Solubilization and Speciation of Iron during Pyrite

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Solubilization and Speciation of Iron during Pyrite Oxidation by Thiobacillus ferrooxidans Antti Vuorinen1, Paula Hiltunen2, Jason C. Hsu3, and Olli H. Tuovinen4* 1 Department of Geology, University of Helsinki, Helsinki, SF-00170, Finland. 2 Department of Microbiology, University of Helsinki, Helsinki, SF-00170, Finland. 3 Department of Statistics, The Ohio State University, Columbus, Ohio. 4 Department of Microbiology, The Ohio State University, Columbus, Ohio. Abstract Various species of soluble iron in pyrite-grown cultures of Thiobacillus ferrooxidans were determined by colorimetry, atomic absorption spectrometry, and ultraviolet spectroscopy. All the cultures were incubated for six weeks before iron analysis. The effects of the following factors were investigated: particle size, initial pH, shaking (aeration), concentration of pyrite, and concentration of yeast extract. Shaking, but not initial pH nor particle size, influenced the relative proportion of different iron species. Polynomial regressions could be used to describe the functional relationship between the different iron species and concentration of pyrite; fewer relationships were ev* To whom reprint requests should be addressed. Received June 10, 1982; revised version accepted September 23, 1982.

Geomicrobiology Journal, Volume 3, Number 2 0149-0451/83/020095-00$02.00/0 Copyright © 1983 Crane, Russak & Company, Inc.

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Vuorinen, Hiltunen, Hsu, and Tuovinen ident with respect to concentration of yeast extract. The variance-covariance matrices indicated a linear dependence among the different iron species. Canonical correlations indicated perfect correlations between group variables of iron, copper, and zinc, with the exception of an absence of significant correlation with the hydroxy complex of iron (FeOH2+). The dissolved ferrous iron (dissociated and weakly chelated) always remained less than 7% of the total iron in solution. The total ferrous iron, which included complexed species, amounted to 7-34% of the total iron in solution. The concentrations of dissociated ferrous and ferric iron and their weak chelates (the dissolved iron) remained mostly constant, irrespective of the concentration of the total iron in solution. Most of the total iron was complexed as ferric species and the amount correlated with culture conditions. The hydroxy complex (FeOH2+), which was indicative of the relative amount of hydrolyzable ferric iron upon dilution in CO2-free water, usually ranged between 60 and 80% of the total iron. The amount of the total iron in uninoculated controls was less than 12% of that solubilized in the presence of iron-oxidizing thiobacilli. T. ferrooxidans was enumerated by a most-probable-number technique after three and six weeks of growth on pyrite. The counts after three weeks indicated an increase in the number of free and loosely attached bacteria, followed by a decline of about one order of magnitude in bacterial numbers after six weeks. The technique for bacterial enumeration was deemed unsatisfactory because it could not account for cells attached on pyrite.

Introduction The bacterial oxidation of iron pyrite can be described with the following sequence of reactions generating acidity and ferric iron: FeS2 Fe2+ FeS2 S +

+ 3.5 O2 + H2O -* Fe2* + 2H+ + 2SO42+ 0.25 O2 + H + -> Fe3+ + 0.5 H2O + 2Fe3+ -»• 3Fe2+ + 2S 1.5 O2 + H2O ~* 2H + + SO42"

The chemical solubilization of pyrite is relatively slow compared with the oxidation rates obtained in the presence of acidophilic


Pyrite Oxidation by T. ferrooxidans

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iron- and sulfur-oxidizing thiobacilli (Hoffmann et al., 1981). Ferrous iron and free sulfur may be detected in the absence of bacteria. In bacterial culture solutions they are present at low concentrations, since both elements are biologically oxidized to ferric iron and sulfuric acid, respectively (Norris and Kelly, 1978; Arkesteyn, 1979). The reaction sequence implies that Fe2"1" and Fe3+ dissociate upon pyrite solubilization. Ferric iron is partly hydrolyzed abiotically with increasing pH values (Sylva, 1972; Byrne and Kester, 1976): Fe3+ + H2O -*• Fe(OH)2+ + H + Fe3+ + 2H2O -> Fe(OH)+ + 2H+ Fe3+ + 3H2O ~> Fe(OH)3° + 3H+ In more concentrated iron solutions a dimerized species predominates (Dutrizac, 1979): 2Fe3+ + 2H2O -»• Fe2(OH)24+ + 2H+ Hydrolysis products, if precipitated, can transform to iron oxyhydroxides and various jarosite-type compounds with anionic components, which are able to form complexes, such as FeSO4+ and Fe(OH)SO4 with soluble iron (Yakhontova et al., 1980): FeSCV + H2O -> Fe(OH)SO4 + H + In addition, bacterial culture solutions contain other anionic components and some organic compounds that have the capacity to form complexes with soluble iron. Total soluble iron, pH, and the formation of sulfate have been used as a measure of bacterially catalyzed oxidation of pyrite, which can be summarized by the equation: 2 FeS2 + 7.5 O2 + H2O -» Fe2(SO4)3 + H2SO4 Moreover, growth of the bacteria with pyrite has been estimated by chemical determinations, according to the following relationship (Hoffmann et al., 1981):

