Enhancement of Copper Dissolution from a Sulfide Ore by Using Thiobacillus thiooxidans

Copper dissolution from a sulfide ore (with covellite as the main copper phase) was investigated in cultures of Thiobacillus ferrooxidans or Thiobacillus thiooxidans and in abiotic controls. In unsupplemented media, T. ferrooxidans was more efficient than T. thiooxidans. In the presence of ferric iron, the dissolution of covellite was not significantly different in cultures inoculated with T. ferrooxidans or T. thiooxidans. However, the most extraction was found in T. thiooxidans cultures supplemented with ferrous sulfate. The first results were explained by the mechanism proposed by Schippers and Sand (Appl Envir Microbiol 65:319-321, 1999), which involves polysulfides and sulfur as intermediates. This mechanism was extended to explain the behavior of T. thiooxidans culture supplemented with ferrous iron.     

Direct zinc sulphide bioleaching by Thiobacillus ferrooxidans and Thiobacillus thiooxidans

Summary Direct bioleaching (no iron(II) present) by Thiobacillus ferrooxidans mainly occurs on the surface of the very insoluble sulphides but is more important in solution when the sulphides are more soluble. In this case, Thiobacillus thiooxidans, normally not able to leach directly insoluble sulphides, has an effective leaching action.     

Bacterial leaching of metals from sewage sludge

Summary Thiobacillus thiooxidans is capable of oxidizing sulfur in digested sludge, while decreasing the pH value from about 5.5 to, say, 1.0 to 1.5. Insoluble metal sulfides can be solubilized through this acidification. Thiobacillus ferrooxidans oxidises pyritic ore in the presence of 6% centrifuged sludge if the pH value is adjusted to about 2.5. When mixing T. thiooxidans and T. ferrooxidans with sludge and 1% sulfur, the former acidifies the sludge and the latter oxidizes metal sulfides; together they solubilize more metal than T. thiooxidans alone. The following metals solubilized from their sulfides have been investigated so far: iron, copper, zinc, nickel, and cadmium. The possibility of recycling metals from sewage sludge with this method is discussed.     

Biochemical rust removal by Thiobacillus ferrooxidans

Summary Biochemical removal of rust from iron surfaces has been investigated. By immersing a rusted iron plate in the culture medium of an iron-oxidizing bacterium, Thiobacillus ferrooxidans, iron adjacent to the rust was dissolved and the rust was peeled off. Since the amount of dissolved iron per unit iron plate surface area correlated with the concentration of ferric iron in the culture medium, the formation of ferric iron is probably involved in dissolving the iron as is the case for bacterial leaching. In the present study, rust removal in a “continuous” system in which the culture medium was circulated from the fermentor to the rust removal vessel and back again to the fermentor, has also been investigated. Although growth inhibition was observed with the formation of ferric iron precipitates during the operation in this system, it was possible to prevent this precipitation by lowering the pH of the medium during the mixed cultivation of T. ferrooxidans and a sulfur-oxidizing bacterium, T. thiooxidans.     

Characterization of arsenopyrite oxidizing Thiobacillus. Tolerance to arsenite,arsenate, ferrous and ferric iron

Two strains of Thiobacillus, T. ferrooxidans and T. thiooxidans, have been isolated from a bacterial inoculum cultivated during a one-year period in a 1001 continuous laboratory pilot for treatment of an arsenopyrite/pyrite concentrate. The optimum pH for the growth of both strains has been found to be between 1.7 and 2.5. Because of the high metal toxicity in bioleach pulps, the tolerance of T. ferrooxidans and T. thiooxidans with respect to iron and arsenic has been studied. The growth of both strains is inhibited with 10 g/l of ferric ion, 5 g/l of arsenite and 40 g/l of arsenate. 20 g/l of ferrous iron is toxic to T. ferrooxidans but 30 g/l is necessary to impede the growth of T. thiooxidans.     

