# Iron isotope fractionation in magnetite produced by the marine magnetotactic bacterium Magnetovibrio blakemorei

Vincent Busigny, François Mathon, Matthieu Amor, François Guyot, Nicolas Menguy, Christopher T. Lefèvre

<figure><img src="image_1.png" /></figure>

## To cite this version:

Vincent Busigny, François Mathon, Matthieu Amor, François Guyot, Nicolas Menguy, et al. Iron isotope fractionation in magnetite produced by the marine magnetotactic bacterium Magnetovibrio blakemorei. Geochimica et Cosmochimica Acta, 2025, 398, pp.83-98. 10.1016/j.gca.2025.03.028. hal-05148603

HAL Id: hal-05148603

https://u-paris.hal.science/hal-05148603v1

Submitted on 7 Jul 2025

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Iron isotope fractionation in magnetite produced by the marine
magnetotactic bacterium *Magnetovibrio blakemorei*

Vincent Busigny¹,*, François P. Mathon¹,², Matthieu Amor³,
François Guyot⁴, Nicolas Menguy⁴, Christopher T. Lefevre²

¹ Université de Paris, Institut de Physique du Globe de Paris, CNRS, F-75005, Paris, France

² Aix-Marseille Université, CEA, CNRS, BIAM, 13115 Saint-Paul-lez-Durance, France

³ Univ. Lyon, ENSL, UCBL, UJM, CNRS, LGL-TPE, 69007 Lyon, France

⁴ Muséum National d'Histoire Naturelle, Sorbonne Université, UMR CNRS 7590. Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie (IMPMC), Paris, France

*For correspondence, e-mail to: busigny@ipgp.fr
**Abstract**

Magnetotactic bacteria (MTB) possess the ability to precipitate intracellular nanosized magnetite. Their emergence may date back to the early Archean (e.g. 3 Ga) but evidence of such a long history has yet to be provided. MTB identification in the rock record relies on magnetofossils, the residual magnetite crystals that may survive in time to rock deformation and low-grade metamorphism. Several criteria such as size, shape and magnetic properties have been proposed to distinguish magnetofossils from other magnetite sources. Recent experimental work on the freshwater strain *Magnetospirillum magneticum* AMB-1 revealed that Fe isotope composition represents a promising additional criterion, but isotopic characterization of distinct MTB strains and of their magnetite needs to be provided to evaluate the robustness of iron isotope signatures for paleontological applications. In the present work, we investigate the marine strain *Magnetovibrio blakemorei* MV-1 under various conditions selected to examine the influence of Fe concentration (50, 100 and 150 µM) and redox state (Fe(II)-ascorbate or Fe(III)-citrate) on bacterial biomineralization and determine associated Fe isotope fractionations. Our results confirm the preferential incorporation of light iron isotopes into magnetite relative to the bacterial growth medium previously observed in AMB-1. Modeling the evolution of the growth medium and magnetite isotope compositions by Rayleigh distillation process yields iron isotope fractionations (i.e. $\Delta^{56}\text{Fe}_{\text{growth medium-magnetite}} = \delta^{56}\text{Fe}_{\text{growth medium}} - \delta^{56}\text{Fe}_{\text{magnetite}}$) ranging between 0.2 and 0.9 ‰ with a mean value of 0.55 (± 0.35) ‰. This isotope fractionation shows no clear relation with iron concentration or redox state. Importantly, the enrichment in light iron isotope of biological magnetite contrasts with the isotopic characteristics of magnetite formed by abiotic precipitation, the latter being enriched in heavy isotopes. This suggests that Fe isotopes could be used as a biosignature for magnetofossils identification in terrestrial or extraterrestrial samples. Finally, in contrast with previous results obtained on AMB-1, no specific enrichment of $^{57}\text{Fe}$ (relative to $^{54}\text{Fe}$, $^{56}\text{Fe}$ and $^{58}\text{Fe}$) could be detected in MV-1 magnetite, which we interpret by differences in iron budgets between the two strains.

Keywords: magnetite; biomineralization; biosignature; magnetotactic bacteria; magnetofossils; iron isotopes
# 1. Introduction

Magnetotactic bacteria (MTB) are ubiquitous microorganisms living in oxic-anoxic transition zones and producing intracellular magnetite (Fe₃O₄) or greigite (Fe₃S₄) nanoparticles in a genetically controlled manner (Uebe and Schüler, 2016). In MTB, a single magnetite or greigite crystal is contained in organelles called magnetosomes and consisting of a bi-layered lipid membrane surrounding the nanoparticle (Bazylinski and Frankel, 2004). Magnetosomes are aligned as chains and provide a permanent magnetic moment to the cell enabling MTB to passively align with the geomagnetic field and optimize their search for microaerophilic conditions in redox-transition zones (Uebe and Schüler, 2016). Phylogenetic reconstructions suggest that MTB emerged as early as the Archean period, possibly around 3 Ga ago (Lin et al., 2017). Archean paleo-environmental conditions must have been ideal for MTB emergence because (i) redox gradients were probably common due to the presence of oxygen oases produced by local oxygenic photosynthetic activity (Riding et al., 2014; Wilmeth et al., 2019), and (ii) the Earth's magnetic field was active and strong enough for magnetosome to confer a selective advantage (Kirschvink, 1982; Biggin et al., 2015). Identification of the fossil remains of MTB (hereafter referred to as magnetofossils) in ancient sedimentary rocks would thus provide strong constraints on the evolution of life and biomineralization processes through geological times, and help reconstructing paleoenvironmental conditions. However, distinguishing magnetofossils from abiotic magnetite remains challenging due to the lack of clear signatures pointing to their biological origin.

After bacterial death, the magnetite nanoparticles can be efficiently preserved in the sedimentary record (Chang and Kirschvink, 1989). Several criteria based on morphology, crystallography, magnetism (Thomas-Keprta et al., 2000; Kopp and Kirschvink, 2008; Jimenez-Lopez et al., 2010; Li et al., 2013; Amor et al., 2020), as well as chemical and isotopic compositions (Amor et al., 2015, 2016, 2018; Mathon et al., 2024) have been proposed to identify magnetofossils. Preliminary work on the freshwater *Magnetospirillum magneticum* strain AMB-1 revealed unique geochemical signatures and iron isotopic compositions that are not shared with any other known source of magnetite (Amor et al., 2015, 2016, 2018; Mathon et al., 2024) and could potentially be used as a biosignature. The δ⁵⁶Fe values of AMB-1 magnetite was found to be depleted in heavy iron isotopes relative to the growth medium (Amor et al., 2016, 2018), by 1.5 to 2.5‰ (δ⁵⁶Fe = [($^{56}$Fe/$^{54}$Fe)sample / ($^{56}$Fe/$^{54}$Fe)standard - 1]×1000, where the standard is IRMM-014, a synthetic metal from the Institute for Reference for Material and
Measurement). The light iron isotope signature observed in biological magnetite may represent a specific feature of MTB since abiotic precipitation of magnetite from aqueous Fe(II) produces a fractionation in the opposite direction, with an enrichment of heavy isotopes by ~+1.5‰ (Friedrich et al., 2014). More importantly, by measuring all iron isotopes, AMB-1 magnetite showed a unique deviation from mass-dependent relationships, with a preferential incorporation of the odd isotope ($^{57}$Fe) relative to even isotopes ($^{54}$Fe, $^{56}$Fe, $^{58}$Fe) (Amor et al., 2016, 2018). This deviation from mass-dependent fractionation was expressed using the capital delta notation $\Delta^{57}\text{Fe}$ (i.e. $\Delta^{57}\text{Fe} = \delta^{57}\text{Fe} - 1000 \times [(1 + \delta^{56}\text{Fe}/1000)^\beta - 1]$, where $\beta$ corresponds to the variation of $^{57}\text{Fe}/^{54}\text{Fe}$ and $^{56}\text{Fe}/^{54}\text{Fe}$ mass ratios, i.e. $\beta = (1/m_{54} - 1/m_{57})/(1/m_{54} - 1/m_{56}) = 1.475$; see Young et al., 2002). Magnetite produced in AMB-1 culture showed $\Delta^{57}\text{Fe}$ values up to +1.56 ‰ (Amor et al., 2016, 2018). The origin of this mass-independent isotopic signature is uncertain but may result from magnetic isotope effects associated with a radical pair mechanism. Iron isotope anomalies could represent a powerful tool for magnetofossil identification, notably because this would survive burial diagenesis and metamorphism even in case of magnetite recrystallization (if not exchanged isotopically and buffered by other Fe-bearing minerals).

