Effect of anions on selective solubilization of zinc and copper in bacterial leaching of sulfide ores

Bacterial leaching of sulfide ores using Thiobacillus ferrooxidans, Thiobacillus thiooxidans, or a combination of the two was studied at various concentrations of specific anions. Selective zinc and copper solubilization was obtained by inhibiting iron oxidation without affecting sulfur/sulfide oxidation. Phosphate reduced iron solubilization from a pyrite (FeS(2))-sphalerite (ZnS) mixture without significantly affecting zinc solubilization. Copper leaching from a chalcopyrite (CuFeS(2))-sphalerite mixture was stimulated by phosphate, whereas chloride accelerated zinc extraction. In a complex sulfide ore containing pyrite, chalcopyrite, and sphalerite, both phosphate and chloride reduced iron solubilization and increased copper extraction, whereas only chloride stimulated zinc extraction. Maximum leaching obtained was 100% zinc and 50% copper. Time-course studies of copper and zinc solubilization suggest the possibility of selective metal recovery following treatment with specific anions.     

Leaching Heavy Metals from Contaminated Soil by Using Thiobacillus ferrooxidans or Thiobacillus thiooxidans

Iron- and sulfur-oxidizing bacteria identified as Thiobacillus ferrooxidans and T. thiooxidans were successfully enriched from various soil samples contaminated with heavy metals and organic compounds. Depending on the growth medium, the soil sample, and the type of contaminant, the indigenous isolates solubilized > 50% of most of the heavy metals present in the solid sample (As, Cd, Co, Cr, Cu, Ni, V, Zn, B, Be). Leaching with T. ferrooxidans strains resulted in total extraction of Cd, Co, Cu, and Ni. With sulfur-oxidizing bacteria > 80% of Cd, Co, Cu, and Zn was mobilized from rainwater sludge. Pb and Ba were not detected in the leachate, given the insolubility of their sulfate compounds. An increase in pulp density up to 20%, indicating 6.6% total organic carbon in the soil and rubble leach experiment (sample 557), did not inhibit the growth of the indigenous T. ferrooxidans strain. In view of these results, bioleaching appears to have some potential for remediation of heavy metal contaminated soils.     

Effect of applied potentials on the activity and growth of Thiobacillus ferrooxidans

The effect of applied DC potentials both in the positive and negative range, on the activity and growth of Thiobacillus ferrooxidans, is discussed. In general, application of positive potentials up to +1000 mV in an acid bioleaching medium was found to be detrimental to bacterial activity, while the impression of negative potentials enhanced both their activity and growth through electrochemical regeneration of ferrous ions and an increase in the biomass. Ferrous-ferric ratios in a bioleaching medium could be monitored through Eh measurements.Among the base sulfide minerals such as pyrite, chalcopyrite, and sphalerite, sphalerite could be selectively bioleached if an impressed potential of -500 mV (SCE) could be maintained in the leaching medium. Electrochemical bioleaching tests carried out under an applied potential of -500 mV with sphalerite in the presence and absence of noble minerals such as pyrite and chalcopyrite indicated enhanced zinc dissolution with negligible copper and iron in solution. Probable mechanisms and advantages of the electrochemical bioleaching process developed in the laboratory are outlined.     

The effects of Fe(II) and Fe(III) concentration and initial pH on microbial leaching of low-grade sphalerite ore in a column reactor

In this study the effects of initial concentration of Fe(II) and Fe(III) ions as well as initial pH on the bioleaching of a low-grade sphalerite ore in a leaching column over a period of 120 days with and without bacteria were investigated. Four different modifications of medium were used as column feed solutions to investigate the effects of initial concentration of Fe(II) and Fe(III) ions on zinc extraction. The experiments were carried out using a bench-scale, column leaching reactor, which was inoculated with mesophilic iron oxidizing bacteria, Acidithiobacillus ferrooxidans, initially isolated from the Sarcheshmeh chalcopyrite concentrate (Kerman, Iran). The effluent solutions were periodically analyzed for Zn, total Fe, Fe(II) and Fe(III) concentrations as well as pH values. Bacterial population was measured in the solution (free cells). Maximum zinc recovery in the column was achieved about 76% using medium free of initial ferrous ion and 11.4 g/L of ferric ion (medium 2) at pH 1.5. The extent of leaching of sphalerite ore with bacteria was significantly higher than that without bacteria (control) in the presence of ferrous ions. Fe(III) had a strong influence in zinc extraction, and did not adversely affect the growth of the bacteria population.     

