# Reduction of ferric green rust by Shewanella putrefaciens

F. Jorand, A. Zegeye, F. Landry, C. Ruby

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F. Jorand, A. Zegeye, F. Landry, C. Ruby. Reduction of ferric green rust by Shewanella putrefaciens. Letters in Applied Microbiology, 2007, 45 (5), pp.515-521. 10.1111/j.1472-765X.2007.02225.x. hal-03210484

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ORIGINAL ARTICLE

# Reduction of ferric green rust by *Shewanella putrefaciens*

F. Jorand, A. Zegeye, F. Landry and C. Ruby

Laboratoire de Chimie Physique et Microbiologie pour l'Environnement (LCPME), UMR 7564 CNRS-UHP, rue de Vandœuvre, Villers-lès-Nancy, France

**Keywords**

biomineralization, green rust, iron reduction, *Shewanella*.

**Correspondence**

Frédéric Jorand, Laboratoire de Chimie
Physique et Microbiologie pour
l'Environnement (LCPME), UMR 7564
CNRS-UHP, 405, rue de Vandœuvre, 54600
Villers-lès-Nancy, France.
E-mail: jorand@pharma.uhp-nancy.fr

2006/0196: received 8 February 2007,
revised and accepted 22 June 2007

doi:10.1111/j.1472-765X.2007.02225.x

**Abstract**

**Aims:** To reduce carbonated ferric green rust (GR*) using an iron respiring bacterium and obtain its reduced homologue, the mixed FeII–FeIII carbonated green rust (GR).

**Methods and Results:** The GR* was chemically synthesized by oxidation of the GR and was incubated with *Shewanella putrefaciens* cells at a defined [FeIII]/[cell] ratio. Sodium methanoate served as the sole electron donor. The GR* was quickly transformed in GR (iron reducing rate = 8·7 mmol l⁻¹ h⁻¹).

**Conclusions:** Ferric green rust is available for *S. putrefaciens* respiration as an electron acceptor. The reversibility of the GR redox state can be driven by bacterial activity.

**Significance and Impact of the Study:** This work suggests that GRs would act as an electronic balance in presence of bacteria. It provides also new perspectives for using iron reducing bacterial activity to regenerate the reactive form of GR during soil or water decontamination processes.

**Introduction**

The mixed valence compound fougerite (IMA 2003-057), a layered FeII–FeIII hydroxysalt called green rust, is found in transitionally oxic and anoxic environments like hydro-morphic soils (Trolard *et al.* 1996; Génin *et al.* 1998). Green rusts are constituted of FeII–FeIII hydroxide sheets separated by interlayers of anions and water molecules balancing the cation layer charge. The formula FeII·₄FeIII₂(OH)₁₂CO₃·3H₂O has been proposed for the stoichiometric mixed FeII–FeIII carbonated green rust (GR) (Hansen 2001). More recently, it was shown that oxidation of GR, by air or H₂O₂, leads to a new compound called ‘ferric green rust’ (GR*) (Refait *et al.* 2003; Legrand *et al.* 2004). The variation of the [FeIII]/[Fe*total*] ratio of the fougerite would correspond to different oxidation states of the green rust compounds (Génin *et al.* 2005; Ruby *et al.* 2006) where the fully oxidized state corresponds to the GR*. GR can be the by-products of iron respiring bacteria when two-line ferrihydrite or lepidocrocite (γ-FeOOH) are the starting FeIII substrate (Parmar *et al.* 2001; Glasauer *et al.* 2002; Ona-Nguema *et al.* 2002; Zachara *et al.* 2002; Zegeye *et al.* 2005).

