Geomicrobiology Journal, 22:389–399, 2005
c Taylor & Francis Inc.
Copyright ISSN: 0149-0451 print / 1362-3087 online
DOI: 10.1080/01490450500248960
Formation of Hydroxysulphate Green Rust 2 as a Single
Iron(II-III) Mineral in Microbial Culture
Asfaw Zegeye
Laboratoire de Chimie Physique et Microbiologie pour l’Environnement (LCPME), UMR 7564
CNRS—Université Henri Poincaré-Nancy 1, F-54600 Villers-lès-Nancy, France
Georges Ona-Nguema
Laboratoire de Chimie Physique et Microbiologie pour l’Environnement (LCPME), UMR 7564
CNRS—Université Henri Poincaré-Nancy 1, F-54600 Villers-lès-Nancy, France and Institut de
Minéralogie et de Physique des Milieux Condensés (IMPMC) UMR 7590 CNRS—Université Paris
6 & 7—IPGP, 75015 Paris, France
Cédric Carteret, Lucie Huguet, Mustapha Abdelmoula, and Frédéric Jorand
Laboratoire de Chimie Physique et Microbiologie pour l’Environnement (LCPME), UMR 7564
CNRS—Université Henri Poincaré-Nancy 1, F-54600 Villers-lès-Nancy, France
GR2(SO2−
4 )
Although
can be easily formed by abiotic synthesis, the biotic formation of hydroxysulphate as a single iron(IIIII) mineral in microbial culture and its characterization was not
achieved. This study was carried out to investigate the sole formation of GR2(SO2−
4 ) during the reduction of γ-FeOOH by a dissimilatory iron-respiring bacterium, Shewanella putrefaciens CIP
8040T . Reduction experiments were performed in a non-buffered
medium devoid of organic compounds, with 25 mM of sulphate and
with a range of lepidocrocite concentrations with H2 as the electron donor under nongrowth conditions. The resulting biogenic
solids, after iron-respiring activity, were characterized by X-ray
diffraction (XRD), transmission Mössbauer spectroscopy (TMS)
and electron microscopy (SEM and TEM). The sulphate has been
identified as the intercalated anion by diffuse reflectance infrared
Fourier transform spectroscopy (DRIFTS). In addition, the structure of this sulphate anion was discussed. Our experimental study
demonstrated that, under H2 atmosphere, the biogenic solid was a
GR2(SO2−
4 ), as the sole iron(II-III) bearing mineral, whatever the
initial lepidocrocite concentration. The crystals of the biotically
formed GR2(SO2−
4 ) are significantly larger than those observed for
Received 25 January 2005; accepted 20 June 2005.
The authors acknowledge S. Borensztajn of the Laboratoire de
Physique des Liquides et Electrochimie (Paris 6 University) for the
SEM-EDS analyses. We thank R. Aissa of the Laboratoire de Chimie
Physique et Microbiologie pour l’Environnement for providing us with
abiotic GR. We also thank I. Bihannic from the Laboratoire Environnement et Minéralurgie for the XRD analyses and J. Ghanbaja (Nancy
I University) for the TEM analyses.
Address correspondence to Frédéric Jorand, LCPME, UMR 7564
CNRS, Université Henri Poincaré-Nancy 1, 405 rue de Vandoeuvre, F-54600 Villers-lès-Nancy, France. E-mail: [email protected]
GR2(SO2−
4 ) obtained through abiotic preparation, >15 µm diameter as against 0.5–4 µm, respectively.
Keywords
hydroxysulphate GR2, Shewanella putrefaciens, biomineralization, bacterial iron reduction, diffuse reflectance
infrared Fourier transform spectroscopy (DRIFTS),
Mössbauer spectroscopy, XRD, MET, lepidocrocite
INTRODUCTION
Green rusts (GRs) are compositionally varied mixed Fe(II)Fe(III) hydroxides belonging to the sjögrenite-pyroaurite class
of minerals (Hansen 1989; Génin et al. 1996) These layered
double hydroxysalts consist of positively charged tri-octahedral
metal hydroxide {FeII (1-x) FeIII x(OH2 )}x+ alternating with a
negatively charged interlayer, {x/n An− ·(m x/n H2 O)}x− , which
includes m water molecules per anions (An− ). The conformation of this intercalated anion governs the GR structure. X-ray
diffraction analysis distinguishes, two types of GR (Bernal et al.
1959): GR1, containing planar or spherical anions (e.g., CO2−
3 ,
Cl− ) (Abdelmoula et al. 1996; Refait et al. 1998) and GR2, con2−
taining three-dimensional anions (e.g., SeO2−
4 , SO4 ) (Hansen
et al. 1994; Refait et al. 2000; Simon et al. 2003).
GRs tend to occur during the aqueous corrosion of iron
(Stampfl 1969) and in reductomorphic soils (Trolard et al. 1997;
Abdelmoula et al. 1998). GR1(CO2−
3 ) can be observed in the
rust covering urban pipes in drinking water networks, while
GR2(SO2−
4 ) occurs during corrosion of iron in seawater (Olowe
et al. 1989; Génin et al. 1993; Refait et al. 2003). Such minerals
play a major role in corrosion and soil science since they are intermediate products of aqueous corrosion during the oxidation
389
390
A. ZEGEYE ET AL.
of iron, and it has been proved that GR as a mineral governs iron
solubility in a soil solution and in groundwater (Bourrié et al.
1999). They also have a significant impact on the mobility of
some elements (O’Loughlin et al. 2003) and are highly reactive
to reducible inorganic and organic pollutants (Erbs et al. 1999;
Loyaux-Lawniczak et al. 2000; Hansen et al. 2001; Lee and
Batchelor 2002; Bond and Fendorf 2003; Legrand et al. 2004).
