Environ. Sci. Technol. 2002, 36, 16-20
Iron(II,III) Hydroxycarbonate Green Rust Formation and Stabilization from Lepidocrocite Bioreduction GEORGES ONA-NGUEMA, MUSTAPHA ABDELMOULA, F R EÄ D EÄ R I C J O R A N D , O M A R B E N A L I , † A N T O I N E G EÄ H I N , JEAN-CLAUDE BLOCK, AND J E A N - M A R I E R . G EÄ N I N * Laboratoire de Chimie Physique et Microbiologie pour l’Environnement (LCPME), UMR 7564 CNRS-Universite´ Henri Poincare´, Nancy 1, France, Equipe Microbiologie et Physique, Faculte´ de Pharmacie-UHP and De´partement Mate´riaux et Structures, ESSTIN-UHP, 405 Rue de Vandoeuvre, F-54600 Villers-le`s-Nancy, France
Bioreduction of the well-crystallized ferric oxyhydroxide γ-FeOOH lepidocrocite was investigated in batch cultures using Shewanella putrefaciens bacterium (strain CIP 8040) at initial pH 7.5 in bicarbonate buffer. The cultures were performed with formate as electron donor without phosphate, in the presence or absence of anthraquinone2,6-disulfonate (AQDS) as electron shuttle. During lepidocrocite reduction, the iron(II,III) hydroxycarbonate green rust GR(CO32-) was characterized by X-ray diffraction, transmission electron microscopy, and transmission Mo¨ ssbauer spectroscopy. The AQDS accelerated the kinetics of GR formation. GR was the major end product when bacterial reduction was not stopped by lack of electron donor, and between 55 and 86% of the iron from γ-FeOOH precipitated in GR(CO32-). However, when the bacterial reduction was stopped by freezing/thawing or the electron donor was exhausted, the large quantity of remaining lepidocrocite induced a transformation of GR into magnetite. This confirms that GR is metastable with respect to magnetite in the presence of γ-FeOOH.
Introduction Solubility, bioavailability, and toxicity of polyvalent elements, among which iron is the most abundant, strongly depend on their redox chemistry (1). In particular, very reactive iron(II,III) layered double hydroxysalt green rusts (GR) that are intermediate products during aqueous corrosion of iron (2, 3) are abundantly found in reductomorphic soils (4, 5). They can reduce pollutants in the laboratory, e.g., NO3- into NH4+ (6), Se(VI) into Se(IV) or Se(0) (7, 8), and Cr(VI) into Cr(III) (9). GR’s structure is constituted of [FeII(1-x)FeIIIx (OH)2]x+ positively charged layers, which alternate with [(x/n)An-‚ (mx/n)H2O]x- negatively charged interlayers comprising m water molecules per An- anion (5). Most GRs are prepared in the laboratory by controlled oxidation of Fe(OH)2 in the presence of anions, Cl-, SO42-, CO32-, ... (5). But the natural GR fougerite mineral (10), which has been proposed to * Corresponding author phone: +33 (0)3-83-91-63-00; fax: +33 (0)3-83-27-54-44; e-mail:
[email protected]. † On leave from De ´ partement de Chimie, Faculte´ des Sciences, Universite´ Ibn Tofail, Kenitra, Morocco. 16
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incorporate OH- ions that led to the formula [Fe(OH)(2+x)‚ (1-x)H2O] (5, 10), has never been synthesized in a laboratory; its formation pathway thus remains open to discussion. Dissimilatory iron-reducing bacteria (DIRB) reduce amorphous hydrous ferric oxide into magnetite, vivianite, and siderite depending on the medium (11, 12). In the presence of phosphorus, a ground mass of poorly differentiated material, which contained a minor fraction of hexagonalshaped crystallites, was claimed to exhibit diffraction maxima consistent with a GR-type compound mixed with wellidentified laths of vivianite (12). In the present study, large GR crystals were observed for the first time from biotic reduction of a most common well-crystallized ferric oxyhydroxide γ-FeOOH lepidocrocite by using Shewanella putrefaciens in anaerobic conditions. Phosphate was prohibited to avoid vivianite formation. X-ray diffraction (XRD), transmission electron microscopy (TEM), and transmission Mo¨ssbauer spectroscopy (TMS) characterized unambiguously the GR that formed. However, this GR was found to exist only in the presence of active bacteria and transformed into magnetite and siderite if the bacteria were inactivated as long as there remained a sufficient amount of lepidocrocite. This biotic stabilization would explain the existence of a GR in natural environments only in active biotic conditions. Finally, the mechanism of microbially influenced corrosion (MIC) where GRs have been observed as corrosion products of cast iron (3) and steels (13) must be revisited from the viewpoint of reducing common ordinary rusts with DIRB.
