Selective Inhibition of the Oxidation of Ferrous Iron or Sulfur in Thiobacillus ferrooxidans

The oxidation of either ferrous iron or sulfur by Thiobacillus ferrooxidans was selectively inhibited or controlled by various anions, inhibitors, and osmotic pressure. Iron oxidation was more sensitive than sulfur oxidation to inhibition by chloride, phosphate, and nitrate at low concentrations (below 0.1 M) and also to inhibition by azide and cyanide. Sulfur oxidation was more sensitive than iron oxidation to the inhibitory effect of high osmotic pressure. These differences were evident not only between iron oxidation by iron-grown cells and sulfur oxidation by sulfur-grown cells but also between the iron and sulfur oxidation activities of the same iron-grown cells. Growth experiments with ferrous iron or sulfur as an oxidizable substrate confirmed the higher sensitivity of iron oxidation to inhibition by phosphate, chloride, azide, and cyanide. Sulfur oxidation was actually stimulated by 50 mM phosphate or chloride. Leaching of Fe and Zn from pyrite (FeS₂) and sphalerite (ZnS) by T. ferrooxidans was differentially affected by phosphate and chloride, which inhibited the solubilization of Fe without significantly affecting the solubilization of Zn.     

Ferrous Iron and Sulfur Oxidation and Ferric Iron Reduction Activities of Thiobacillus ferrooxidans Are Affected by Growth on Ferrous Iron, Sulfur, or a Sulfide Ore

Eight strains of Thiobacillus ferrooxidans (laboratory strains Tf-1 [= ATCC 13661] and Tf-2 [= ATCC 19859] and mine isolates SM-1, SM-2, SM-3, SM-4, SM-5, and SM-8) and three strains of Thiobacillus thiooxidans (laboratory strain Tt [= ATCC 8085] and mine isolates SM-6 and SM-7) were grown on ferrous iron (Fe²⁺), elemental sulfur (S⁰), or sulfide ore (Fe, Cu, and Zn). The cells were studied for their aerobic Fe²⁺ - and S⁰-oxidizing activities (O₂ consumption) and anaerobic S⁰-oxidizing activity with ferric iron (Fe³⁺) (Fe²⁺ formation). Fe²⁺-grown T. ferrooxidans cells oxidized S⁰ aerobically at a rate of 2 to 4% of the Fe²⁺ oxidation rate. The rate of anaerobic S⁰ oxidation with Fe³⁺ was equal to the aerobic oxidation rate in SM-1, SM-3, SM-4, and SM-5, but was only one-half or less that in Tf-1, Tf-2, SM-2, and SM-8. Transition from growth on Fe²⁺ to that on S⁰ produced cells with relatively undiminished Fe²⁺ oxidation activities and increased S⁰ oxidation (both aerobic and anaerobic) activities in Tf-2, SM-4, and SM-5, whereas it produced cells with dramatically reduced Fe²⁺ oxidation and anaerobic S⁰ oxidation activities in Tf-1, SM-1, SM-2, SM-3, and SM-8. Growth on ore 1 of metal-leaching Fe²⁺-grown strains and on ore 2 of all Fe²⁺-grown strains resulted in very high yields of cells with high Fe²⁺ and S⁰ oxidation (both aerobic and anaerobic) activities with similar ratios of various activities. Sulfur-grown Tf-2, SM-1, SM-4, SM-6, SM-7, and SM-8 cultures leached metals from ore 3, and Tf-2 and SM-4 cells recovered showed activity ratios similar to those of other ore-grown cells. It is concluded that all the T. ferrooxidans strains studied have the ability to produce cells with Fe²⁺ and S⁰ oxidation and Fe³⁺ reduction activities, but their levels are influenced by growth substrates and strain differences.     

Growth of Thiobacillus ferrooxidans on Elemental Sulfur

Growth kinetics of Thiobacillus ferrooxidans in batch cultures, containing prills of elementary sulfur as the sole energy source, were studied by measuring the incorporation of radioactive phosphorus in free and adsorbed bacteria. The data obtained indicate an initial exponential growth of the attached bacteria until saturation of the susceptible surface was reached, followed by a linear release of free bacteria due to successive replication of a constant number of adsorbed bacteria. These adsorbed bacteria could continue replication provided the colonized prills were transferred to fresh medium each time the stationary phase was reached. The bacteria released from the prills were unable to multiply, and in the medium employed they lost viability with a half-life of 3.5 days. The spreading of the progeny on the surface was followed by staining the bacteria on the prills with crystal violet; this spreading was not uniform but seemed to proceed through distortions present in the surface. The specific growth rate of T. ferrooxidans ATCC 19859 was about 0.5 day⁻¹, both before and after saturation of the sulfur surface. The growth of adsorbed and free bacteria in medium containing both ferrous iron and elementary sulfur indicated that T. ferrooxidans can simultaneously utilize both energy sources.     

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.     