Thus, GRs are believed to play a key role in the redox cycling
of Fe and in the speciation of metals in anoxic sediments and
groundwater. As such, they could serve as a soft method for
getting rid of pollution in humid soils.
In order to better depict these minerals, and define the parameters necessary for their formation, the synthesis of GR needs
to be optimized. In abiotic laboratory systems, the nature of GR
is determined by the anionic composition of the medium. For
instance, GR2(SO2−
4 ) is prepared by Fe(OH)2 oxidation (Olowe
and Génin 1991), by co-precipitation of Fe(II) and Fe(III) cations
(Géhin et al. 2002) and by electrochemical oxidation (Peulon
et al. 2003) in the presence of sulphate anions. In biotic systems, GR production results from the reduction of ferrihydrite
(Parmar et al. 2001; Zachara et al. 2002; Glasauer et al. 2003;
Kukkadapu et al. 2004) or lepidocrocite (Ona-Nguema et al.
2002a, 2004) by Shewanella putrefaciens an iron-respiring bacteria (IRB). With regards to the hydroxycarbonate GR1, the interlayer carbonate anion is provided by a carbonate buffer (OnaNguema et al. 2002a, 2002b, 2004) and/or by oxidation of the
organic compound throughout the respiratory metabolism (OnaNguema et al. 2004).
In this condition, the GR1(CO2−
3 ) was the major iron(II)bearing mineral and the single GR type. In the Ona-Nguema et al.
(2004) study, the biotic reduction of lepidocrocite in presence
of sulphate and with sodium methanoate (NaHCO2 ) as the elec2−
tron source leads to a mixture of GR2(SO2−
4 ) and GR1(CO3 )
2−
but never to a single GR2(SO4 ). When the ratio of bicarbon2−
ate and sulphate anions (R = HCO−
3 /SO4 ) is lower than 0.17,
2−
GR2(SO4 ) formed (Ona-Nguema et al. 2004). Even with a high
concentration of sulphate (up to 175 mM), GR1(CO2−
3 ) is formed
as long as bicarbonate anions are present in the medium. This
is due to its high affinity for carbonate over sulphate during GR
precipitation (Refait et al. 1997). Therefore, in order to obtain
only a hydroxysulphate GR2 by microbial activity, the medium
should be free of carbonate anions. To achieve this, it would be
necessary to use an inorganic electron source instead of an organic one, so that its oxidation throughout bacterial respiration
does not generate CO2 in the medium.
The aim of the present study is to define the appropriate conditions that lead to the formation of hydroxysulphate GR2 by the
activity of S. putrefaciens, a facultative anaerobic bacterium capable of utilizing O2 , Fe(III), and a number of other compounds
as terminal electron acceptors for carbon metabolism. H2 is used
as the electron source instead of methanoate in order to avoid the
presence of carbonate in the medium and thus prevent the for2−
mation of GR1(CO2−
3 ). The formation of a GR2(SO4 ) which
is not mixed with any type of GR allows a better characteri-
zation of the mineral. The GR2(SO2−
4 ) that we formed in this
way was then well characterized by combining X-ray diffraction
(XRD), transmission Mössbauer spectroscopy (TMS), Diffuse
reflectance infrared Fourier transform spectroscopy (DRIFTS),
scanning electron microscopy (SEM) and transmission electron
microscopy (TEM).
EXPERIMENTAL METHODS AND PROCEDURES
Preparation of Shewanella putrefaciens Inoculum
Shewanella putrefaciens CIP 8040T (equivalent to ATCC
8071), the reference strain originally isolated from tainted butter
(Derby and Hammer 1931), was obtained from the Collection Institut Pasteur (Paris, France). Frozen cells from a stock (frozen
in 20% glycerol at −80◦ C) were revived under aerobic conditions on trypcase soy agar (TSA, BioMérieux, 51044). They
were transferred twice to remove glycerol, and subsequently, the
colonies were used to prepare a suspension with a target optical
density of 0.55 ± 0.01 (λ = 600 nm). Then, 20 mL of this suspension were inoculated in 200 mL of trypcase soy broth (TSB,
BioMérieux, 51019) in order to initiate the liquid preculture.
Cells were grown, continuously agitated at 300 rpm and at 30◦ C,
harvested after 14 h of culture by centrifugation (10,000 × g at
20◦ C for 10 min) and concentrated in 30 mL of TSB. 20 mL of
this concentrated suspension were inoculated into a 2-L batch
reactor containing 1.5 L of TSB to initiate the main culture. The
reactor was continuously agitated at 300 rpm and flushed by
sterilised air through a 0.22-µm filter (Millex FG50, Millipore)
at 30◦ C. Cells were grown to a stationary growth phase (24 h)
and harvested by centrifugation, washed twice with sterile NaCl
0.7% and concentrated in 40 mL of the same medium. Cells
were purged for 30 min by bubbling with N2 (alphagaz 1, Air
Liquide) sterilized by filtration through a membrane of pore size
0.2 µm (Millex FG50, Millipore) and used to inoculate batches
with lepidocrocite as the sole electron acceptor.
Preparation of γ-FeOOH Lepidocrocite
Lepidocrocite was prepared and characterized as previously
described by (Ona-Nguema et al. 2002a) by aerobic oxidation of
FeCl2 in sodium hydroxysulfate solution. The mineral suspension was washed three times with sterile osmosed water to remove salts and dispersed by ultrasonic treatment (19 mm probe,
40 s, in 20 mL, 40 W, VCX 600, Vibracell) to decrease particle
size (d50 = 1.1 µm). The suspension of lepidocrocite was then
dried at 105◦ C; 88.8 mg of this dry suspension was assumed to
correspond to 1 mM of γ -FeOOH.