Oxidation of gallium sulfides by Thiobacillus ferrooxidans.

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

Chemolithotrophic Bacteria in Copper Ores Leached at High Sulfuric Acid Concentration

Extensive bacterial growth was observed when copper sulfide ores were leached with 0.6 N sulfuric acid. The bacterial population developed in this condition was examined by characterization of the spacer regions between the 16S and 23S rRNA genetic loci obtained after PCR amplification of the DNA extracted from the leached ore. The spacers observed had the sizes found in strains of "Leptospirillum ferrooxidans" and Thiobacillus thiooxidans, except for a larger one, approximately 560 bp long, that was not observed in any of the strains examined, including those of Thiobacillus ferrooxidans. The bacteria with this last spacer were selected after culturing in mineral and elemental sulfur media containing 0.7 N sulfuric acid. The spacer and the 16S ribosomal DNA of this isolate were sequenced and compared with those in species commonly found in bioleaching processes. Though the nucleotide sequence of the spacer showed an extensive heterologous region with T. thiooxidans, the sequence of its 16S rDNA gene indicated a close relationship (99.85%) with this species. These results indicate that a population comprised of bacterial strains closely related to T. thiooxidans and of another strain, possibly related to "L. ferrooxidans," can develop during leaching at high sulfuric acid concentration. Iron oxidation in this condition is attributable to "L. ferrooxidans" and not T. ferrooxidans, based on the presence of spacers with the "L. ferrooxidans" size range and the absence of spacers characteristic of T. ferrooxidans.     

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.     

The potential of mining slag as a substrate for microbial growth and the microbiological analysis of slag and slag seepage

The potential of a Cu/Ni mining slag to act as a substrate for the growth of the bacteria Thiobacillus ferrooxidans, Thiobacillus thiooxidants, and Thiobacillus thioparus was examined. As well, slag and seepage samples were screened for the presence of the Thiobacillus species. For the 28 samples employed in the environmental recovery studies, T. ferrooxidans was recovered in 25 samples, T. thiooxidans in 19 samples, and T. thioparus in 27 samples. For T. ferrooxidans, the development of a colour change in the medium corresponded with the presence of motile bacilli as detected microscopically. For T. thiooxidans and T. thioparus, a decrease in culture pH of greater than 0.2 units usually corresponded with the presence of motile bacilli. The potential for growth on slag was determined by adding slag samples to media (devoid of an electron donor) appropriate for the growth of the three Thiobacillus species. All pulverized slag samples supported the growth of the three species.     

Cultural and phylogenetic analysis of mixed microbial populations found in natural and commercial bioleaching environments.

A range of autotrophic and heterotrophic enrichment cultures were established to determine the cultural bacterial diversity present in samples obtained from the acidic runoff of a chalcocite overburden heap and from laboratory-scale (1- to 4-liter) batch and continuous bioreactors which were being used for the commercial assessment of the bioleachability of zinc sulfide ore concentrates. Strains identified as Thiobacillus ferrooxidans, Thiobacillus thiooxidans, "Leptospirillum ferrooxidans," and Acidiphilium cryptum were isolated from both the natural site and the batch bioreactor, but only "L. ferrooxidans," a moderately thermophilic strain of T. thiooxidans, and a moderately thermophilic iron-oxidizing bacterium could be recovered from the continuous bioreactor running under steady-state conditions. Sequence analysis of the 16S rRNA genes of 33 representative strains revealed that all of the strains were closely related to strains which have been sequenced previously and also confirmed the phylogenetic diversity of bacteria present in bioleaching environments.     

