Oxidation of galena by Thiobacillus ferrooxidans and Thiobacillus thiooxidans

Oswaldo Garcia, Jr., Jerry M. Bigham, and Olli H. Tuovinen

Abstract: The objective of this work was to determine solution- and solid-phase alterations associated with galena (PbS) oxidation by Thiobacillus ferrooxidarzs and Thiobacillus thiooxidui~s. In T. ferroo-xidans experiments with 2.5-5% (w/v) galena, the pH remained almost coilstant at pH 2, whereas the pH increased in uninoculated controls. In T. thiooxidans cultures, the pH initially increased from 2 to 4. This initial increase was comparable to the pH change in an abiotic control, but the oxidation reaction in T , thioo,xiclarzs cultures subsequently became acid producing. Anglesite (PbS04) was detected by X-ray diffraction as a solid-phase product of galena decomposition in both abiotic and inoculated experiments. When ferrous sulfate was added as a supplementary energy source for T. ferrooxillans, jarosite (MFe3(S04)2(oW)6) was detected as a new solid phase. Elemental S was not detected in the residues.

Key words: anglesite, bioleaching of galena, galena oxidation, jarosite, lead sulfide oxidation, Thiohacill~ls ferrou,xidans, Thiobacillus thiooxidans.

Resume : Le but de ce travail Ctait de dkterminer les changements sui-venant entre la phase liquide et la phase solide lors de l'oxydation de la galitne (PbS) par ThioBucillus furrooxida~~.~ et Thiohacillus thiooxidans. Lors des essais avec T.J;.rrooxicicms en presence de 2,5-5% (p/v) de galitne, le pH demeurait B peu prks constant 7,2 alors qu'il augmentait dans les contr8les non-inoculks. Dans les cultures de T. thiooxidaiz.~, le pH passait initialement de 2 B 4. Cette augmentation de depart Ctait comparable a la variation de pH dans un contrale abiotique, mais la rkaction d'oxydation dans les cultures de T. thiooxidar~s devenait substquemment productrice d'acide. L'anglCsite (PbS04) a 6tC dCtectCe par diffraction des rayons X comme produit de phase solide de la d6coinposition de la galitne dans les essais abiotiques et inoculks. Lorsque du sulfate fenreux est ajoutC comne source supplCmentaire d'Cnergie pour T.ferroo,xidans la jarosite (MFe3(S04)2(0H)6) a CtC dCtectCe comme nouvelle phase solide. Le soufre ClCmentaire S n'a pas CtC dttectCe dans les rCsidus.

Mots cxle's : anglksite, lixiviation de la galhe, oxydation de la galitne, jarosite, oxydation du sulfure de plomb, Thiohucillus~ferro~~ridans, Thiobacillus thiooxiduils. [Traduit gar la Redaction]

Introduction produce sulfuric acid and dissolved iron. Silver and Toma (1974) observed oxygen uptake activity by Tferrooxidans

Galena (PbS) is relatively common as a minor mineral constitu- ent in sulfide ore materials. Torma and Subramanian (1974) and with synthetic PbS as the sole substrate and detected anglesite

Kingma and Silver (1980) demonstrated the dissolution of (PbS04) as the major mineral product in the residue. Anglesite pbs concentrates by ~ ~ i ~ ~ b n c i ~ ~ u s ~ c n . o o X i ~ a n , r in an iron-free has also been identified as a product in sulfide mineral bio- medium, but the concentrates contained several other sulfide leaching systems containing galena (Ballester et al. 1989). minerals that could potentidly support bacterial growth and Complete oxidation of galena and the formation of anglesite

should involve no net change in the hydrogen ion activity:

1995. Accepted February 15, 1995. [I] PbS t 202 + Pb2+ + SO4" # PbS04

0. Garcia, Jr.' and O.H. T u o ~ i n e n . ~ Department of Microbiology, The Ohio State University, 484 West 12th Tomizuka and Yagisawa (1 978) suggested that So could also Avenue, Columbus, OH 432 10- 1292, U.S.A. be a product of galena oxidation in accordance with the fol- J.M. Bigham. School of Natural Resources, The Ohio State University, 2021 Coffey Road, Columbus, OH 43210-1085, lowing chemical reactions:

Present address: Departamento de Bioquimica, Institute de [2] PbS + H2SO4 + 0.502 + PbS04 + W 2 0 + SO

Quimica. Universidade Estadual Paulista, Caixa Postal 355, Araraquwa, SP, CEP.14.800, Brazil. "uthor to whom all correspondence should be addressed. [31 PbS + Fe?(So~)q + PbS04 + 2FeSOd + So - . -

Can. J. Microbial. 41: 508-514 (1995). hinted in Canada / lmprim6 au Canada.