Preliminary results obtained on iron isotopes are promising for identifying biogenic magnetite but need to be validated in additional MTB strains. Moreover, the results obtained on AMB-1 differed from those of a pioneer work, where no iron isotope fractionation was observed during magnetite biomineralization in the magnetotactic strains MV-1 and MS-1 (Mandernack et al., 1999). The origin of this discrepancy is unclear but may derive from different strains and/or culture conditions. A possible key parameter explaining these apparent contradictory results is the iron concentration in the external solution that could control the intracellular iron mass balance: AMB-1 cultures were prepared at higher iron concentrations (150 µM) than MS-1 and MV-1 cultures (30-70 µM). Accordingly, iron isotope fractionation associated with magnetite biomineralization in MTB needs to be investigated further in experimental studies, through cultures under various Fe concentrations.

The main objective of the present work is to establish whether iron isotopes can be used as a proxy to identify magnetofossils in the rock record. More specifically, we aim at (1) expanding the isotopic features determined in AMB-1 cultures (low $\delta^{56}\text{Fe}$ values in magnetite relative to the growth medium and positive $\Delta^{57}\text{Fe}$ values) to other MTB strains, and (2) constraining the impact of iron conditions (i.e., concentration and oxidation state) on the iron isotope signatures
in MTB. We selected the marine MTB strain, *Magnetovibrio blakemorei* MV-1 (Bazylinski et al., 2013). MV-1 belongs to the *Alphaproteobacteria* class of the *Pseudomonadota* phylum (like AMB-1) and is a facultative microaerophilic bacterium with the ability to grow anaerobically while respiring nitrous oxide (N₂O) (Bazylinski et al., 1988). Bacteria were grown in media containing either Fe(II)-ascorbate or Fe(III)-citrate supplemented at different concentrations (50, 100 and 150 µM). The cellular components of MV-1 were separated and purified prior to iron isotope analyses by mass spectrometry. The results confirmed the preferential incorporation of light iron isotopes into biological magnetite. However, no Δ⁵⁷Fe anomaly could be detected in MV-1 cultures, contrasting with the results obtained on the strain AMB-1. The results are interpreted in terms of biological mechanisms and bacterial iron mass balance, and their implications for microbial paleontology are finally discussed.

## 2. Material and methods

### 2.1. Culture and cell components extraction

#### 2.1.1. MV-1 cultures

The cultures of *Magnetovibrio blakemorei* strain MV-1 (DSMZ #18854) were undertaken under anoxic and reducing conditions in an artificial seawater mixed with a modified Wolfe's mineral solution (Wolin et al., 1963; Frankel et al., 1997; Mathon, 2021, Mathon et al., 2024). All experiments were performed in 1-L Schott DURAN™ Pressure Plus flasks containing the growth medium solution as described in Bazylinski et al. (2013). Two groups of experiments were designed by supplying either Fe(II)-ascorbate or Fe(III)-citrate to the growth medium, at concentrations of 50, 100 and 150 µM. Molecular oxygen (O₂) was replaced by nitrous oxide (N₂O), also used by bacteria as the terminal electron acceptor (Bazylinski et al., 2013). A redox indicator (resazurin) was added to the medium in order to detect potential O₂ introduction. The sources of organic carbon used as electron donor were sodium succinate and sodium acetate. Three replicates were prepared from 9 bottles of 1L for all conditions. Before cultures, 10 mL of the initial growth medium were sub-sampled to determine the iron concentration and isotope composition of the initial growth medium. Iron concentration and redox state were monitored every 24 h during cultivation, and determined using the ferrozine method (Viollier et al., 2000). The magnetic cell density, estimated from the measurement of optical density at 565 nm (OD565) and the C<sub>mag</sub> coefficient (Schüler et al., 1995), were monitored by spectrophotometry
throughout experiments. C<sub>mag</sub> relies on the differential measurement of a culture's optical density when a magnet is oriented either vertically or horizontally close to the cell suspension. It is defined as the ratio of the maximum and minimum optical density and quantifies the capacity of bacteria to orient along an external magnetic field. Previous work demonstrated from C<sub>mag</sub> and Fe uptake relationships that C<sub>mag</sub> can be used as a proxy for magnetite formation (Schüler and Baeuerlein, 1998).

2.1.2. *Cell components extraction and magnetosome/magnetite purification*

Initial cell density after inoculation ranged between 2.3 and 6.6 × 10⁸ cell/mL (Mathon et al., 2024), determined from calibrated optical density (calibration preliminary obtained from cell counting using Malassez counting chamber after cultures of MV-1 with Fe concentrations ranging from 30 to 150 µM, n=24; Mathon, 2021). The cultures were stopped when cell density reached > 5.4 × 10⁹ cell/mL and C<sub>mag</sub> became roughly constant, indicating that no additional magnetosomes were produced. Forty mL of the final growth medium were sub-sampled and filtered at 0.22 µm for subsequent measurement of iron concentration and isotope composition. The magnetosomes were separated and purified using a procedure described in previous studies (Ginet et al., 2011; Mériaux et al., 2015). In brief, cells were separated from the growth medium in Teflon flasks of 500 mL by centrifugation at 4500 rpm and 4°C for 45 min. The bacterial pellets were then pooled in 50 mL polypropylene Falcon tubes, centrifuged again at 4500 rpm and 4°C for 20 min. The final pellets were suspended in a cold buffer (hereafter named B1), containing HEPES (20 mM, pH = 7.5), NaCl (0.9 wt%), EDTA (1 mM), and glycerol (8 vol%) (Mériaux et al. 2015), as well as a protease inhibitors cocktail (ref. P8849 Sigma-Aldrich; 20 µl of cocktail in 10 mL of B1), at 4°C to prevent sample degradation by enzymatic activity. Cells were lysed with a French Press operating at 7 MPa. Three lysis cycles were run (with no rinsing treatment between cycles) to ensure complete disruption of the bacteria. Magnetosomes were magnetically recovered from the bacterial lysates by placing a magnet against the 50-mL Falcon tube containing the magnetosome suspension. Tubes were left for 1.5 hour at 4°C before collecting the bacterial lysates. Magnetic recovery was repeated four times for 30 min in a buffer (B1), and 5 times in a second buffer (B2) consisting of B1 buffer without EDTA. Half of the magnetic samples was collected for analysis of whole magnetosomes. The other half was treated further to remove the magnetosome membrane and analyze only the purified magnetite crystals. Digestion of the magnetosome membrane was performed using a protocol adapted
from a previous study (Amor et al., 2015). Magnetosomes were sonicated at 100 Hz in a TES
solution (0.5 vol% Triton X, 10 mM EDTA, 1 vol% SDS) at 60°C for one hour. Then, they
were washed twice with Milli-Q water and recovered with a magnet. This operation was
repeated 5 times before collecting and storing purified magnetite in Milli-Q water at -20°C.

