,当初始铁浓度为8g/L和10g/L时,完全氧化所需时间相同.     

Production and Regeneration of Thiobacillus ferrooxidans Spheroplasts

We have isolated a metabolite of territrem, designated territrem B', from the chloroform extract of a rice culture of Aspergillus terreus 23-1 by using the same isolation procedure as that for territrems A, B, and C. The present isolation procedure gave about 10 mg of territrem B' from 4 kg of rice culture per batch. Analysis of the high-resolution mass spectrum showed that the molecular composition of territrem B' is C29H34O10 (found, 542.2167; required, 542.200). Some results of physicochemical and acute tests are presented in this paper. Single-crystal X-ray diffractometry of territrem B' indicated that the three-dimensional structure of territrem B' has not changed significantly from that of territrem B except for the insertion of one oxygen atom into territrem B to make an additional pyron ring in the E ring. The tremorgenic activity of territrem B' is greatly reduced as tested by intraperitoneal injection in mice.     

EPR study and structural aspects of ferredoxins obtained from Thiobacillus ferrooxidans

A comparison of iron-sulfur proteins obtained from Thiobacillus ferrooxidans was carried out. The microorganisms were grown on iron(II)- or sulfur-containing nutrients. In both cases different, broad elctron paramagnetic resonance (EPR) lines, originating from an iron(III) compound, were detected. Additional EPR lines of tetrahedral iron(III) and free radicals were observed. The UV spectra of these compounds also differ. Received: 15 July 1998 / Received revision: 8 October 1998 / Accepted: 16 October 1998     

Importance of Extracellular Polymeric Substances from Thiobacillus ferrooxidans for Bioleaching

Leaching bacteria such as Thiobacillus ferrooxidans attach to pyrite or sulfur by means of extracellular polymeric substances (EPS) (lipopolysaccharides). The primary attachment to pyrite at pH 2 is mediated by exopolymer-complexed iron(III) ions in an electrochemical interaction with the negatively charged pyrite surface. EPS from sulfur cells possess increased hydrophobic properties and do not attach to pyrite, indicating adaptability to the substrate or substratum.     

Growth Kinetics of Thiobacillus ferrooxidans Isolated from Arsenic Mine Drainage

Thiobacillus ferrooxidans is found in many Alaskan and Canadian drainages contaminated by metals dissolved from placer and lode gold mines. We have examined the iron-limited growth and iron oxidation kinetics of a T. ferrooxidans isolate, AK1, by using batch and continuous cultures. Strain AK1 is an arsenic-tolerant isolate obtained from placer gold mine drainage containing large amounts of dissolved arsenic. The steady-state growth kinetics are described with equations modified for threshold ferrous iron concentrations. The maximal specific growth rate (μ_max) for isolate AK1 at 22.5°C was 0.070 h⁻¹, and the ferrous iron concentration at which the half-maximal growth rate occurred (K_μ) was 0.78 mM. Cell yields varied inversely with growth rate. The iron oxidation kinetics of this organism were dependent on biomass. We found no evidence of ferric inhibition of ferrous iron oxidation for ferrous iron concentrations between 9.0 and 23.3 mM. A supplement to the ferrous medium of 2.67 mM sodium arsenite did not result in an increased steady-state biomass, nor did it appear to affect the steady-state growth kinetics observed in continuous cultures.     

Competitive Inhibition of Ferrous Iron Oxidation by Thiobacillus ferrooxidans by Increasing Concentrations of Cells

The oxidation of ferrous iron (Fe²⁺) to ferric iron (Fe³⁺) with dioxygen (O₂) by various strains of Thiobacillus ferrooxidans was studied by measuring the rate of O₂ consumption at various Fe²⁺ concentrations and cell concentrations. The apparent K_m values for Fe²⁺ remained constant at different cell concentrations of laboratory strains ATCC 13661 and ATCC 19859 but increased with increasing cell concentrations of mine isolates SM-4 and SM-5. The latter results are explained by the competitive inhibition of the Fe²⁺-binding site of a cell by other cells in the reaction mixture. Possible mechanisms involving cell surface properties are discussed.     

Crystallization and preliminary X-ray crystallographic studies of rusticyanin from Thiobacillus ferrooxidans.

Rusticyanin is a 16.5 kDa type I blue copper protein isolated from Thiobacillus ferrooxidans. This organism can grow on Fe2+ as its sole energy source. Rusticyanin is thought to be a principal component in the iron respiratory electron transport chain of T. ferrooxidans. As a component of the periplasmic space of an acidophilic bacterium, rusticyanin is remarkably stable at acidic pH. It is redox-active down to pH 0.2. Crystals of rusticyanin have been grown from solutions of PEG 8000 by the hanging-drop vapor diffusion method. The crystals are orthorhombic, space group P2(1)2(1)2(1), with unit cell dimensions a = 32.36 A, b = 60.37 A, c = 74.60 A. The crystals diffract to 2.0 A resolution and they are stable in the X-ray beam for at least two days.     

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.     

A molecular analysis of a broad-host-range plasmid isolated from Thiobacillus ferrooxidans

Abstract: A 12.4-kb plasmid, pTF-FC2, that was isolated from Thiobacillus ferrooxidans and which is capable of replication in a wide range of Gram-negative bacteria, has been sequenced. The extent of the regions involved in both replication and mobilization have been delineated. The site of initiation of replication ( oriV ) has been localized on a 185-bp fragment and the origin of transfer ( oriT ) on a 138-bp fragment. Three proteins that were essential for replication and four that were essential for mobilization have been identified. The origin of replication was clearly similar to that of the IncQ plasmids although no complementation or incompatibility between pTF-FC2 and the IncQ plasmid, R300B, was detected. There was a clear similarity in the size,location and amino acid sequence of the proteins of the pTF-FC2 mobilization region with those of the TraI region of the IncP plasmids, RP4 and R751.Two inverted repeated sequences which had 37/38-bp and 38/38-bp sequence identity with the Tn 21 transposon were identified. The C-terminal part of a transposase and the N-terminal portion of a resolvase were located between the inverted repeats. These open reading frames are most likely the remnants of a defective transposon. A protein with homology to a mercury- resistance regulator was also present within the transposon-like element although no gene encoding for mercury reductase could be indentified.     

Use of Thiobacillus ferrooxidans for iron oxidation and precipitation

Fe(II) oxidation reaction was carried out using an acidophilic microorganism, Thiobacillus ferrooxidans. Four different parameters such as pH, Fe(II), Fe(III) and biomass concentration were studied. The oxida-tion reaction follows a pseudo first order rate equation. Apparent reaction rate constants were calculated. Unified rate equation was developed using the four parameters. Along with oxidation, a part of the iron also was precipitated. The extent of Fe(III) precipitation in each case was calculated. © Rapid Science 1998