Bioreduction Cultures
The culture medium for the lepidocrocite bioreduction assays contained hydrogen as the electron donor, and lepidocrocite as the electron acceptor (from 30 to 100 mM, depending on the assay). H2 , 100% in headspace, was estimated at
0.8 mM according to the molar fraction solubility of hydrogen
MICROBIAL FORMATION OF GREEN RUST 2
(1.35 × 10−5 mol L−1 ) at 303 K. The atmosphere inside the
bottles was maintained at a near constant concentration of H2
by adding H2 (100%) daily and after each sample collection.
Sulphate ions (25 mM) were added to the culture medium (at
the close level found in seawater). The culture medium was
constituted by (purity grade for analysis) NH4 Cl 22 mM, NaCl
1.5 mM, KCl 1.2 mM, MgSO4 · 7 H2 O 1.1 mM, Nitrilotriacetic acid (NTA) 0.71 mM, CaCl2 0.67, MnSO4 · H2 O 0.27
mM, ZnCl2 86 µM, CoSO4 7H2 O 38 µM, FeSO4 · 7 H2 O 32
µM, Na2 MoO4 · 2 H2 O 9.3 µM, NiCl2 · 6 H2 O 9.1 µM, Na2 WO4
2 H2 O 6.8 µM, CuSO4 · 5 H2 O 3.6 µM, AlK(SO4 )2 · 12 H2 O
1.9 µM, H3 BO3 1.5 µM, and 100 µM of anthraquinone-2,6disulfonate acid (AQDS) (ACROS, 10495–1000) (Ona-Nguema
et al. 2004). The medium was heat sterilized, except for the
sodium anthraquinone-2,6-disulfonate AQDS (100 µM) which
was sterilized by filtration through a filter pore size 0.2 µm.
No specific buffer was added in the medium. The medium was
also purged with filter-sterilized H2 (100%) and dispensed into
sterile 100 mL flasks, with butyl rubber stoppers and then crimp
sealed.
S. putrefaciens cell suspensions were added to obtain final
concentrations of 1.0 × 1010 cells mL−1 , corresponding to 1.8 ×
109 colony-forming units (CFU) mL−1 . The pH, measured after
all components had been mixed, was 7.2. Cultures were incubated at 30◦ C in darkness. The microbial reduction of γ -FeOOH
was mostly monitored for 14 days (and in some case up to 30
days). During this incubation period, aliquots were removed at
selected time intervals to measure Fe(II) production and cell
concentration.
Cell and CFU Counts
Cells were counted by epifluorescence microscopy using 4 ,6-diamidino-2-phenylindole (DAPI) fluorochrome (Saby
et al. 1997). The number of CFU was determined by plate counting. 1 mL of the bacterial suspension adequately diluted in sterile
NaCl 0.7% solution was incorporated into TSA and incubated in
an N2 /H2 (97/3) anaerobic chamber (Coy Laboratory products).
Calculation of Iron (III) Reduction Initial Rate
We assume that the bioreduction of Fe(III) follows a pseudo
first-order kinetic. The course of Fe(III) reduction was then fitted
according to the equation of a first-order chemical reaction [1],
Vi = k[Fe(II)]max
Chemical Analysis
HCl-extractable Fe(II) was obtained by placing 1 mL of the
sample directly into 1 mL of 2 N HCl. The concentration of
Fe(II) was determined after 1 week using the modified 1,10phenanthroline method (Fadrus and Maly 1975).
X-ray Diffraction (XRD)
Once filtered (filter of pore size 0.45 µm, under an N2 atmosphere), the wet paste was spread out on a glass plate and coated
with glycerol to prevent oxidation (Hansen 1989). The XRD
data were collected with a D8 Bruker diffractometer, equipped
with a monochromator and position-sensitive detector. The
X-ray source was a Co anode (λ = 0.17902 nm). The diffractogram was recorded in the 3–64 2θ range, with a 0.0359◦ step
size and collecting time of 3 seconds per point.
Transmission Mössbauer Spectroscopy Analysis
Mössbauer spectra were obtained from a constant acceleration Mössbauer spectrometer connected to a 512 multichannel
analyzer. The amplifier output and the drive mechanism were
manufactured by Halder Electronics GmbH. The detector consisted of an NaI(Tl) scintillation counter. The source of 50 mCi
57
Co in Rh matrix was maintained at room temperature. The
spectrometer was calibrated with a 25-µm foil of α-Fe at room
temperature and isomer shifts were given relative to this reference. Aliquots were taken from the well-homogenized medium
and the iron particles were concentrated by filtration (0.45-µm
pore size) under an N2 atmosphere inside a glove box. The sample absorbers were immediately cooled to the liquid nitrogen
temperature and maintained until measurements were taken. To
prevent oxidation, they were quickly transferred to the cryostat under an inert He atmosphere before measuring at 77 K
or ambient temperature. TMS was performed in a Mössbauer
cryostat (Cryo Industries of America). The maximum area density of Fe in the samples was approximately 10 mg cm−2 , and
thickness effects were assumed to be negligible. Computer fittings were performed using Lorentzian-shape lines based on the
least-squares method. The parameters, like those resulting from
any computer fitting, must be mathematically significant (χ 2
minimization) and their number small enough using contrast to
have a physical meaning. Full width at half-maximum must, for
example, be in the order of 0.3 mm s−1 .
[1]
where Vi is the initial reduction rate of iron (III), Fe(II)max is
the concentration of iron (II) at time infinite, and k is the rate
constant (h−1 ). The integration of equation [1] give the variation
of Fe(II) concentration ([Fe(II)]t as a function of time [2] from
which the constant k and [Fe(II)]max can be extracted to calculate
Vi
[FeII]t = [Fe(II)]max (1 − e−kt )
391
[2]
Diffuse Reflectance Infrared Fourier Transform
Spectroscopy (DRIFTS)
The hydroysulphate GR2 was recovered by centrifugation
and subsequently washed twice with oxygen-free 18 M cm−1
nano-pure water. This washing procedure removed adsorbed sulphate from the GR. X-ray diffraction analysis indicated that the
GR2(SO2−
4 ) was not subject to transformation during this washing process. The GR2 was then dried for 24 h away from oxygen
in an anaerobic chamber under a 90% relative humidity.