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Vuorinen, Hiltunen, Hsu, and Tuovinen


dxldt = Yd[FeS2)ldt where x is the amount of biomass and Y is the cell-yield coefficient. Both ferrous and ferric species (Felll, Fell) prevail in acid leach solutions (Bhappu et al., 1969; Schlitt and Jackson, 1981). In a previous study, the analysis of soluble iron by different methods indicated that iron in solution has a tendency to form chelated or complexed species in Thiobacillus ferrooxidans cultures growing on pyrite (Hiltunen et al., 1981). The present communication extends these observations by determining the relative concentrations of iron ionic species by colorimetry, atomic absorption spectrometry, and ultraviolet spectroscopy. In support of the experimental results, the effects of growth conditions on the relative concentrations of iron species are analyzed statistically.

Materials and Methods Bacteria A strain of Thiobacillus ferrooxidans (Hiltunen et al., 1981) previously grown with ferrous sulfate was used in all experiments. Cultures (100 ml) were grown in 250-ml conical flasks on ?. gyratory shaker (180 rpm) at 28°C in the dark. Unless otherwise indicated, the pyrite culture medium contained (per liter): pyrite, 10 g; K2HPO4, 0.4 g; (NH4)2SO4, 0.4 g; MgSO4.7H2O, 0.4 g; yeast extract (Difco), 0.2 g; pH 2 adjusted with sulfuric acid. The medium with pyrite was sterilized by autoclaving. The inoculum consisted of 10 ml of actively growing bacterial cultures per 100 ml of medium. A most-probable-number (MPN) technique was employed for bacterial enumeration with five replicate tubes of each tenfold dilution. For MPN enumeration, both static and shake flasks were shaken manually and the pyrite suspended in the medium was allowed to settle for about 20 min before the solution phase was sampled. The MPN medium contained (per liter of 0.005 N H2SO4): FeSO4.7H2O, 33.3 g; K2HPO4, 0.4 g; (NH4)2SO4, 0.4 g;



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MgSO4.7H2O, 0.4 g. The ferrous sulfate and the minimal salts were sterilized separately by membrane filtration and autoclaving, respectively. The same medium was also used for subcultures of T. ferrooxidans on ferrous iron with the exception that the pH was adjusted to pH 1.5 by using 0.11 N H2SO4 for the mineral salts solution.

Pyrite The pyrite sample was received from the Pyhasalmi sulfide ore deposit (Finland) and contained over 90% FeS2 (46% Fe, 50.6% S) and small amounts of sphalerite (ZnS) and chalcopyrite (CuFeS2) in the matrix. The sample was finely ground and sieved to the following size fractions: +100, -100, +200, -200, +325, and -325 mesh (particle diameter (0), 0>174 ^m, 74 0.894; Table 2). Fed2+, Fed, and Fed3+ appeared to be quadratic functions of concentration of pyrite (Table 2). A cubic polynomial of concentration of pyrite appeared to best describe FeT2+ (R2 = 0.967).

