APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1994, p. 3268-3274 Vol. 60, No. 90099-2240/94/$04.00+0Copyright © 1994, American Society for Microbiology

Oxidative Dissolution of Arsenopyrite by Mesophilic andModerately Thermophilic Acidophilest

OLLI H. TUOVINEN,1* TARIQ M. BHAITI,1t JERRY M. BIGHAM,2 KEVIN B. HALLBERG,3OSWALDO GARCIA, JR.,1§ AND E. BORJE LINDSTROM,3T

Department of Microbiology' and Department ofAgronomy,2 The Ohio State University, Columbus, Ohio 43210, andDepartment ofApplied Cell and Molecular Biology, University of Umea', S-901 87 Umea, Sweden3

Received 2 May 1994/Accepted 30 June 1994

The purpose of this work was to determine solution- and solid-phase changes associated with the oxidativeleaching of arsenopyrite (FeAsS) by ThiobaciUlus ferrooxidans and a moderately thermoacidophilic mixedculture. Jarosite [KFe3(SO4)2(OH)6], elemental sulfur (SO), and amorphous ferric arsenate were detected byX-ray diffraction as solid-phase products. The oxidation was not a strongly acid-producing reaction and wasaccompanied by a relatively low redox level. The X-ray diffraction lines ofjarosite increased considerably whenferrous sulfate was used as an additional substrate for T. ferrooxidans. A moderately thermoacidophilic mixedculture oxidized arsenopyrite faster at 45°C than did T. ferrooxidans at 22°C, and the oxidation wasaccompanied by a nearly stoichiometric release of Fe and As. The redox potential was initially low butsubsequently increased during arsenopyrite oxidation by the thermoacidophiles. Jarosite, S0, and amorphousferric arsenate were also formed under these conditions.

Thiobacillusferrooxidans can oxidize many iron sulfides, suchas pyrite (FeS2) and pyrrhotite (Fe1,S), as the sole sources ofenergy. The bacterial oxidation of pyrite is an acid-producingreaction, releasing ferric iron and sulfate to solution. Withpyrrhotite, the initial dissolution reaction is nonoxidative and isaccompanied by the acid-consuming oxidation of dissolvedFe2 . Pyrrhotite oxidation becomes a net acid-producing re-action upon hydrolysis of Fe3" and the bacterial oxidation ofthe intermediate S (1). These biogeochemical reactions havea major impact on acid mine drainage as well as on metaldissolution in heap and dump leaching processes involvinglow-grade ore materials.

Arsenopyrite (FeAsS) is also a potential substrate for T.ferrooxidans and is a factor in the biological pretreatment ofAu-containing sulfide ore concentrates (9). Extraction of goldfrom sulfide minerals by hydrometallurgical methods is usuallybased on the formation of a soluble Au complex with cyanide.Pyrite and arsenopyrite decrease the efficacy of cyanidationbecause they physically block the contact of cyanide solutionwith Au inclusions (8). Additionally, arsenopyrite interfereswith cyanidation because it competes with cyanide for molec-ular oxygen (10). Biological pretreatment of Au-containingores tends to substantially increase the subsequent gold recov-ery by cyanidation (3, 9, 12). The role of bacteria in this processis to oxidize pyrite and arsenopyrite, leading to mineraldissolution and thereby to the exposure of gold disseminated inthe sulfide matrix. Arsenic-containing minerals are relatively

* Corresponding author. Mailing address: Department of Microbi-ology, The Ohio State University, 484 West 12th Ave., Columbus, OH43210-1292. Phone: (614) 292-3379. Fax: (614) 292-8120. Electronicmail address: otuovine@magnus.acs.ohio-state.edu.

t Article 75-94 of the Ohio Agricultural Research and DevelopmentCenter, The Ohio State University.

t Present address: National Institute for Biotechnology and GeneticEngineering, Faisalabad, Pakistan.

§ Present address: Departamento de Bioquimica, Instituto de Quimica,Universidade Estadual Paulista, Araraquara-SP-CEP.14.800, Brazil.

¶ Present address: Department of Microbiology, University of Umea,S-901 87 Ume'a, Sweden.

common in many sulfide ore deposits, and the solubilization ofAs compounds is also of considerable interest with a view tothe transport and environmental toxicity of this element. In thepresent work, the biological oxidation of arsenopyrite wasinvestigated with T. ferrooxidans and a moderately thermoaci-dophilic mixed culture as the test organisms and with emphasison solution- and solid-phase changes.

