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).     

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.     

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 (FeS(2)) 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.     

In vitro loading of apoferritin.

This study compared the effect of loading apoferritin either with ferrous ammonium sulfate in various buffers or with ceruloplasmin and chelated ferrous iron. It was shown that loading of apoferritin with ferrous ammonium sulfate was dependent on buffer and pH, and was directly related to the rate of iron autoxidation. The ceruloplasmin-dependent loading of apoferritin, however, was unaffected by these factors. Isoelectric focusing and amino acid analysis of the differently loaded ferritins showed that ferrous ammonium sulfate loading of apoferritin resulted in the depletion of the basic amino acids, lysine and histidine, probably as a result of protein oxidation. No significant differences in amino acid composition was noted for ceruloplasmin-loaded ferritin. Furthermore, ferritin loaded with ferrous ammonium sulfate released more iron than either native or ceruloplasmin-loaded ferritin when either paraquat or EDTA was used as an iron mobilizing agent. We suggest that the loading of apoferritin with ferrous ammonium sulfate occurred as a result of iron autoxidation and may result in oxidation of amino acids and loss of integrity of the protein, and that ceruloplasmin may act as a catalyst for the incorporation of iron into apoferritin in a manner more closely related to that occurring in vivo.     

Two pathways of iron uptake in bovine spleen apoferritin dependent on iron concentration

Iron incorporation by bovine spleen apoferritin either with ferrous ammonium sulfate in different buffers or with ferrous ammonium sulfate and phosphate was studied. Iron uptake and iron autoxidation were recorded spectrophotomerically. The buffers [4-(2-hydroxyethyl)-1-piperazinyl]ethanesulphonic acid (Hepes) and tris(hydroxymethyl)aminoethane (Tris) exhibited pH-dependent iron autoxidation, with Tris showing less iron autoxidation than Hepes. An Eadie-Scatchard plot (v/[s] versus v) of the iron uptake rate in Hepes was a curved rather than a straight line, suggesting that there are two iron uptake pathways. On the other hand, the Eadie-Scatchard plots of Tris and of Hepes after the addition of phosphate showed a straight line. Phosphate accelerated the iron uptake rate. The iron loading kinetics of apoferritin in Hepes was dependent on apoferritin concentration. The K_m value obtained from iron uptake kinetics was 4.5 M, corresponding to the physiological iron concentration. These results demonstrate that iron loading of apoferritin was accomplished at physiological iron concentrations, which is essential for iron uptake, via two uptake pathways of dependent on iron concentration.     

Kinetics of phosphate-mediated oxidation of ferrous iron and formation of 8-oxo-2'-deoxyguanosine in solutions of free 2'-deoxyguanosine and calf thymus DNA

Formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) in solutions of free 2'-deoxyguanosine (dG) and calf thymus DNA (DNA) was compared for the diffusion-dependent and localised production of oxygen radicals from phosphate-mediated oxidation of ferrous iron (Fe2+) to ferric iron (Fe3+). The oxidation of Fe2+ to Fe3+ was followed at 304 nm at pH 7.2 under aerobic conditions. Given that the concentration of Fe2+ >or=phosphate concentration, the rate of Fe2+ oxidation was significantly higher in DNA-phosphate as compared for the same concentration of inorganic phosphate. Phosphate catalysed oxidation of ferrous ions in solutions of dG or DNA led through the production of reactive oxygen species to the formation of 8-oxo-dG. The yield of 8-oxo-dG in solutions of dG or DNA correlated positively with the inorganic-/DNA-phosphate concentrations as well as with the concentrations of ferrous ions added. The yield of 8-oxo-dG per unit oxidised Fe2+ were similar for dG and DNA; thus, it differed markedly from radiation-induced 8-oxo-dG, where the yield in DNA was several fold higher.For DNA in solution, the localisation of the phosphate ferrous iron complex relative to the target is an important factor for the yield of 8-oxo-dG. This was supported from the observation that the yield of 8-oxo-dG in solutions of dG was significantly increased over that in DNA only when Fe2+ was oxidised in a high excess of inorganic phosphate (50 mM) and from the lower protection of DNA damage by the radical scavenger (hydroxymethyl)aminomethane (Tris)-HCl.     