Analysis of molybdenite bioleaching by Thiobacillus ferrooxidans in the absence of iron (II)

Summary Thiobacillus ferrooxidans attachment on MoS² and Mo dissolution are increased by the addition of the tensioactive agent Tween 80 in absence of iron(II), which suggests that the poor bioleaching of MoS² is caused by its hydrophobic character. Additionally, inhibition ofThiobacillus ferrooxidans growth by the presence of MoO₄ ^2– and the effect of variable amounts of Tween 80 on bacteria growth and on MoS² bioleaching are considered in this paper. Data confirm the need of bacterial attachment to insoluble substrate for bioleaching by the direct mechanism.     

The role of exopolymers in the bioleaching of a non-ferrous metal sulphide

Exocellular polysaccharides were extracted from Thiobacillus ferrooxidans cells grown in the presence of iron. Cells without these compounds could not adhere to covellite. The loss of the layer of exocellular polysaccharides also affected the direct mechanism of bioleaching of covellite in a negative way. This ability to attach to and leach covellite was restored within a few hours when exopolymeric material was produced again. The addition of exocellular compounds to cells stripped of exocellular polymers also restored their ability to the same level as that of untreated cells. Thiobacillus thiooxidans was not able to attach to and leach covellite even when exocellular compounds from Thiobacillus ferrooxidans were added. Received 2 June 1998/ Accepted in revised form 8 January 1999     

Genomic and physiological diversity amongst strains of Thiobacillus ferrooxidans,and genomic comparison with Thiobacillus thiooxidans

Twenty-three strains of Thiobacillus ferrooxidans of known pedigree were examined. Thirteen strains survived 65° C for 5 min and 7 of these for 10 min, but sporulation was never observed. All strains grew between 25° C and 35° C and some strains grew at 5° and 40° C. They were genomically diverse, comprising 7 DNA homology groups, and the GC content varied from 55–65 mol %. Correlation between genomic group and growth temperature was noted. All strains grew on ferrous sulfate as energy source, but some failed to utilize elemental sulfur. Acidified thiosulfate supported growth of most of the strains examined but it was judged to be a poor substrate upon which to base taxonomic conclusions because of decomposition of thiosulfate in acid. Six strains of Thiobacillus thiooxidans showed negligible genomic affinity to T. ferrooxidans, and they comprised 2 DNA homology groups and their GC content varied from 52–62 mol%. Anomalies due to contaminants in cultures of T. ferrooxidans were resolved, and the contaminants were identified.     

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.     

Bioleaching of arsenic from medicinal realgar by pure and mixed cultures

The aim of this paper is to determine the efficiency of bioleaching of arsenic in realgar, a Chinese mineral drug, using pure cultures of Acidithiobacillus ferrooxidans or Acidithiobacillus thiooxidans and a mixed culture of A. ferrooxidans and A. thiooxidans. The experiments were carried out in shaker flasks, at 150 rpm, 30 °C at a culture pH of 1.80. To investigate the mechanism of the bioleaching in realgar, media with and without ferrous iron were chosen for the experiments. The results showed that the leaching rate of arsenic in realgar after 20 days was higher (43%) in A. ferrooxidans cultures with ferrous iron compared to cultures without ferrous iron (10%), and the leaching rate of A. thiooxidans cultures only increased from 21% to 23% in the presence of ferrous iron. The leaching rate of arsenic in mixed culture with ferrous iron was greatly enhanced from 16% to 56%, indicating that bioleaching in mixed culture is preferable for the dissolution of realgar.     