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FIG. 1. Changes in pH and redox potential during oxidative leach-ing of arsenopyrite in the presence of T. ferrooxidans (A) and in sterilecontrol flasks (B). The inoculated and uninoculated flasks eachreceived an aliquot of fresh medium following the sampling on day 30.Symbols: 0 and 0, pH in the presence and absence of 120 mM ferroussulfate; V and V, redox potential in the presence and absence of 120mM ferrous sulfate.

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BIOLOGICAL OXIDATION OF ARSENOPYRITE 3269

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FIG. 2. Changes in the concentration of dissolved arsenic (A) andiron (B) during oxidative leaching of arsenopyrite in the presence of T.ferrooxidans and in sterile control flasks. Symbols: 0 and 0, Asd in thepresence and absence of 120 mM ferrous sulfate in T. ferrooxidanscultures; * and O, ASd in the respective sterile control flasks; v and V,Fed in T. ferrooxidans cultures and in sterile control flasks withoutadditional ferrous sulfate.

MATERUILS AND METHODS

A research-grade sample of arsenopyrite was obtained froma commercial source (Ward's Natural Science Establishment,Inc., Rochester, N.Y.) and was ground and sieved to a -200-mesh size (<74 ,um) fraction. An X-ray amorphous ferricarsenate was prepared according to the method of Krause andEttel (7) by combining 1.0 liter of 0.3 M Fe(NO3)3 with 25 g ofAs as As2O5 followed by heating at 80°C for 24 h. The purityof the powdered specimens was established by X-ray diffrac-tion (XRD) analysis.Two test cultures of acidophilic bacteria, T. ferrooxidans

(mesophilic) and a moderately thermophilic mixed culture,were used in this work. T. ferrooxidans (strain TFI-35) wasgrown in a mineral salts solution which contained 0.4 g each of(NH4)2S04, MgSO4- 7H20, and K2HPO4 per liter of distilledwater. The medium was acidified with H2SO4 to pH 2 andamended with 2.5 g of arsenopyrite per 100 ml. Supplementalferrous sulfate (120 mM FeSO4 - 7H20) was also used in someT. ferrooxidans experiments in which the cultures were incu-bated at 22 ± 2°C. The moderately thermophilic consortiumwas composed of iron- and sulfur-oxidizing acidophiles. Theconsortium contained Thiobacillus caldus (strain KU), a re-cently characterized, S-oxidizing, moderate thermoacidophile(5), as one of the predominant organisms. The mixed culturewas grown in the same mineral salts medium at both 22 ± 2and 45 ± 1°C.The redox potential (a Pt electrode against an Ago/AgCl

reference) and pH of the leach solutions were monitoredduring arsenopyrite oxidation. In the T. ferrooxidans experi-ments, the dissolved As (Asd) and Fe (Fed) in supernatants ofcentrifuged leach solutions were measured by inductively

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FIG. 3. X-ray diffractograms of arsenopyrite before leaching (A)and after leaching for 40 days in sterile unsupplemented mineral saltssolution (B) and in sterile mineral salts solution supplemented with120 mM ferrous sulfate (C). Diagnostic XRD lines for arsenopyriteand elemental sulfur are identified by the letter designations A and S,respectively. Numerical values, when given, are in angstroms. Thebroad primary diffraction band (2.25 to 4.0 A [ca. 0.23 to 0.40 nm]) ofX-ray amorphous ferric arsenate is highlighted. Question marks indi-cate unassigned diffraction lines detected in the untreated arsenopyritesample. The scale of relative counts is indicated with bars for eachX-ray diffractogram.

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3270 TUOVINEN ET AL.

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FIG. 4. X-ray diffractograms of arsenopyrite leached in unsupple-mented T. ferrooxidans cultures for 10 (A), 30 (B), and 40 (C) days.The broad diffraction bands (2.25 to 4.0 and 1.35 to 1.85 A [0.23 to 0.40and 0.14 to 0.19 nm]) of X-ray amorphous ferric arsenate arehighlighted. The cultures received fresh media following sampling onday 30. A, arsenopyrite; J, jarosite; S, elemental sulfur.

coupled plasma emission spectroscopy. Asd and Fed reflect thetotal amounts of As and Fe in solution and do not discriminatebetween different redox species of these elements.