392
A. ZEGEYE ET AL.
A Fourier transform infrared spectrometer, the Perkin-Elmer
2000, equipped with a KBr beam splitter and a DTGS detector,
was used for sample characterization by vibrational spectroscopy. The spectral resolution and the total acquisition time
were 4 cm−1 and 5 min respectively. FTIR spectra in diffuse
reflectance mode were collected using Harrick DRA-2CI equipment. To perform the analysis, the samples were first diluted
in a KBr matrix (5 wt%). The samples were mixed very gently with KBr in an agate mortar, so that the mixtures were not
subjected to any elevated pressures. It should be noted that a
great deal of caution was exercised to avoid exposure to oxygen, indeed the samples were prepared in an anaerobic chamber (Coy Laboratory Products) and enclosed in an airtight Harrick HVC-DRP cell with KRS-5 windows for infrared analysis.
The reflectances Rs of the sample and Rr of pure KBr, used
as a nonabsorbing reference powder, were measured under the
same conditions. Iron oxide-hydroxide reflectance is defined
as R = Rs /Rr . The spectra are shown in pseudo-absorbance
(−logR) mode.
In one figure, we compare the spectra of abiotically and biotically formed GR2. The abiotically GR2 were synthesized
according to the method described in Aissa et al. (2004).
Electron Microscopy
Scanning electron microscopy (SEM) was conducted using a
LEICA Stereoscan 440 scanning electron microscope equipped
with an energy dispersive spectrometer (EDS) for chemical analysis (20 kV, 150 pA).
Transmission electron microscopy (TEM) was conducted using a CM20/STEM Philips transmission electron microscope,
with a voltage of 200 kV. One drop of the suspension was laid
on an amorphous carbon-coated grid and loaded into the analysis
holder of the microscope under 10−8 Torr vacuum.
FIG. 1. The monitoring of biogenically produced Fe(II) and cell numbers in
experiment 5. All experiments were performed at 30◦ C with an initial pH of 7.2.
RESULTS
Bioreduction of Lepidocrocite
In order to investigate the formation of hydroxysulphate GR2,
iron reduction experiments with S. putrefaciens were conducted
and monitored with different concentrations of lepidocrocite
(from 30 to 100 mM), as the electron acceptor and H2 as the
sole electron source.
The reduction of γ -FeOOH was immediately initiated after the inoculation of bacteria without a lag phase. Fe(II)
increased with time, thus reflecting the progression of lepidocrocite reduction (Figure 1). Furthermore, the initial rate of
lepidocrocite reduction increased slightly with the iron concentration from 0.05 to 0.12 mM Fe(II) h−1 (Table 1). The extent
TABLE 1
Parameters and data for lepidocrocite bioreduction by S. putrefaciens in the presence of sulphate (25 mM)
and hydrogen as the electron donor
Experiment no.
γ -FeOOH (mM)
Initial rate of Fe(II) production
(mM h− 1)
Total Fe(II) produced (mM) at
14 days
Final products
Control
1
2
3
4
5
6
40
0
30
0.050
40
0.076
50
0.080
60
0.110
80
0.120
100
0.123
0
9
10
12.7
13.7
21.0
23.7
γ -FeOOH
GR2
GR2
GR2
GR2
GR2
GR2
Coefficient of variation % ([(standard deviation/mean) × 100]) of the initial rate ≤5%.
Experiments were conducted with different lepidocrocite concentrations (30–100 mM) and anthraquinone-2,6-disulfonate
(100 µM). All were performed at 30◦ C in the absence of added bicarbonate ions in order to avoid the formation of hydroxycarbonate GR1. The initial pH was 7.2, and the final pH reached 8.4 after 14 days of incubation. Final products were characterized
by XRD after 14 days of culture. The control was performed without addition of inoculum.
393
MICROBIAL FORMATION OF GREEN RUST 2
of bacterial lepidocrocite reduction increased linearly with the
iron oxide concentration (r2 = 0.996, slope = 0.247). The ratio Fe(II)/Fe(III) is around 0.25 (data from Table 1) for any
concentration of lepidocrocite (30–100 mM) after 14 days of incubation. The results presented here show that S. putrefaciens is
able to couple the oxidation of H2 with lepidocrocite reduction.
This reaction is represented by the following equation [3].
2γ -FeOOH + H2 + 4H+ → 2Fe2+ + 4H2 O
[3]
The pH of the iron suspension increased from 7.2 to a final
value of 8.4 ± 0.2 probably due to proton consumption from
equation (3).
Plate counts were periodically carried out, in triplicate, during
the iron-reduction experiments (Figure 1). The initial cell density
was 1.8 × 109 CFU mL−1 , which decreased to 108 CFU mL−1
during the first day and stabilised at around 106 CFU mL−1 for
the remaining time of the assays.
Characterization of the Microbially Formed GR2
X-ray Diffraction Analysis. The greenish solid phases
formed from γ -FeOOH bioreduction were characterised by Xray diffraction (XRD). In order to ascertain the nature of these
new phases, the XRD diffractogram of the control was contrasted
with the XRD diffractograms of the solids formed by the microbial reduction of lepidocrocite (Figure 2). The XRD patterns of
products obtained after 14 days and 3 months of incubation in
experiments 5 and 6, respectively, exhibit lines of GR2 and lepidocrocite. For instance, after 14 days of culture, prominent GR2
peaks at d001 = 1.11 nm, d002 = 0.554 nm, d003 = 0.368 nm,
d004/100 = 0.268 nm, and d102 = 0.221 nm are easily observed.