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A A

18 20 90 40 50 6Q 7 0

" 2 8 CuKa

Fig. 1. X-ray diffractograms of galena before leaching (A) and after 29 days of leaching in abiotic controls containing 5.0% galena in unsupplemented mineral salts solution (B), or supplemented with 30 mM ferrous sulfate (C) or with filter-sterilized spent-culture medium containing 30 rnM Fe3+ (D). All controls were held at approximately pH 2. Diagnostic XRD lines for galena and anglesite are identified by the letter designations G and A, respectively. Line spacings are given in .&ngstroms. The vertical bars show the scale of relative counts.

In actual biological leaching experiments, SO was not detected by CS2 extraction of solid residues from T. f~rrooxiduns cul- tures grown in an iron-free medium, whereas trace amounts of SO were found when the culture growing with galena was supplemented with ferric iron (Tomizuka and Yagisawa 1978). The presence of SQ was not confirmed by other methods of analysis.

T/~iohcrcillus ferrooxidarzs is sometimes used in mixed cul- ture with Tlziohacillus thiooxidans to enhance the oxidation of sulfide minerals. Thiobacillus tlziooxidans is a sulfur-oxidizing acidophile, distinctly different from T. ferroo-xidans because it is not capable of oxidizing Fe(I1). Thiobacillus thiooxidans may oxidize some S compounds at pH values ranging from acidic to near neutral, although growth is possible only under acidophilic conditions (Kelly 1989; Suzuki et al. 1992). This sulfur oxidizer occurs commonly in association with T. ferro- oxidans in biological leaching situations and is involved in the oxidation of So and other inorganic S compounds (thiosulfate, polythionates) formed during oxidative dissolution of sulfide minerals. For example, So is an intermediate in the oxidation of pyrrhotite and arsenopyrite by T. ferroo-xidans (Bhatti et al. 1993; Tuovinen et al. 1994). Another acidophile, Thiobacillus plumhophilus, has been demonstrated to oxidize synthetic PbS (Drobner et al. 1992), but the end products of the reaction have not been characterized and little additional information Is available on this bacterium.

The purpose of the present work was to investigate solid- phase transformations and changes in solution-phase composi- tion associated with the oxidation of natural, research-grade galena as the sole mineral substrate by acidophilic, Fe- and S-oxidizing (T. ferrooxidcrns) and S-oxidizing (T. thiooxidans) bacteria under biological leaching conditions. In some experi- ments, chemical controls and T. ferrooxidans cultures also received additional ferrous or ferric iron in an effort to enhance the abiotic and biological leaching of galena.

Materials and methods

Mineral sample A natural specimen of galena was ground in a disc mill and sieved to 99% <0.5 mm. Purity of the galena sample was verified by X-ray diffraction (XRD) analysis.

Galena oxidation experiments Thiobacillus ferrooxiduns TFI-35 (Tuovinen et al. 1994) and T. thiooxidans FGOl (Garcia 1991) were used throughout this study. Both organisms were initially adapted to grow with galena by successively replacing ferrous sulfate (for T. ferro- oxidans) or sulfur (for T thioo-xiclans) in a mineral salts solution

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Can. J. Micrsbisl. Vol. 41, 1995

Fig. 2. Nomalized peak intensities for galena, d220 = 2.10 A, and anglesite, dI22 = 2.07 A, in abiotic (pH 2.0) and inoculated experiments with 5.0% galena. Maximum intensities of the specified peaks were assigned a value of 100. 1, abiotic control in unsupplemented mineral salts solution; 2, T. thiooxidans in unsupplemented mineral salts solution; 3, T.fc.rrooxicktizs in unsupplemented mineral salts solution; 4, abiotic control with 30 mM Fe2+; 5, abiotic control in filter-sterilized spent-culture medium containing 30 mM Fe3 +; 6, T. ferrooxidaras containing 30 mM Fe2'.

Legend: rn d a y 0; arm d a y 8; w d a y 29

that contained 0.4 g each of (NH4)2S04, MgS04. 7H20, and K2HP04 per litre of distilled water. The medium was acidified with H2SO4 to pH 2.0 and amended with either 2.5 or 5.0% (w/v) galena. Galena in the mineral salts medium was equi- librated for 48 h to allow for the initial acid demand before adjustment to pH 2. After the initial acid demand was satisfied, the flasks were sterilized by autoclaving (30 min, 120°C) followed by inoculation (5% v/v) with T.$~rrooxidar~s or T. thiooxidans. The carry-over ferric iron concentration was negligible because the inocula were taken from cultures grow- ing with galena as the sole substrate. The experiments were cmied out in 100-mL cultures in 250-mL shake flasks that were incubated at 150 rpm and at 22 5 2°C. In some T. ferrooxiduns experiments 30 mM FeS04. 7H20 was used as a supplement- ary energy source.