Monitoring of bacteria in acid mine environments by reverse sample genome probing

A variety of microorganisms can exist in acid mine drainage (AMD) environments, although their contribution to AMD problems is unclear. Environmental strains of Thiobacillus ferrooxidans and Thiobacillus acidophilus were purified by repeated plating and single-colony isolation on iron salts and tetrathionate media, respectively. Thiobacillus thiooxidans was enriched on sulfur-containing media. For the isolation of Leptospirillum ferrooxidans, iron salts and pyrite media were inoculated with environmental samples. However, L. ferrooxidans was never recovered on solid media. Denatured chromosomal DNAs from type and (or) isolated strains of T. ferrooxidans, T. acidophilus, T. thiooxidans, and L. ferrooxidans were spotted on a master filter for their detection in a variety of samples by reverse sample genome probing (RSGP). Analysis of enrichments of environmental samples by RSGP indicated that ferrous sulfate medium enriched T. ferrooxidans strains, whereas all thiobacilli grew in sulfur medium, T. thiooxidans strains being dominant. Enrichment in glucose medium followed by transfer to tetrathionate medium resulted in the selection of T. acidophilus strains. DNA was also extracted directly (without enrichment) from cells recovered from AMD water or sediments, and was analyzed by RSGP to describe the communities present. Strains showing homology with T. ferrooxidans and T. acidophilus were found to be major community components. Strains showing homology with T. thiooxidans were a minor community component, whereas strains showing homology with L. ferrooxidans were not detected.     

Alterations in surfaces and textures of minerals during the bacterial leaching of a complex sulfide ore

Abstract

The purpose of the work was to characterize changes in surface textures of minerals during the biological leaching of a complex sulfide ore. The ore contained pyrrhotite (Fe_I__xS), pyrite (FeS₂), sphalerite (ZnS), pentlandite [(Ni,Fe,Co)₉S₈], and chalcopyrite (CuFeS₂). Several mixed cultures were initially screened using the ore material as the sole substrate. Shake flask leaching experiments showed no major differences among test cultures, which were all derived by enrichment techniques using environmental samples collected from a mine site. Leached pyrrhotite surfaces were invariably surrounded by a dark rim of elemental S. A reaction zone was also associated with leached sphalerite grains. Chemical analyses of leach solutions indicated that the relative ranking of biological leaching of the sulfide minerals was Zn > Ni > Co > Cu. Microscopic observations were in keeping with this rankin     

Biological oxidation of sulfide raw material using a culture of Thiobacillus ferrooxidans under various conditions of leaching

Major parameters of the first stage of leaching of a copper-zinc sulfide product by a culture of Thiobacillus ferrooxidans have been studied, including the effects of solid phase concentration, Fe2+ and Fe3+ ions, pH, and the intensity of mixing. The first stage of leaching of the sulfide raw material is optimum under the following conditions: pH of the original leaching solution equal to 1.6; Fe3+ concentration of order of 10 g/l; and vigorous mixing of the suspension at solid phase concentrations of 30-35%. A theoretical substantiation of the observed dependences is proposed.     

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.     

Microbiological Leaching of Metallic Sulfides

The percentage of chalcopyrite leached in percolators by Thiobacillus ferrooxidans was dependent on the surface area of the ore but not on the amount. Typical examples of ore leaching, which demonstrate the role of the bacteria, are presented. In stationary fermentations, changes in KH(2)PO(4) concentration above or below 0.1% decreased copper leaching as did reduction in the MgSO(4).7H(2)O and increase in the (NH(4))(2)SO(4) concentration. Bacterial leaching of chalcopyrite was more effective than nonbiological leaching with ferric sulfate; ferric sulfate appeared to retard biological leaching, but this effect was likely caused by formation of an insoluble copper-iron complex. Ferrous sulfate and sodium chloride singly accentuated both bacterial and nonbiological leaching of chalcocite but jointly depressed bacterial action. Sodium chloride appeared to block bacterial iron oxidation without interfering with sulfide oxidation. Bacterial leaching of millerite, bornite, and chalcocite was greatest at pH 2.5. The economics of leaching a number of British Columbia ore bodies was discussed.     