In experiments with the moderately thermophilic mixedculture, Asd and Fed were determined by flame atomic absorp-tion spectrometry in supernatants of centrifuged samples.Aliquots (0.2 ml) of supernatants were mixed with 1.8 ml of 5M HCl and then diluted in 0.3 M HCl for atomic absorptionspectrometry. Dissolved Fe2+ was also determined by titrationwith ceric sulfate (6). In this procedure, samples (0.2 ml) ofsupernatants were mixed with 1 ml of 1 M H2S04 and 0.2 mlof indicator solution (15 mM 1,10-phenanthroline in 5 mMH2SO4) and titrated with ceric sulfate [1 mM H4Ce(SO4)4 in 1M H2SO4]. The endpoint was indicated by a change of thecolor from pink to blue due to the formation of a ferriciron-phenanthroline complex. The titration is based on thefollowing reaction: Fe2+ + Ce4 -> Fe3" + Ce3+.

Total Fe (FeHCI) and total As (ASHCI) in the thermophilicsuspensions were measured by atomic absorption spectrometryafter acid digestion (4.5 M HCI, 30 min at 65°C) and dilutionin 0.3 M HCl. This digestion selectively dissolved jarosite andferric arsenate precipitates that were formed during arsenopy-rite oxidation. In the culture flasks, a residual Fe(III) precip-itate adhered to the glass wall and could not be retrieved uponsampling. Control experiments with arsenopyrite suspensionsindicated that the HCI extraction released less than 1% of theFe and As content of the reference arsenopyrite.

Samples of leach residues were air dried and gently groundwith an agate mortar for XRD analysis. All samples wereanalyzed with CuKa radiation and a wide-range Philips PW1316/90 goniometer equipped with a diffracted-beam mono-chromator and a 0 compensating slit. Step scans were con-ducted from 10 to 70020 in 0.05'20 increments, using a 4-sstep time. Powdered specimens from the T. ferrooxidans exper-

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FIG. 5. Plots of normalized peak intensities for arsenopyrite, d2O = 2.66 A (ca. 0.27 nm) (A); jarosite, d,13 = 3.09 A (ca. 0.31 nm) (B); andelemental sulfur, d222 = 3.86 A (ca. 0.39 nm) (C). Maximum intensities of the specified peaks were assigned a value of 100. The data were calculatedfrom arsenopyrite leaching experiments with T. ferrooxidans in the presence (0) and absence (0) of 120 mM ferrous sulfate.

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BIOLOGICAL OXIDATION OF ARSENOPYRITE 3271

iments were prepared as standard top-fill mounts, using Alholders. Residues from the thermophilic mixed cultures werelimited in quantity and were mounted with a single-crystalsilicon holder (Dow Coming model C-12). Although XRD isnot a quantitative method, relative peak heights can be used asindicators of changes in the abundance of minerals in amixture. The intensities of the 2.66-A (ca. 0.27-nm) arsenopy-rite peak, the 3.09-A (ca. 0.31-nm) jarosite peak, and the3.86-A (ca. 0.39-nm) sulfur peak were monitored for thispurpose.

RESULTS

Arsenopyrite oxidation by T. ferrooxidans. Arsenopyrite ox-idation in mineral salts medium inoculated with T. ferrooxidanswas accompanied by a relatively low redox potential and aminor, transient decrease in pH for the first 30 days (Fig. 1A).Amendment of the culture media with 120 mM ferrous sulfatedid not substantially change this pattern. On day 30, 20 ml offresh mineral salts solution (pH 1.5) was added to the T.ferrooxidans cultures and the respective sterile controls, andthe cultures were incubated for an additional 10 days. Afterthis addition, the redox potential increased, suggesting thatactive Fe oxidation took place in both unamended and Fe-amended cultures. The redox potential and pH values in thesterile controls showed little change throughout the timecourse except for the low pH caused by the addition of fresh,sterile media (Fig. 1B). Negligible Fe2" oxidation was appar-ent from the low redox potential in the sterile control flasks.The solubilization of As was enhanced in cultures supple-

mented with ferrous sulfate (Fig. 2A). Up to 7% of the Ascontent of arsenopyrite was found in solution after 30 days ofleaching in T. ferrooxidans cultures. The concentration of Fedafter a 30-day contact time (Fig. 2B) was equivalent to about8.5% dissolution of added arsenopyrite in cultures which didnot receive ferrous sulfate. There was a difference in theapparent rate of dissolution of Fed and Asd (Fig. 2A and B),but this difference cannot be properly assessed because of theconcurrent formation of secondary Fe- and As-containing solidphases.

Mineralogical analysis revealed no major sulfide impuritiesin the arsenopyrite sample used in these experiments (Fig. 3A);however, several minor diffraction lines in the range of 5.68 to3.88 A (ca. 0.57 to 0.39 nm) and 1.51 to 1.40 A (0.15 to 0.14nm) could not be assigned. Leaching of the sample understerile conditions resulted in a general decrease in arsenopyritepeak intensities and the formation of So and X-ray amorphousferric arsenate (Fig. 3B and C). Comparative XRD data fromsynthetic ferric arsenate are presented within the context of thethermophilic leaching experiments.