Magnetosome and magnetite samples were observed by transmission electron microscopy (TEM) at each extraction/purification step using a JEOL JEM-2100F microscope operating at 200 kV. Magnetite size and shape were measured with ImageJ (i) inside the cells after harvesting bacteria, (ii) after magnetosome extraction from the bacterial lysates, and (iii) after digestion of the magnetosome membrane (Mathon et al., 2024). We assessed the efficiency of magnetite nanoparticles recovery by calculating iron mass balance between initial and final states, and by monitoring the magnetite size throughout the purification procedure (Mathon, 2021). Both approaches indicated consistent results and a good recovery rates with no evidence for magnetite alteration and/or loss.

## 2.2. Iron chemistry and isotope composition analysis

### 2.2.1 Chemistry

Iron was analyzed in four types of samples: growth media (initial and final states), bacterial lysates, magnetosomes and purified magnetite. Organic compounds were first digested from samples using successive HNO₃ treatments. The removal of organic matter was required because iron can form organic complexes and therefore lead to an incomplete recovery during separation by ion chromatography, with possible isotope fractionation (Anbar et al., 2000).

Bacterial lysates and growth media particularly enriched in organic compounds were digested with additional HNO₃ and H₂O₂ treatments at 95°C to ensure complete oxidation. After complete removal of organic materials, lysate and growth medium samples were treated to separate iron from the salty solutions, corresponding to the synthetic marine solution for growth media and buffer solution used for lysate extraction. We adapted a protocol previously developed for seawater chemistry (Chou and Moffat, 2000). Our samples were reacted with HClO₄ for 2h at 120°C and evaporated, to ensure iron oxidation into Fe³⁺. Then, they were dissolved in 1M HNO₃, and basified with NH₄OH to precipitate ferric oxyhydroxides. They were finally centrifuged for 20 min at 4000 rpm, the supernatant was eliminated and the precipitate was re-dissolved in concentrated HNO₃ for further chemistry. For all types of samples, iron was separated from matrix elements by anion-exchange chromatography in HCl medium (Strelow, 1980; Dauphas et al, 2004; Busigny et al, 2014; Amor et al., 2018). The
procedural yield was always >90%. The blank level of the present protocol has been evaluated by systematic analysis in all sample series, prepared as described above but without any sample. It was always below 50 ng iron and represented less than 1% of the bulk Fe in the sample.

### 2.2.2. Isotope analysis

Iron isotope measurements were performed on a ThermoFischer Scientific™ Neptune Plus MC-ICP-MS (Multiple Collector - Inductively Coupled Plasma - Mass Spectrometer) at IPGP. The method was described in previous contributions (Busigny et al., 2014; Amor et al., 2018) and is summarized hereafter. The four iron isotopes were measured simultaneously at masses 54, 56, 57 and 58. The contributions of Cr and Ni on masses 54 and 58 were monitored and corrected by measuring masses 53 and 60. High-resolution mode was used to resolve iron isotopes from argide interferences (Weyer and Schwieters, 2003). Sample solutions were nebulized and introduced into the mass spectrometer using the Stable Introduction System (SIS). Samples were analyzed in 0.3 M HNO₃ at iron concentrations ranging between 2 to 3 ppm, providing a signal intensity between 15 and 20 Volts on ⁵⁶Fe. Instrumental mass bias was corrected using the sample-standard bracketing method (Albarède and Beard, 2004). The ⁵⁶Fe/⁵⁴Fe, ⁵⁷Fe/⁵⁴Fe and ⁵⁸Fe/⁵⁴Fe ratios were expressed in the δ notation (in ‰) as:

$$ \delta^{5x}Fe_{sample} = [(^{5x}Fe/^{54}Fe)_{sample}/(^{5x}Fe/^{54}Fe)_{IRMM-014} - 1] \times 1\,000 \quad (\text{Eq. 1}) $$

where 5x represents either 56, 57 or 58, and the standard IRMM-014 is a pure iron metal from the Institute for Reference Materials and Measurements. The precision of iron isotope measurements was estimated through replicate analyses of multiple preparations of the international rock geostandard IF-G (Dauphas et al., 2009; Craddock and Dauphas, 2011), a 3.8 Ga old Banded Iron Formation from Isua, Greenland, and an internal laboratory standard labelled ISVB, corresponding to a pure iron solution. The average δ⁵⁶Fe, δ⁵⁷Fe and Δ⁵⁷Fe values were obtained with precisions better than ± 0.05, ± 0.08 and ± 0.06 ‰, respectively (2SD external reproducibility).

## 3. Results

### 3.1. Transmission electron microscopy observations

Representative TEM images of (i) MV-1 bacteria harvested after laboratory cultures, (ii) extracted chains of magnetosomes and (iii) magnetite nanocrystals for which organic
membrane has been chemically removed are shown in Figure 1. Importantly, the crystal surfaces and well-defined edges are indistinguishable before and after chemical treatments, indicating that magnetite was unaffected. High-resolution TEM observation confirms that the organic membrane surrounded magnetite nanocrystals has been efficiently removed. Finally, the elongation of MV-1 magnetite (i.e. length) shows identical distribution for all conditions and at all steps of the chemical treatments, indicating that no magnetite was dissolved or lost during sample preparation (Fig. S2 in Mathon et al., 2024).

## 3.2. Iron redox state in MV-1 cultures

Iron concentrations and redox states of the growth media (GM) were monitored during cultures to better model bacterial iron uptake (Table S1, Fig. S1). In Fe(II)-ascorbate experiments, iron was maintained in its ferrous form in the GM and its concentration progressively decreased with time (Fig. S1). In Fe(III)-citrate experiments, the GM contained a mixing of ferric and ferrous iron that formed immediately after adding the iron source, implying partial reduction of Fe(III) to Fe(II) similar to what has been observed in AMB-1 cultures (Amor et al, 2020a). Reducing compounds such as organic carbon sources added to MTB growth medium have the potential to reduce a fraction of the ferric iron. In our MV-1 cultures, citrate was used as an Fe(III) chelator to maintain its ferric state but cysteine necessary for MV-1 growth (Bazylinski et al., 2013a) likely participated in its partial reduction. Moreover, a degassing step purging dissolved O₂ with N₂O (used as terminal electron acceptor) generated anoxic conditions. Such reducing conditions led to the observed partial Fe(III) reduction. Comparison of the results obtained from the various Fe(III)-citrate experiments indicated that the lowest Fe(II) fractions were obtained at higher initial Fe(III)-citrate concentrations. On average, Fe(III) reduction represented ~75%, 50% and 20% for initial Fe(III)-citrate concentrations of 50, 100 and 150 µM, respectively (Fig. S1).

## 3.3. Iron distribution between growth medium, lysate, magnetosome and magnetite

The iron fractions in the different subcellular reservoirs were determined from mass balance calculation based on measurements (Fig. 2). We checked that the total amount of iron recovered at the end of experiments was similar to the amount introduced at the initial stage (Mathon, 2021). The proportions reported here correspond to the average values obtained from replicates of all culture conditions, which show highly reproducible and consistent results (Table S2).
For Fe(II)-ascorbate conditions, Figure 2 shows a significant rise in the proportion of Fe left in the final GM (from 11 to 69 %) with increasing initial iron concentration (50, 100 and 150 µM). This effect is not observed in Fe(III)-citrate condition, where final GM represent proportions of 62, 20 and 86 % for Fe concentrations of 50, 100 and 150 µM (Fig. 2). For initial iron concentrations of 50 and 150 µM, bacteria assimilated more iron in Fe(II) than in Fe(III) experiments. In contrast, experiments performed at 100 µM gave opposite results with more Fe left in the GM for Fe(II) than Fe(III) experiments (42% vs 20%; Fig. 2). For a given experimental condition, magnetite represented either identical or slightly lower fraction of the total iron when compared to magnetosomes (Fig. 2). This indicates that the cultures were efficiently split in two near-identical fractions but also that a small fraction of Fe may be contained in the magnetosome membrane. A rough calculation from average data presented in Figure 2 suggests that about 85 % (76 to 100%) of magnetosome iron is in magnetite versus 15 % in membrane. The proportion of iron contained in magnetite and magnetosomes tends to decrease with increasing initial iron concentration (average of 14.5 to 4% in Fe(III)-citrate experiments and 38 to 12.5% in Fe(II)-ascorbate experiments when the bacteria were grown from 50 to 150 µM). Regardless of the initial condition, the iron fraction contained in the bacterial lysate never exceeded 14% of the total iron. Iron accumulated in the cell is thus dominantly sequestered in the magnetite/magnetosome fraction (>70 %), with an average of 84 ± 7 % (Table 2). Only one culture condition provided with 150 µM Fe(III)-citrate showed lower iron content in magnetite/magnetosome, with proportions between 29 and 71 % for replicate analyses (Table 2). Overall, the low iron accumulation in MV-1 lysate contradicts the previous results obtained on strain AMB-1, where iron of the lysate represented around 70 % of the total cellular iron (Amor et al., 2020a).