Yeast Extract In the presence of increasing concentrations of yeast extract, the level of total ferrous iron (FeT2+) increased while the concentrations of dissolved ferrous iron (Fed2+) gradually decreased (Table 4). This indicated a tendency of ferrous iron to become increasingly complexed in the presence of yeast extract. The dissolved iron (Fed) concentration remained constant. The total iron (FeT) concentration decreased with increasing amounts of yeast extract present in the solution (Table 4), indicating an inhibition of bacterial pyrite solubilization in the presence of >0.05% yeast extract. A similar trend was observed with the ferric iron species with the exception of dissolved ferric iron (Fed3+),


TO

1 Table 1 Influence of Pyrite (Substrate) Concentration on Iron Solubilization and Speciation in T. ferrooxidans Culture Solutions.3 Concentration of pyrite g/liter

Fe2d+

fi-complex

Fef

Fed

Fe3/

2 4 6 8 10 15 20

16 11 25 29 34 113 374

106 102 172 135 155 88 108

122 113 197 164 189 201 482

455 459 482 470 463 415 351

439 448 457 441 429 302 0

a

~3 3"

Species of iron (mg/L) Fe complex

741 352 1980 2740 2780 3680 4930

Fef+

Fec

FeT

1180 890 1240 740 910 800 2440 1980 2640 3180 2430 3340 3210 2900 3400 3980 4120 4180 4930 5180 5410

FeOH2+ 1080 1380 1750 2000 2280 2830 3290

Notations of iron species: Fe^+ = dissolved ferrous iron; Fe^ mplex = complexed ferrous iron; Fey+ = total ferrous iron; Fed + dissolved iron; Fe\+ = dissolved ferric iron; F e l ^ , ^ = complexed ferric iron; Fe^1" = total ferric iron; F e c = complexed and dissolved iron; FeT = total iron; FeOH 2+ = hydroxy complex of ferric iron.

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Table 2

Regression Analysis of the Concentration of Pyrite.


Variable"^ Fed+ Fe T +

Fed Fec Fe T FeOH 2 + FeT+ Fe d + r c

complex

Znc Zn T Cu T

Linear

Quadratic

Cubic

0.7666 0.7425 0.7244 0.9750 0.9077 0.9859 0.8937 0.7679 0.9239 0.9917 0.9855 0.9883

0.9742 0.8724 0.9796 0.9784 0.9278 0.9992 0.9268 0.9907 0.9373 0.9920 0.9877 0.9970

0.9985 0.9667 0.9874 0.9794 0.9324 0.9994 0.9290 0.9996 0.9400 0.9922 0.9885 0.9977

a

The notations of iron species are defined in the footnote to Table 1. b Z n c = complexed and dissolved zinc; Zn T = total zinc; Cu T = total copper.

Table 3

Regression Analysis of the Concentration of Pyrite: Functions of Best Fit. Coefficients for T^

j

L

9\** n

Variable"-" (y) 2+

Fe Fe T + Fe d Fec Fe T FeOH 2 + Fe T + Fed+ Fp3+ . rc complex

Znc

Zn T Cu T

h

Intercept

X

X*

x3

-12.01 14.52 425.07 223.44 -181.68 695.97 -196.20 437.08 -633.28 -1.34 2.67 2.15

15.99 57.40 16.36 219.94 582.32 195.64 524.91 0.37 524.54 2.61 3.73 0.63

-2.48 -6.65 -1.52 8.06 -29.02 -4.28 -22.37 0.96 -23.33 0.08 -0.09 0.10

0.13 0.25 0.03 -0.34 0.69 0.05 0.44 -0.11 0.54 -0.002 0.004 -0.002

"The notations of iron species are defined in the footnote to Table 1. b The notations of zinc and copper are defined in the footnote to Table 2.


Table 4 Effect of Yeast extract on Iron Solubilization and Speciation in T. ferrooxidans Culture Solutions."

0 0.1 0.2 0.5 1.0 1.5 2.0 2.5 a

?

Species of iron (mg/liter)

Yeast extract (g/liter)

§.

5" 5

Fed+ 85 85 42 37 84 27 30 30

2+ Fecomplex ,

Fef

58 154 191 267 272 373 364 536

143 239 233 304 356 400 394 566

1 c

d

Fe3/

490 500 490 485 498 496 496 492

405 415 448 447 414 469 466 462

Fe

rK

complex

Fe3T+

Fec

FeT

FeOH*+

3270 3640 3000 2900 2340 1720 1450 1090

3240 3390 2930 2930 2470 2360 1930 1760

3210 3880 3230 3200 2700 2120 1840 1660

2410 2560 2170 2070 1690 1690 1330 1340

2870 3230 2550 2450 1930 1250 984 628

The notations of iron species are defined in the footnote to Table 1.

