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.     

Relative contributions of biological and chemical reactions to the overall rate of pyrite oxidation at temperatures between 30 degrees C and 70 degrees C

The stoichiometry and kinetics of the spontaneous, chemical reaction between pyrite and ferric iron was studied at 30, 45, and 70 degrees C in shake flasks at pH 1.5 by monitoring the ferrous iron, total iron, elemental sulfur, and sulfate concentration profiles in time. It was found that the sulfur moiety of pyrite was oxidized completely to sulfate. Elemental sulfur was not produced in detectable amounts. The iron moiety of pyrite was released as ferrous iron. All observed initial reaction rates could be fitted into an empirical equation. This equation includes the concentrations of ferric iron and pyrite, and a constant which is dependent on the temperature and the nature of the main anion present. It was observed that ferrous iron formed during the reaction slowed down the oxidation of pyrite by ferric iron. The extent of this effect decreased with increasing temperature. With the aid of the empirical equation, the contribution of the chemical oxidation of pyrite by ferric iron to the overall oxidation in a hypothetical plug-flow reactor, in which biologically mediated oxdidation of pyrite and ferrous iron by oxygen also takes place, can be assessed. At 30, 45, and 70 degrees C, respectively, 2, 8-17, and 43% of the pyrite was oxidized chemically by ferric iron. Therefore, it is expected that only in reactors operating at high temperatures with extremely thermophilic bacteria, will chemical oxidation cause a significant deviation from the apparent first order overall kinetics of biological pyrite oxidation.     

Leaching of Pyrites of Various Reactivities by Thiobacillus ferrooxidans

Wide variations were found in the rate of chemical and microbiological leaching of iron from pyritic materials from various sources. Thiobacillus ferrooxidans accelerated leaching of iron from all of the pyritic materials tested in shake flask suspensions at loadings of 0.4% (wt/vol) pulp density. The most chemically reactive pyrites exhibited the fastest bioleaching rates. However, at 2.0% pulp density, a delay in onset of bioleaching occurred with two of the pyrites derived from coal sources. T. ferrooxidans was unable to oxidize the most chemically reactive pyrite at 2.0% pulp density. No inhibition of pyrite oxidation by T. ferrooxidans occurred with mineral pyrite at 2.0% pulp density. Experiments with the most chemically reactive pyrite indicated that the leachates from the material were not inhibitory to iron oxidation by T. ferrooxidans.     

A mathematical model for the bacterial oxidation of a sulfide ore concentrate

The effect of dilution rate and feed solids concentration on the bacterial leaching of a pyrite/arsenopyrite ore concentrate was studied. A mathematical model was developed for the process based on the steady-state data collected over the range of dilution rates (20 to 110 h) and feed solids concentrations (6 to 18% w/v) studied. A modified Monod model with inhibition by arsenic was used to model bacterial ferrous ion oxidation rates. The model assumes that (i) pyrite and arsenopyrite leaching occurs solely by the action of ferric iron produced from the bacterial oxidation of ferrous iron and (ii) bacterial growth rates are proportional to ferrous ion oxidation rate. The equilibrium among the various ionic species present in the leach solution that are likely to have a significant effect on the bioleach process were included in the model. (c) 1994 John Wiley & Sons, Inc.     

Modeling continuous bioleach reactors

The results of recent research have shown that the bioleaching of sulfide minerals occurs via a two‐step mechanism. In this mechanism, the sulfide mineral is chemically oxidized by the ferric‐iron in the bioleaching liquor. The ferrous‐iron produced is subsequently oxidized to ferric‐iron by the microorganism. Further research has shown that the rates of both the ferric leaching and ferrous‐iron oxidation are governed by the ferric/ferrous‐iron ratio (i.e., the redox potential). During the steady‐state operation of a bioleach reactor, the rate of iron turnover between the chemical ferric leaching of the mineral and the bacterial oxidation of the ferrous‐iron will define the rate and the redox potential at which the system will operate. The balance between the two rates will in turn depend on the species used, the microbial concentration, the residence time employed, the nature of the sulfide mineral being leached, and its active surface area. The model described proposes that the residence time and microbial species present determine the microbial growth rate, which in turn determines the redox potential in the bioleach liquor. The redox potential of the solution, in turn, determines the degree of leaching of the mineral; that is, conversion in the bioleach reactor. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 671–677, 1999.     