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.     

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.     

Kinetics of phosphate-mediated oxidation of ferrous iron and formation of 8-oxo-2′-deoxyguanosine in solutions of free 2′-deoxyguanosine and calf thymus DNA

Formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) in solutions of free 2′-deoxyguanosine (dG) and calf thymus DNA (DNA) was compared for the diffusion-dependent and localised production of oxygen radicals from phosphate-mediated oxidation of ferrous iron (Fe²⁺) to ferric iron (Fe³⁺). The oxidation of Fe²⁺ to Fe³⁺ was followed at 304 nm at pH 7.2 under aerobic conditions. Given that the concentration of Fe²⁺ ≥phosphate concentration, the rate of Fe²⁺ oxidation was significantly higher in DNA-phosphate as compared for the same concentration of inorganic phosphate. Phosphate catalysed oxidation of ferrous ions in solutions of dG or DNA led through the production of reactive oxygen species to the formation of 8-oxo-dG. The yield of 8-oxo-dG in solutions of dG or DNA correlated positively with the inorganic-/DNA-phosphate concentrations as well as with the concentrations of ferrous ions added. The yield of 8-oxo-dG per unit oxidised Fe²⁺ were similar for dG and DNA; thus, it differed markedly from radiation-induced 8-oxo-dG, where the yield in DNA was several fold higher.For DNA in solution, the localisation of the phosphate ferrous iron complex relative to the target is an important factor for the yield of 8-oxo-dG. This was supported from the observation that the yield of 8-oxo-dG in solutions of dG was significantly increased over that in DNA only when Fe²⁺ was oxidised in a high excess of inorganic phosphate (50 mM) and from the lower protection of DNA damage by the radical scavenger (hydroxymethyl)aminomethane (Tris)–HCl.     

Calcification in insects: The dwelling-tube and midgut of machaerotid larvae (Homoptera)

The dwelling-tubes of machaerotid larvae consist of a mineralized organic scaffolding of mucofibrils. The mineral component accounts for 85 per cent of the dry weight and is composed of calcium, ferrous iron, manganese, magnesium, potassium, sodium, phosphate, carbonate, and chloride and of these the major ions are calcium and carbonate. Ferric iron in the form of ferritin is probably also present.Calcium, manganese, magnesium, and phosphate are derived from spherites secreted by a specialized region of the midgut. Calcium and phosphate are present in the spherites, probably as amorphous tricalcium phosphate. Subsequent to secretion the spherites are slowly dissolved and the calcium is incorporated into the dwelling-tube as calcium carbonate. Thus it appears that within the dwelling-tube calcium phosphate is converted to calcium carbonate.Ferritin and ferrous iron are secreted by another specialized region of the midgut and are also incorporated into the dwelling-tube.     

Mineral and iron oxidation at low temperatures by pure and mixed cultures of acidophilic microorganisms

An enrichment culture from a boreal sulfide mine environment containing a low-grade polymetallic ore was tested in column bioreactors for simulation of low temperature heap leaching. PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing revealed the enrichment culture contained an Acidithiobacillus ferrooxidans strain with high 16S rRNA gene similarity to the psychrotolerant strain SS3 and a mesophilic Leptospirillum ferrooxidans strain. As the mixed culture contained a strain that was within a clade with SS3, we used the SS3 pure culture to compare leaching rates with the At. ferrooxidans type strain in stirred tank reactors for mineral sulfide dissolution at various temperatures. The psychrotolerant strain SS3 catalyzed pyrite, pyrite/arsenopyrite, and chalcopyrite concentrate leaching. The rates were lower at 5 degrees C than at 30 degrees C, despite that all the available iron was in the oxidized form in the presence of At. ferrooxidans SS3. This suggests that although efficient At. ferrooxidans SS3 mediated biological oxidation of ferrous iron occurred, chemical oxidation of the sulfide minerals by ferric iron was rate limiting. In the column reactors, the leaching rates were much less affected by low temperatures than in the stirred tank reactors. A factor for the relatively high rates of mineral oxidation at 7 degrees C is that ferric iron remained in the soluble phase whereas, at 21 degrees C the ferric iron precipitated. Temperature gradient analysis of ferrous iron oxidation by this enrichment culture demonstrated two temperature optima for ferrous iron oxidation and that the mixed culture was capable of ferrous iron oxidation at 5 degrees C.     

