Vol. 54, No. 12

Characterization of Jarosite Formed upon Bacterial Oxidation ofFerrous Sulfate in a Packed-Bed Reactort

SERGEI I. GRISHIN,'t JERRY M. BIGHAM,2 AND OLLI H. TUOVINEN1*Departments of Microbiology1 and Agronomy,2 The Ohio State University, Columbus, Ohio 43210

Received 29 June 1988/Accepted 31 August 1988

A packed-bed bioreactor with activated-carbon particles as a carrier matrix material inoculated withThiobacillusferrooxidans was operated at a pH of 1.35 to 1.5 to convert ferrous sulfate to ferric sulfate. Despitethe low operating pH, trace amounts of precipitates were produced in both the reactor and the oxidized effluent.X-ray diffraction and chemical analyses indicated that the precipitates were well-ordered potassium jarosite.The chemical analyses also revealed a relative deficiency of Fe and an excess of S in the reactor samplecompared with the theoretical composition of potassium jarosite.

Thiobacillus ferrooxidans is an autotrophic bacteriumwhich usually lives at a pH range from 1.5 to 4.5 and whichoxidizes inorganic compounds of iron and sulfur for energy.

The bacterium proliferates in acid mine sites, where exposedore and coal surfaces provide a substratum. The mainoxidation end products are ferric iron and sulfate, which are

problematic because of their environmental impact in acidmine drainage. Other metals may also be released in theacid-leaching process. The bacterium can be used in mineralprocessing to solubilize sulfide minerals and to assist in theleaching of uranium ores. It also is a candidate organism forbiological desulfurization of pyritic coals, although no pilot-scale developments in this context have been reported. Animportant component in these biological leaching applica-tions is the oxidation of ferrous iron by T. ferrooxidans.

4Fe2+ + 02 + 4H+ -* 4Fe3+ + 2H20 (1)

Ferric iron thus formed hydrolyzes in aqueous solutions.

Fe3+ + H20 -> FeOH2+ + H+ (2)

Fe3+ + 2H20 -> Fe(OH)2+ + 2H+ (3)

Fe3+ + 3H20-* Fe(OH)3 + 3H+ (4)

The extent of the ferric iron hydrolysis is dependent on thepH; in general, ferric iron has an extremely low solubility ata pH of >2.5. A competing reaction for the hydrolysis is theformation of basic ferric sulfates which have the generalformula XFe3(SO4)2(OH)6, where X = K+ (potassium jaro-site), Na+ (natrojarosite), NH4+ (ammoniojarosite), or

H30+ (hydronium jarosite). Jarosite precipitation is also an

acid-producing reaction:

3Fe3+ + X+ + 2HS04 + 6H20-- XFe3(SO4)2(OH)6+ 8H+ (5)

Jarosites can be found in sediments of acid mine drainage,sometimes in co-occurrence with Fe(III) oxyhydroxides (1).Jarosite precipitation is dependent on the pH and on theionic composition and concentration of the medium (5).

* Corresponding author.t Paper no. 280-88, Ohio Agricultural Research and Development

Center, The Ohio State University.t Present address: Moscow Steel and Alloys Institute, 117465

Moscow, USSR.

Jarosite-type precipitates have previously been produced inexperimental systems involving ferrous iron oxidation by T.ferrooxidans (6, 11, 14). These systems utilized pH values of>2, at which extensive precipitation of ferric iron occurs.Additionally, clay minerals have been included in someexperiments (7, 8, 12) in attempts to simulate processesoccurring in acid sulfate soils. While jarosite formation alsooccurs in the presence of clay minerals, the mineralogicalfeatures in such systems are complex due to undefinedsource materials and are difficult to interpret for biogenicand chemical differences in solid phases and mineralproducts.

In the present study, iron-oxidizing bacteria were main-tained under low-pH conditions (pH <1.5) in a packed-bedbioreactor which displayed extremely low amounts of ironprecipitation. At this pH, iron oxides and oxyhydroxidesshould be soluble, and even jarosite-type compounds shouldbe only marginally stable (4). The composition and mineral-ogy of the precipitates are of interest because secondary ironcompounds are undesirable products in leach mines due tothe fact that they form rims around ore particles, therebyaltering the diffusion characteristics and decreasing the rateof leaching reactions occurring on the mineral surface. Theobjective of this study was to characterize the secondaryiron precipitates occurring both in the effluent and within theworking column of a lab-scale bioreactor after 3 months ofcontinuous operation.