Experimental Methods Mineral Preparation and Microbial Culture. Lepidocrocite was prepared as follows: FeCl2‚4H2O (0.228 M) solution was mixed with NaOH (0.4 M) to precipitate Fe(OH)2. Suspension was aerated by magnetic stirring, ensuring homogeneous oxidation. Formed lepidocrocite was characterized by XRD, washed three times with sterile Milli-Q water to remove electrolytes, and added to the sterile medium to 80 or 300 mM Fe(III). HCl-extractable Fe(II) was obtained by placing 1 mL of suspension directly into 1 mL of 1 M HCl. After at least 1 h of contact, Fe(II) was determined by using the modified 1,10-phenanthroline method (14). S. putrefaciens is a Gram-negative mobile rod with obligatory respiration and facultative anaerobe, isolated from a wide variety of environment (15) and showing several strains with different properties (16). The strain S. putrefaciens CIP 80.40 (Collection Institut Pasteur, Paris, France) used was first isolated at the surface of tainted butter corresponding to the ATCC 8071 strain. It was cultivated aerobically in trypcase-soy broth (BioMe´rieux-51019) at 25 °C. Cells were harvested at late log phase by centrifugation (16000g at 20 °C for 10 min) and washed twice with sterile Milli-Q water. The final suspension was purged 15 min with sterilized N2 (Azote C, Air Liquide), and cells were enumerated by epifluorescence microscopy (17). Cell suspensions, reaching a final concentration of 109 to 8 × 109 cells mL-1, were added to a mineral medium, close to that of “experiment 1” in the Fredrickson et al. study (12), but neither organic buffer nor phosphate were added. Moreover, instead of lactate and hydrous ferric oxide, the culture contained sodium formate as electron donor (50 or 75 mM) and lepidocrocite as acceptor (80 to 300 mM). Composition of the basic medium was as follows: 27 mM NaHCO3, 22 mM NH4Cl, 1.5 mM NaCl, 1.2 mM KCl, 1.1 mM MgSO4‚7H2O, 0.71 mM NTA, 0.67 mM CaCl2, 0.27 mM MnSO4‚ H2O, 86 µM ZnCl2, 38 µM CoSO4‚7H2O, 32 µM FeSO4‚7H2O, 9.3 µM Na2MoO4‚2H2O, 9.1 µM NiCl2‚6H2O, 6.8 µM Na2WO4‚ 10.1021/es0020456 CCC: $22.00
2002 American Chemical Society Published on Web 11/28/2001
FIGURE 1. Bioreduction of various initial concentrations of lepidocrocite (80 and 300 mM) and formate (75 mM) in the presence of 100 µM AQDS and S. putrefaciens inoculum of 8 × 109 cells mL-1. 2H2O, 3.6 µM CuSO4‚5H2O, 1.5 µM H3BO3, 1.9 µM AlK(SO4)2‚ 12H2O, in the absence or presence of 100 µM AQDS (ACROS, 10495-1000) depending on the assays. Formate and bicarbonate in solution were quantified by using capillary electrophoresis (Waters Capillary Ion Analyser) under the following conditions: fused silica capillary, 60 cm total length (52 cm to the detector) and 75 µm i.d.; electrolyte, 4.6 mM chromate containing 0.5 mM Waters OFM-OH at pH 8.0; 25 °C; 20 kV (negative polarity); hydrostatic injection for 30 s; indirect detection UV at 254 nm. Samples were injected after filtration (0.22 µm) and quantified from external calibration. Product Characterization. Samples for TMS and XRD analyses were extracted by filtration (0.45 µm) of 10 mL of slurry. They were prepared for TMS under N2 atmosphere in a glovebox and quickly transferred to the cryostat under inert He atmosphere before measuring at 77 or 12 K. TMS was performed from 12 to 295 K with a closed helium cycle variable temperature Mo¨ssbauer cryostat equipped with vibration isolation stand manufactured by Cryo Industries of America, utilizing a constant acceleration Mo¨ssbauer spectrometer with a 50 mCi source of 57Co in Rh and calibrated with a 25 µm thick R-iron foil at ambient temperature. Spectra were fitted using Lorentzian-shape lines, and parameters were mathematically (minimization of χ2) and physically