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.     

Anaerobic Growth of Thiobacillus ferrooxidans

The obligately autotrophic acidophile Thiobacillus ferrooxidans was grown on elemental sulfur in anaerobic batch cultures, using ferric iron as an electron acceptor. During anaerobic growth, ferric iron present in the growth media was quantitatively reduced to ferrous iron. The doubling time in anaerobic cultures was approximately 24 h. Anaerobic growth did not occur in the absence of elemental sulfur or ferric iron. During growth, a linear relationship existed between the concentration of ferrous iron accumulated in the cultures and the cell density. The results suggest that ferric iron may be an important electron acceptor for the oxidation of sulfur compounds in acidic environments.     

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.     

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.     

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures was examined, using on-line off-gas analyses to measure the oxygen and carbon dioxide consumption rates continuously. A cell suspension from continuous cultures at steady state was used as the inoculum. It was observed that a dynamic phase occurred in the initial phase of the experiment. In this phase the bacterial ferrous iron oxidation and growth were uncoupled. After about 16 h the bacteria were adapted and achieved a pseudo-steady state, in which the specific growth rate and oxygen consumption rate were coupled and their relationship was described by the Pirt equation. In pseudo-steady state, the growth and oxidation kinetics were accurately described by the rate equation for competitive product inhibition. Bacterial substrate consumption is regarded as the primary process, which is described by the equation for competitive product inhibition. Subsequently the kinetic equation for the specific growth rate, μ, is derived by applying the Pirt equation for bacterial substrate consumption and growth. The maximum specific growth rate, μ _max, measured in the batch culture agrees with the dilution rate at which washout occurs in continuous cultures. The maximum oxygen consumption rate, q _O2,max, of the cell suspension in the batch culture was determined by respiration measurements in a biological oxygen monitor at excess ferrous iron, and showed changes of up to 20% during the course of the experiment. The kinetic constants determined in the batch culture slightly differ from those in continuous cultures, such that, at equal ferric to ferrous iron concentration ratios, biomass-specific rates are up to 1.3 times higher in continuous cultures. Received: 8 February 1999 / Accepted: 17 February 1999     

Development of an optimal medium for continuous ferrous iron oxidation by immobilized Acidothiobacillus ferrooxidans cells

This study was aimed at developing an immobilized bioreactor system in which long-term continuous ferrous iron oxidation can be realized with no formation of jarosite, which causes clogging of support pores and reactor lines. For this purpose, a medium with no jarosite formation was developed first by selecting optimal nitrogen and phosphate sources and their concentrations. Then with the developed medium containing ammonium phosphate instead of ammonium sulfate and potassium phosphate, repeated batch and continuous operations of ferrous iron oxidation by Acidothiobacillus ferrooxidans cells immobilized in a depth filter were successfully performed for an extended period of time. For about 510 h of operation including 450 h of continuous operation at dilution rates of 0.1, 0.2, and 0.3 h(-)(1), no formation of jarosite and thus no clogging of the reactor system were observed. The maximum ferrous iron oxidation rate was as high as 2.6 g/(L.h) at a dilution rate of 0.3 h(-)(1).     

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.     

A kinetic model for biological oxidation of ferrous iron by Thiobacillus ferrooxidans

The kinetics of bacterial oxidation of ferrous iron in the presence of Thiobacillus ferrooxidans cells were studied using an initial-rate method. Measurements of the redox potential of the solution during the oxidation of ferrous iron were used to assess the initial rate of the reaction. Effects on the rate of reaction were determined for ferrous iron concentration in the range 0.25 to 30 kg m(-3), bacterial concentration in the range 3.25 x 10(7) to 4.47 x 10(8) cells mL(-1), and temperature in the range 20 to 35 degrees C. Using these experimental results and an approach based on Michaelis-Menten kinetics, a model for biological oxidation of ferrous iron was developed. The model, which incorporates terms for the effect of temperature and substrate and cell inhibition, was successfully used to simulate the full range of experimental data obtained. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 478-486, 1997.     

Nutritional Requirements of the Micro-organisms Active in the Oxidation of Ferrous Iron in Acid Mine Leach Liquors

The nutritional requirements for the oxidation of ferrous iron in acid mine water by fixed films of micro-organisms were investigated. Supplementing the mine leach liquor with additional ammonium, phosphate and potassium did not improve the efficiency of ferrous iron oxidation. The minimum non-limiting requirement of the ferrous iron oxidizing organisms for these nutrients was (mg/l): ammonium, 10; phosphate, 45, and potassium, 2·5. A strain of Thiobacillus was isolated which was able to grow on a medium to which only dihydrogen potassium phosphate, ferrous sulphate and agar had been added.     