By coupling the organic carbon oxidation to the iron oxide reduction, the iron respiring bacteria are well-known to make the connection between the biogeochemical cycles of carbon and iron. Thus, such bacteria contribute to bioremediation of contaminated soils by removing the hydrocarbons in anoxic environment for example (Anderson *et al.* 1998). But these bacteria can also contribute to decontamination in other ways, i.e. by producing reactive FeII species during the FeIII respiration. As GRs are considered to be the most reactive FeII-bearing compounds in reaction with organic and inorganic pollutants (Myneni *et al.* 1997; Erbs *et al.* 1999; Loyaux-Lawniczak *et al.* 2000; Refait *et al.* 2000; O'Loughlin *et al.* 2003; Elsner *et al.* 2004), it is therefore relevant to control their biotic synthesis for future prospects in soil or water remediation applications. Currently, GR formation from γ-FeOOH bioreduction by *Shewanella putrefaciens* can be controlled by adjusting the bacterial inoculum size. In particular, the formation of the thermodynamically stable compound magnetite was avoided (Zegeye *et al.* 2007). GR* represents also a potential ferric substrate for iron reducing bacteria and GR formation. However, its bacterial reducibility and the resulting products have not yet
been examined. One may expect that the formation of GR from GR\* will occur at a high reduction rate due to the similarity between their crystalline structure.

Therefore, the purpose of this work was to demonstrate that GR\* can serve as an electron acceptor for iron reducing bacteria and be transformed into a mixed FeII–FeIII green rust. A fast reduction of the GR\* by S. *putrefaciens* and a subsequent transformation into GR was effectively observed, suggesting that green rust compounds could act as an electronic balance in the environment or during diverse water or soil remediation processes. These findings suggest that GR\* is a very good substrate for the GR bio-(re)generation.

## Materials and methods

### Preparation of the oxyhydroxycarbonate green rust

The fully GR\* has been prepared by fast oxidation of a GR by using a hydrogen peroxide solution (Legrand *et al.* 2004; Ruby *et al.* 2006). The GR has been prepared by the co-precipitation of FeII and FeIII species in a basic solution containing NaOH and Na₂CO₃ (Ruby *et al.* 2003).

The GR\* suspension was centrifuged (10 000 g, 10 min) and washed with purified water (MilliQ+/Helix40; Millipore, Billerica, MA, USA). The pellet was dried at ambient temperature and then crushed in a mortar to obtain a fine and homogenous powder. Finally, the powder was added to the culture medium and treated with ultrasound (1 min, 40 W, with a probe of 13-mm diameter) in aliquots of 25 ml. As green rust compounds are not stable at high temperature (80°C), only the culture medium was heat sterilized prior to the mineral addition.

### Culture conditions and cell preparation

Shewanella putrefaciens CIP80-40 (Collection of Institut Pasteur, Paris, France) suspension was prepared according to Zegeye *et al.* (2007). After 24 h of aerobic growth in tryptic soy broth (30 g l⁻¹, 51019; BioMérieux, Marcy l'Étoile, France) the cells, exhibiting a typical salmon colour, were washed (centrifugation 10 000 g, 10 min, 20°C) and resuspended in NaCl solution (7 g l⁻¹ in purified water). The cells (1·1 × 10¹⁰ cell ml⁻¹, final concentration) were added to serum bottles (sealed with thick butyl rubber stoppers, Bellco Glass Inc., Vineland, NJ, USA) containing the anaerobic incubation medium (O₂ free, flushed with 100% N₂ gas) composed of the following constituents (per litre of basal mineral medium described previously, Zegeye *et al.* 2007) : GR\* [Fe₆O₄(OH)₈·CO₃·3H₂O], 30 g (i.e. 277 mmol FeIII l⁻¹); sodium met-hanoate (as electron donor), 10·9 g (i.e. 160 mmol l⁻¹);

anthraquinone disulfonate (as electron shuttle), 100 µmol l⁻¹. All incubations were at 30°C in the dark and conducted in triplicate.

### Analytical techniques

FeIII reduction was monitored by measuring the FeII accumulation over time. The amount of FeII extracted by HCl 1 mol l⁻¹ after 1 week, was determined with ortho-phenanthroline (Fadrus and Maly 1975). As previously described (Zegeye *et al.* 2007), the initial rate of reduction was computed from the first derivative of a nonlinear curve fit for FeII vs time data to the following equation: $Fe_t^{II} = Fe_{max}^{II} [1 - \exp(-k_{obs}t)]$, where $Fe_t^{II}$ is the concentration of total FeII produced at time t, $Fe_{max}^{II}$ is the maximum FeII concentration observed at the end of the reduction period, and $k_{obs}$ is the pseudo-first order constant.