MATERIALS AND METHODS

The experimental system was a packed-bed reactor (16mm x 320 mm) with activated carbon (701- to 841-,um-sizefraction, Union Carbide Corp.) as the carrier matrix. Theworking volume of the column was 50 ml, and the specificsurface area was 38 cm2/ml of reactor volume. Inlets forfresh medium and air were at the bottom of the column,whereas outlets for exhaust air and effluent were at the top.The reactor was operated at 230C. The column influentsolution contained 120 mM ferrous sulfate, which was con-

verted to ferric sulfate by bacteria (T. ferrooxidans) presenton the surfaces of activated-carbon particles. Other mineralsalts used in the nutrient solution were 2.3 mM K2HPO4, 1.6mM MgSO4, and 3.0 mM (NH4)2SO4. The bacteria were

inoculated to the column while it was operated as a batch

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3102 GRISHIN ET AL.

reactor. Once >95% Fe2" oxidation was established, thereactor column was changed to a continuous mode ofoperation. The influent pH was 1.35, and the effluent pH was1.5. Iron was oxidized at 50 to 99% efficiency depending onthe flow rate, which was controlled with a peristaltic pump.The rate of abiotic iron oxidation at this pH range wasnegligible. Iron oxidation was monitored by titration ofeffluent samples with 2 mM KMnO4.Two samples of iron precipitates were obtained for anal-

ysis. Sample 1 was concentrated from 80 liters of effluentpassed through the packed-bed reactor during 1 month ofoperation. The precipitates were collected by sedimentationfollowed by filtration through glass fiber filters. The precip-itates were washed four times each with 0.1 N sulfuric acidand distilled water and then dried at 104°C to a constantweight. The amount of iron precipitates in the efflu-itaveraged 3 mg/liter (total dry weight, 240 mg). Sample 2 wasa composite of iron precipitates collected from the inside ofthe reactor column at the termination of the experiment (3months). The activated-carbon particles were suspended in200 ml of 1.0 N sulfuric acid and vigorously shaken to stripthe cells and precipitates from the surfaces. The carbonparticles were then collected by filtration on a screen andrinsed with 100 ml of 0.1 N sulfuric acid. The filtratecontaining the cells and precipitates was allowed to settle for3 h before it was decanted, and the precipitate-containingportion was filtered through polycarbonate membrane filters(0.2 ,um; Nuclepore). The precipitates on the filter weredried at 104°C to a constant weight. A total of 410 mg ofprecipitate (dry weight) was recovered from the initial 50 mlof activated-carbon bioreactor volume.

Colors of the dried precipitates were determined by usingMunsell color charts under natural sunlight. Total chemicalanalyses were conducted using inductively coupled plasmaemission spectroscopy. X-ray diffraction analyses of randompowder mounts of the precipitates were obtained by usingCuKao radiation and a Philips PW 1316/90 wide-range goni-ometer fitted with a theta-compensating slit, a 0.2-mm re-ceiving slit, and a diffracted-beam graphite monochromator.Samples were step scanned from 10 to 650 20 at 0.100 20increments using a counting time of either 10 or 40 s perincrement. The digital data were stored on floppy discs byusing an IBM personal computer, reformatted for compati-bility with the Lotus 1-2-3 graphics package, and plottedusing an IBM XY 749 plotter.For scanning electron microscopy (SEM), samples of

activated-carbon particles were removed from the reactorcolumn, washed gently with 0.1 N sulfuric acid, and fixedwith 2.5% glutaraldehyde for 1 h at pH 7 followed by OS04fixation for 1 h. The samples were then dehydrated andcritical-point dried before being mounted on specimen stubswith a silver paint. After being gold coated, the samples wereexamined under a scanning electron microscope (HitachiS-500) at 20 kV.Two different instruments were used for X-ray microanal-

ysis of the fixed biofilm on activated-carbon particles. Forqualitative analysis, samples were washed, air dried, placedin specimen holders, and analyzed under air without a filterat 10 kV with a Tracor Spectrace Model 4050 X-ray ana-lyzer. For quantitative analysis, samples were prepared asfor SEM, except that carbon stubs and carbon coating wereused. The samples were analyzed with an EDAX 9100WX-ray system at 20 kV attached to a JEOL JSM-35Ascanning electron microscope. Low-atomic-number (<10)elements were excluded due to the method used by instru-ment.