After 3 months of incubation, no noticeable change is observed,
except that the intensity of the diffraction lines increased, indicating the relative stability of biogenic GR2. Some minor peaks
of lepidocrocite remain after 90 days (Figure 2).
Experimental GR2 d-spacings are similar to those previously
obtained in biotic and abiotic conditions for (Table 2). Moreover,
FIG. 2. X-ray diffractograms of the solid phases from control, assays 5 and 6.
“L” denotes lepidocrocite peaks, “GR2” denotes GR2 peaks. Analyses of solid
phases were performed after 14 days of incubation, except assay 6, which was
performed after 3 months. Co-Kα radiation was used (λ = 0.17902 nm).
supplementary lines are observed with respect to GR2(SO2−
4 )
obtained by Ona-Nguema et al. (2004). These results show the
formation of hydroxysulphate GR2. No other minerals were detected by XRD in microbial assays, indicating that, when S.
putrefaciens couples the lepidocrocite reduction with hydrogen
oxidation in a sulphated medium, only a GR2 is obtained.
Electronic Microscopy. The SEM micrograph of the greenish phase from experiment 5 (Table 1) displays the characteristic hexagonal plates of GR (>15 µm of equivalent diameter)
(Figure 3A). EDS analysis shows the presence of S and O
(Figure 3B), suggesting the presence of SO2−
4 anions at the surface and/or in the interlayer of the GR2 formed. The TEM micrograph of a GR particle from experiment 4 (Figure 3C) also
presents large hexagonal crystal measuring about 17–19 µm
TABLE 2
dhkl parameters of synthetic and biogenic GR2
dhkl (nm)
Relative
intensity
100
80
60
20
30
30
30
GR2
hkl
001
002
003
004/100
101
102
005/103
Exp. 5
∗a
1.11
0.554
0.368
0.268
—
0.221
—
Exp. 6
∗b
1.11
0.557
0.367
0.268
—
0.221
—
refc
(abiotic)
refd
(abiotic)
refe
(biotic)
1.09
0.545
0.362
0.275
0.266
0.245
0.219
1.10
0.549
0.366
0.275
0.226
—
0.2195
1.092
0.551
0.366
—
—
—
—
∗
This work: characterisation after a 14 days and b 90 days of incubation c (Simon et al. 1997); d (Hansen et al. 1994); e (Ona-Nguema et al.
2004).
394
A. ZEGEYE ET AL.
FIG. 3. (A) SEM micrograph of solids produced during bacterial reduction of lepidocrocite in the presence of hydrogen as the electron donor; (B) composition
of hexagonal crystal as determined by EDS (experiment 5) and (C) TEM image and corresponding electron diffraction pattern of the GR hexagonal crystal obtained
from bacterial reduction of lepidocrocite in the presence of H2 as the electron donor (experiment no. 4).
in diameter. The electron diffraction pattern of the [001] zone
(caption to Figure 3C), indexed in the trigonal representation of
the P 3̄m1 space group, yielded the same parameter a as that
obtained by XRD.
Transmission Mössbauer Spectroscopy Analysis. In this
study, the Mössbauer spectrum of the greenish phase sampled
from experiment 6 (Table 1) is a well-resolved spectrum at 77 K.
It can reasonably be fitted with three paramagnetic quadrupole
doublets D1 , D2 , and DL (Figure 4). The doublets D1 (36%)
and D2 (19%) correspond to GR (55%) and the doublet DL
is assigned to paramagnetic Fe3+ in lepidocrocite (45%), with
small isomer shifts δ (0.52 mm s−1 ) and quadrupole splitting (0.55 mm s−1 ) values at 77 K (Murad and Schwertmann 1984).
The doublet D1 with a large δ (1.26 mm s−1 ) and a large (2.93 mm s−1 ) corresponds to the ferrous state, whereas the last
D2 with smaller δ (0.35 mm s−1 ) and (0.61 mm s−1 ) values
corresponds to the ferric state. These characteristic features indicate that these components are associated with ordered arrangements of Fe2+ and Fe3+ in the brucite sheet. The Fe(II)/Fe(III)
ratio in the GR formed is, as expected, around 2, being the ratio
of the doublet intensities D1 and D2 .
Diffuse Reflectance Infrared Fourier Transform Spectroscopy. In order to ascertain the nature of the mineral phase
formed during the microbial reduction of lepidocrocite, the
infrared absorption spectrum of the biotically formed mineral is compared with the spectrum of the abiotically synthesised hydroxysulphate GR2 (Figure 5). These minerals were
dried for one day in the anaerobic chamber under 90% relative humidity before IR analysis. The spectra of biotic and abiotic samples (Figure 5a and 5b) exhibit the typical features of
MICROBIAL FORMATION OF GREEN RUST 2
395
FIG. 4. TMS spectrum of solid phase obtained in experiment 6, measurement
at 77 K after 3 months of incubation.
hydroxysulphate GR2 (Peulon et al. 2003; Ona-Nguema et al.
2004): bands arising in the brucite-like sheets at 515, 780, 880,
and 1550 cm−1 , and bands due to the intercalated sulphate at
around 620/660 and 1105/1138 cm−1 . In addition to these GR
absorptions, the spectrum of the biotic sample reveals the presence of lepidocrocite, especially with the sharp absorption at
1022 cm−1 . Finally around 50% of the initial amount of lepidocrocite was not reduced by microbial activity.