The soluble fraction of FeII was performed on the solution filtered through 0·22 µm filter before extraction by HCl. The cell numbers were determined by the epifluorescence microscopy technique (Hobbie *et al.* 1977) modified by Saby *et al.* (1997).

The solids were characterized by X-ray diffraction (XRD) and transmission electron microscopy (TEM). Briefly, the suspension was filtered under N₂ atmosphere, mixed with glycerol to avoid oxidation and poured on a sample carrier to be analysed by XRD. For TEM observation, one drop of the suspension was laid on an amorphous carbon-coated grid and the sample was loaded into the microscope (CM20/STEM; Philips Electronics, Eindhoven, Netherlands). For observation, using optical microscopy (BX-51; Olympus, Tokyo, Japan) a droplet of the suspension was deposited on a glass slide, covered with a cover slip and observed under natural light using phase contrast.

## Results

### Characterization of the starting material

Optical microscopy observation of the chemically synthesized GR\* shows brown solids of different size up to 20 µm, but the resolution of the microscope is too low to allow the crystal shape to be distinguished (Fig. 1a). The GR\* observed by TEM displays crystal length around 100 nm and exhibits a typical hexagonal geometry (Fig. 1b). Electron diffraction and XRD indicate $d_{hkl}$ parameters specific of a GR\* (Table 1, Fig. 1b,c).

### Ferric green rust reduction

Total FeII concentration measurement (i.e. both in the solid and aqueous phase) showed a rapid increase as soon
<figure><img src="image_2.png" /><figcaption>**Figure 1** Characterization and observation of the ferric carbonated green rust by (a) optic microscopy in natural light; (b) transmission electronic microscopy; and (c) X-ray diffraction. The *d*<sub>hkl</sub> values are indicated on each peak of the diffractogram, these values are characteristic of the structure of the solid and here are specific of the GR* compounds. The inset image represents the electron diffraction of the crystals, the *d*<sub>hkl</sub> values have been calculated by using the radius of the diffraction pattern (Table 1).</figcaption></figure>

as the bacterial cells were added to the mineral suspension (Fig. 2). The initial rate of reduction was 8.7 mmol l⁻¹ h⁻¹ (SD = 5%). To our knowledge, this is the fastest reduction rate for iron bioreducing systems leading to FeII–FeIII GR formation.

The GR* reduction ceased when approx. 50% of the FeIII (277 mmol l⁻¹ FeIII were initially added) have been reduced, i.e. after 92 h (around 4 days) of incubation when a plateau was reached at 140 mmol l⁻¹ of FeII. The soluble fraction of FeII at the end of the incubation time indicated that only 70 ± 17 µmol l⁻¹ remained in the

<figure><img src="image_3.png" /><figcaption>**Figure 2** Production of Fe(II) (both in the soluble and solid phases) during incubation of 46 mmol l⁻¹ of the GR* (277 equivalent millimolar iron concentration) with the *Shewanella* cells and with 160 mmol l⁻¹ of methanoate as the electron donor (closed circle). The blank (open circle) was performed without cells. The initial pH was comprised between 6.7 and 7.2. Data correspond to mean values of triplicate experiments ± SD.</figcaption></figure>

solution confirming that FeII species were essentially present in the solid state. A blank incubated in the same conditions without *Shewanella* cells, did not exhibited any FeII increase during the time of the experiment (Fig. 2), the soluble fraction of FeII measured was below the detection limit (<20 µmol l⁻¹).