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.     

An ultraviolet spectrophotometric method for the determination of pyrite and ferrous ion oxidation by Thiobacillus ferrooxidans

Summary An ultraviolet spectrophotometric method was used to monitor the formation of soluble ferric iron in acid culture solutions of Thiobacillus ferrooxidans. This methodology was demonstrated to be applicable for determining both pyrite and ferrous ion oxidation. Kinetic parameters of Fe²⁺ oxidation determined with the use of this method were in close agreement with those previously obtained by measurement of oxygen uptake rates.     

Degradation of massive pyrite: Physical,chemical, and bacterial effects

Massive pyrite was shown to produce soluble iron, hydrogen, and sulfate ions on exposure to air and water. The rate of this process was directly proportional to the surface area of the mineral; it was unaffected by a drop in the pH and the presence of the ferrous and sulfate ions formed. Cupic ion had no effect but ferric ion accelerated pyrite degradation until all the ferric ion was consumed, in accordance with FeS₂ + 2Fe³⁺ —>‐3Fe²⁺ + 2S°. Thiobacillus ferrooxidans increased pyrite degradation considerably; the presence of Thiobacillus thiooxidans had no influence on pyrite degradation.     

Physiological diversity amongst some moderately thermophilic iron-oxidising bacteria

Abstract Three strains of moderately thermophilic and acidophilic bacteria capable of oxidising ferrous iron, isolated from different sources, were compared with each other and with an earlier isolate, TH1. The isolates displayed different rates of carbon dioxide fixation and incorporation of glucose and glycine; only one could be subcultured continuously in organic-free ferrous sulfate medium. Two of the isolates readily oxidised elemental sulfur, and all were capable of solubilising pyrite and chalcopyrite, though at different rates; there was no correlation between rates of ferrous iron and pyrite oxidation. Reduction of ferric iron by two of the isolates was observed in unshaken cultures containing 10 mM glucose. The DNA base composition of the isolates varied from 43–68 mol% G + C.     

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.     

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.     

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.     

Reduction of Soluble Iron and Reductive Dissolution of Ferric Iron-Containing Minerals by Moderately Thermophilic Iron-Oxidizing Bacteria