Figure 6 illustrates the spectral changes that occur through
dehydration of the GR mineral. After one hour under 10−6 Torr,
a significant amount of water was evacuated as evidenced in
the infrared spectrum (Figure 6b), by decreasing water absorption to 1650 cm−1 . It is worth noting that all the characteristic
wavenumbers of GR shift depend on the amount of water. Since
the water molecules were either H-bonded to FeO-H or in the
FIG. 5. IR spectra of (a) biotic GR, (b) abiotic GR, and (c) lepidocrocite.
Before analysis each mineral was kept in an anaerobic chamber for 24 h under
90% relative humidity.
FIG. 6. Influence of the hydration state of the GR on its infrared spectrum.
(a) sample was kept in an anaerobic chamber for 24 h under 90% relative humidity, (b) sample was evacuated for 1 hour under 10−6 Torr.
hydration shell of the sulphate, sulphate absorptions, as well as
the brucite-like sheet absorptions, were affected by the drying
procedure. This sheds light on the phenomenon of the infrared
spectrum of GR being highly dependent on the “drying” procedure used to prepare the analysed sample.
DISCUSSION
Bacterial γ-FeOOH Reduction and GR2(SO2−
4 )
Biomineralization
The effect of bacterial activity on the formation of mineral is
well accepted and the interest of the scientific community in this
topic continues. However, the environmental conditions needed
for this formation have remained unclear principally because
they are so difficult to reproduce in the laboratory. With low cell
and nutriment concentrations, conditions close to those found
in the environment, the amount of mineral transformed during
the incubation of Shewanella cells remains weak and heterogeneous (Glasauer et al. 2003). The experimental conditions we
have selected, allow the production of very large and dense GR1
and GR2 crystals (Ona-Nguema et al. 2002a, 2004). To get significant iron (II) production, we should use a very large inoculum
size (109 CFU mL−1 ). In this way, Shewanella cells, harvested
from the stationary phase in an aerobic and rich medium, are also
able to convert γ -FeOOH in hydroxysulphate GR2 by using H2
instead of methanoate as the sole electron donor.
As previously noted by (Glasauer et al. 2003) iron (III) reduction is correlated with CFU decline confirming the non-growth
conditions of the medium. This could be the result of nutritional
stresses since neither organic carbon nor phosphate were added
(as in the present study) or weakly available because under a
stable mineral phase like vivianite (Glasauer et al. 2003). Alternatively, due to their negative charge at circumneutral pH,
396
A. ZEGEYE ET AL.
the interaction of cells with (oxyhydr)oxides would result in
the formation of mineral-cell aggregates (Caccavo et al. 1997;
Glasauer et al. 2001) and consequently could have introduced
inaccuracy into the CFU determination. In addition, the intimate
association between bacteria and the mineral could affect cell
viability, as suggested by (Glasauer et al. 2003). However, the
presence of such clusters in our assays was never observed. The
cause of this decline remains unclear, but it should be noted
that there obviously remained sufficient active cells to reduce
the γ -FeOOH. As well, the cell amount cannot be considered
as the limiting factor for iron reduction in the system, since
the increase in iron (III) leads to an increase in the iron reduction rate. The extent of Fe(II) production at 14 days was effectively linearly correlated with the initial iron (III) oxide reduction
(from Table 1 data).
In order to stimulate iron reduction, we added 100 µM of
AQDS (anthraquinone-2,6-disulfonate) to the media. AQDS is
a typical component, of the quinone and hydroquinone structure
in humic acids, and acts as an electron shuttle between the
microbial transport chain and iron oxide surface, obviating
the need for direct contact between the cells and the Fe(III) oxide
(Lovley et al. 1996, 1998; Royer et al. 2002). As shown previously, AQDS is not necessary to form the GR mineral (Parmar
et al. 2001; Ona-Nguema et al. 2002a).
The Fe(II) formed during γ -FeOOH reduction intervenes in
the formation of hydroxysulphate GR2 according to the following reaction:
+
II
III
6γ -FeOOH + 2H2 + SO2−
4 + 2H → Fe4 Fe2 (OH)12 SO4 [4]
In the current lepidocrocite reduction reaction, GR2(SO2−
4 )
was observed to be the sole solid phase formed in the media, as
shown by TMS, XRD and DRIFTS. Only half the lepidocrocite
was transformed into hydroxysulphate GR2, thereby suggesting
that the cells did not use all the crystalline Fe supplied as the terminal electron acceptor. Nonetheless, as was previously shown
(Ona-Nguema et al. 2002a), a large amount (≈90%) of the lepidocrocite (80 mM) can be transformed into GR in the presence
of methanoate (75 mM) as the electron source. Previous experiments that have investigated the bioreduction of γ -FeOOH in the
presence of sulphate anions (75–175 mM) showed a strong negative linear correlation between sulphate anion concentration
and the extent of lepidocrocite reduction (Ona-Nguema et al.
2004). The sorption of sulphate anions onto γ -FeOOH might
impede further reduction, resulting in a lower overall extent of
the reduction. However, in the present study, the sulphate anions
were present in lower concentration (25 mM) in the medium
and no relationship was observed between SO2−
4 /Fe(III) and
Fe(II)/Fe(III), thus indicating that sulphate anions do not play
a significant role in Fe(III) reduction within the range of lepidocrocite concentration tested. Additional research is needed to
precisely determine the role played by SO2−
4 anions during the
microbial reduction of lepidocrocite.