## Secondary mineral by-product characterization

The solid phase was sampled after 6 days of incubation and characterized by XRD and TEM. XRD gave typical diffractogram of GR(CO₃²⁻) with a very intense peak at *d* = 0.7553 nm (Fig. 3b). TEM observation indicates a mixed population of large and small hexagonal crystals,

<table><caption>**Table 1** *d*<sub>hkl</sub> and a (mesh) parameters of the ferric GR (GR*) and of the stoichiometric GR from this study and from literature</caption><thead><tr><th>Mineral</th><th>(hkl)</th><th>d<sub>hkl</sub> calculated (nm) from electronic diffractogram data</th><th>Cell parameter a (nm) calculated from d<sub>hkl</sub></th><th>Cell parameter a (nm) from literature</th></tr></thead><tbody><tr><td rowspan="2">GR*(CO<sub>3</sub><sup>2-</sup>)</td><td>(100)</td><td>0.251 (0.006)</td><td>0.290 (0.007)</td><td rowspan="2">0.301*</td></tr><tr><td>(120)</td><td>0.147 (0.002)</td><td>0.295 (0.003)</td></tr><tr><td rowspan="2">GR(CO<sub>3</sub><sup>2-</sup>)</td><td>(100)</td><td>0.264 (0.007)</td><td>0.305 (0.008)</td><td rowspan="2">0.3175788†</td></tr><tr><td>(120)</td><td>0.154 (0.002)</td><td>0.308 (0.003)</td></tr></tbody></table>

*d*<sub>hkl</sub> is the interplanar distance between the crystallographical plans named '(hkl)'.

Values in parenthesis denote standard error.

*Génin *et al.* (2006).

†Aissa *et al.* (2006).
<figure><img src="image_4.png" /><figcaption>**Figure 3** Characterization and observation of the Fe<sup>II</sup>–Fe<sup>III</sup> carbonated green rust by (a) transmission electronic microscopy, arrows indicate the lower sized hexagonal crystals, the inset image represents the electron diffraction of the larger crystals at the top of the picture; (b) X-ray diffraction diagram, each annotated peak is specific of GR; (c) picture from optical microscopy in natural light (phase contrast); and (d) picture of bacteria adhered on a GR crystal, observed by optical microscopy under epifluorescence (bacteria were marked by SYTO9®; Molecular Probes).</figcaption></figure>

some crystals appeared fractured in particular the smaller ones, and others appeared notched especially the larger ones as also illustrated by optical microscopy (Fig. 3a,c). No other crystal shape was found. The electronic diffraction of the large hexagonal crystals confirms the nature of the GR (Fig. 3a). The GR cell parameter *a* is higher than the one measured for GR* (Table 1) because the deprotonation induces a global compaction of the structure (Génin *et al.* 2006). The small sized crystals were mainly associated in aggregates and were constituted of perfectly hexagonal crystals, similar in shape to the starting GR* (Fig. 1), and by fractured crystals. Electronic diffraction of these crystal aggregates gives a series of rings corresponding to both GR and GR* (not shown). In addition, a Mössbauer spectroscopy analysis was performed (at ambient temperature) on the GR* bioreduction products, it confirms the presence of both Fe<sup>II</sup> and Fe<sup>III</sup> oxidation states in the solid product (not shown).

A physical association was revealed between bacteria and the green hexagonal crystals, which was assumed to be the Fe<sup>II</sup>–Fe<sup>III</sup> GR. As it can be seen on optical microscopy, especially when bacteria have been coloured by SYTO9 (S34854, Molecular Probes, Invitrogen Corp., Eugene, OR, USA), the GR crystals are partially covered by rod-shaped bacteria attached end-on to the crystals by their apical side (Fig. 3d).

## Discussion

We demonstrate in the present study that the ferric oxyhydroxide GR* can serve as an electron acceptor for *Shewanella* cells. In addition, the GR* is the third ferric crystalline solid, after lepidocrocite and ferrihydrite, being transformed into GR by bacterial activity (Parmar *et al.* 2001; Ona-Nguema *et al.* 2002). The reduction proceeds at a particularly high reduction rate (near 9 mmol l<sup>-1</sup> h<sup>-1</sup>). In comparison, the well crystallized ferric oxyhydroxide lepidocrocite (γ-FeOOH), common in intermittently anaerobic soils (Cornell and Schwertmann 2003), is reduced at a much lower rate (<1 mmol l<sup>-1</sup>) under the same experimental conditions (Zegeye 2006; Zegeye *et al.* 2007). As the solubility of iron oxides is a key factor in determining their reduction rate (Bonneville *et al.* 2004), the high reduction rate observed could be explained by the relatively high solubility of the GR (Bourrié *et al.* 1999).