Comparison of ferric iron generation by different species of acidophilic bacteria immobilized in packed-bed reactors

Flooded packed-bed bioreactors, prepared by immobilizing four different species of acidophilic iron-oxidizing bacteria on porous glass beads, were compared for their ferric iron-generating capacities when operated in batch and continuous flow modes over a period of up to 9 months, using a ferrous iron-rich synthetic liquor and acid mine drainage (AMD) water. The bacteria used were strains of Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans, a Ferrimicrobium-like isolate (TSTR) and a novel Betaproteobacterium (isolate PSTR), which were all isolated from relatively low-temperature mine waters. Three of the bacteria used were chemoautotrophs, while the Ferrimicrobium isolate was an obligate heterotroph. Greater biomass yields achievable with the Ferrimicrobium isolate resulted in greater iron oxidation efficiency in the newly commissioned bioreactor containing this bacterium, though long-term batch testing with organic carbon-free solution resulted in similar maximum iron oxidation rates in all four bioreactors. Two of the bioreactors (those containing immobilized L. ferrooxidans and Ferrimicrobium TSTR) were able to generate significantly lower concentrations of ferrous iron than the others when operated in batch mode. In contrast, when operated as continuous flow systems, the bioreactor containing immobilized PSTR was superior to the other three when challenged with either synthetic or actual AMD at high flow rates. The least effective bacterium overall was At. ferrooxidans, which has previously been the only iron-oxidizer used in the majority of reports describing ferric iron-generating bioreactors. The results of these experiments showed that different species of iron-oxidizing acidophiles have varying capacities to oxidize ferrous iron when immobilized in packed-bed bioreactors, and that novel isolates may be superior to well-known species.     

Inhibition of growth, iron, and sulfur oxidation in Thiobacillus ferrooxidans by simple organic compounds.

Iron and sulfur oxidation by Thiobacillus ferrooxidans as well as growth on ferrous iron were inhibited by a variety of low molecular weight organic compounds. The influences of chemical structure of the organic inhibitors, pH, temperature, physical treatment of cells, and added inhibitory or stimulatory inorganic ions and iron oxidation suggest that a major factor contributing to the inhibitory effects on iron oxidation is the relative electronegativity of the organic molecule. The data also suggest that inhibitory organic compounds may (i) directly affect the iron-oxidizing enzyme system, (ii) react abiologically with ferrous iron outside the cell, (iii) interfere with the roles of phosphate and sulfate in iron oxidation, and (iv) nonselectively disrupt the cell envelope or membrane.     

Isolation of thermophilic iron-oxidising bacteria from sulfidic waste rock

Summary A thermophilic, rod-shaped, iron-oxidising bacterium was isolated by enrichment culture of rock samples from an overburden dump at the Rum Jungle mine site in Australia's Northern Territory. Oxidation of ferrous iron and sulfur occurred at 50–55°C, with a temperature maximum of 60°C. The isolate required yeast extract for growth. The pH optimum for iron oxidation at 50°C was 1.4. Rapid iron-oxidation occurred at a pH as low as 0.35, but little or no oxidation occurred at or above pH 2.2.     

Iron and carbon metabolism by a mineral-oxidizing Alicyclobacillus-like bacterium

A novel iron-oxidizing, moderately thermophilic, acidophilic bacterium (strain “GSM”) was isolated from mineral spoil taken from a gold mine in Montana. Biomolecular analysis showed that it was most closely related to Alicyclobacillus tolerans, although the two bacteria differed in some key respects, including the absence (in strain GSM) of ϖ-alicyclic fatty acids and in their chromosomal base compositions. Isolate GSM was able to grow in oxygen-free media using ferric iron as terminal electron acceptor confirming that it was a facultative anaerobe, a trait not previously described in Alicyclobacillus spp.. The acidophile used both organic and inorganic sources of energy and carbon, although growth and iron oxidation by isolate GSM was uncoupled in media that contained both fructose and ferrous iron. Fructose utilization suppressed iron oxidation, and oxidation of ferrous iron occurred only when fructose was depleted. In contrast, fructose catabolism was suppressed when bacteria were harvested while actively oxidizing iron, suggesting that both ferrous iron- and fructose-oxidation are inducible in this acidophile. Isolate GSM accelerated the oxidative dissolution of pyrite in liquid media either free of, or amended with, organic carbon, although redox potentials were significantly different in these media. The potential of this isolate for commercial mineral processing is discussed.     