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.     

High-rate ferrous iron oxidation by immobilized Acidithiobacillus ferrooxidans with complex of PVA and sodium alginate

By four different methods, Acidithiobacillus ferrooxidans cells were immobilized by the complex of PVA and sodium alginate. The beads formed by these different methods were evaluated in terms of relative mechanical strength, biological activity, dilatability, and so on. The results indicate that the technique utilizing the complex of PVA and sodium alginate crosslinked with Ca(NO(3))(2) is more appropriate for the immobilization of A. ferrooxidans than any others. So the PVA-calcium nitrate beads were used in batch and continuous culture. A maximum ferrous iron oxidation rate of 4.6 g/l/h was achieved in batch culture. Long-time performance of packed-bed bioreactor was evaluated systematically over 40 days, depending on the conversion ratio of ferrous iron and the residence time. At a residence time of 2.5 h, 96% of the initial ferrous iron was oxidized. This study shows this new immobilization technique will be a feasible and economical method for A. ferrooxidans.     

Inhibitory effects of particulate materials in growing cultures of Thiobacillus ferrooxidans

Particulate materials (flowers of sulfur, fluorapatite, glass beads, and pyrite) inhibited the growth of Thiobacillus ferrooxidans on ferrous iron at pH 1.5. The degree of inhabitation varied with the type of the particulate and culture conditions. The inhibition caused by pyrite involved a soluble toxic component. The inhibition caused by sulfur particles and glass beads was alleviated by incubation under static culture conditions (i.e., no shaking) but oxidation rates declined because of lack of aeration an increase in the pH above 2 also relieved the inhibition. The nature of inhibition. The nature of inhibition by different particles varied from declined iron oxidation rates to prolonged lag periods depending on the concentration of the test material. Thus, the results indicate that the bacterial activity at the liquid-solid interface is different from that in the bulk liquid phase and is influenced by the proton concentration and by the physiochemical properties of the solids.     

Oxidation of Ferrous Iron and Elemental Sulfur by Thiobacillus ferrooxidans

The oxidation of ferrous iron and elemental sulfur by Thiobacillus ferrooxidans that was absorbed and unabsorbed onto the surface of sulfur prills was studied. Unadsorbed sulfur-grown cells oxidized ferrous iron at a rate that was 3 to 7 times slower than that of ferrous iron-grown cells, but sulfur-grown cells were able to reach the oxidation rate of the ferrous iron-adapted cells after only 1.5 generations in a medium containing ferrous iron. Bacteria that were adsorbed to sulfur prills oxidized ferrous iron at a rate similar to that of unadsorbed sulfur-grown bacteria. They also showed the enhancement of ferrous iron oxidation activity in the presence of ferrous iron, even though sulfur continued to be available to the bacteria in this case. An increase in the level of rusticyanin together with the enhancement of the ferrous iron oxidation rate were observed in both sulfur-adsorbed and unadsorbed cells. On the other hand, sulfur oxidation by the adsorbed bacteria was not affected by the presence of ferrous iron in the medium. When bacteria that were adsorbed to sulfur prills were grown at a higher pH (ca. 2.5) in the presence of ferrous iron, they rapidly lost both ferrous iron and sulfur oxidation capacities and became inactive, apparently because of the deposition of a jarosite-like precipitate onto the surface to which they were attached.     

Enzymic comparisons of the inorganic sulfur metabolism in autotrophic and heterotrophic Thiobacillus ferrooxidans.

Activities of enzymes which mediate the oxidation of thiosulfate to sulfate and the assimilation of sulfate to sulfide were assayed in various cell-free fractions of Thiobacillus ferrooxidans grown autotrophically on either ferrous iron or thiosulfate or heterotrophically on glucose. There was no activity of the thiosulfate-oxidizing enzyme in extracts of bacteria grown with ferrous iron. Comparable activities for ATP-sulfurylase (EC 2.7.7.4), ADP-sulfurylase (EC 2.7.7.5), and adenylate kinase (EC 2.7.4.3) were found in the bacteria grown autotrophically with either Fe2+ or S2O32- or heterotrophically with glucose.     