Bacterial Community in Copper Sulfide Ores Inoculated and Leached with Solution from a Commercial-Scale Copper Leaching Plant

Most copper bioleaching plants operate with a high concentration of sulfate salts caused by the continuous addition of sulfuric acid and the recycling of the leaching solution. Since the bacteria involved in bioleaching have been generally isolated at low sulfate concentrations, the bacterial population in ores leached with the high-sulfate solution (1.25 M) employed in a copper production plant was investigated. The complexity of the original population was assessed by the length pattern of the spacer regions between the 16S and 23S rRNA genes, observed after PCR amplification of the DNA extracted from the leached ore. Six main spacers were distinguished by electrophoretic migration, but they could be further resolved into eight spacers by nucleotide sequence homology. The degree of homology was inferred from the electrophoretic migration of the heteroduplexes formed after hybridization. One of the spacers was indistinguishable from that found in Thiobacillus thiooxidans, four could be related to Thiobacillus ferrooxidans, and three could be related to Leptospirillum ferrooxidans. Only five of the spacers in the original sample could be recovered after culturing in media containing different inorganic energy source. Altogether, the results indicate that the bacteria in the leached ore formed a community composed of at least three species: a fairly homogeneous population of T. thiooxidans strains and two heterogeneous populations of T. ferrooxidans and L. ferrooxidans strains.     

A novel mineral flotation process using Thiobacillus ferrooxidans.

Oxidative leaching of metals by Thiobacillus ferrooxidans has proven useful in mineral processing. Here, we report on a new use for T. ferrooxidans, in which bacterial adhesion is used to remove pyrite from mixtures of sulfide minerals during flotation. Under control conditions, the floatabilities of five sulfide minerals tested (pyrite, chalcocite, molybdenite, millerite, and galena) ranged from 90 to 99%. Upon addition of T. ferrooxidans, the floatability of pyrite was significantly suppressed to less than 20%. In contrast, addition of the bacterium had little effect on the floatabilities of the other minerals, even when they were present in relatively large quantities: their floatabilities remained in the range of 81 to 98%. T. ferrooxidans thus appears to selectively suppress pyrite floatability. As a consequence, 77 to 95% of pyrite was removed from mineral mixtures while 72 to 100% of nonpyrite sulfide minerals was recovered. The suppression of pyrite floatability was caused by bacterial adhesion to pyrite surfaces. When normalized to the mineral surface area, the number of cells adhering to pyrite was significantly larger than the number adhering to other minerals. These results suggest that flotation with T. ferrooxidans may provide a novel approach to mineral processing in which the biological functions involved in cell adhesion play a key role in the separation of minerals.     

Sulfide oxidation by spheroplasts of Thiobacillus ferrooxidans.

Thiobacillus ferrooxidans is an acidophilic organism important to metal leaching of low-grade ores. The aforementioned importance is related to the ability of the bacterium to oxidize reduced iron and sulfur, principally found in nature as pyrite (FeS2). The present study dealt with sulfide oxidation at low pH values and the involvement of the cell envelope in the process of the inorganic oxidations. Sulfide oxidation was noted in spheroplasts of T. ferrooxidans prepared by enzymatic and chemical treatments and partially purified by differential centrifugation. No enzyme activities were noted in membrane fractions containing enrichments of lipopolysaccharide symbolic of outer membrane material or in membrane vesicles containing (or associated with) higher levels of proteins. Results to date indicate that in an acid milieu the envelope structure containing both the outer membrane and the intact inner cytoplasmic membrane is required for sulfide oxidation.     