significant. Mineral residues were also coated with glycerol under anaerobic conditions, and the wet paste was spread out on a glass plate before Bragg-Brentano XRD analysis at ambient using Co KR1 radiation (λ ) 0.17889 nm). A Philips CM 20 electron microscope operating at 200 kV, equipped with an energy-dispersive spectrometer, was used to observe the solid phases. A suspension was quickly dispersed in air on an amorphous carbon-coated grid and loaded into the analysis holder of the microscope. Products were identified from selected-area diffraction patterns and energy-dispersive analysis.
Results Microbial reduction of lepidocrocite was accompanied by the consumption of formate (Figure 1) and production of bicarbonate (not shown) when pH increased from 7.5 to 9.5. With an initial concentration of 8 × 109 cells mL-1 and 100 µM AQDS, the formate (75 mM initially) was completely removed from the solution when 300 mM γ-FeOOH was added to the culture whereas 135 mM iron was reduced (Figure 1). In contrast, when 80 mM γ-FeOOH was added initially, 55 mM iron was reduced and only 31 mM formate was removed (Figure 1). According to the stoichiometry of the redox equation, i.e., 1 formate oxidized by 2Fe(III), the bacteria oxidized 67.5 mM formate with 135 mM Fe(III) and
oxidized 27.5 mM formate with 55 mM Fe(III) for assays with 300 and 80 mM of γ-FeOOH, respectively (Figure 1). Thus, only 7.5 and 3.5 mM formate cannot be attributed to its oxidation by Fe(III), probably due to the adsorption onto lepidocrocite or incorporation into biomass. Control experiments were investigated: cells (4 × 109 cells mL-1) without formate, cells (1010 cells mL-1) killed by heat at 120 °C during 20 min, or no cell. The quantity of Fe(II) in all these controls remained constantly low. During bioreduction of γ-FeOOH, the color of the suspensions turned from orange to dark green after 3 d. In addition, for the assay with 300 mM γ-FeOOH, the color turned to black after 6 d, and the precipitate adhered to a magnetic rod. For assays performed with 80 mM (Figure 1) or 160 mM γ-FeOOH (not shown), the green precipitate formed but never became black. Characterization analyses of reduction products for the assay where 80 mM γ-FeOOH was added (Figure 1) are displayed in Figures 2, 3a, and 4 for TMS, XRD, and TEM, respectively. Mo¨ssbauer spectra of precipitates sampled during bioreduction after 1 and 6 d (Figure 1) and measured at 77 K exhibited four quadrupole doublets (Figure 2a,b; Table 1), the difference concerning only abundance areas. Doublets D1, D2, and D3 are typical of a GR spectrum at 77 K (5, 18). D3 is due to high-spin Fe3+ in octahedral sites with small values of isomer shift δ and quadrupole splitting ∆, whereas D1 and D2 with larger δ and ∆ values are due to high-spin Fe2+ in octahedral sites. Doublet Dγ corresponds to Fe(III) with small δ and ∆ values, as small as those found for ferric oxyhydroxide paramagnetic at 77 K, e.g., γ-FeOOH with ∆ ) 0.57 mm s-1 (19). Phases present after 6 d of bioreduction were also analyzed by TMS at 12 K (Figure 2c). The spectrum consists of magnetically split components for γ-FeOOH and paramagnetic doublets for GR (5, 18). Asymmetrically broadened absorption lines are fitted with three sextets; the main outer sextet Sγ has a field of 449 kOe (19). Doublet Dγ (Figure 2b) and sextet Sγ (Figure 2c) are both due to unreduced lepidocrocite. Dγ displays a larger abundance after 1 d than after 6 d (Figure 2a,b; Table 1) confirming the decrease of γ-FeOOH by reduction. Information is obtained from spectra of Figure 2a,b concerning the Fe(II)/Fe(III) ratio inside the GR. Assuming equal Lamb-Mo¨ssbauer factors f for all sites, the abundance of each iron site