Development of a Laboratory-Scale Leaching Plant for Metal Extraction from Fly Ash by Thiobacillus Strains

Semicontinuous biohydrometallurgical processing of fly ash from municipal waste incineration was performed in a laboratory-scale leaching plant (LSLP) by using a mixed culture of Thiobacillus thiooxidans and Thiobacillus ferrooxidans. The LSLP consisted of three serially connected reaction vessels, reservoirs for a fly ash suspension and a bacterial stock culture, and a vacuum filter unit. The LSLP was operated with an ash concentration of 50 g liter⁻¹, and the mean residence time was 6 days (2 days in each reaction vessel). The leaching efficiencies (expressed as percentages of the amounts applied) obtained for the economically most interesting metal, Zn, were up to 81%, and the leaching efficiencies for Al were up to 52%. Highly toxic Cd was completely solubilized (100%), and the leaching efficiencies for Cu, Ni, and Cr were 89, 64, and 12%, respectively. The role of T. ferrooxidans in metal mobilization was examined in a series of shake flask experiments. The release of copper present in the fly ash as chalcocite (Cu₂S) or cuprite (Cu₂O) was dependent on the metabolic activity of T. ferrooxidans, whereas other metals, such as Al, Cd, Cr, Ni, and Zn, were solubilized by biotically formed sulfuric acid. Chemical leaching with 5 N H₂SO₄ resulted in significantly increased solubilization only for Zn. The LSLP developed in this study is a promising first step toward a pilot plant with a high capacity to detoxify fly ash for reuse for construction purposes and economical recovery of valuable metals.     

Intermediary sulfur compounds in pyrite oxidation: implications for bioleaching and biodepyritization of coal

Accumulation of elemental sulfur during pyrite oxidation lowers the efficiency of coal desulfurization and bioleaching. In the case of pyrite bioleaching by Leptospirillum ferrooxidans, an iron(II)-ion-oxidizing organism without sulfur-oxidizing capacity, from the pyritic sulfur moiety about 10% elemental sulfur, 2% pentathionate, and 1% tetrathionate accumulated by a recently described cyclic pyrite oxidation mechanism. In the case of pure cultures of Thiobacillus ferrooxidans and mixed cultures of L. ferrooxidans and T. thiooxidans, pyrite was nearly completely oxidized to sulfate because of the capacity of these cultures to oxidize both iron(II) ions and sulfur compounds. Pyrite oxidation in acidic solutions, mediated chemically by iron(III) ion, resulted in an accumulation of similar amounts of sulfur compounds as obtained with L. ferrooxidans. Changes of pH to values below 2 or in the iron ion concentration are not decisive for diverting the flux of sulfur compounds. The literature on pyrite bioleaching is in agreement with the findings indicating that the chemistry of direct and indirect pyrite leaching is identical. Received: 20 April 1998 / Received revision: 27 August 1998 / Accepted: 3 September 1998     

Thiobacillus ferrooxidans response to copper and other heavy metals: growth, protein synthesis and protein phosphorylation

Respirometric experiments demonstrated that the oxygen uptake by Thiobacillus ferrooxidans strain LR was not inhibited in the presence of 200 mM copper. Copper-treated and untreated cells from this T. ferrooxidans strain were used in growth experiments in the presence of cadmium, copper, nickel and zinc. Growth in the presence of copper was improved by the copper-treated cells. However, no growth was observed for these cells, within 190 h of culture, when cadmium, nickel and zinc were added to the media. Changes in the total protein synthesis pattern were detected by two-dimensional polyacrylamide gel electrophoresis for T. ferrooxidans LR cells grown in the presence of different heavy metals. Specific proteins were induced by copper (16, 28 and 42 kDa) and cadmium (66 kDa), whereas proteins that had their synthesis repressed were observed for all the heavy metals tested. Protein induction was also observed in the cytosolic and membrane fractions from T. ferrooxidans LR cells grown in the presence of copper. The level of protein phosphorylation was increased in the presence of this metal.     