Sulfide oxidation by spheroplasts of Thiobacillus ferrooxidans.

Thiobacillus ferrooxidans is an acidophilic organism important to metal leaching of low-grade ores. The aforementioned importance is related to the ability of the bacterium to oxidize reduced iron and sulfur, principally found in nature as pyrite (FeS2). The present study dealt with sulfide oxidation at low pH values and the involvement of the cell envelope in the process of the inorganic oxidations. Sulfide oxidation was noted in spheroplasts of T. ferrooxidans prepared by enzymatic and chemical treatments and partially purified by differential centrifugation. No enzyme activities were noted in membrane fractions containing enrichments of lipopolysaccharide symbolic of outer membrane material or in membrane vesicles containing (or associated with) higher levels of proteins. Results to date indicate that in an acid milieu the envelope structure containing both the outer membrane and the intact inner cytoplasmic membrane is required for sulfide oxidation.     

Kinetic study of sulfide leaching by galvanic interaction between chalcopyrite, pyrite, and sphalerite in the presence of T. ferrooxidans (30 degrees C) and a thermophilic microorganism (55 degrees C)

A systematic, kinetic study and comparison of the leaching of mixed metal sulfides by galvanic conversion and in the presence of bacteria has been carried out for the first time using both powder (-100 to -400 mesh) and larger (bulk) specimen systems. The rates of dissolution of copper from chalcopyrite and zinc from sphalerite as single, electrically isolated (separate) systems were compared with electrically contacting (galvanically coupled) systems involving CuFeS(2)/FeS(2) and ZnS/FeS(2), with and without bacteria and at temperatures of 30 and 55 degrees C. The dissolution of Cu was observed to increase by a factor of 4.6 when the galvanic leaching of CuFeS(2)/FeS(2) was compared to CuFeS(2) leaching at 30 degrees C. When bacteria were present, Cu dissolution increased by an additional factor of 2.1 in the CuFeS(2)/FeS(2) system. At 55 degrees C, the corresponding ratios for Cu were 4.3 and 2.7, respectively. The galvanic leaching of Zn in the ZnS/FeS(2) system compared to ZnS leaching increased by a factor of 2 at 30 degrees C; in the presence of bacteria the dissolution of Zn from the ZnS/FeS(2) system increased by an additional factor of 1.3 at the same temperature. By comparison, the ratio of Cu dissolution from CuFeS(2) in acid-bacterial medium and sterile controls (without bacteria) was 5.5. The corresponding ratio for Zn from ZnS was 2.2 at both 30 and 55 degrees C. The order of reaction was found to be essentially first order for the leaching of powder systems at both 30 and 55 degrees C (with T. Ferrooxidans and thermophilic microorganisms, respectively). The corresponding reaction rate constants were observed to be 12.6 and 22.9 for T. ferrooxidans and the thermophilic microorganisms, respectively. Activation energies for the various systems were also determined.     

SEM and AFM images of pyrite surfaces after bioleaching by the indigenous<Emphasis Type="Italic"> Thiobacillus thiooxidans</Emphasis>

The bioleaching mechanism of pyrite by the indigenous Thiobacillus thiooxidans was examined with the aid of scanning electron microscopy (SEM) and atomic force microscopy (AFM) images of the pyrite surface. The presence of pyrite eliminated the lag phase during growth of this microorganism. This was due to the stimulatory effect on cell growth of the slight amount of Cu2+ that had leached from the pyrite. Zn2+ was found to be much more readily solubilized than Cu2+. The efficiency of bioleaching was four times higher than that of chemical leaching. SEM images provided evidence of direct cell attachment onto the pyrite surface, thereby enhancing the bioleaching rate. Furthermore, extracellular polymeric substances (EPSs) were found on the pyrite surface after 4 days of oxidation. AFM images showed that the pyrite surface area positively correlated with the oxidation rate. A combination of direct and indirect mechanism is probably responsible for the oxidation of pyrite by T. thiooxidans.