### 3.4. Iron isotope compositions in MV-1 culture products

The MV-1 cultures performed in bottles were individually analyzed (Table 1) but the results presented in this section correspond to mean values. The first and second paragraphs summarize the δ<sup>56</sup>Fe values obtained in Fe(II)-ascorbate and Fe(III)-citrate conditions, respectively, while the third one gives a brief overview of Δ<sup>57</sup>Fe values of magnetite and magnetosomes produced in all experiments.
In Fe(II)-ascorbate conditions, the mean $\delta^{56}$Fe value of the initial growth medium (GM) was -0.12 (± 0.03) ‰ and can be taken as a reference. The final GM was isotopically fractionated relative to the initial GM and yielded higher $\delta^{56}$Fe values in all experiments (Fig. 3a). Specifically, the $\delta^{56}$Fe values of final GM are 0.98 (± 0.02), 0.55 (± 0.04), and 0.18 (± 0.02) ‰ for cultures with initial concentrations of 50, 100, and 150 µM respectively. The $\delta^{56}$Fe values of magnetite and magnetosomes are generally lower than those of the initial GM, except for the condition with 50 µM Fe(II)-ascorbate where they are undistinguishable (Fig. 3a), likely due to the strong transfer of Fe in magnetite/magnetosome (77 % of the initial total Fe; Fig. 2a). Pairs of magnetite and magnetosomes of Fe(II)-ascorbate experiments have similar $\delta^{56}$Fe values (Table 1) although the 150 µM experiment shows a moderate depletion in heavy isotopes by ~0.1‰ in magnetosomes relative to the associated magnetite. For initial iron conditions of 100 and 150 µM, $\delta^{56}$Fe values in magnetosomes are -0.55 (± 0.02) and -0.60 (± 0.02) ‰, and in magnetite -0.52 (± 0.02) and -0.49 (± 0.02) ‰, respectively. The bacterial lysates have intermediate values between the final GM and magnetite/magnetosomes pairs (Fig. 3a).

In Fe(III)-citrate condition, the mean $\delta^{56}$Fe value of initial GM was 0.43 (± 0.03) ‰ (Fig. 3b). Iron isotopes in the final GM at 50 and 100 µM were fractionated with respect to initial GM, and recorded a higher $\delta^{56}$Fe value as observed in the Fe(II)-ascorbate experiment. The $\delta^{56}$Fe values were 0.76 (±0.02) and 1.20 (±0.06) ‰ for initial iron concentrations of 50 and 100 µM, respectively. In contrast, the final GM of the experiment with 150 µM Fe(III)-citrate had a $\delta^{56}$Fe value (0.45 ± 0.01 ‰; Fig. 3b) undistinguishable from the initial GM. All magnetite and magnetosome samples had lower $\delta^{56}$Fe values than the initial GM. For the experiments conducted with 100 and 150 µM Fe(III)-citrate, magnetite and magnetosome were indistinguishable within uncertainties, with values of 0.33 (±0.02) and 0.32 (±0.01) ‰ at 100 µM, and -0.05 (±0.01) and -0.08 (±0.03) ‰ at 150 µM. When bacteria were grown with 50 µM Fe(III)-citrate, magnetite was isotopically lighter than magnetosome, with $\delta^{56}$Fe values of 0.11 (±0.04) and 0.29 (±0.02) ‰, respectively. For the 100-µM condition, the bacterial lysate had an intermediate isotopic composition between the final GM and magnetite/magnetosomes, with $\delta^{56}$Fe value of 0.60 (±0.02) ‰. In cultures provided with 50 and 150 µM Fe(III)-citrate, $\delta^{56}$Fe of the lysate had more erratic values (Fig. 3b). At 50 µM, the lysate $\delta^{56}$Fe was lighter than the initial GM (0.28 ± 0.03 ‰) similar to magnetosomes. At 100 and 150 µM, the lysate was heavier than the initial GM, with $\delta^{56}$Fe value up to 1.08 (±0.01) ‰ for 150 µM.
In order to identify any deviation from mass-dependent fractionation laws, we have calculated $\Delta^{57}$Fe values for magnetite and magnetosome samples (see definition in the introduction). Figure 4 reports the $\Delta^{57}$Fe values as a function of $\delta^{56}$Fe for magnetite and magnetosomes extracted after experiments with Fe(II)-ascorbate and Fe(III)-citrate. It reveals that $\Delta^{57}$Fe values are not different from zero within uncertainties (±0.06‰ obtained from external reproducibility on standards), regardless of the initial iron concentration and redox state (see also data in Table 1). Therefore, magnetite produced by MV-1 displayed no detectable deviation from the mass-dependent fractionation law, in contrast with previous findings obtained from magnetite produced by AMB-1 cultivated with Fe(III) sources (Amor et al., 2016, 2018).

# 4. Discussion

## 4.1. Iron isotope fractionations in MV-1 cultures

Microbial cultures under controlled conditions enable quantification of Fe isotope fractionations in biological systems, as exemplified by Fe-oxidizing bacteria either anaerobically or through $O_2$-production (e.g. Croal et al., 2004; Balci et al., 2007; Swanner et al., 2017), dissimilatory Fe-reducing bacteria (Crosby et al., 2007), Fe uptake by diazothrophic bacteria (Wasylenski et al., 2007), or processes of Fe assimilation and intracellular magnetite production in MTB (Mandernack et al., 1999; Amor et al., 2016, 2018). The iron isotope composition of magnetite nanocrystals produced by MV-1 was previously examined in laboratory cultures by Mandernack et al. (1999). The authors concluded that no isotope fractionation occurred under several iron concentrations ranging from 28 to 71 µM at three different temperatures (4, 28 and 32°C). This pioneer work was based on isotope measurement by thermo-ionization mass spectrometry (TIMS), a technique associated with relatively large instrumental uncertainty on $\delta^{56}$Fe, around ± 0.3 ‰ (Mandernack et al., 1999). Additionally, uncertainty associated with replicate analyses in this previous study differed up to ~0.6 ‰, making identification of potential isotope fractionation challenging considering the total range of $\delta^{56}$Fe values obtained in magnetite from the present contribution (i.e. ~0.6‰ for either Fe(II)-ascorbate or Fe(III)-citrate conditions, Fig. 5b). We examined MV-1 iron isotope fractionation by investigating a larger range of iron concentration and by working with both reduced (Fe(II)-ascorbate) and oxidized (Fe(III)-citrate) iron sources. The precision of our analytical measurements is also strongly improved by the use of MC-ICP-MS, with uncertainties below
± 0.05 ‰ (2σ). Our results on MV-1 evidence a significant isotope fractionation related to iron uptake into the cell and subsequent magnetite formation. In the following sub-sections, we discuss, model and/or quantify these Fe isotope fractionations.