Kinetics of Iron Oxidation by Thiobacillus ferrooxidans

A statistical relationship between the rate of ferric ion production by a strain of Thiobacillus ferrooxidans and various levels of cell concentration, Fe²⁺ concentration, Na⁺ concentration, and temperature was studied by a direct colorimetric method at 304 nm. The relationship was linear (90 to 93%), cross-product (3 to 4%), and quadratic (1 to 2%). The levels of cell concentration and Fe²⁺ concentration and their respective interactions with one another and the other factors had the most significant effects on the regression models. The solution of the quadratic response surface for optimum oxidation was a saddle point, and the predicted critical levels of temperature, cell concentration, Fe²⁺ concentration, and Na⁺ concentration ranged between −6 and 2°C, 0.43 and 0.62 mg/ml, 72 and 233 mM, and 29.6 mM, respectively.     

Reduction of Cupric Ions with Elemental Sulfur by Thiobacillus ferrooxidans

In anaerobic or aerobic conditions in the presence of 5 mM sodium cyanide, an inhibitor of iron oxidase, cupric ion (Cu²⁺) was reduced enzymatically with elemental sulfur (S⁰) by washed intact cells of Thiobacillus ferrooxidans AP19-3 to give cuprous ion (Cu⁺). The rate of Cu²⁺ reduction was proportional to the concentrations of S⁰ and Cu²⁺ added to the reaction mixture. The pH optimum for the cupric ion-reducing system was 5.0, and the activity was completely destroyed by 10-min incubation of cells at 70°C. The activity of Cu²⁺ reduction with S⁰ by this strain was strongly inhibited by inhibitors of hydrogen sulfide: ferric ion oxidoreductase (SFORase), such as α,α′-dipyridyl, 4,5-dihydroxy-m-benzene disulfonic acid disodium salts, and diazine dicarboxylic acid bis-(N, N-dimethylamide). A SFORase purified from this strain, which catalyzes oxidation of both hydrogen sulfide and S⁰ with Fe³⁺ or Mo⁶⁺ as an electron acceptor in the presence of glutathione, catalyzed a reduction of Cu²⁺ by S⁰, and the Michaelis constant of SFORase for Cu²⁺ was 7.2 mM, indicating that a SFORase catalyzes the reduction of not only Fe³⁺ and Mo⁶⁺ but also Cu²⁺.     

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

Oxidation of ferrous iron by Thiobacillus ferrooxidans SM-4 was inhibited competitively by increasing concentrations of ferric iron or cells. A kinetic analysis showed that binding of one inhibitor did not exclude binding of the other and led to synergistic inhibition by the two inhibitors. Binding of one inhibitor, however, was affected by the other inhibitor, and the apparent inhibition constant increased with increasing concentrations of the other inhibitor.     

氧化亚铁硫杆菌铁氧化系统分子生物学研究进展

氧化亚铁硫杆菌（Thiobacillus ferrooxidans,简称T.f）是目前研究最多、最具经济价值的浸矿微生物。由于该菌的能量代谢对于生物浸矿起决定作用,因此其机制的研究必然能促进对该菌生理特性的认识及其遗传改造。氧化亚铁硫杆菌生长方式代表了迄今所知的能够进行生长的热力学极限,可供该菌生长利用的△Eh仅有340mV,氧化Fe2+所能得到的能量很少。在 Fe2+氧化过程中,电子通过电子传递链最终传递给氧,     

Oxidation of Elemental Sulfur to Sulfite by Thiobacillus thiooxidans Cells

Thiobacillus thiooxidans cells oxidized elemental sulfur to sulfite, with 1 mol of O₂ consumption per mol of sulfur oxidized to sulfite, when the oxidation of sulfite was inhibited with 2-n-heptyl-4-hydroxyquinoline N-oxide.     

Molybdenum Oxidation by Thiobacillus ferrooxidans

Thiobacillus ferrooxidans AP19-3 oxidized molybdenum blue (Mo⁵⁺) enzymatically. Molybdenum oxidase in the plasma membrane of this bacterium was purified ca. 77-fold compared with molybdenum oxidase in cell extract. A purified molybdenum oxidase showed characteristic absorption maxima due to reduced-type cytochrome oxidase at 438 and 595 nm but did not show absorption peaks specific for c-type cytochrome. The optimum pH of molybdenum oxidase was 5.5. The activity of molybdenum oxidase was completely inhibited by sodium cyanide (5 mM) or carbon monoxide, and an oxidized type of cytochrome oxidase in a purified molybdenum oxidase was reduced by molybdenum blue, indicating that cytochrome oxidase in the enzyme plays a crucial role in molybdenum blue oxidation.     

Ferrous Iron Oxidation by Thiobacillus ferrooxidans: Inhibition with Benzoic Acid, Sorbic Acid, and Sodium Lauryl Sulfate

Benzoic acid, sorbic acid, and sodium lauryl sulfate at low concentrations (5 to 10 mg/liter) each effectively inhibited bacterial oxidation of ferrous iron in batch cultures of Thiobacillus ferrooxidans. The rate of chemical oxidation of ferrous iron in low-pH, sterile batch reactors was not substantially affected at the tested concentrations (5 to 50 mg/liter) of any of the compounds.     

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.     