In unsupplemented T. ferrooxidans cultures, arsenopyritediffraction peaks weakened throughout the time course, butcomplete dissolution did not occur (Fig. 4A to C; Fig. SA).Both jarosite and X-ray amorphous ferric arsenate appeared toincrease in abundance over time (Fig. SB). So lines were clearlypresent after 10 days and increased in intensity throughout thetime course of 40 days (Fig. SC).

In T. ferrooxidans cultures which were supplemented withferrous sulfate, S0 was formed in amounts comparable tounsupplemented samples (Fig. 5C; Fig. 6A to C); however,jarosite was precipitated in greater quantities (Fig. SB) andarsenopyrite lines were markedly weakened, indicating accel-erated decomposition during the early stages of the timecourse (Fig. SA). Once again, weak, broad diffraction bandsfrom 2.25 to 4.0 A (ca. 0.23 to 0.40 nm) and 1.35 to 1.85 A (ca.0.14 to 0.19 nm) were evident in X-ray diffractograms of the

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FIG. 6. X-ray diffractograms of arsenopyrite leached in ferroussulfate-supplemented T. ferrooxidans cultures for 10 (A), 30 (B), and40 (C) days. The broad diffraction bands (2.25 to 4.0 and 1.35 to 1.85A [ca. 0.23 to 0.40 and 0.14 to 0.19 nm]) of X-ray amorphous ferricarsenate are highlighted. The cultures received fresh media followingsampling on day 30. A, arsenopyrite; J, jarosite; S, elemental sulfur.

leach residues collected from ferrous sulfate-supplemented T.ferrooxidans cultures (Fig. 6B and C), suggesting the presenceof amorphous ferric arsenate. The crystallinity of this phase didnot improve upon heat treatment (24 h at 160°C) or autoclav-ing (8 h at 120°C).

Arsenopyrite oxidation by the moderately thermoacidophilicmixed culture. The moderately thermophilic consortium oxi-dized arsenopyrite slowly at 22°C compared with incubation at45°C (Fig. 7 and 8). The concentrations of FeHcl and ASHCIleached from the arsenopyrite were comparable, suggestingcongruent dissolution of FeAsS. The concentrations of Fed andAsd were also comparable at 22°C but not at 45°C. Thedifference between FeHCl and Fed represents iron precipitationwhich increased with time (Fig. 7 and 8). Similarly, these datasuggest that arsenic precipitated concurrently with iron.The bacterial oxidation of Fe2+ was negligible at 22°C,

whereas it was rapidly oxidized at 45°C (Fig. 9). The entire

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FIG. 7. Dissolution of iron and arsenic from arsenopyrite by themoderately thermophilic mixed culture incubated at 22°C. Symbols: *,FeHCI; 0, Fed; *, AsHCI; O, ASd-

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FIG. 9. Concentration of ferrous iron during arsenopyrite oxida-tion by the moderately thermophilic mixed culture incubated at 22°C(V) and 45°C (v).

time course of 51 days at 22°C was characterized by a relativelylow redox potential (Fig. 10), indicating the lack of ferrous ironoxidation. By contrast, the redox potential increased in cul-tures incubated at 45°C, indicating an increase in the Fe3"/Fe2+ ratio. The net reaction was acid producing at 45°C andacid consuming at 22°C (Fig. 10).

Diffraction data showed enhanced decomposition of ar-

senopyrite at both 22 and 45°C in the thermophilic culturescompared with those prepared with T. ferrooxidans (Fig. 11Aand B). Conversion of the arsenopyrite to So, jarosite, andamorphous ferric arsenate was almost complete at 45°C. At45°C, two broad diffraction bands from approximately 2.25 to4.0 A (ca. 0.23 to 0.40 nm) and 1.35 to 1.85 A (ca. 0.14 to 0.19nm) attributable to amorphous ferric arsenate are clearlypresent and are similar in shape and relative intensity to thoseobtained from a pure, synthetic specimen (Fig. 11C). Theabundance of jarosite and amorphous ferric arsenate in thesolid residues sampled from the 45°C cultures (Fig. 11B) isconsistent with solution-phase changes observed as an increasein redox potential, active oxidation of Fe2+, and a decrease inthe Fed concentration.