02e,Cu KcaFIG. 1. X-ray diffraction spectra for pooled effluent (sample 1) (A)

and reactor (sample 2) (B). Numerical values are in Angstroms(lA=0.1 nm).

RESULTS AND DISCUSSIONPooled effluent sample. The color of the dried, pooled

effluent sample (no. 1) was yellow (5Y 8/6) and was withinthe range of pale to straw yellow colors (2.5-SY 8/3-8/6)commonly reported for natural jarosites (15). The X-rayprofile for the specimen (Fig. 1A) was well defined andcontained all lines considered diagnostic for basic ferricsulfates. The symmetric, well-resolved character of thediffraction lines and the low background counts furtherindicate that the precipitate was well crystallized (despite avery fine grain size) and free of significant amounts ofcoprecipitated compounds such as iron oxyhydroxides (e.g.,ferrihydrite or feroxyhite). The purity of the specimen alsosupports the results of earlier abiotic synthesis experiments(3), which indicate that jarosite-type compounds form di-rectly from solution without a precursor solid phase. TheX-ray diffraction spectra in Fig. 1 displayed no unique linesthat could be specifically attributed to the biological natureof the samples. It should be noted that abiotic synthesis ofjarosite-type compounds from ferrous sulfate at pHs of 1.35to 1.5 is not possible due to the extremely slow rate of ironoxidation in the absence of a catalyst.On the basis of equilibrium stability calculations, Van

Breemen (15) concluded that natural jarosites should onlyform in oxidized environments (Eh >400 mV) with pHs inthe range of 2 to 4. The extremely low pH maintained in thepresent study therefore represents a somewhat anomalouscondition for the formation of potassium jarosite and raisesthe question of whether the precipitate is a derivativecompound, such as hydronium jarosite. The occurrence ofboth K and NH4 salts in the reaction medium also invites thepossibility of a mixed jarosite.

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JAROSITE FORMATION IN A BIOREACTOR 3103

TABLE 1. Inductively coupled plasma emission spectroscopydata on the elemental composition of the bulk jarosite samples

used for X-ray diffraction

Concentration (ppm)Element"

Sample 1 Sample 2

Fe 281,242 209,732K 61,226 47,149P 2,662 3,189Na 1,611 663Ca 404 337Mg 258 291Zn 246 46Pb 223 135Al 188 131B 122 70Sn 62 64Mo 40 26Cu 26 23Ba 21 33a Mn, Be, Sr, Cd, V, and Ti were present at <20 ppm.

Numerous investigators have demonstrated that a solidsolution series exists among jarosites by virtue of substitu-tions occurring in the monovalent cation site, especiallybetween K+, Na+, NH4' and H30O (2, 7). The (003) X-raydiffraction line for the basic ferric sulfates is sensitive to thetype of cation and shifts progressively towards larger valueswith increasing ionic radii of the substituting cations. Thus,the reported spacings for the (003) lines of hydronium-,natro-, potassium-, and ammonio-jarosites are 0.554, 0.557,0.572, and 0.580 nm, respectively (12). The observed spacingfor the dOO3 line from the pooled-effluent precipitate is 0.568nm and is indicative of a potassium jarosite with perhapssome replacement of K+ by Na+ or H30O. Despite theabundance ofNH4+ in the reaction medium and the reportedpreference for NH4' over Na+ and H30 (7), there is no

X-ray evidence for NH4 substitution in the mineral struc-ture. The spacings of the doublet produced by the intensed021 and d113 lines at 0.38 and 0.311 nm, respectively, arealso consistent with the presence of potassium jarosite.Brown (3) obtained potassium jarosites from solutions withaK+/aH+ as low as 0.3 and concluded that a very strongpreference exists for K+ over H30+ under ambient condi-tions. In the present study aK+/aH+ is approximately 0.1. Ourresults therefore support the findings of Shiskin (13), whoreported that hydronium jarosite can be synthesized only inthe absence of alkali in the temperature range of 25 to1700C.