is proportional to the area under the peaks, and the Fe(II)/Fe(III) ratio is equal to ratio {D1 + D2}/D3. From fittings (Table 1), it is 32/33 and 44/41 for the samples obtained after 1 and 6 d of bioreduction, respectively. XRD (Figure 3a) and TEM (Figure 4) suggested the product of γ-FeOOH reduction by S. putrefaciens to be hydroxycarbonate GR(CO32-). The XRD pattern resulted from the superimposition of peaks due to the remaining lepidocrocite and to the formed green rust one with trigonal symmetry R3h m and computed parameters a ) 0.317(1) nm and c ) 2.272(6) nm (18, 20). Lines attributed to the GR are comparable to those of hydroxycarbonate GR(CO32-) as found by Drissi et al. (18) where a ) 0.316 nm and c ) 2.245 nm, excluding some other GRs (5). After 6 d, TEM image of GR particles (Figure 4) presented large hexagonal crystals measuring about 10 µm in diameter, typical of GR (21). The electron diffraction pattern of [001] zone (caption to Figure 4), indexed in the hexagonal representation of R3h m space group, yielded the same parameter a as that obtained by XRD. The presence of AQDS enhanced the rate of lepidocrocite reduction. In Figure 5a, the kinetics of Fe(II) production is presented for initial concentrations of 80 mM γ-FeOOH, 75 mM formate, and an inoculum of 109 cells mL-1, in the absence or presence of 100 µM AQDS. Without AQDS, the quantity of reduced Fe(III) was close to half that obtained in VOL. 36, NO. 1, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Mo1 ssbauer spectra of products obtained during reduction of γ-FeOOH (80 mM) with formate (75 mM) in the presence of AQDS (100 µM) in bacterial cultures (initially 8 × 109 cells mL-1). Measurements at 77 K after (a) 1 and (b) 6 d of culture (Table 1) (cf. Figures 3b and 4); (c) measurement at 12 K after 6 d of culture. (d) Spectrum measured at 290 K of the products obtained by bioreduction of lepidocrocite during 30 h and after freezing (250 K) and thawing (295 K) of the mixture. (•), experimental curves; (-‚-), global computed curve; s, elementary components.
FIGURE 3. XRD patterns of the products obtained by bacterial reduction of γ-FeOOH. (a) Initially 80 mM γ-FeOOH, 75 mM formate, and 100 µM AQDS; after 6 d of bioreduction (cf. Figures 2b and 4). (b) Initially 300 mM γ-FeOOH, 75 mM formate, and 100 µM AQDS; after 3 d of bioreduction. (c) Initially 300 mM γ-FeOOH, 75 mM formate, and 100 µM AQDS; after 6 d of bioreduction. GR, green rust indexed in its hexagonal representation (20); γ, γ-FeOOH lepidocrocite; M, magnetite. Co Kr1 radiation (λ ) 0.17889 nm). its presence (Figure 5a). The Mo¨ssbauer spectra can be compared (Figure 5b,c; Table 2): after 12 d of bioreduction, 18
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FIGURE 4. TEM image and corresponding electron diffraction pattern of GR hexagonal crystals mixed with a minor fine-grained phase (γ-FeOOH) obtained after bacterial reduction of lepidocrocite during 6 d (initially 80 mM γ-FeOOH, 75 mM formate, and 100 µM AQDS; cf Figures 2b and 3a). Electron diffraction pattern along [001] zone axis. 55 and 86% of GR, 11 and 13% of siderite, and 34 and 1% of remaining lepidocrocite were observed in the absence and presence of AQDS, respectively. The stability of the bioreduction products was studied. After about 1 yr at ambient temperature, the sample corresponding to Figure 1 (80 mM γ-FeOOH) did not change
FIGURE 5. (a) HCl-extractable Fe(II) in the absence or presence of 100 µM AQDS during γ-FeOOH reduction (80 mM) with formate (75 mM) in bacterial cultures initially 1 × 109 cells mL-1. Mo1 ssbauer spectra measured at 77 K of products obtained after 12 d of reduction (Table 2) (b) in the absence and (c) in the presence of 100 µM AQDS.