4.1.1. Isotope fractionation upon iron uptake: an estimation from growth media evolution

In this section, we first estimate the isotope fractionation associated with iron assimilation by MV-1 using the growth medium data from all individual culture conditions, and then discuss the effect of variable Fe redox state on this isotope fractionation. All samples studied in the present work were collected at the initial or final stages of MV-1 cultures. Bacteria were grown in closed system (i.e. bottles) and the final iron concentration in the growth media records a range of iron depletion due to variable assimilation and magnetite precipitation in the cell. In all conditions, the δ⁵⁶Fe values of the residual growth media were increased compared to initial conditions, indicating that MV-1 favors the assimilation of light isotopes. Assuming that iron taken up into the cell was irreversibly removed from the growth medium and that precipitated magnetite could not re-equilibrate (i.e. no isotope exchange) further with the growth medium, the system can be described by a Rayleigh distillation process. We have thus calculated a Rayleigh model and compared it with the growth media data measured in all bottles of Fe(II)-ascorbate and Fe(III)-citrate conditions (Fig. 5a). The equation representing the growth medium evolution can be written as (see for instance Sharp, 2017):

$$ \delta^{56}\text{Fe}_{\text{GM}} = (\delta^{56}\text{Fe}_{\text{GM initial}} + 1000) \times F^{\alpha-1} - 1000 \quad (\text{Eq. 2}) $$

where α is the isotopic fractionation factor associated with iron assimilation, F is the fraction of initial iron remaining in the growth medium, and the subscript GM stands for growth medium. Figure 5a shows that MV-1 data fit well with a Rayleigh distillation process, and that the isotope compositions of the growth media measured in Fe(III)-citrate conditions are more scattered than in Fe(II)-ascorbate conditions which is in good agreement with previous results in AMB-1 (Amor et al. 2018). Interestingly, the model illustrates a restricted and overlapping range of isotope fractionation factors (α) in the two conditions, Fe(II)-ascorbate and Fe(III)-citrate. For Fe(II) experiments, the α values best matching the data range from 0.9992 to 0.9996, while in Fe(III) experiments, α values range from 0.9991 to 0.9997 (Fig. 5a). This implies that Fe taken up by MV-1 is associated with an isotope fractionation ranging between 0.3 and 0.9
‰, with a mean value around 0.6 ± 0.3 ‰ similar between Fe(II) and Fe(III) sources. Data obtained from the same experimental conditions do not always align on the same Rayleigh distillation curve (Fig. 5a). A source of isotope variability in the Rayleigh plots could result from Fe(II) back-diffusion from the cell to the growth medium, as already observed in AMB-1 cultures (Amor et al., 2018). Alternatively, this may be due to slightly different Fe concentrations and bacterial growth evolution, as well as non-perfect reproducibility of Fe redox state from one bottle to another. In particular, the results of Fe(III)-citrate experiments exhibit larger dispersion in the Rayleigh curves (Fig. 5), indicating that Fe redox variability could explain part of the observed variations. Specifically, diffusion of intracellular reduced iron to Fe(III)-containing growth medium could trigger isotope fractionation between Fe(II) and Fe(III).

It is important to evaluate the impact of Fe redox state on the global isotope fractionations determined here. Indeed, part of the fractionation assigned to the assimilation process might be derived from the abiotic reduction in the growth medium. We have therefore calculated the isotope composition of Fe(II) and Fe(III) in solution using their measured abundances (Table S1) and the isotope composition of the total dissolved Fe (Table 1) at the starting point of the experiments. We assumed that the isotope exchange between dissolved Fe(II) and Fe(III) reached equilibrium, and considered a $\Delta^{56}\text{Fe}_{\text{Fe(III)-Fe(II)}}$ value of 2.8 ‰ at room temperature (Welch et al., 2003). In the case of Fe(II)-ascorbate experiments, 93 to 99 % of the total Fe is in the form of Fe(II) (Table S1, Fig. S1). The $\delta^{56}\text{Fe}$ values calculated for dissolved Fe(II) (-0.12 to -0.34 ‰) are close to those of total Fe (-0.09 to -0.11 ‰) (Table 3), which is consistent with its predominance in solution. In contrast, the $\delta^{56}\text{Fe}$ values of the poorly-abundant dissolved Fe(III) are markedly different, with positive values between 2.48 and 2.68 ‰ (Table 3). Two conclusions can be derived from these calculations. First, preferential assimilation of Fe(III) is unlikely because it should induce a progressive decrease of the $\delta^{56}\text{Fe}$ values in the growth medium, and the opposite is observed (Fig. 5a). Second, the isotope effect of redox exchange on Fe(II) can produce $\delta^{56}\text{Fe}$ values slightly lower (-0.03 to -0.22‰; Table 3) than the total dissolved Fe but cannot explain a depletion of 0.3 to 0.9 ‰ as observed in the global trends of the growth medium (Fig. 5a). An additional isotope fractionation related to Fe uptake is clearly required. In the case of Fe(III)-citrate experiments, the starting points contain a fraction of only 26, 69 and 61% of Fe(III), the rest being in the form of Fe(II) (Table 3). The $\delta^{56}\text{Fe}$ values of the total Fe were identical within uncertainty for all conditions (0.41 to 0.45 ‰; Table 3). The
calculated δ⁵⁶Fe values for dissolved Fe(II) and Fe(III) were very distinct, with values from -0.32 to -1.65‰ and +1.28 to +2.53 ‰, respectively. Once again, a preferential assimilation of Fe(III) is incompatible with the increasing δ⁵⁶Fe trend observed in the growth medium (Fig. 5a). The direction and range of isotope fractionation determined to fit the Rayleigh model (0.3 to 0.9 ‰) are however compatible with a preferential assimilation of isotopically-light dissolved Fe(II), a result in good agreement with previous conclusions obtained from AMB-1 cultures (Amor et al., 2018). The abiotic reduction of Fe(III) in the growth medium may thus have played a significant role in the Fe(III)-citrate conditions. A surprising but striking observation is however that the mean isotope fractionations determined for Fe(II) and Fe(III) experiments (0.6‰, α = 0.9994, Fig. 5a) are the same for both conditions. If not a coincidence, this possibly indicates that assimilation (rather than reduction) was the dominant process of Fe isotope fractionation in our MV-1 cultures.

4.1.2. Similar isotope composition in magnetosome and magnetite

Until recently, it was generally assumed that intracellular iron in MTB was dominated by the
magnetite fraction even though other intracellular iron phases had been identified (e.g., Frankel
et al, 1983; Faivre et al, 2007). However, laboratory cultures of AMB-1 followed by chemical
and magnetic analyses showed that a significant pool of iron, distinct from magnetite, could
accumulate in the cell and represent the major cellular iron reservoir (Amor et al., 2020a). Iron
enrichment has also been revealed in the magnetosome membrane of both cultured and
environmental MTB by scanning transmission electron microscopy at sub-nanometric
resolution (Werckmann et al. 2017) although this has never been confirmed by other methods.
The present data obtained on MV-1 support this latter suggestion since iron in the magnetosome
fraction is higher than in the purified magnetite (Fig. 2), except in one condition with identical
values. A mass balance calculation based on MV-1 data indicates that ~85% of the
magnetosome iron is in magnetite and 15% in membrane. Although minor, it is important to
evaluate if the magnetosome membrane carries an isotope signature different from the
magnetite crystals. Figure 6 plots the iron isotope composition of magnetite versus
magnetosomes for pairs of identical initial conditions (i.e. 3 bottles distributed in two fractions
for each condition, i.e. 1.5 bottles for magnetosome and 1.5 bottles for magnetite). The data
show a good linear correlation close to the 1:1 line. The difference between magnetite and
magnetosome $\delta^{56}$Fe values ranges between -0.07 and 0.17‰, with an average of 0.06 ± 0.18 ‰ (2σ, n=12), not significantly different from zero. This demonstrates that within uncertainty, magnetite and magnetosome have similar Fe isotope composition, and therefore that iron contained in the membrane has an isotope composition similar to magnetite. In the following section, we will consider that both magnetite and magnetosome isotope signatures are representative of the magnetite fraction and therefore will call them “magnetite”.