The Effect of the Facultative Chemolithotrophic Bacterium Thiobacillus acidophilus on the Leaching of Low-Grade Cu-Ni Sulfide Ore by Thiobacillus ferrooxidans

The purpose of this investigation was to determine the effect of Thiobacillus acidophilus on the leaching of a low-grade Cu-Ni sulfide ore by Thiobacillus ferrooxidans. A sample of low-grade Cu-Ni sulfide ore containing 0.36% Cu, 0.48% Ni, and 7.87% Fe was pulverized and initially leached for a 21-day period using two different pure cultures of T. ferrooxidans, an environmental strain (F2) and a strain from the American Type Culture Collection (ATCC 23270). Samples of the ore slurries were drawn and the pH was monitored over the course of the leaching period. The concentrations of Cu and Ni leached by each strain were determined and compared. No significant differences were observed in the concentrations of Cu and Ni leached by the two pure cultures of T. ferrooxidans. Subsequently, the ore was leached with mixed cultures of T. ferrooxidans and T. acidophilus to determine the effect of the latter on the concentrations of Cu and Ni leached from the ore. The environmental strain F2 of T. ferrooxidans was used in combination with both a type strain (ATCC 27807) and an environmental strain (64) of T. acidophilus. After 21 days, the mixed cultures of T. ferrooxidans and T. acidophilus leached significantly greater amounts of copper than the pure strain alone, but no such difference was observed for the leaching of nickel.     

Bioleaching of chalcopyrite by pure and mixed cultures of Acidithiobacillus spp. and Leptospirillum ferriphilum

Bioleaching is an economical method for the recovery of metals that requires low investment and operation costs. Furthermore, it is generally more environmentally friendly than many physicochemical metal extraction processes. The bioleaching of chalcopyrite in shake flasks was investigated with pure and mixed cultures of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Acidithiobacillus caldus, and Leptospirillum ferriphilum. The mixed cultures containing both iron- and sulfur-oxidizing bacteria were more efficient than the pure culture alone. The presence of sulfur-oxidizing bacteria positively increased the dissolution rate and the percentage recovery of copper from chalcopyrite. Mixed cultures consisting of moderately thermophilic L. ferriphilum and A. caldus leached chalcopyrite more effectively than mesophilic A. ferrooxidans pure and mixed cultures. The decrease of the chalcopyrite dissolution rate in leaching systems containing A. ferrooxidans after 12–16 days coincided with the formation of jarosite precipitation as a passivation layer on the mineral surface during bioleaching. Low pH significantly reduces jarosite formation in pure and mixed cultures of L. ferriphilum and A. caldus.     

Specific Dot-Immunobinding Assay for Detection and Enumeration of Thiobacillus ferrooxidans

A specific and very sensitive dot-immunobinding assay for the detection and enumeration of the bioleaching microorganism Thiobacillus ferrooxidans was developed. Nitrocellulose spotted with samples was incubated with polyclonal antisera against whole T. ferrooxidans cells and then in ¹²⁵I-labeled protein A or ¹²⁵I-labeled goat antirabbit immunoglobulin G; incubation was followed by autoradiography. Since a minimum of 10³ cells per dot could be detected, the method offers the possibility of simultaneous processing of numerous samples in a short time to monitor the levels of T. ferrooxidans in bioleaching operations.     

Investigation of energy gene expressions and community structures of free and attached acidophilic bacteria in chalcopyrite bioleaching

In order to better understand the bioleaching mechanism, expression of genes involved in energy conservation and community structure of free and attached acidophilic bacteria in chalcopyrite bioleaching were investigated. Using quantitative real-time PCR, we studied the expression of genes involved in energy conservation in free and attached Acidithiobacillus ferrooxidans during bioleaching of chalcopyrite. Sulfur oxidation genes of attached A. ferrooxidans were up-regulated while ferrous iron oxidation genes were down-regulated compared with free A. ferrooxidans in the solution. The up-regulation may be induced by elemental sulfur on the mineral surface. This conclusion was supported by the results of HPLC analysis. Sulfur-oxidizing Acidithiobacillus thiooxidans and ferrous-oxidizing Leptospirillum ferrooxidans were the members of the mixed culture in chalcopyrite bioleaching. Study of the community structure of free and attached bacteria showed that A. thiooxidans dominated the attached bacteria while L. ferrooxidans dominated the free bacteria. With respect to available energy sources during bioleaching of chalcopyrite, sulfur-oxidizers tend to be on the mineral surfaces whereas ferrous iron-oxidizers tend to be suspended in the aqueous phase. Taken together, these results indicate that the main role of attached acidophilic bacteria was to oxidize elemental sulfur and dissolution of chalcopyrite involved chiefly an indirect bioleaching mechanism.     