Inductively coupled plasma emission spectroscopy results(Table 1) confirm that Fe and K are the main cationicconstituents in the pooled effluent sample, but both elementsare below the stoichiometric levels for potassium jarosite.Theoretically, jarosite with the formula KFe3(SO4)2(OH)6contains 78,000 ppm (7.8% [wt/wt]) of K and 334,000 ppm(33.4% [wt/wt]) of Fe. The amounts of K and Fe in sample 1(Table 1) were 78 and 84% of the theoretical level, respec-tively. The observed K:Fe ratio is 0.22, compared with thetheoretical ratio of 0.23.

Reactor sample. The precipitate isolated from the packed-bed reactor after 3 months of operation (sample 2) was

markedly different in appearance from the pooled effluentspecimen due to a distinctive greenish-gray (SGY 6/1) color.Despite its unusual pigmentation, this specimen yielded an

X-ray diffraction profile (Fig. 1B) and a chemical analysis(Table 1) virtually identical to that of the pooled effluent

8 10

keVFIG. 2. Elemental composition of activated-carbon particles re-

moved from the packed-bed reactor. The sample was analyzed byenergy-dispersive X-ray analysis before (upper curve) and after(lower curve) being washed with 0.1 N sulfuric acid.

precipitate. While the reason for the observed color varia-tion is uncertain, a reasonable hypothesis might be that theFe2'-rich environment in the reactor may have inducedsome substitution of Fe2+ for Fe3" in the jarosite structure.Kubisz and Zabinski (9) reported the presence of up to 0.3%Fe2+ in some natural jarosites, and one of the authors(J.M.B.) has observed jarosites with similar coloration inprecipitates from acid mine waters.Energy dispersive X-ray analysis of activated-carbon par-

ticles collected from the exit port yielded S, K, and Fe as

major elements (Fig. 2). The presence of an Ar peak in Fig.2 is a result of sample analysis under air. The unlabeled peakbetween S and Ar is ascribed to Cl (Ka, 2.5 keV) but cannotbe verified with certainty because of the presence of Ar (K,,,3.0 keV; K., 3.2 keV), which masks the 2.8-keV K, peak ofCl. The peaks for potassium are 3.3 keV (K,,,) and 3.6 keV(KP).Over the 12-week period of operation, 50 to 99% of the 120

mM influent FeSO4 was oxidized in the bioreactor, depend-ing on the flow rate. The effluent zone in the bioreactor wascharacterized by turbulent solution flow and high Fe3'/Fe2+ratios. The middle section was characterized by less turbu-lence and Fe3'/Fe2+ ratios which are intermediate betweenthe influent and effluent values. No effort was made toquantitatively describe turbulence and redox conditions dur-ing the bioreactor operation. SEM of the precipitate formedon activated carbon revealed a varied morphology (Fig. 3),depending upon location within the bioreactor. Samplestaken near the effluent zone of the bioreactor after 12 weeksof operation contained aggregates and bacterial cells as wellas precipitates that had a regular pattern in shape (Fig. 3A).These precipitates had a distinct rosette morphology (Fig.3B). In contrast, jarosites formed under natural conditionsare usually reported to have a pseudocubic morphology (16).Specimens secured from the middle section of the bioreactorexhibited a rounded or botryoidal form (Fig. 3C and D).Similar morphological features were observed by Lazaroff etal. (10) for potassium and rubidium jarosites formed from

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3104 GRISHIN ET AL.