Of significance is that the starting and the final products present a similar crystalline structure as indicated by XRD and TEM. Specifically, the morphology of the crystal is hexagonal and the diffraction lines are conserved, for example the 003 line (Figs 1c and 3b). This suggests that the bacteria reduce the solid without any dissolution-recrystallization processes and that such a direct
transformation could be energetically more favourable,
and could also explained the high reduction rate.

The question of how electrons are transferred inside
the mineral arises. The bacteria are systematically local-
ized at the surface of the GR crystals by their apical
region, indicating a close association between cells and
GR crystal during its reduction. The same behaviour is
usually observed when lepidocrocite was the starting
material (Zegeye *et al.* 2006), indicating that the *de novo*
GR formation, or GR\* reduction into GR in that parti-
cular case, occurs closely to the bacterial cells. This
contrasts to what has been previously suggested with 2-line
ferrihydrite as the Fe\*\*\* substrate (Glasauer *et al.* 2002).

Shewanella oneidensis MR1 can transfer electrons to
iron oxides through pili-like appendages serving as elec-
tron-wires (Gorby et al. 2006). It is probable that S. pu-
trefaciens CIP80.40 can proceed similarly. However, in
our experimental conditions, we did not identify any such
appendages, probably because our culture conditions were
not the same as those used for such pili-like expression,
or simply because cell suspension preparation (centrifuga-
tion, agitation, washing step) broke down these structures
(Gorby et al. 2006). In this study, the transfer of electrons
probably proceeds through direct contact between the cell
surface and the solid and/or via the electron shuttle
(AQDS) as previously suggested (Myers and Myers 1997;
Lovley et al. 1998). However, both bacteria and AQDS
are too large to diffuse between the hydroxide sheets, sug-
gesting that an electron transfer occurred inside the solid
as it was recently suggested in regards to pollutants
reduction by structural Fe²⁺ of GR (Williams and Scherer
2004). On the other hand, a de novo GR formation from

<figure><img src="image_5.png" /><figcaption>**Figure 4** A possible mechanism of a nondestructive oxidation of the green rust by oxidants (Ox-Mn⁺) (Ruby *et al.* 2006) and its reduction (i.e. recovery of its reactivity) by bacterial reduction (this study). The oxidative green rust is GR\* (in brown) and the reduced form is GR (in green).</figcaption></figure>

a dissolution-recrystallization mechanism should not be
discarded. In that case, the GR would be formed by co-
precipitation of Fe^III^ and Fe^II^ as shown by Géhin *et al.*
(2002).

The GR\* could be the best substrate to produce fouge-
rite or its GR homologous. Firstly because it has crystal-
line structure closely related to the Fe\*\*-Fe\*\*\* GR (Génin
et al. 2005). Secondly, it was previously shown that the
oxidation of GR can directly yield to GR\* (Ruby et al.
2006). Therefore, the green rusts would play the role of
an electron balance sometimes having the function of oxi-
dant, sometimes that of reductant. As the transformation
from one redox state to another occurs inside a unique
crystalline structure as illustrated in the Fig. 4, reactions
with fast kinetics are expected. In that way, the reducing
power of the very reactive Fe\*\*-bearing mineral green rust
(Elsner et al. 2004) would be quickly restored.

The results presented here provide the first step to an
applied research aiming to bacterially drive the forma-
tion/regeneration of these very reactive and versatile min-
erals, which are able to alternatively serve as an electron
donor for redox reactions with oxidized pollutants and
an electron acceptor for iron reducing bacterial activity.
As GR activity is known to occur in permeable reactive
barriers for soil remediation (e.g. Gaber et al. 2002), the
bacterially driven transformation described here could aid
greatly in a better understanding the mechanisms of such
GR-related processes.

Acknowledgements

The authors would like to thank Dr Jaafar Ghanbaja
(Université Henri Poincaré, Nancy, France), and Dr Isa-
belle Bihannic (LEM UMR 7569, CNRS-INPL, Nancy,
France) for TEM and DRX analysis respectively.

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