TABLE 1. Mo1 ssbauer Hyperfine Parameters of Spectra Measured at 77 K of Products Obtained during Reduction of γ-FeOOH Lepidocrocite (80 mM) with Formate (75 mM) in the Presence of AQDS (100 µM) in Bacterial Cultures (Initially 8 × 109 Cells mL-1) after 1 and 6 d of Culture (Figure 2a,b)a 1 day
D1 D2 D3 Dγ
6 day
δ (mm s-1)
∆ (mm s-1)
RA (%)
δ (mm s-1)
∆ (mm s-1)
RA (%)
1.27 1.27 0.59 0.42
2.89 2.51 0.56 0.57
29 3 33 35
1.27 1.28 0.51 0.35
2.90 2.58 0.51 0.56
41 3 41 15
a δ, isomer shift taking R-iron as reference at ambient; ∆, quadrupole splitting; RA, relative abundance.
TABLE 2. Mo1 ssbauer Hyperfine Parameters of Spectra Measured at 77 K of Products Obtained after 12 d of Reduction of γ-FeOOH Lepidocrocite (80 mM) with Formate (75 mM) in Bacterial Cultures (Initially 1 × 109 Cells mL-1) in the Absence and Presence of AQDS (Figure 5b,c)a absence of AQDS
D1 D2 D3 DS Dγ
presence of AQDS
δ (mm s-1)
∆ (mm s-1)
RA (%)
δ (mm s-1)
∆ (mm s-1)
RA (%)
1.29 1.28 0.51 1.29 0.42
2.92 2.59 0.67 2.23 0.51
22 13 20 11 34
1.28 1.27 0.49 1.29 0.44
2.94 2.64 0.48 2.22 0.66
36 24 26 13 1
a δ, isomer shift taking R-iron as reference at ambient; ∆, quadrupole splitting; RA, relative abundance.
at all. However, even though the initial greenish color did not change by freezing a sample at 255 K after 30 h of culture, its color turned from green to black when it reached 295 K by thawing. A similar sample, i.e., 80 mM lepidocrocite, 50 mM formate, and the same inoculum size, remained stable
after freezing and thawing with finally 12 mM unreduced γ-FeOOH. TMS analysis of the black product under helium atmosphere at 295 K exhibited sextets SA and SB with hyperfine magnetic fields H of 485 and 451 kOe due to magnetite (91%) and doublets Dγ and DS due to lepidocrocite (7%) and siderite (2%), respectively (Figure 2d). In anoxic conditions and after freezing at 255 K, the mixture of GR(CO32-) and lepidocrocite became a mixture of magnetite, siderite, and lepidocrocite after thawing at 300 K. The products were also characterized when the initial quantity of lepidocrocite was 300 mM and that of formate was 75 mM (Figure 1): after 3 d, a mixture of GR and γ-FeOOH was observed (Figure 3b); then after 6 d, the mixture evolved toward magnetite as checked by TMS (not shown) and XRD (Figure 3c).