FIG. 3. SEM of precipitates on activated-carbon particles collected from the packed-bed reactor. The specimens were gold coated beforeSEM examination. (A and B) Samples removed near the effluent zone. Bars, 5 ,um, (A) and 1 ,um (B). (C and D) Samples removed from themiddle section. Bars, 5 ,um each.

bacterially oxidized FeSO4 solutions. Samples shown in Fig.3C and D were separated by an interval of about 7 weeks.After 5 weeks of bioreactor operation, discrete crystals witha regular, smooth, and half-round appearance occurred inassociation with bacterial cells and activated-carbon surface(Fig. 3C). After 12 weeks of operation, a layer or crust ofprecipitate had formed on the particle surface (Fig. 3D).

Despite the massive character of the crust, individual crys-tals were still visible with a rounded shape but smaller in sizecompared with those in Fig. 3C.To confirm that the precipitate was jarosite, a carbon-

coated specimen (Fig. 4) was analyzed by using energy-dispersive X-ray analysis. The composition of both individ-ual crystals (samples 1 and 2 in Table 2) on the same stub and

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JAROSITE FORMATION IN A BIOREACTOR 3105

FIG. 4. SEM of precipitates on activated-carbon particles col-lected from the packed-bed reactor after 11 weeks of operation. Thespecimen was carbon coated before SEM examination. Bar, 5 ,um.Energy-dispersive X-ray analysis data on this specimen are pre-sented in Table 2.

the entire specimen (sample 3 in Table 2) were determined.In all cases, S, Fe, and K were the major elements (Table 2),and their relative concentrations were compatible with ajarosite formula which had a K:Fe:S ratio of 0.9:2.3:2.3instead of the theoretical one of 1:3:2. Thus, the precipitatedeviated from known jarosite composition by having excessS and a deficiency of K and, especially, Fe. Sample 1additionally contained 11.5% Na, but it was deemed to resultfrom contamination because Na was not detected in the twoother samples.

Concluding remarks. The precipitates analyzed in thiswork were collected from a low-pH (1.35 to 1.5) reactorsystem in which no visible turbidity was evident in the finaleffluent. The precipitates consisted of well-ordered potas-sium jarosite with distinct, sharp X-ray diffraction peaks.Fe(III) P04 was also present, based on the relative concen-tration of P in the samples. Cu and Zn were essentially

TABLE 2. Elemental composition of reactor precipitates andcomparison with that of potassium jarosite [KFe3(SO4)2(OH)6]a

Weigh.composTheoretical compositionElement in potassium Jarosite

Sample 1 Sample 2 Sample 3 Weight (%) Atomic (%)

P 2.38 2.46 2.94S 25.87 25.65 26.94 23.7 33.3K 11.49 14.65 12.82 14.4 16.7Fe 42.08 52.62 48.37 62.0 50.0Cu 4.28 0.62 5.81Zn 2.34 NDa 3.11a The specimen was carbon coated and is shown as an SEM micrograph in

Fig. 4.b ND, Not detected.

impurities in the bioreactor system, originating from the useof ferrous sulfate as the substrate. Only trace amounts ofsecondary iron precipitates were produced in the bioreactorover a 3-month operating period; however, SEM observa-tions demonstrated that crusting had developed on theactivated carbon matrix by the end of the experiment. Whileno decrease in efficiency of the reactor was observed overthe time course of the study, accumulations of jarosite-typeprecipitate could eventually lead to plugging and reducedactivity of similar reactors operated on either a laboratory orcommercial scale. Our results indicate that potassium jaro-site can form even at aK,/aH+ ratios as low as 0.1. Eliminat-ing or reducing the amount of K in nutrient solutionsemployed with low-pH bioreactors might further reduce oreliminate undesirable secondary Fe precipitates.

ACKNOWLEDGMENTS

We thank the International Research and Exchange Board,Princeton, New Jersey, for partial support of the work. Salary andresearch support to J.M.B. were provided by state and federal fundsappropriated to the Ohio Agricultural Research and DevelopmentCenter, The Ohio State University.We thank Deanna Anderson, John Hanson, Clare McDonald,

Robert Pfister, and David Stutes for technical advice and assistancein microanalytical and SEM techniques, and Laurie Haldeman fortyping the manuscript. The inductively coupled plasma emissionspectroscopy data were obtained through the Research and Exten-sion Analytical Laboratory, Ohio Agricultural Research and Devel-opment Center, The Ohio State University.