4.1.3. Iron isotope fractionation associated to cellular cycling and magnetite precipitation

The fate and storage of iron after assimilation in MV-1 are poorly known, but have been
extensively investigated and discussed for AMB-1 and MSR-1 strains (see for instance review
in Amor et al., 2020 and references therein). From an iron isotope study in AMB-1, it was
suggested that iron was stored as Fe(III) species in the intracellular medium whatever the Fe(II)
or Fe(III) sources provided to the bacteria (Amor et al., 2018). Such Fe(III) was proposed to be
stored in proteins such as ferritins, bacterioferritins and DNA-binding proteins (Baumgartner
et al., 2013; Fdez-Gubied et al, 2013; Uebe et al., 2019). Oxidized iron could then be partially
reduced and transferred to magnetosomes as labile species for subsequent magnetite formation
(Frankel et al., 1983; Faivre et al., 2007; Amor et al., 2018, 2020). Here, iron isotope
distribution in initial and final growth medium, lysate and magnetite/magnetosome samples
from MV-1 cultures can be used to provide constraints on the cellular iron cycling and
magnetite biomineralization processes. A comparison of δ<sup>56</sup>Fe values in lysate with those of
magnetite and magnetosomes shows that the former is always higher than the later, with
Δ<sup>56</sup>Fe<sub>lysate-mag</sub> ranging between 0.10 and 1.34 ‰ (Fig. 7, Table 2). This isotopic distribution is
compatible with a storage as Fe(III) in the cell and a partial reduction step before transfer to the
magnetosome vesicle and magnetite formation, as previously proposed for AMB-1 (Amor et
al., 2018). We note however that, in the case of AMB-1, the difference in δ<sup>56</sup>Fe between
bacterial lysate and magnetite was higher, with values between 1.97 and 2.3 ‰ (Amor et al.,
2018). A major difference between the present results on MV-1 and those obtained previously
on AMB-1 is that intracellular iron in AMB-1 was mostly contained in the magnetite-free
cellular fraction (lysates) while magnetite represented at most 30% of the total intracellular iron
(Amor et al., 2020a). The opposite picture is observed in MV-1 cultures, since the magnetite
fraction largely dominates the iron budget accumulated in the cells. With that in mind, our MV-
1 samples can be divided into two groups. The first one includes most samples and corresponds
to $\Delta^{56}\text{Fe}_{\text{lysate-mag}}$ between 0.10 and 0.53 ‰, with average of 0.33 ± 0.15 ‰ (n=16). For these samples, more than 70 % of the cellular iron is contained in magnetite (mean value of 84 ± 7 %; Fig. 7, Table 2). A second group of four samples corresponding to cultures with 150 µM Fe(III)-citrate, shows a range of $\Delta^{56}\text{Fe}_{\text{lysate-mag}}$ between 0.93 to 1.34 ‰, with a mean at 1.13 ± 0.19 ‰ (n=4). In these four samples, the fraction of iron in magnetite is variable, from 29 to 71%, and averages 53 ± 21 % of the total cellular iron. Although there is no obvious correlation between $\Delta^{56}\text{Fe}_{\text{lysate-mag}}$ values and the fraction of intracellular iron in magnetite (Fig. 7), these results suggest that a large isotope fractionation between lysate and magnetite can only exist when sufficient iron is left in the lysate. In cases where almost all the Fe assimilated in the cell is transferred to magnetite, the apparent isotope fractionation will tend to lower values, close to 0. This can be explained by mass balance considerations. When iron entering into the cell is efficiently transferred to magnetite, its accumulation in the lysate is limited and buffered by fluxes from external solution into the cell. This process leads a decrease of $\delta^{56}\text{Fe}$ values in the lysate of most experimental conditions as compared to the 150 µM Fe(III)-citrate condition. Such explanation is not only compatible with the present data on MV-1 but also with previous results obtained on AMB-1 (Amor et al., 2018).

### 4.1.4. Assessing isotope fractionation upon iron uptake from magnetite composition

The large range of $\delta^{56}\text{Fe}$ values measured in MV-1 magnetite and magnetosome (Fig. 3) can be used as another mean to estimate the isotope fractionation associated with iron assimilation process. Although an Fe isotope fractionation is clearly detected between lysate and magnetite, the fraction of intracellular Fe in MV-1 cultures is dominated by magnetite (> 70% of the total Fe for 18 over 20 samples; e.g. Fig. 7). We can therefore neglect the lysate fraction and assume that intracellular Fe is made up entirely of magnetite. This simplification allows us to apply a Rayleigh distillation model developed above (section 4.1.1) to magnetite. In that case, magnetite and magnetosome represent the same cumulative products formed during the culture. The Fe isotope composition of magnetite and magnetosome can be related to that of the growth medium from the following mass balance equation, describing initial and final states as:

$$ \delta^{56}\text{Fe}_{\text{GM initial}} = F \times \delta^{56}\text{Fe}_{\text{GM}} + (1-F) \times \delta^{56}\text{Fe}_{\text{mag}} \quad (\text{Eq. 3}) $$

where F is the fraction of iron remaining in the growth medium, and the subscripts GM and mag stand for the growth medium and cumulated magnetite/magnetosomes, respectively.
Equation 3 can then be modified to express the isotope composition of magnetite/magnetosome as:

$$ \delta^{56}Fe_{mag} = \frac{1}{1-F} \times (\delta^{56}Fe_{GM initial} - \delta^{56}Fe_{GM} \times F) \quad (\text{Eq. 4}) $$

In the model, $\delta^{56}Fe_{GM}$ is calculated from Equation 2 and includes the fractionation factor $\alpha$ between growth medium and cell as a free parameter. Rayleigh distillation curves are illustrated in Figure 5b with corresponding $\alpha$ values and compared with the experimental results on MV-1. Cultures with Fe(II)-ascorbate and Fe(III)-citrate are compatible with $\alpha$ values between growth medium and cell ranging from 0.9991 to 0.9997, and from 0.9992 to 0.9998, respectively (Fig. 5b). Again, $\alpha$ values determined for Fe(II) and Fe(III) conditions largely overlap. The Fe isotope fractionation from the magnetite cumulative products range from 0.2 to 0.9 ‰, with a mean value of 0.55 ± 0.35 ‰, which is similar to the value determined above from growth medium modelling (0.3 to 0.9 ‰, mean value of 0.6 ± 0.3 ‰; Fig. 5a). This demonstrates the overall validity of our approximation that intracellular iron is mostly contained in magnetite. Since iron taken up by MV-1 cells is dominantly transferred to magnetosomes, the isotopic fractionation determined from cumulative fraction modelling represents another assessment of iron uptake fractionation. Accordingly, our calculations offer two independent methods for determining the isotope fractionation associated with iron uptake by MV-1: one based on the growth medium evolution (section 4.1.1) and the other based on magnetite/magnetosomes record. The combination of these two assessments gives an overall isotope fractionation between growth medium and magnetite around 0.55 ± 0.35 ‰.

A similar model was built for $\delta^{56}Fe$ value of the whole cells using all available lysate and magnetite data in order to test if a more precise estimate of the isotope fractionation could be determined (supplementary material). However, some lysate samples were lost during preparation and much less lysate than magnetite/magnetosome data were available. Overall, the results of this calculation gave $\alpha$ values between growth medium and cell similar to those obtained here using magnetite/magnetosome data only. The $\alpha$ values ranged from 0.9991 to 0.9997 and from 0.9993 to 0.9999 for Fe(II)-ascorbate and Fe(III)-citrate experiments, respectively (Fig. S2). Yet, two samples cultivated with 150 µM Fe(III)-citrate had $\delta^{56}Fe$ values of the whole cell higher than initial growth medium, which was unpredicted. They may reflect preferential incorporation of isotopically heavy Fe(III) into the cell since Fe(II) and Fe(III) were
both present in the growth medium for this condition (Table S1), and Fe(III) was likely enriched
in heavy isotope by ~3‰ relative to Fe(II) (Welch et al., 2003; Anbar et al., 2005).