DISCUSSION

The FeHCl and ASHC1 data indicated that the moderatelythermophilic consortium more or less completely dissolved thearsenopyrite at 45°C, which is a temperature prohibitive tomesophilic cultures of T. ferrooxidans (11). About 40 to 50%dissolution of arsenopyrite occurred at 22°C in the moderatelythermophilic mixed culture, whereas only about 7% of theinitial As content was leached by the mesophilic T. ferrooxidanswithin 7 weeks. The moderately thermophilic consortium was

As resistant and had been previously subcultured once in a

medium which contained arsenical pyrite as the substrate. Tferrooxidans had been neither adapted to elevated levels ofdissolved arsenate or arsenite in culture media nor subculturedwith arsenopyrite before these experiments. However, there

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VOL60,1994~~~~~~~BIOLOGICAL OXIDATION OF ARSENOPYRITE 3273

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pyrite and arsenopyrite. Oxidative leaching of arsenical pyritedecreased the pH values to <1, which is within the Eh-pHstability field of scorodite (4) but is prohibitively low for Tferrooxidans. The pH values of >1.7 in the present work wereapparently outside the scorodite stability field.

In the moderately thermophilic mixed culture at 220C, theconcentrations of ASd and Fed were similar for the first 37 daysof incubation. Neither ferric arsenate nor jarosite precipitatedin significant quantities during this period because the Fed wasprimarily in the ferrous form, as also evidenced by the lack ofincrease in the redox potential. Only amorphous ferric arse-nate, So, and residual arsenopyrite were detected in solidresidues at the termination (51 days) of the experiment,evidence for equimolar precipitation of Fe and As. In contrast,jarosite was a major sink for Fe(III) in solid residues from theT' ferrooxidans at 220C and the mixed culture at 450C evenwhen ferric arsenate was not detected. These cultures activelyoxidized iron, and the ASd and Fed concentrations weredissimilar, indicating that both ferric arsenate and jarositeprecipitated but in different amounts and rates. Thus, thebiological oxidation of arsenopyrite produces mixtures of ferricarsenate, jarosite, and elemental S depending on the extent ofarsenopyrite oxidation, pH and ionic composition of the leachsolution, and the temperature. Arsenopyrite oxidation alonedoes not produce sufficient amounts of sulfuric acid to lowerthe pH to the stability field of scorodite, but it can be formedin mixtures of arsenopyrite and pyrite because pyrite oxidationis a strongly acid-producing reaction (2).Amorphous ferric arsenate and well-crystalline scorodite are

metastable and can redissolve incongruently. Under ambientsurface conditions, the resolubilization results in the formationof Fe(III) oxides and oxyhydroxides and the release of arsenateions (4). The mobility of arsenic species in bioleaching effluentsis greatly influenced by the activities of Fe , S042, H3O+,and AsO43 as well as by biological reactions influencing theirconcentrations. In heap and dump leaching, the redissolutionof ferric arsenates may continually mobilize As species intoseepage. Thus, precipitates from arsenopyrite bioleaching may,with time, become a pool of soluble As depending on environ-mental conditions.

ACKNOWLEDGMENTS

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We thank Siv Saaf for skillful assistance with the bioleachingexperiments carried out with the moderately thermophilic culture.

_________________Liisa Carlson kindly prepared the amorphous ferric arsenate specimen.50 60 70 This work is part of a cooperative program (O.H.T.) funded by the

National Science Foundation International Programs (INT 9113347).nopyrteater 1 das of

Partial funding for the work was also received from the U.S. Agencynopyrite afte451C days ofe for International Development and the Government of Pakistan'chinbrat diffrct(B) byathe (T.M.B.); the Narings-och teknikutvecklingsverket, Boliden Mineralhe4brand diffration01 bnds AB, Nordisk Industrifond, and Styrelsen for U-landsforskning, Sweden

0.40and0.1 to0.1 nm) (K.B.H. and E.B.L.); and the Fundagdo de Amparo A Pesquisa doilighted. A synthetic sample Estado de Sao Paulo, Brazil (0.G.). Salary and research support were[red as described by Krause provided to J.M.B. by state and federal funds appropriated to the Ohioarsenopyrite; J, jarosite; 5, Agricultural Research and Development Center, The Ohio State

University.

were too many cultural variables to unequivocally suggest that

arsenic resistance enhanced arsenopyrite oxidation.

Well-ordered scorodite (FeAsO4 - 2H20) has been previ-

ously detected in solid residues from bioleaching experimentscarried out with arsenical pyrite (2). In the present study, no

evidence of the presence of scorodite was obtained in any of

the samples examined. This difference is likely a result of pHdifferences between the experiments conducted with arsenical

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