Stability of the Biogenic GR2(SO2−
4 )
Some authors have shown that a mixture of Fe(III) oxides
and GR turned into black magnetite (Benali et al. 2001; OnaNguema et al. 2002a) due to the fact that magnetite is thermodynamically more stable than GR (Génin et al. 1998). This
transformation results from the sorption of Fe2+
aq , which was in
equilibrium with the GR (Génin et al. 1998; Bourrié et al. 1999),
on the iron oxyhydroxide surface (Tamaura et al. 1983; Tronc
et al. 1992), which leads to a topotactic transformation of lepidocrocite in magnetite. The Fe2+
aq concentration decreases in the
medium that accelerates the dissolution of the GR. In the present
study, the mixture (50% of γ -FeOOH and 50% of GR2(SO2−
4 ))
was not transformed into the inverse spinel phase, as shown
by X-ray diffraction and TMS after 90 days of incubation. For
any concentration of lepidocrocite tested (30–100 mM), there
was an excess of sulphate anions, assuming that the ratio of
Fe/S in a hydroxysulphate GR is 6 (Simon et al. 2003). The
dissolution of GR2(SO2−
4 ) could be inhibited by the surplus
of sulphate. The sorption of sulphate anions onto γ -FeOOH
could prevent the sorption of Fe2+
aq , which remains in equilib),
thus
avoiding
the dissolution of the
rium with the GR2(SO2−
4
mineral. In addition, the possibility of sulphate sorption onto
the hydroxysulphate GR has to be taken into account in order
to explain the stability of biogenic mineral in the presence of
lepidocrocite.
Fe Speciation by Transmission Mössbauer Spectroscopy
Numerous papers on synthetic GRs making use of Mössbauer
spectroscopy have since been published, and this technique has
provided decisive data which led to the identification of a natural occurrence of GR in reductomorphic soil in Brittany (Trolard
et al. 1996, 1997; Abdelmoula et al. 1998; Génin et al. 1998). In
a recent paper by Ona-Nguema et al. (2004), Mössbauer spectroscopy did not distinguish GR1 structure from that of GR2.
However, Ona-Nguema et al. (2004) prove that the fit of the
contributions due to Fe(II) sites is satisfactory through the use
of two quadrupole doublets. In contrast, the Mössbauer spectrum
of the GR2(SO2−
4 ) presented in this work (not mixed with GR1),
is composed of only one ferrous and one ferric doublet. The presence of only one ferrous site in GR2(SO2−
4 ) in the present study
thus indicates an ordered organisation of the sulphate ions and
water molecules inside the interlayers (Simon et al. 2003), in
contrast to what is observed in GR1 (Refait et al. 1998). In addition, the Fe(II)/Fe(III) ratio keeps a constant value of 2 for
the GR2, whereas for the GR1 we observed a certain evolution
and variation of this ratio (Refait et al. 1998; Ona-Nguema et al.
2002a).
Sulphate Coordination in the GR Interlayer
To discuss the structure and bonding of sulphate from infrared spectra, we first look at the vibrational characteristics of
this anion. The sulphate anion is characterised by four vibrational modes named ν1 , ν2 , ν3 , and ν4 , which are respectively
assigned to the symmetrical stretching, symmetrical bending,
MICROBIAL FORMATION OF GREEN RUST 2
asymmetrical bending, and asymmetrical stretching vibrations.
In various sulphate minerals and solutions, these modes have
been observed in the spectral ranges 960–1020 cm−1 , 415–
515 cm−1 , 1000–1250 cm−1 , and 590–700 cm−1 , for ν1 , ν2 , ν3 ,
and ν4 , respectively (Farmer 1974; Nakamoto 1978; Hug 1997;
Wijnja and Schulthess 2000). As a free sulphate anion has tetragonal symmetry and belongs to the point group Td , only two
peaks due to the triply degenerate ν3 and ν4 modes (symmetry
species T2 ) are observed in the infrared spectrum; all modes are
observed in the Raman spectrum since, in addition to ν3 and
ν4 , the ν1 (symmetry species A1) and ν2 (symmetry species E)
modes are Raman active. For example, a free sulphate anion
in solution exhibits an infrared absorption spectrum with two
bands, at 1105 (ν3 ) and 611 (ν4 ) cm−1 and a Raman diffusion
spectrum with four bands, at 983 (ν1 ), 450 (ν2 ), 1105 (ν3 ), and
611 (ν4 ) cm−1 .
Given the fact that, in the outer-sphere complex, the anion
would retain its hydration shell and would not form a direct
chemical bond with the metal ion, it is expected that the symmetry of the outer-sphere sulphate complex remains tetrahedral,
like the sulphate ion in solution. In the case of an inner-sphere
complex, the splittings and shifts of bands compared to the spectrum of the free sulphate provide valuable information about the
coordination state of the anion. After coordination with a metal
ion in an inner-sphere complex, the Td symmetry of SO4 is lowered: monodentate coordination lowers the symmetry to C3v ,
whereas bidentate coordination lowers the symmetry to C2v . As
a result, each of the triply degenerate ν3 and ν4 modes splits
into two bands (C3v ) and three bands between 1030 and 1250
(C2v ), while the ν1 band becomes infrared active at about 975
cm−1 and around 1000 cm−1 . Sample drying strongly favors the
inner-sphere complex and, in addition, shifts one of the ν3 peaks
from 1170 to 1200 cm−1 (Parfitt and Smart 1978; Hug 1997;
Eggleston et al. 1998; Peak et al. 1999).
In the GR sample spectrum (Figure 5), we observe two peaks,
at 1104 and 1137 cm−1 . Peulon et al. (2003) have also observed
two bands, at 1100 and 1145 cm−1 , for electrochemically formed
GR2 on inert gold substrate, and assigned them to the split ν3 SO4
mode. According to these authors, this splitting may indicate a
lowering of the symmetry of the intercalated sulphate due to a
nonsymmetrical interaction with layers of Fe(OH)6 octahedra
and intercalated water molecules. However, they conclude that
the deviation from Td symmetry may be very small since no
activation of the normally IR-forbidden ν1 mode was observed
in their GR(SO4 ) samples. Although we report the same spectral features of intercalated sulphate, our conclusion on sulphate
coordination differs from the conclusion put forward by Peulon
et al. (2003). Indeed, since the ν1 is not observed in GR2, intercalated sulphate should form an outer-sphere complex with
sulphate coordination close to that of the free sulphate in solution (Td symmetry). If there is no lowering of the sulphate
symmetry, the ν3 mode cannot be reasonably split. In our GR2
spectrum, the ν3 absorption is sharp and characterised by two
well-defined peaks with a wavenumber gap of only 33 cm−1 (45
397
cm−1 in Peulon et al. 2003). This may indicate the presence of
two intercalated sulphate anions (symmetry Td ) per unit GR2
cell. These two anions can vibrate in-phase or out-of-phase, inducing two peaks for the ν3 mode.