4.2. *Comparison of iron uptake in MV-1 and AMB-1*

Modeling of iron isotope data in MV-1 culture yielded an isotope fractionation of 0.55 (± 0.35) ‰ between growth medium and magnetite, attributed essentially to iron uptake process. Such isotope fractionation favors light iron isotope enrichment in the cell, leaving the growth medium progressively enriched in heavy isotopes (Fig. 5). Interestingly, a preferential assimilation of light iron isotopes was also deduced from analyses of dissolved iron in phytoplankton blooms from several open ocean regions (Radic et al., 2011; Elwood et al., 2014). However, this isotopic behavior differs from results obtained previously on AMB-1 (Amor et al., 2018). Indeed, when single iron sources (either ferrous or ferric iron) were supplemented to its growth media, AMB-1 preferentially assimilated heavy iron isotopes, leaving the residual growth medium enriched in light isotopes (Amor et al, 2016, 2018). The origin of the opposite behaviors between MV-1 and AMB-1 is unclear but several hypotheses can be examined. For instance, the contrasting isotope fractionation upon Fe uptake by MV-1 and AMB-1 might be related to a variability of Fe redox state in the growth media (a mixing between Fe(II) and Fe(III) species; Fig. S1). Equilibrium fractionation in aqueous solutions enriches Fe(III) species by 3‰ in δ⁵⁶Fe relative to Fe(II) species (Johnson et al, 2002; Anbar et al, 2005). Moreover, assimilation of ferric and ferrous compounds by microorganisms are known to rely on distinct biological processes yielding contrasting isotope fractionations (Wasylenski et al., 2007; Radic et al., 2011; Elwood et al., 2014; Mulholland et al., 2015; Amor et al, 2018). Dissolved Fe(II) is generally considered to easily enter the cell, by a diffusion-like process, while Fe(III) is actively but more slowly incorporated into bacteria (Schüler and Bauerlein, 1996; Andrews et al, 2003; Suzuki et al, 2006). The case of mixed Fe(II)/Fe(III) sources in AMB-1 cultures led to progressive enrichment of heavy isotopes in the residual growth medium. This was interpreted as a preferential uptake of ferrous species enriched in light isotopes owing to Fe(II)/Fe(III) isotope equilibration (Amor et al, 2018). Although a similar scenario could be proposed for MV-1, the culture with Fe(II)-ascorbate was mostly composed of Fe(II) (i.e. Fe(II)/Fe<sub>tot</sub> ranging from 0.92 to 0.99, see Table S1), implying that δ⁵⁶Fe of Fe(II) was identical to the bulk, and could not impart a heavy isotope signature to Fe(III) in solution. We conclude that this process cannot explain the heavy signature of the residual growth medium, at least for
Fe(II)-ascorbate condition. More complex mechanisms related to differences in Fe uptake by AMB-1 and MV-1 may be at play and explain some of the isotopic variability. Molecular biology studies showed evidences for active Fe assimilation by excretion of siderophore in AMB-1 (Calugay et al., 2006), and both ferric and ferrous transporter proteins in the case of Magnetospirillum magneticum MS-1 (Taoka et al., 2009). A comparative study between MV-1 and one of its non-magnetic mutant revealed the role of proteins in Fe uptake (Dubbels et al., 2004). Therefore, distinct Fe isotope fractionation in MV-1 and AMB-1 likely resulted from various strategies of Fe(II) and Fe(III) assimilation processes.

### 4.3. Iron isotopes as a biosignature of magnetite produced by MTB

Among the various criteria used for magnetofossils identification (Thomas-Keprta et al., 2000; Kopp and Kirschvink, 2008), iron isotopes have yet only been explored in the strain AMB-1 (Amor et al., 2016, 2018). The isotope signatures proposed to be used as a tool for magnetofossils identification were (i) light isotope composition relative to the growth medium ($\delta^{56}$Fe values lower than -1‰ and reaching -2.1‰) and (ii) unique mass-independent fractionation on the odd isotope ($^{57}$Fe) (Amor et al., 2016, 2018). In the present work, we have confirmed the enrichment in light iron isotopes of MTB magnetite relative to the growth medium but have not been able to identify any $\Delta^{57}$Fe anomaly (Fig. 4). The production of a $\Delta^{57}$Fe anomaly in AMB-1 magnetite was proposed to occur during iron oxidation and/or reduction in the cell and associated radical pair mechanisms (Amor et al, 2018). Exchange of iron and its isotopes between AMB-1 cells and the external medium was also shown to mask mass-independent signatures in magnetite, by diffusion of Fe carrying no anomaly. The lack of mass-independent variations in MV-1 can thus be explained by the iron budget in the cell: a complete, or almost complete, transfer of iron in the lysate to magnetite would mask potential mass-independent fractionations generated in the cell. However, we cannot exclude that distinct oxidation and/or reduction processes in MV-1 could generate mass-dependent fractionations only, in particular because the two samples with the highest fraction of cellular Fe in the lysate show no $\Delta^{57}$Fe anomaly (samples magnetosome #2 and magnetite #2 in the cultures with 150 µm Fe(III)-citrate, Table 1).

The light iron isotope signature of magnetite produced by MTB, both AMB-1 and MV-1, represents an important finding. Indeed, this result contrasts with experimental precipitation of abiotic magnetite, which is systematically enriched in heavy isotopes relative to the solution
(Frierdich et al., 2014). The iron isotope fractionations measured between abiotic magnetite and aqueous solutions are around +1.5 ‰ (Δ⁵⁶Fe_magnetite-solution), and therefore differ by at least 1.7 ‰ with the isotope fractionation determined between MTB and growth medium (i.e. Δ⁵⁶Fe_magnetite-growth medium from -2.5 to -0.2 ‰; Amor et al., 2018, this study). This also differs from the isotope fractionation associated with iron oxide precipitation by iron-metabolizing bacteria (Δ⁵⁶FeFe-oxide-solution > 1 ‰; Anbar, 2004; Bullen et al., 2001; Croal et al., 2004; Swanner et al., 2017). Finally, magnetite can also precipitate from the biological activity of dissimilatory iron-reducing bacteria (DIRB) capable of oxidizing organic matter while using ferric oxyhydroxides as electron acceptor. Experimental work showed that the magnetite produced by DIRB was enriched in heavy isotopes relative to dissolved Fe(II), with Δ⁵⁶Fe_magnetite-aqueous Fe(II) of +1.3 ‰ near equilibrium (Johnson et al., 2005). Interestingly this isotope fractionation value is very close to the one determined for abiotic precipitation of magnetite (Frierdich et al., 2014) and is again clearly distinct from the values produced by MTB. Magnetite biomineralized by DIRB could carry a negative δ⁵⁶Fe value similar to MTB magnetite only if the fluid has even more negative δ⁵⁶Fe value, which might be the case in the very early steps of partial ferric oxyhydroxides reduction (Crosby et al., 2005). Therefore, the isotope composition of magnetite produced by MTB can potentially be distinguished from other sources of abiotic and biological magnetite providing that the fluid composition is available or can be estimated. For instance, iron-bearing mineral phases such as siderite or other accessory minerals for which isotope fractionation between mineral and solution are known (Blanchard et al., 2009) could be used as proxies for reconstructing the iron isotope composition of the paleofluid. In the case of absence of other iron phases, an alternative could be to measure magnetite from several nearby samples and estimate if an isotopic reservoir evolution allows the modeling of isotope fractionation values (Havas et al., 2021). Importantly, magnetite with negative δ⁵⁶Fe values could potentially be produced abiotically by so-called reservoir effect, i.e. successive precipitation of magnetite enriched in heavy isotope driving the fluid towards lighter isotope composition. In that case, isotope composition comparable to MTB magnetite could be produced. This kind of situation could be examined and identified by analyzing a distribution of magnetite in rock samples. Another way of avoiding reservoir effect would be to work on sediments deposited in Fe-rich conditions, where the dissolved reservoir can be considered as constant. This may be the case in ancient Archean sediments deposited in Fe-rich waters. We conclude that Δ⁵⁶Fe_magnetite-fluid could potentially be used as a biomarker of the magnetotactic
origin of magnetite. This will need to be tested further on environmental MTB where both
bacteria and fluid can be collected (Li et al., 2010; Busigny et al., 2021).