Ferrous iron oxidation and uranium extraction by Thiobacillus ferrooxidans

The microbiological oxidation of ferrous iron in batch and continuous systems has been investigated in relation to uranium extraction from a low-grade ore by Thiobacillus ferrooxidans. The influence of the parameters, agitation, and aeration on oxygen saturation concentration, rate of oxygen mass transfer, and rate of ferrous iron oxidation was demonstrated. The kinetic values, V_max and K were determined using an adapted Monod equation for different dilution rates and initial concentrations of ferrous iron. The power requirements for initial leaching conditions were also calculated. Uranium extraction as high as 68% has been realized during nine days of treatment. Regrinding the leach residue and its subsequent leaching yielded 87% uranium solubilization.     

Effect of Water Potential on Growth and Iron Oxidation by Thiobacillus ferrooxidans

The effect of water potential on the growth of two strains of Thiobacillus ferroxidans was determined by adding defined amounts of sodium chloride or glycerol to the culture medium. The two strains differed slightly, and the most tolerant strain had a minimum water potential for growth of -15 to -32 bars when sodium chloride was used and -6 bars when glycerol was used. In another approach, the limiting water potential was determined by equilibrating small amounts of culture medium with atmospheres of relative humidities equivalent to specific water potentials, and the ability of the organism to grow and oxidize ferrous iron was determined. Under these conditions, which are analogous to those which might control water potential in a coal refuse pile or copper leaching dump, the lower limit at which iron oxidation occurred was -23 bars. The water potential of some coal refuse materials in which T. ferrooxidans was present were determined, and it was found that the water potentials at which the organism was active in these habitats were similar to those at which it was able to grow in culture. However, marked variation in water potential of coal refuse materials was found, presumably due to differences in clays and organic materials, and some coal refuse materials would probably never have water potentials at which the organism could grow. Some literature on the water potentials in copper leach dumps is reviewed, and it is concluded that control of water potential is essential to maximize the success of leaching operations. Because adequate drainage is necessary in a leach dump to ensure sufficient aeration, in many cases water availability in leach dumps may restrict the development of the bacterium necessary for the process.     

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。     

Role of a Ferric Ion-Reducing System in Sulfur Oxidation of Thiobacillus ferrooxidans

The properties of a ferric ion-reducing system which catalyzes the reduction of ferric ion with elemental sulfur was investigated with a pure strain of Thiobacillus ferrooxidans. In anaerobic conditions, washed intact cells of the strain reduced 6 mol of Fe³⁺ with 1 mol of elemental sulfur to give 6 mol of Fe²⁺, 1 mol of sulfate, and a small amount of sulfite. In aerobic conditions, the 6 mol of Fe²⁺ produced was immediately reoxidized by the iron oxidase of the cell, with a consumption of 1.5 mol of oxygen. As a result, Fe²⁺ production was never observed under aerobic conditions. However, in the presence of 5 mM cyanide, which completely inhibits the iron oxidase of the cell, an amount of Fe²⁺ production comparable to that formed under anaerobic conditions was observed under aerobic conditions. The ferric ion-reducing system had a pH optimum between 2.0 and 3.8, and the activity was completely destroyed by 10 min of incubation at 60°C. A short treatment of the strain with 0.5% phenol completely destroyed the ferric ion-reducing system of the cell. However, this treatment did not affect the iron oxidase of the cell. Since a concomitant complete loss of the activity of sulfur oxidation by molecular oxygen was observed in 0.5% phenol-treated cells, it was concluded that the ferric ion-reducing system plays an important role in the sulfur oxidation activity of this strain, and a new sulfur-oxidizing route is proposed for T. ferrooxidans.     

Ferrous ion oxidation by Thiobacillus ferrooxidans immobilized in calcium alginate

Summary Thiobacillus ferrooxidans was immobilized by entrapment into calcium alginate matrix. The immobilized bacteria were used in packed-bed column reactors for the continuous oxidation of ferrous ion at pH 1.5. The presence of mineral salts resulted in a shorter lag period before a steady-state of about 95% iron oxidation was achieved. Parallel shake flask experiments were used to evaluate pH, mineral salts, and alginate toxicity as factors influencing biological iron oxidation. Manometric experiments indicated that the previous growth history of T. ferrooxidans was important in determining the rate of iron oxidation. Scanning electron microscopy and energy dispersive analysis of X-rays were used to characterize bacteria entrapped in calcium alginate and the enrichment of iron in the matrix.     

Oxidation of gallium sulfides by Thiobacillus ferrooxidans.

The bacterial oxidation of naturally occurring gallium-bearing chalcopyrite concentrate and a pure synthetic gallium (III) sulfide has been investigated at pH 1.8 and 35 degree C, using an active culture of Thiobacillus ferrooxidans. This oxidation process may proceed by direct or by indirect bacterial action. The highest dissolved gallium and copper concentrations were about 2.2 and 40.2 g/l, respectively. The order of the specific rate of oxygen uptake by T. ferrooxidans in approximately CuFES2 greater than or equal to gallium-bearing CuFeS2 greater than FeS2 greater than Cu2S greater than Cu2S greater than Ga2S3.