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which showed little change at various levels of yeast extract. Total ferrous iron (FeT2+) appeared to be a cubic polynomial of the amount of yeast extract in the culture medium (Tables 5 and 6). No functional relationship between Fed, Fed2+, Fed3+, and the amount of yeast extract was discerned (R2 < 0.523). Fec, FeT, Fe3+complex, and FeT3+ could be expressed as linear functions of yeast extract (R2 > 0.937), with little improvement by using quadratic or cubic polynomial models (Tables 5 and 6). FeOH2+ was a quadratic function of yeast extract (R2 = 0.944). No functional relationship between zinc, copper, and yeast extract was indicated (Table 5).

Table 5 Regression Analysis of the Concentration of Yeast Extract.

Variable"'11

Linear

Quadratic

Cubic

Fe d + Fe T + Fe d Fec

0.4101 0.8975 0.0354 0.9541 0.9369 0.9006 0.9596 0.5038 0.9581 0.0938 0.1527 0.4088

0.4192 0.8980 0.0773 0.9606 0.9469 0.9437 0.9678 0.5234 0.9667 0.3054 0.2995 0.5338

0.4251 0.9746 0.2368 0.9612 0.9534 0.9449 0.9688 0.5234 0.9676 0.6049 0.7514 0.5361

FeT

FeOH 2 + Fe T + Fe d + Fe|^mp]H

Znc Zn T Cu T

"The notations of iron species are defined in the footnote to Table 1. b The notations of zinc and copper are defined in the footnote to Table 2.


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Table 6 Regression Analysis of the Concentration of Yeast Extract: Functions of Best Fit. Coefficients for


a b

Variable -

3+ - complex

Fec

FeT

FeOH2+

903

500 409

I' iis I

I

The notations of iron species are defined in the footnote to Table 1.

I



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colorimetric analysis of the total ferrous iron (FeT2+) since at that level of acidity the treatment preserved the divalent state of ferrous iron. In atomic absorption spectrometry, hot acid-digested iron can be considered to fully atomize in an air-acetylene flame. Diluted undigested samples represented dissociated iron and weak chelates as well as complexed forms, which atomize in the air-acetylene flame. The difference between the total iron (FeT) and Fe c is due to iron polymerization, often in colloidal series resisting the atomization effect of the air-acetylene flame without a prior hot-acid digestion. The data indicated that polymerized iron compounds such as iron colloids were present only in small quantities. The relative amount of hydrolyzed iron (FeOH2+) is probably indicative of the tendency of iron to react in the solution as the pH increases (Byrne and Kester, 1976). With strongly complexed iron species, much less hydrolyzed ferric iron would have been detectable since strong complexes can effectively resist the hydrolysis. In previous work (Hiltunen et al., 1981), the data did not allow differentiation between organic and inorganic iron complexes. The present data clearly demonstrate that an increase in the amount of organic substances (yeast extract) does not increase the relative concentration of complexed ferric iron. Instead, the ferrous iron was increasingly complexed with yeast extract in the medium. Theis and Singer (1973) previously demonstrated that organic substances can stabilize ferrous iron through the formation of Fe(II)organic complexes in aqueous solutions. Moreover, the effect of pH on the pattern of iron solubilization in the bacterial cultures suggested that inorganic iron species were formed. This is because it is highly unlikely that organic substances secreted by the bacteria could account for complexation of such large amounts of ferric iron. Inorganic chelates can be formed from sulfate and phosphate ions, which were both medium constituents. The pK values of the stability constants for sulfate complexes have been reported (Krajnov and Shvets, 1980): FeSO4° = Fe2+ + SO42FeSO/ = Fe3+ + SO42-

pK = 2.3 pK = 4.2


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The sulfate concentration, though not analyzed, should have increased in parallel with the soluble iron, since, owing to the action of thiobacilli, pyrite solubilization generates sulfuric acid. The tendency of iron to form complex species was also indicated by the level of dissolved iron (Fed) which did not increase in parallel with other iron species. The trend of iron solubilization in the static cultures (initial pH, particle size) indicated slow oxidation of pyrite. In parallel shake flask experiments, the solubilization of iron was enhanced, indicating that the pyrite oxidation was probably under diffusion control by oxygen and carbon dioxide as previously discussed by Myerson (1981). Total soluble iron is commonly used as a measure of bacterial oxidation of pyritic material. The present work shows that a pattern of iron speciation can be determined. The data indicate that the chemical speciation of iron can be manipulated by changes in pH and solution composition. The viable counts of T. ferrooxidans were deemed imprecise because of cell sorption to the solids, but overall trends were observed after three and six weeks of incubation. Cell sorption and detachment remain to be quantitatively determined before bacterial enumeration can be reliably used for monitoring purposes. The statistical analysis of the data indicate that predictions can be made on the formation of various iron species. Linear dependence among iron species was indicated by singular variance-covariance matrices and was resolved by analysis of canonical correlations of group variables. This suggests that in future analyses of relationships among the variables, it may be worthwhile to examine combinations of variables. Since ferric iron is an important oxidant and precipitant in extractive metallurgy, further work is warranted to elucidate the composition, kinetics, and thermodynamics of iron species in synthetic and commercial leach solutions.