The following medium formulations were used as chemical controls in time course experiments with (approx. pH 2) or without pH adjustment: (i) mineral salts solution, (ii) mineral salts solution supplemented with 30 mM ferrous sulfate, and (iii) mineral salts solution amended with filter-sterilized (0.22 bm) spent medium from a T.ferrooxidans culture con- taining about 30 mM Fe3+. The purpose of the spent-culture filtrate containing ferric iron was to initiate an abiotic leaching experiment at a relatively high redox potential and to establish whether the biologically produced filtrate contained substances

that could enhance the abiotic leaching of galena. Selected experiments were replicated to confirm the chemical and mineralogical trends.

The mobilization of Pb is often an environmental concern. Because Pb phosphates are highly insoluble compounds, a final leaching experiment was carried out in which the concentration of phosphate was increased from 2.3 to 50 mM by using phosphoric acid for pH adjustment. The 50 mM phosphate - mineral salts solution contained 2.5% galena and was inoculated with T. fel-roo.ui&ns.

Analytical methodology Samples were aseptically withdrawn from the flasks at inter- vals for measurement of pH, redox potential (a Pt electrode against an Ago/AgC1 reference), and the concentration of Fe2+ and total soluble Fe. For Fe2+, the samples (I mL) were acidified with 1 mL of a 1 : 1 mixture of concentrated H3P04 and H2SO4 and diluted to 25 mL with H20. Fe2+ was titrated with 0.5-1 mM K2Cr207, using 0.1 mL of 0.3% stock solution of diphenylamine sulfonic acid (sodium salt) as an indicator. For total soluble Fe, the samples (1 mL) were acidified with 1 mL of 1 M HC1, heated to just below the boiling point, and then reduced with a few drops of Sn(II)-chloride (130 g SnC12 dissolved in 250 mL concentrated HC1 and diluted to 1 L of H20) until colorless. After cooling, the samples received 2 mL

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Fig. 3. Changes in pH (A) and redox potential (B) in T. ferro- axidans and 9: thic~o.xidans cultures growing with galena as the sole source of energy. 0, T. fcrrooxidans with 2.5% galena; @, 9: ferroo~~idans with 5.0% galena: V, T. thiooxidans with 5.0% galena; r. abiotic control with 5.0% galena.

Time (days)

Fig. 4. Changes in pH (A) and redox potential (B) in T. fel-8.0- oxidans cuItures growing with galena in mineral salts solution supplemented with 30 mM ferrous sulfate and in sterile control flasks. G, lC jerrooxiduns with 2.5% galena and 30 mM FeD; @, T. ferrooxidans with 5.0% galena and 30 mM Fe2+; V, abiotic control with 5.0% galena and 30 mM Fe2+; r, abiotic control with 5.0% galena supplemented with filter-sterilized spent-culture medium containing 30 mM Fe3+.

I i

of saturated HgG12 solution to coprecipitate the excess Sn2+ before titration. The concentration of Fe" was calculated as the difference between total soluble Fe and Fe2+. Suspended solids were recovered by centrifugation (5000 x 8 for 10 min), and the supernatants were preserved in 1 M HG1 for subsequent analysis of Pb by atomic absorption spectrometry (AAS). The solid residues were washed with distilled water, air dried, and gently ground with an agate mortar for XRB analysis. Powdered specimens were prepared as standard topfill mounts using Si holders. The samples were analyzed with CuKa radia- tion and a wide-range goniometer equipped with a diffiicted- beam monochromator and a @ compensating slit. Step scans were conducted from 10 to 70Q20 in 0.05"20 increments using a 4-s step time. Relative peak heights were used as indicators of changes in the abundance of minerals in solid residues. The intensities of the 2.10-8. (d220) galena peak and the 2.07-A (dI22) anglesite peak (1 A = 0.1 nm) were monitored for this purpose.

Results and discussion

The research-grade galena sample used in this work did not contain mineral impurities detectable by XRD (Fig. IA). Abiotic oxidation of galena was evident from the presence of anglesite (PbS04) in sterile samples leached at pH 2.0 for up to 29 days with or without FeD or Fe3+ (Figs. 1B-1D; treat- ments 1, 4, and 5 in Fig. 2). Maximal oxidation of galena

0 10 20 38

Tlme (days)

(anglesite formation) in abiotic controls was achieved in the presence of supplemental FeG (treatment 5 in Fig. 2).