Five moderately thermophilic iron-oxidizing bacteria, including representative strains of the three classified species (Sulfobacillus thermosulfidooxidans, Sulfobacillus acidophilus, and Acidimicrobium ferrooxidans), were shown to be capable of reducing ferric iron to ferrous iron when they were grown under oxygen limitation conditions. Iron reduction was most readily observed when the isolates were grown as mixotrophs or heterotrophs with glycerol as an electron donor; in addition, some strains were able to couple the oxidation of tetrathionate to the reduction of ferric iron. Cycling of iron between the ferrous and ferric states was observed during batch culture growth in unshaken flasks incubated under aerobic conditions, although the patterns of oxidoreduction of iron varied in different species of iron-oxidizing moderate thermophiles and in strains of a single species (S. acidophilus). All three bacterial species were able to grow anaerobically with ferric iron as a sole electron acceptor; the growth yields correlated with the amount of ferric iron reduced when the isolates were grown in the absence of oxygen. One of the moderate thermophiles (identified as a strain of S. acidophilus) was able to bring about the reductive dissolution of three ferric iron-containing minerals (ferric hydroxide, jarosite, and goethite) when it was grown under restricted aeration conditions with glycerol as a carbon and energy source. The significance of iron reduction by moderately thermophilic iron oxidizers in both environmental and applied contexts is discussed.Moderately thermophilic acidophilic bacteria that catalyze the dissimilatory oxidation of ferrous iron are distinct both phylogenetically and in aspects of their physiology. They differ from the known acidophilic mesophilic iron oxidizers (gram-negative, nonsporulating chemolithotrophic bacteria) and the extremely thermophilic iron oxidizers (certain archaea) in several fundamental ways, including cellular morphology (they are gram-positive rods that often form endospores) and growth temperature optima, which are typically 45 to 55°C (15). In addition, the moderately thermophilic iron-oxidizing acidophiles characteristically have a highly versatile metabolism (18) and may grow as autotrophs (e.g., in media containing ferrous iron or reduced sulfur), heterotrophs (e.g., on yeast extract), mixotrophs (e.g., in media containing both ferrous iron and glucose, in which both CO₂ and glucose are used as carbon sources), or chemolithoheterotrophs (e.g., in ferrous iron-yeast extract medium, in which iron acts as the energy source and yeast extract is the carbon source). Isolates have been obtained from a range of thermal acidic environments, such as geothermal areas, self-heating mine waste spoils, and commercial mineral-processing operations (2a, 5, 14). There are currently two recognized genera of these bacteria. All but one Sulfobacillus species are iron- and sulfur-oxidizing, gram-positive, sporulating rods. Two such species have been described, Sulfobacillus thermosulfidooxidans and Sulfobacillus acidophilus, which may be distinguished by their different chromosomal DNA base compositions and by their abilities to grow autotrophically on reduced sulfur (16). The genus Acidimicrobium currently contains a single species, Acidimicrobium ferrooxidans. This organism differs from Sulfobacillus spp. by its greater capacity to fix CO₂, by its lower tolerance of ferric iron, by its apparent lack of spore formation (although it is also gram positive), and by its chromosomal DNA base composition (4). Analysis of 16S rRNA sequences has also differentiated this moderate thermophile from Sulfobacillus spp. (9).The small amount of energy associated with the oxidation of ferrous iron (−30 kJ mol⁻¹ at pH 2) can serve as the exclusive source of energy for moderately thermophilic iron-oxidizing acidophiles when they are growing autotrophically with oxygen as the terminal electron acceptor. Under limited aeration conditions, ferric iron, which is often abundant and present in a soluble form in extremely acidic environments, is a thermodynamically attractive alternative electron sink (electrode potential [E′], +780 mV). Ferric iron reduction by mesophilic chemolithotrophic and heterotrophic acidophiles has been observed previously (5, 7, 17). Some moderately thermophilic, acidophilic, heterotrophic bacteria (Alicyclobacillus-like isolates) (5a) and the extremely thermophilic archaeon Sulfolobus acidocaldarius (3) can also reduce iron. While many neutrophilic microorganisms are also able to reduce ferric iron, the ability to conserve energy to support growth by coupling organic matter oxidation exclusively to ferric iron reduction appears to be more restricted among neutrophilic bacteria (11).In this paper, we describe the dissimilatory reduction of ferric iron by representative isolates of different species of iron-oxidizing moderate thermophiles with both an organic electron donor (glycerol) and an inorganic electron donor (tetrathionate), and we also describe the reductive dissolution of ferric iron-containing minerals by a Sulfobacillus isolate.     

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     

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.     

Selective Adhesion of Thiobacillus ferrooxidans to Pyrite

Bacterial adhesion to mineral surfaces plays an important role not only in bacterial survival in natural ecosystems, but also in mining industry applications. Selective adhesion was investigated with Thiobacillus ferrooxidans by using four minerals, pyrite, quartz, chalcopyrite, and galena. Escherichia coli was used as a control bacterium. Contact angles were used as indicators of hydrophobicity, which was an important factor in the interaction between minerals and bacteria. The contact angle of E. coli in a 0.5% sodium chloride solution was 31°, and the contact angle of T. ferrooxidans in a pH 2.0 sulfuric acid solution was 23°. E. coli tended to adhere to more hydrophobic minerals by hydrophobic interaction, while T. ferrooxidans selectively adhered to iron-containing minerals, such as pyrite and chalcopyrite. Ferrous ion inhibited the selective adhesion of T. ferrooxidans to pyrite competitively, while ferric ion scarcely inhibited such adhesion. When selective adhesion was quenched by ferrous ion completely, adhesion of T. ferrooxidans was controlled by hydrophilic interactions. Adhesion of E. coli to pyrite exhibited a liner relationship on langmuir isotherm plots, but adhesion of T. ferrooxidans did not. T. ferrooxidans recognized the reduced iron in minerals and selectively adhered to pyrite and chalcopyrite by a strong interaction other than the physical interaction.     