In a previous study (Ona-Nguema et al. 2004), the authors had
discussed the competitive formation of hydroxycarbonate GR1
versus hydroxysulphate GR2. The hydroxysulphate GR2 had
been observed only for sulphate concentrations above 100 mM
when methanoate was used as the electron source. At these high
sulphate concentrations, sulphate adsorption and/or precipitation on the GR surface occurred. Owing to the spectral difference
between two selected spectra (Figure 4B in Ona-Nguema et al.
2004), the authors assumed that they would extracting sulphate
wavenumbers at 1012, 1085, 1109, 1137, and 1180 cm−1 , arising
from the intercalated sulphate involved in a C2v inner-complex.
In light of our new data, the coordination of intercalated sulphate
is not C2v , since only the absorptions at 1109 and 1137 should
be assigned to intercalated sulphate. Thus, in Ona-Nguema et al.
(2004), the absorptions of adsorbed or precipitated sulphate are
mixed with the absorptions of intercalated sulphate peaking near
1105 and 1140 cm−1 . In the present paper, we have taken some
precautions to avoid the presence of adsorbed or precipitated
sulphate in the infrared spectrum of biogenic hydroxysulphate
GR. First, the use of H2 as the electron donor for lepidocrocite
bioreduction allows the observation of hydroxysulphate GR2
formation for sulphate concentration as low as 25 mM, which
is four times less than in the previous paper. Secondly, prior to
the infrared analysis, the hydroxysulphate green rust is washed
twice by oxygen free 18 M cm nano-pure water to eliminate
possible adsorbed sulphate.
When GR is highly hydrated, intercalated sulphate is surrounded with a hydration shell. The sulphate does not form a
direct chemical bond with the Fe3+ metal ion, but it is expected
that the symmetry of the outer-sphere sulphate complex will
remain tetrahedral like the sulphate ion in solution. When, the
GR is dehydrated (Figure 6b), water molecules in the sulphate
hydration shell are eliminated. The sulphate coordinates with a
Fe3+ metal ion, as suggested by Simon et al. (2003) in an innersphere complex. The Td symmetry of SO2−
4 is lowered: ν1 mode
becomes infrared-active with a low intensity and the ν3 mode
splits into peaks at 1035, 1135, and 1180. It is worth noting that
these peaks are not well defined for the biotic sample since unreduced lepidocrocite absorbs at 1025 cm−1 and the adsorption of
ν3 SO2−
4 is large with a high level of recovery of the components. The mode of coordination of intercalated sulphate in GR
is, consequently, highly dependent on the hydration/dehydration
state of the mineral.
Morphologic Properties of Biogenic GR2(SO2−
4 ) Crystals
The SEM and TEM micrographs show that the crystals
of hydroxysulphate GR formed by microbial reduction of
lepidocrocite are large (>15µm). These samples present a similar XRD pattern which is typical of layered compounds, with
398
A. ZEGEYE ET AL.
FIG. 7.
A possible representation of the relationship between the various GR crystal sizes and the rate of iron(II) production in the medium.
maximum intensity for the (00l) reflections. The relative sharpness of the lines is characteristic of crystals presenting exceptional coherence length (L ∼ 1/, with : width half maximum).
In contrast, in abiotic methods, smaller GR2(SO2−
4 ) crystals
are observed. In the coprecipitation methods, medium-sized is
around 0.5 µm (Géhin et al. 2002); this could be explained by the
high Fe(II) output kinetics in the solution during the synthesis.
The formation, with electrochemical oxidation of soluble Fe(II),
can lead to crystals reaching 2–4 µm in diameter. This value is
the largest observed in abiotic synthesis (Peulon et al. 2003).
It is probable that, the operating mode could explain the difference in morphological properties observed between abiotic
and biotic synthesis of GR2(SO2−
4 ). Indeed, the low kinetics of
Fe(II) production could allow the germination and nucleation
of larger GR crystals (Figure 7). Thus, the biotically formed
GR2(SO2−
4 ) crystals are significantly larger than those observed
for GR2(SO2−
4 ) obtained through abiotic preparation, >15 µm
diameter as against 0.5–4 µm.
CONCLUSION
The experiments reported in this manuscript have focused on
the formation and characterization of GR2(SO2−
4 ) as a single
iron(II-III) bearing mineral resulting from the microbial reduction of lepidocrocite. Accordingly, around 50% of the γ -FeOOH
was transformed into GR2(SO2−
4 ) by Shewanella putrefaciens
under an H2 atmosphere as the sole electron source. TEM and
SEM reveal a perfectly hexagonal crystal size larger than 15
µm. Mössbauer analysis indicates that the Fe(II)/Fe(III) ratio of
the biotically formed GR2(SO2−
4 ) is in the range of 2. Moreover,
the presence of two intercalated sulphate anions per unit cell of
GR2(SO2−
4 ) is proposed according to its infrared spectrum.
In light of our study, the possibility of GR2(SO2−
4 ) formation
by iron-respiring bacteria has to be taken into account to explain
the occurrence of this mineral in anaerobic environments, where
both H2 and SO2−
4 are present, and associated with low organic
and inorganic sources of carbon.
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