The oxidation of galena by T. fsrroo.xidans and T. thio- oxidans in uilsupplemented media showed no net change in acidity, although there were transient pH changes during the experiment (Fig. 3A). In contrast, the pH in the abiotic control increased to around 4.0. The redox potential increased to around 500 mV in inoculated T. ferrooxidcins cultures, and this increase was faster with the lower pulp density (2.5 vs. 5%) (Fig. 3B). The increase in redox potential was relatively slow in the T. thiooxidans culture. The abiotic control remained at around 300 mV. These results indicate that galena acted as a strong reductant in the leaching system. Both organisms could effectively leach galena albeit at different rates. Although crystalline material can usually be detected to a level of at least 1-2% (w/w) by XRB, SO was not identified in the solid resi- dues. Therefore, the transient pH increase in the T. tl~iooxidans culture suggested the formation of an uncharacterized S inter- mediate through acid-consuming steps before final oxidation to sulfate. In the corresponding abiotic control, the oxidation to sulfate apparently did not occur to the same extent because the pH did not decrease toward the end of the time course.

The pH changes were again minor when galena was inoculated with T. fen-roo-xidons and supplemented with 40 mM ferrous sulfate (Fig. 4A). In the corresponding control flasks

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Fig. 5. Changes in the concentration of Fe2+ (A) and Fe3+ (B) during oxidative leaching of galena in T. ferrooxidans cultures and in sterile control flasks. 0, T. ferrooxiduns with 2.5% galena and 30 mM Fe2+; 0, T. ferrooxidans with 5.0% galena and 30 mM Fe2+; 7, abiotic control with 5.0% galena and 30 mM Fe2 +; V, abiotic control with 5.0% galena supplemented with filter-sterilized spent-culture medium containing 30 mM Fe3 +.

0 40 20 30

Time (days)

amended with 30 mM Fe2+ or Fe", the pH values increased to 4.0, again indicating incomplete oxidation of the S entity of PbS to sulfate. Oxidation of Fe2+ by T. feri-ooxidans caused an increase in the redox potential to 600 mV and was faster at the lower (2.5%) pulp density (Fig. 4B). The difference in the redox profile with pulp density was comparable to that shown in Fig. 3B, indicating that the change in the redox potential was dependent on the concentration of galena as a reductant. The redox potential remained unchanged in abiotic controls containing 5% galena and 30 mM iron added as Fe2+. In the abiotic control experiment with 30 mM Fe3+ addition, the initial redox potei~tial of 530 mV decreased rapidly to 200 mV as a result of the redox reaction with PbS (Fig. 4B). The low redox potential reached in the control galena supplemented with 30 mM Fe3+ confirmed that galena acted as a reductant and thereby completely depleted Fe3+ from solution.

Ferrous iron oxidation by Tfi~rroo.xiduns occurred at both pulp densities, but was somewhat slower with 5% galena (Fig. 5A), perhaps because of the concurrent reductive reaction of Fe" with PbS. Ferrous iron was the predominant dissolved iron species in the abiotic controls. The dissolved Fe3+ con- centrations were negligible, in keeping with the initial, rapid reduction of ferric iron by PbS (Fig. 5B). The formation of Fe3+ as a result of bacterial oxidation of 30 mM F e u was slow

Pig. 6. X-ray diffractograms of solids from galena experiments after 29 days of leaching in T. thioo.xiduns cultures containing 5 % galena in unsupplernented mineral salts solution (A) and in T.ferroosidans cultures containing 5% galena in unsupple- mented inineral salts solution (B). Diagnostic XRB lines for galena and anglesite are designated with G and A. respectively.

at the higher pulp density (Fig. 5B). The slow oxidation, and also the gradual decrease in the Fe'+ concentration seen with 2.5% pulp density (Fig. 5B), may reflect Fe(II1) precipitation over time owing to the formation of jarosite or Fe oxides.

Anglesite (PbS04) was the only new solid phase detected by XRD analysis of leach residues in the experiments with galena as the sole energy source for T. thiooxidans and T. ferro- oxidurzs (Figs. 6A and 6B). Additionally, jarosite was detected within 8 days from solids generated by T. Jerrooxiclans cultures grown for 29 days with 2.5% galena and 30 mM ferrous sulfate (Fig. 7A). With 5% galena under comparable conditions, only traces of jarosite were detected by XRD after 29 days of leaching (Fig. 7B). The precipitation of jarosite was in accord with the data in Fig. 5B, which showed Fe3+ precipitation in T. ferrooxidans cultures growing with 2.5% galena, whereas iron oxidation was slow and the Fe3+ concentration continued

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to increase in cultures supplemented with 5% galena. Jarosite was not detected in any of the abiotic controls that were initially amended with Fe2+ or Fej+ (Figs. 1B and 1C).