Different iron sources to study the physiology and biochemistry of iron metabolism in marine micro-algae

We compared ferric EDTA, ferric citrate and ferrous ascorbate as iron sources to study iron metabolism in Ostreococcus tauri, Phaeodactlylum tricornutum and Emiliania huxleyi. Ferric EDTA was a better iron source than ferric citrate for growth and chlorophyll levels. Direct and indirect experiments showed that iron was much more available to the cells when provided as ferric citrate as compared to ferric EDTA. As a consequence, growth media with iron concentration in the range 1–100 nM were rapidly iron-depleted when ferric citrate—but not ferric EDTA was the iron source. When cultured together, P. tricornutum cells overgrew the two other species in iron-sufficient conditions, but E. huxleyi was able to compete other species in iron-deficient conditions, and when iron was provided as ferric citrate instead of ferric EDTA, which points out the critical influence of the chemical form of iron on the blooms of some phytoplankton species. The use of ferric citrate and ferrous ascorbate allowed us to unravel a kind of regulation of iron uptake that was dependent on the day/night cycles and to evidence independent uptake systems for ferrous and ferric iron, which can be regulated independently and be copper-dependent or independent. The same iron sources also allowed one to identify molecular components involved in iron uptake and storage in marine micro-algae. Characterizing the mechanisms of iron metabolism in the phytoplankton constitutes a big challenge; we show here that the use of iron sources more readily available to the cells than ferric EDTA is critical for this task.     

Attachment of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum cultured under varying conditions to pyrite, chalcopyrite, low-grade ore and quartz in a packed column reactor

The attachment of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum spp. grown on ferrous medium or adapted to a pyrite mineral concentrate to four mineral substrata, namely, chalcopyrite and pyrite concentrates, a low-grade chalcopyrite ore (0.5 wt%) and quartzite, was investigated. The quartzite represented a typical gangue mineral and served as a control. The attachment studies were carried out in a novel particle-coated column reactor. The saturated reactor containing glass beads, which were coated with fine mineral concentrates, provided a quantifiable surface area of mineral concentrate and maintained good fluid flow. A. ferrooxidans and Leptospirillum spp. had similar attachment characteristics. Enhanced attachment efficiency occurred with bacteria grown on sulphide minerals relative to those grown on ferrous sulphate in an ore-free environment. Selective attachment to sulphide minerals relative to gangue materials occurred, with mineral adapted cultures attaching to the minerals more efficiently than ferrous grown cultures. Mineral-adapted cultures showed highest levels of attachment to pyrite (74% and 79% attachment for A. ferrooxidans and L. ferriphilum, respectively). This was followed by attachment of mineral-adapted cultures to chalcopyrite (63% and 58% for A. ferrooxidans and L. ferriphilum, respectively). A. ferrooxidans and L. ferriphilum exhibited lower levels of attachment to low-grade ore and quartz relative to the sulphide minerals.     

Characteristics of a moderately thermophilic and acidophilic iron-oxidizing Thiobacillus

Summary Thiobacillus TH1 is an acidophilic chemolithotrophic heterotroph growing at temperatures up to about 50°C on media containing ferrous iron or pyrite when supplemented with yeast extract or glutathione. Virtually no carbon dioxide fixation occurred during growth on iron with yeast extract. Its DNA contains 48 mol % guanine + cytosine. The organism effects the thermophilic leaching of metals from pyrite, chalcopyrite, CuS, and copper concentrates. Oxidation of soluble ferrous iron at pH 1.6 was competitively inhibited by ferric iron and had a K_m of 7.3 mM FeSO₄.