Growth of Thiobacillus ferrooxidans on Formic Acid

A variety of acidophilic microorganisms were shown to be capable of oxidizing formate. These included Thiobacillus ferrooxidans ATCC 21834, which, however, could not grow on formate in normal batch cultures. However, the organism could be grown on formate when the substrate supply was growth limiting, e.g., in formate-limited chemostat cultures. The cell densities achieved by the use of the latter cultivation method were higher than cell densities reported for growth of T. ferrooxidans on ferrous iron or reduced sulfur compounds. Inhibition of formate oxidation by cell suspensions, but not cell extracts, of formate-grown T. ferrooxidans occurred at formate concentrations above 100 μM. This observation explains the inability of the organism to grow on formate in batch cultures. Cells grown in formate-limited chemostat cultures retained the ability to oxidize ferrous iron at high rates. Ribulose 1,5-bisphosphate carboxylase activities in cell extracts indicated that T. ferrooxidans employs the Calvin cycle for carbon assimilation during growth on formate. Oxidation of formate by cell extracts was NAD(P) independent.     

Bioleaching of pyrite by acidophilic thermophile Acidianus brierleyi

The kinetics of bioleaching of pyrite (FeS(2)) by the acidophilic thermophilic bacterium Acidianus brierleyi was studied in a well-mixed batch reactor. Experiments were done at 65 degrees C and pH 1.5 on adsorption of A. brierleyi onto pyrite particles, liquid-phase oxidation of ferrous iron by A. brierleyi, and microbial leaching of pyrite. The adsorption of A. brierleyi was a fast process; equilibrium was attained within the first 30 min of exposure to pyrite. The adsorption equilibrium data were well correlated with the Langmuir isotherm. The oxidation of ferrous iron was markedly accelerated in the presence of A. brierleyi, and the growth yield on ferrous iron was determined. The bioleaching of pyrite by A. brierleyi was found to take place with a direct attack by adsorbed cells on the surface of pyrite, the chemical leaching of pyrite by ferric iron being insignificant. Rate data collected under a wide variety of operating variables were analyzed to determine kinetic and stoichiometric parameters for the microbial pyrite leaching. The specific growth rate on pyrite for A. brierleyi was about four times that for the mesophilic bacterium, Thiobacillus ferrooxidans, whereas the growth yields on pyrite for the two microbes were approximately equal to one another in magnitude. A comparison of A. brierleyi with T. ferrooxidans for pyrite leachability demonstrated the thermophile to be much more effective. (c) 1995 John Wiley & Sons, Inc.     

Bio-oxidation of iron using Thiobacillus ferrooxidans

The effects of pH, ferrous and ferric ion concentrations on iron oxidation by Thiobacillus ferrooxidans were examined. The initial temperature and bacterial concentration were maintained at 37°C and 2±1×104cells/ml, respectively. The iron oxidation rate increased with increased initial ferrous iron concentration to 4g/l and thereafter decreased. The presence of iron(III) showed a negative effect on the bacterial iron oxidation rate. The increase of pH also showed an increase in the oxidation rate up to pH 1.75. The oxidation rate followed first order kinetics for the parameters studied. A rate equation has been developed.     

Bio-oxidation of iron using Thiobacillus ferrooxidans

The effects of pH, ferrous and ferric ion concentrations on iron oxidation by Thiobacillus ferrooxidans were examined. The initial temperature and bacterial concentration were maintained at 37°C and 2±1×104cells/ml, respectively. The iron oxidation rate increased with increased initial ferrous iron concentration to 4g/l and thereafter decreased. The presence of iron(III) showed a negative effect on the bacterial iron oxidation rate. The increase of pH also showed an increase in the oxidation rate up to pH 1.75. The oxidation rate followed first order kinetics for the parameters studied. A rate equation has been developed.     

Growth of Thiobacillus ferrooxidans: a Novel Experimental Design for Batch Growth and Bacterial Leaching Studies

The concentrations of ferrous and ferric ions change dramatically during the course of the batch experiments usually performed to study the kinetics of the bacterial oxidation of ferrous ions and sulfide minerals. This change in concentration of the iron species during the course of the experiment often makes it difficult to interpret the results of these experiments, as is evidenced by the lack of consensus concerning the mechanism of bacterial leaching. If the concentrations of ferrous and ferric ions were constant throughout the course of the batch experiment, then the role of the bacteria could be easily established, because the rate of the chemical leaching should be the same at a given redox potential in the presence and in the absence of bacteria. In this paper we report an experiment designed to obtain kinetic data under these conditions. The redox potential is used as a measure of the concentrations of ferrous and ferric ions, and the redox potential of the leaching solution is controlled throughout the experiment by electrolysis. The effects of ferrous, ferric, and arsenite ions on the rate of growth of Thiobacillus ferrooxidans on ferrous ions in this redox-controlled reactor are presented. In addition, the growth of this bacterium on ferrous ions in batch culture was also determined, and it is shown that the parameters obtained from the batch culture and the redox-controlled batch culture are the same. An analysis of the results from the batch culture indicates that the initial number of bacteria that are adapted to the solution depends on the concentrations of ferrous and arsenite ions.