Phosphate rock bioleaching

Summary Microbiological acid solutions produced byThiobacillus ferrooxidans andThiobacillus thiooxidans on pyritiferous concentrate were used to solubilize phosphate rock with a high grade in P₂O₅. Five different mixtures of pyritiferous concentrate and phosphate rock, in different proportions, were used in adequate liquid culture media. Phosphate solubilization ranged from 12% to 100% when 9K nutrients medium was used and from 12% to 89% when medium contained only 3.0g/l ammonium sulphate.     

An Immunological Strategy To Monitor In Situ the Phosphate Starvation State in Thiobacillus ferrooxidans

Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. During the process of ore bioleaching, the microorganisms are subjected to several stressing conditions, including the lack of some essential nutrients, which can affect the rates and yields of bioleaching. When T. ferrooxidans is starved for phosphate, the cells respond by inducing the synthesis of several proteins, some of which are outer membrane proteins of high molecular weight (70,000 to 80,000). These proteins were considered to be potential markers of the phosphate starvation state of these microorganisms. We developed a single-cell immunofluorescence assay that allowed monitoring of the phosphate starvation condition of this biomining microorganism by measuring the increased expression of the surface proteins. In the presence of low levels of arsenate (2 mM), the growth of phosphate-starved T. ferrooxidans cells was greatly inhibited compared to that of control nonstarved cells. Therefore, the determination of the phosphorus nutritional state is particularly relevant when arsenic compounds are solubilized during the bioleaching of different ores.     

Lipopolysaccharides of Thiobacillus species containing lipid A with 2,3-diamino-2,3-dideoxyglucose

Lipopolysaccharides were isolated from two strains of Thiobacillus ferrooxidans and one strain each of Thiobacillus thiooxidans, Thiobacillus novellus and Thiobacillus sp. IFO 14570. Neutral sugars, 2-keto-3-deoxyoctonate, fatty acids and the rare 2,3-diamino-2,3-dideoxyglucose were detected in all lipopolysaccharides. Lipopolysaccharides of both T. ferrooxidans strains contained l-glycero-d-manno-heptose, whereas that of T. thiooxidans contained both l-glycero-d-manno-heptose and d-glycero-d-manno-heptose. On the other hand, heptoses were absent in lipopolysaccharides of T. novellus and Thiobacillus sp. IFO 14570. Lipid A of T. ferrooxidans and T. thiooxidans contained both glucosamine and 2,3-diamino-2,3-dideoxyglucose, in contrast, lipid A of T. novellus and the Thiobacillus sp. IFO 14570 most likely contain only 2,3-diamino-2,3-dideoxyglucose as backbone sugar. Deoxycholate polyacrylamide gel electrophoresis revealed S-type character for all lipopolysaccharides studied. The significance of the lipopolysaccharide composition for taxonomic and phylogenetic questions with regard to thiobacilli is discussed.Abbreviations DAG 2,3-diamino-2,3-dideoxyglucose - DOC sodium deoxycholate - GC gas-liquid chromatography - GC/MS gas-liquid chromatography/mass spectrometry - d,d-Heptose d-glycero-d-manno-heptose - l,d-Heptose l-glycero-d-manno-heptose - KDO 2-keto-3-deoxyoctonate - LPS lipopolysaccharide - 3-OH-14:0 3-hydroxy-tetradecanoic acid - PAGE polyacrylamide gel electrophoresis - PCP phenol-chloroform-petroleum ether