LITERATURE CITED

1. Brady, K. S., J. M. Bigham, W. R. Jaynes, and T. J. Logan.1986. Influence of sulfate on Fe-oxide formation: comparisonswith a stream receiving acid mine drainage. Clays Clay Miner.34:266-274.

2. Brophy, G. P., and M. F. Sheridan. 1965. Sulfate studies IV: thejarosite-natrojarosite-hydronium jarosite solid solution series.Am. Mineral. 50:1595-1607.

3. Brown, J. B. 1970. A chemical study of some synthetic potas-sium-hydronium jarosites. Can. Mineral. 10:696-703.

4. Brown, J. B. 1971. Jarosite-goethite stabilities at 25°C, 1 atm.Mineral. Depos. 6:245-252.

5. Dutrizac, J. E. 1984. The behavior of impurities during jarositeprecipitation, p. 125-169. In R. G. Bautista (ed.), Hydrometal-lurgical process fundamentals. Plenum Publishing Corp., NewYork.

6. Ivarson, K. C. 1973. Microbiological formation of basic ferricsulfates. Can. J. Soil Sci. 53:315-323.

7. Ivarson, K. C., G. J. Ross, and N. M. Miles. 1979. Themicrobiological formation of basic ferric sulfates. II. Crystalli-zation in the presence of potassium-, ammonium-, and sodium-salts. Soil Sci. Soc. Amer. J. 43:908-912.

8. Ivarson, K. C., G. J. Ross, and N. M. Miles. 1982. Microbiolog-ical transformations of iron and sulfur and their applications toacid sulfates soils and tidal marshes, p. 57-75. In J. A. Kittrick,D. S. Fanning, and L. R. Hossner (ed.), Acid sulfate weather-ing. Soil Science Society of America, Madison, Wis.

9. Kubisz, J., and W. Zabinski. 1958. The jarosites from theSilesia-Cracow zinc and lead ore deposits. Bull. Acad. Pol. Sci.Ser. Sci. Chim. Geol. Geogr. 6:793-797.

10. Lazaroff, N., L. Melanson, E. Lewis, N. Santoro, and C. Pues-chel. 1985. Scanning electron microscopy and infrared spectros-copy of iron sediments formed by Thiobacillus ferrooxidans.Geomicrobiol. J. 4:231-268.

11. Lazaroff, N., W. Sigal, and A. Wasserman. 1982. Iron oxidationand precipitation of ferric hydroxysulfates by resting Thioba-cillus ferrooxidans cells. Appl. Environ. Microbiol. 43:924-938.

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12. Ross, G. J., K. D. Ivarson, and N. M. Miles. 1982. Microbialformation of basic ferric sulfates in laboratory systems and insoils, p. 77-94. In J. A. Kittrick, D. S. Fanning, and L. R.Hossner (ed.), Acid sulfate weathering. Soil Science Society ofAmerica, Madison, Wis.

13. Shiskin, N. V. 1951. The oxonium ion in the crystal lattice ofinorganic compounds. J. Gen. Chem. 21:456-457.

14. Toro, L., B. Paponetti, and C. Cantalini. 1988. Precipitateformation in the oxidation of ferrous ions in the presence ofThiobacillus ferrooxidans. Hydrometallurgy 20:1-9.

15. Van Breemen, N. 1982. Genesis, morphology and classificationof acid sulfate soils in coastal plains, p. 95-108. In J. A. Kittrick,D. S. Fanning, and L. R. Hossner (ed.), Acid sulfate weather-ing. Soil Science Society of America, Madison, Wis.

16. Wagner, D. P., D. S. Fanning, J. E. Foss, M. S. Patterson, andP. A. Snow. 1982. Morphological and mineralogical featuresrelated to sulfide oxidation under natural and disturbed landsurfaces in Maryland, p. 109-125. In J. A. Kittrick, D. S.Fanning, and L. R. Hossner (ed.), Acid sulfate weathering. SoilScience Society of America, Madison, Wis.

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