Anglesite is a relatively insoluble compound with a log K,, , of -7.85. Accordingly, dissolved Pb concentrations in cul- ture supernatants averaged only l .5 + 0.3 mg Pb2+/L (n = 8) after 29 days of leaching of galena, and Pb2+ levels were in the range of 1.1-5.0 mg Pb2+/L throughout the time course in all experiments. Lead phosphates are even more insoluble and should effectively compete with anglesite for Pb released during galena dissolution. For example,

PbHP04 ++ Pb2+ + HP042- log K,, = -1 1.57

Pb3 (P04)2 ++ 3Pb2+ + 2P043- log K,, = -44.5

To investigate the possible formation of Pb phosphates, the phosphate concentration was increa~ed from 2.3 to 50 mM in T. ferrooxidans media containing 2.5% galena, yielding an approximate initial molar ratio of 1 sulfate : 0.7 phosphate in solution. Additional sulfate from the oxidation of 2.5% (105 mM) galena should not be overlooked in the mass ratio, although it would not significantly change the solution-solid phase ratio if the product precipitates as anglesite. Except for anglesite, no additional solid-phase products of galena oxida- tion were detected by XRD analysis of residues from these experiments after 8 and 29 days contact time.

The relative peak intensities for galena and anglesite in solid residues from T. fel-rooxidans and T. thiooxidans cultures are shown in Fig. 2. Anglesite formation and concurrent galena dissolution were more pronounced in T. ferrooxidan experi- ments than in T. thioodxida?zs cultures or the abiotic controls. The relative peak intensities showed that the biological leaching of galena declined toward the end of the incubation. This trend suggests that anglesite, and jarosite in Fe-amended cultures, may precipitate on the surface of galena particles so that further oxidation becomes diffusion limited. Elemental S was not detected in solids from any of the galena leaching experiments sampled at 0 ,8, 17, and 29 days.

Figure 8 presents a possible scheme for galena oxida- tion based on the solution chemistry and XRD data collected in the abiotic and bacterial experiments. Without an Fe3+-regenerating system, galena leaching is based on acid dissolution (abiotic oxidation) or on direct biological oxidation of the S entity (T. thioo.xidans. T. f~rrooxidans), as shown in equation 1 and in Fig. 8. In the presence of dissolved iron, galena oxidation by T. ferrooxidans is enhanced owing to an Fewf-mediated redox reaction (equation 3), as also demon- strdted by Bang et al. (1995). Because So was not detected in the leach residues, it can be concluded that So was concurrently oxidized to sulfate by T. fel-rooxieluns and T. thiooxiduns (equa- tion 4), and by excess Fe3+ while T.fe?-mn.~i&ns regenerates Fe3+, as shown in equations 5 and 6:

Fig. 7. X-ray diffractograrns of solids from galena experiments after 29 days of leaching in T. ferrooxidarzs cultures in mineral salts soIution supplemented with 30 mM Fe2+ and either 2.5% (A) or 5% galena (B). Line spacings for jarosite are given in Angstroms. Diagnostic XRD lines for galena, anglesite, and jarosite are designated with G, A, and J, respectively.

The kinetics of galena oxidation by bacteria have not been investigated. In view of the solid-phase products found in this work, the rate limitation in bacterial leaching systems for galena may be due to the formation of mineral surface coatings (anglesite, jarosite) that hinder the diffusion of reactants and products and thereby slow the rates of some abiotic or bio- logical oxidation steps involving O2 and Fe2+ as the available electron acceptors.

Acknowledgements

Partial funding for the work was received from the Fundagiio de Amparo a Pesquisa do Estado de Siio Paulo, Brazil (O.G.). Salary and research support were provided to J.M.B. by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. This is journal article No. 187-94 of the Ohio Agricultural Research and Development Center, The Ohio State University.

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Fig. 8. Schematic of the oxidative dissolution of galena with Fe3 + and O2 as primary oxidants. The Q2-coupled oxidation may be abiotic or catalyzed by T. thiooxidans or T. ferrooxidans. The Fe3+-mediated dissolution involves the reoxidation of Fe2+ by T, ferr-soxidans.

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