Anaerobic and aerobic oxidation of ferrous iron at neutral pH by chemoheterotrophic nitrate-reducing bacteria

Nine out of ten anaerobic enrichment cultures inoculated with sediment samples from various freshwater, brackish-water, and marine sediments exhibited ferrous iron oxidation in mineral media with nitrate and an organic cosubstrate at pH 7.2 and 30° C. Anaerobic nitrate-dependent ferrous iron oxidation was a biological process. One strain isolated from brackish-water sediment (strain HidR2, a motile, nonsporeforming, gram-negative rod) was chosen for further investigation of ferrous iron oxidation in the presence of acetate as cosubstrate. Strain HidR2 oxidized between 0.7 and 4.9 mM ferrous iron aerobically and anaerobically at pH 7.2 and 30° C in the presence of small amounts of acetate (between 0.2 and 1.1 mM). The strain gained energy for growth from anaerobic ferrous iron oxidation with nitrate, and the ratio of iron oxidized to acetate provided was constant at limiting acetate supply. The ability to oxidize ferrous iron anaerobically with nitrate at approximately pH 7 appears to be a widespread capacity among mesophilic denitrifying bacteria. Since nitrate-dependent iron oxidation closes the iron cycle within the anoxic zone of sediments and aerobic iron oxidation enhances the reoxidation of ferrous to ferric iron in the oxic zone, both processes increase the importance of iron as a transient electron carrier in the turnover of organic matter in natural sediments. Received: 24 April 1997 / Accepted: 22 September 1997     

Mineralogical characteristics of residues produced in microbiological treatment of acidic mine drainages

Inorganic residues produced on the disks of rotating biological contactor (RBC) wastewater treatment units during microbiological oxidation of the ferrous iron in both natural and synthetic acidic mine drainages were analyzed using x‐ray energy‐dispersive microanalysis, x‐ray diffraction, and scanning electron microscopy. The predominant mineral forms were x‐ray amorphous ferric hydroxysulfates. In addition, jarosites were detected in RBC units which treated natural mine drainages, and magnetite was detected in solids generated during treatment of a synthetic acidic mine drainage. Bacteria were observed on the surface of the inorganic residues and in gelatinous films seen microscopically following chemical dissolution of the iron crust.     

Microbiological Oxidation of Ferrous Iron at Low Temperatures

Acidophilic iron-oxidizing bacteria were enriched from mine water samples with ferrous sulfate as the substrate at incubation temperatures in the range of 4 to 46°C. After several subcultures at each test temperature except 46°C, which was prohibitive to growth, the rates of iron oxidation were determined in batch cultures. The results yielded linear rates in a semilogarithmic scale. The rate constants of iron oxidation by growing cultures were fitted into the Arrhenius equation, which displayed linearity in the 4 to 28°C range and yielded an activation energy value of 83 ± 3 kJ/mol.     

Ferric iron reduction by Thiobacillus ferrooxidans at extremely low pH-values

When ferrous iron and sulfur were supplied, cells of T. ferrooxidans in a well-aerated medium started growth by oxidizing ferrous iron. After ferrous iron depletion a lagphase followed before sulfur oxidation started. During sulfur oxidation at pH-values below 1.3 (±0,2) the ferrous iron concentration increased again, although the oxygen saturation of the medium amounted to more than 95%. The number of viable cells did not increase. Thus resting cells of T. ferrooxidans, which are oxidizing sulfur to maintain their proton balance, reduce ferric to ferrous iron. The ferrous iron-oxidizing system seemed to be inhibited at pH-values below 1.3. At a pH-value of 1.8 the ferrous iron was reoxidized at once. A scheme for the linkage of iron- and sulfur metabolism is discussed.