Acknowledgments The work was funded under a research contract with the Ministry of Trade and Industry, Finland (O.H.T., P.H.). Partial support


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(O.H.T.) was also received from the Ohio Coal Research Laboratories Association (contract OCRLA-6) and the Office of Water Research and Technology (grant No. 14-34-0001-2137). J.C.H. was supported in part by the National Cancer Institute, DHEW (grant No. 1 RO2 CA26254-03). References Arkesteyn, G. J. M. W. 1979. Pyrite oxidation by Thiobacillus ferrooxidans with special reference to the sulphur moiety of the mineral. Antonie van Leeuwenhoek. 45:423-435. Bhappu, R. B., P. H. Johnson, J. A. Brierley, and D. H. Reynolds. 1969. Theoretical and practical studies on dump leaching. Trans. Soc. Min. Engin. AIME. 244:307-320. Brown, M., and D. R. Kester. 1980. Ultraviolet spectroscopic study of ferric iron solutions. Appl. Spectrosc. 34:377-380. Byrne, R. H., and D. R. Kester. 1976. Solubility of hydrous ferric oxide and iron speciation in seawater. Mar. Chem. 4:255-274. Dutrizac, J. E. 1979. The physical chemistry of iron precipitation in the zinc industry, p. 532-564. In J. M. Cigan, T. S. Mackey, and T. J. O'Keefe (eds.), Lead-Zinc-Tin '80. AIME, Warrendale, Pennsylvania. Heaney, S. I., and W. Davison. 1977. The determination of ferrous iron in natural waters with 2,2'-bipyridyl. Limnol. Oceanogr. 22:753-760. Hiltunen, P., A. Vuorinen, P. Rehtijärvi, and O. H. Tuovinen. 1981. Bacterial pyrite oxidation: release of iron and scanning electron microscopic observations. Hydrometallurgy. 7:147-157. Hoffmann, M. R., B. C. Faust, F. A. Panda, H. H. Koo, and H. M. Tsuchiya. 1981. Kinetics of the removal of iron pyrite from coal by microbial catalysis. Appl. Environ. Microbiol. 42:259-271. Irving, H., and D. H. Mellor. 1962. The stability of metal complexes of 1,10phenanthroline and its analogues. Part 1. 1, 10-Phenanthroline and 2, 2'bipyridyl. J. Chem. Soc. pp. 5222-5237. Krajnov, S. R., and V. M. Shvets. 1980. Osnovy Geokhimii Podzemnyh Vod. Nedra, Moskva. Myerson, A. S. 1981. Oxygen mass transfer requirements during the growth of Thiobacillusferrooxidans on iron pyrite. Biotechnol. Bioeng. 23:1413-1416. Norris, P. R., and D. P. Kelly. 1978. Dissolution of pyrite (FeS2) by pure and mixed cultures of some acidophilic bacteria. FEMS Microbiol. Lett. 4:143-146. Schlitt, W. J., and J. J. Jackson. 1981. In situ generation of acid during dump leach production of copper. In Situ. 5:103-131. Sylva, R. N. 1972. The hydrolysis of iron(III). Rev. Pure Appl. Chem.


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22:115-132. Theis, T. L., and P. C. Singer. 1973. The stabilization of ferrous iron by organic compounds in natural waters, pp. 303-320. In: P. C. Singer (ed.), Trace Metals and Metal-Organic Interactions in Natural Waters. Ann Arbor Science Publishers, Inc., Ann Arbor, Michigan. Yakhontova, L. K., L. G. Nesterovich, and A. P. Grudev. 1980. The bacterial oxidation of pyrite. Moscow Univ. Geol. Bull. 35:51-57 (English Translation).