Discussion Nature of Intercalated Anions. The nature of intercalated anions in GR is strongly suspected to be carbonate ions in view of lattice parameters (18, 20). This was confirmed unambiguously by the Fourier transformed infrared spectrum (not shown). Moreover, the Fe(II)/Fe(III) ratio found from TMS varied, in contrast to previous studies where the ratio was always equal to 2 (5, 18) and the formula [FeII4FeIII2(OH)12]2+‚[CO32-‚2H2O]2- obtained by monitored oxidation of Fe(OH)2 in carbonate. Therefore, a general formula must be now attributed to GR(CO32-), [FeII(1-x)FeIIIx(OH)2]x+‚ [(x/2)CO32-‚yH2O]x-, with y e [1 - (3/2)x], since the Fe(II)/Fe(III) ratio varies, as also recently observed by voltammetry (22). Stabilization of Green Rust by Bacterial Activity. During bacterial culture, the partial or total consumption of formate depended upon the initial amount of lepidocrocite (Figure 1). Part of produced bicarbonate was available for incorporation into forming GR(CO32-) and siderite. Considering that formate mainly got oxidized by Fe(III), the remaining formate could be incorporated into biomass or adsorbed onto lepidocrocite. The inactivation of bacterial reduction was obtained either by freezing or by exhausting the source of electrons with an VOL. 36, NO. 1, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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initial excess of lepidocrocite (300 mM). In the two cases, the green mixture of GR-lepidocrocite turned to black magnetite. It came from the reaction between lepidocrocite and Fe2+ ions that were adsorbed on its surface (23, 24). Since the GR is in equilibrium with the Fe2+(aq) (5, 25), the adsorption of Fe2+ induces the dissolution of GR. This kind of transformation of the mixture [GR + FeOOH] in anoxic conditions was already observed recently (26). In contrast, in other assays (80 and 160 mM γ-FeOOH with 75 mM formate initially), almost all lepidocrocite was reduced in agreement with the stoichiometry of redox equation. The transformation of the mixture [GR + FeOOH] into spinel phase was no longer possible. Among extracellular Fe(II)-bearing minerals widely reported to be induced by microbial iron reduction (11, 12, 27-29), GR(CO32-) must be now considered to be one of the possible major end products; mineral stabilization would be determined by bacterial activity. For instance, lepidocrocite was used recently by Cooper et al. (29) to perform cultures with S. putrefaciens, which only produced magnetite. In contrast, in the present report, biogenesis of GR from lepidocrocite reduction was unambiguosly observed for the first time. Cycling of Iron in the Field. Reduction of ferric minerals contributes to iron cycling, and bacterial activity in anoxic conditions reduces Fe(III) at the surface of (oxyhydr)oxides into Fe(II) ions in solution using humic substances as reduction mediator (30). Some of these Fe2+ ions form iron(II,III) GR, by incorporating the available dissolved surrounding anions. Since it is now shown that GR(CO32-) has a variable Fe(II)/Fe(III) ratio (this work), the assumption that fougerite GR mineral cannot be GR(CO32-) (4, 5, 10) must be discarded. The equilibrium assuming a hydroxyhydroxide GR mineral Fe(OH)(2+x) can be revisited (5, 10, 31, 32). However, GR is only a transient state during bacterial reduction of ferric iron and is converted to more stable forms (magnetite and siderite) upon cessation of microbial activity in the presence of ferric oxyhydroxide reservoir. Thus, the possibility of “green rust in the lab and in the soil” to challenge the reduction of pollutants directly mediated by bacteria (33), e.g., Se(VI) (7, 8), must be evaluated. However, since the GR mineral comes from ferric species due to the activity of DIRB, all pathways are still biotic, although either direct or indirect. Finally, the bluish green fougerite mineral turning ochrous on exposure to the air, which monitors dissolved iron in soil solutions and aquifers (5, 10, 25), could well be generated by the dissimilatory microbial reduction of ferric (oxyhydr)oxides favored by the presence of humic substances.
Acknowledgments We thank Dr. Claire Louis, Mr. Michel Paris, Dr. Jaafar Ghanbaja, and Dr. Bernard Humbert of the University Henri Poincare´ of Nancy for capillary electrophoresis, XRD analysis, TEM identification, and IR spectroscopy, respectively. A review by Pr. Adrien Herbillon (Universities Henri Poincare´ of Nancy, France, and of Louvain-la Neuve, Belgium) was very useful.
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Received for review December 28, 2000. Revised manuscript received September 24, 2001. Accepted October 4, 2001. ES0020456