**5. Conclusion**

In the present contribution, we cultivated the marine strain Magnetovibrio blakemorei MV-1 under various conditions to measure the iron isotope composition of its magnetite and of different cell compartments. A range of iron concentrations (50, 100 and 150 µM) and redox states (Fe(II)-ascorbate or Fe(III)-citrate) was explored to evaluate their impact on iron isotope fractionation and biomineralization. In contrast with a pioneer work on MV-1 (Mandernack et al., 1999), our results demonstrate a significant isotope fractionation between magnetite and the bacterial growth medium. In all conditions, light iron isotopes are preferentially taken up into the cell, with fractionation values between 0.2 and 0.9 ‰ (average ~ 0.55 ± 0.35 ‰), and transferred to magnetite. Overall, iron accumulated in MV-1 cells is dominantly stored in the magnetite/magnetosomes fraction (>70 %) even though one culture condition (150 µM Fe(III)-citrate) showed accumulation in distinct intracellular reservoirs.

Previous cultures on AMB-1 also showed magnetite enriched in light isotopes relative to growth medium, even though the magnitude of this enrichment was higher. Accordingly, light iron isotope composition of MTB magnetite relative to aqueous medium may be used as a biosignature, in comparison with abiotic magnetite which is enriched in heavy isotope (Frierdich et al., 2014).

The $\Delta^{57}$Fe anomaly observed on AMB-1 magnetite was not detected here in MV-1 despite exploration of a wide range of iron concentrations and Fe(II)/Fe(III) conditions. The absence of $\Delta^{57}$Fe anomaly does not exclude a biological origin while its presence provides a strong support. Future laboratory experiments should re-examine AMB-1, as well as other MTB strains, to understand the reasons for producing an isotope anomaly and determine its conditions of applicability as a biosignature.

Finally, the results obtained from laboratory cultures on AMB-1 and MV-1 strains need to be
tested on environmental MTB harvested in natural systems. New methods have been developed
for the collection and purification of environmental MTB samples in either water column or
sediments, and can yield large quantities of MTB, thus meeting the requirements for future geochemical analyses (Busigny et al., 2020).

### CRediT authorship contribution statement

**Vincent Busigny:** Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft. **François P. Mathon:** Investigation, Formal analysis, Visualization, Writing – review & editing. **Matthieu Amor:** Methodology, Validation, Writing – review & editing. **François Guyot:** Conceptualization, Methodology. **Nicolas Menguy:** Methodology, Resources. **Christophe Lefèvre:** Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Validation, Writing – review & editing.

### Data availability

Data are available through IPGP Research Collection at https://doi.org/10.18715/IPGP.2025.m88ndizi

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgments

Colleagues from IPGP and BIAM-CEA Cadarache are thanked for analytical assistance and discussions. In particular, we thank Delphine Limmois for helping in clean room chemistry, and Pascale Louvat, Dimitri Rigoussen and Tu-Han Luu for supporting mass spectrometry analyses. This work was funded by the French National Research Agency (SIGMAG: ANR-18-CE31-0003 and BIOMAG: ANR-23-CE49-0007) and the Institut de Physique du Globe de Paris. V.B. thanks the Institut Universitaire de France (IUF#2017-2021) and INSU-CNRS Origines program (AAP2023) for funding. Part of this work was supported by IPGP multidisciplinary program PARI and by Région Île-de-France SESAME Grant no. 1015908. MA is supported by grant through the CNRS Tellus/INTERRVIE program (grant #MagBio).

## Appendix A. Supplementary material

Supplementary material includes a file with one figure illustrating the iron concentration and speciation monitored during MV-1 cultures (Fig. S1), as well as two tables reporting the iron
concentrations and redox states of the growth media monitored during MV-1 cultures (Table S1), and the iron distribution between final growth medium, lysate, magnetosome and magnetite fractions for all culture conditions (Table S2).

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<caption>Table 1</caption>

Iron isotope compositions of the growth media (GM) and cellular compartments (lysate, magnetosome, magnetite) in MV-1 cultures.

<table><thead><tr><th>Replic.</th><th>Sample type*</th><th>[Fe]<sup>**</sup><br>μM</th><th>δ<sup>56</sup>Fe<br>‰</th><th>±1SD<br>‰</th><th>δ<sup>57</sup>Fe<br>‰</th><th>±1SD<br>‰</th><th>δ<sup>58</sup>Fe<br>‰</th><th>±1SD<br>‰</th><th>Δ<sup>57</sup>Fe<br>‰</th><th>±1SD<br>‰</th></tr></thead><tbody><tr><td colspan="11"><strong>50 μM Fe(III)-citrate</strong></td></tr><tr><td></td><td>Initial GM</td><td>51</td><td>0.45</td><td>0.01</td><td>0.65</td><td>0.01</td><td>0.79</td><td>0.33</td><td>-0.02</td><td>0.02</td></tr><tr><td># 1</td><td>Magnetosome (mg)</td><td>21</td><td>0.15</td><td>0.02</td><td>0.21</td><td>0.02</td><td>-0.06</td><td>0.18</td><td>0.00</td><td>0.01</td></tr><tr><td># 1</td><td>Magnetite (mt)</td><td>20</td><td>0.29</td><td>0.02</td><td>0.38</td><td>0.04</td><td>0.45</td><td>0.52</td><td>-0.04</td><td>0.02</td></tr><tr><td># 1</td><td>Lysate mg</td><td>6</td><td>0.25</td><td>0.03</td><td>0.38</td><td>0.07</td><td>0.54</td><td>0.15</td><td>0.01</td><td>0.03</td></tr><tr><td># 1</td><td>Lysate mt</td><td>5</td><td>0.43</td><td>0.08</td><td>0.63</td><td>0.09</td><td>0.94</td><td>0.21</td><td>0.00</td><td>0.03</td></tr><tr><td># 1</td><td>Final GM mg</td><td>28</td><td>0.73</td><td>0.04</td><td>1.08</td><td>0.07</td><td>1.49</td><td>0.26</td><td>0.01</td><td>0.02</td></tr><tr><td># 1</td><td>Final GM mt</td><td>20</td><td>0.98</td><td>0.01</td><td>1.45</td><td>0.04</td><td>1.58</td><td>0.59</td><td>0.01</td><td>0.02</td></tr><tr><td># 1</td><td>Final GM</td><td>37</td><td>0.67</td><td>0.01</td><td>0.99</td><td>0.01</td><td>1.74</td><td>0.58</td><td>0.00</td><td>0.01</td></tr><tr><td># 2</td><td>Magnetosome (mg)</td><td>19</td><td>-0.09</td><td>0.06</td><td>-0.14</td><td>0.06</td><td>-0.50</td><td>0.46</td><td>-0.02</td><td>0.05</td></tr><tr><td># 2</td><td>Magnetite (mt)</td><td>18</td><td>0.07</td><td>0.02</td><td>0.13</td><td>0.02</td><td>-0.06</td><td>0.13</td><td>0.02</td><td>0.06</td></tr><tr><td># 2</td><td>Lysate mg</td><td>9</td><td>0.25</td><td>0.03</td><td>0.33</td><td>0.01</td><td>0.41</td><td>0.26</td><td>-0.03</td><td>0.04</td></tr><tr><td># 2</td><td>Lysate mt</td><td>7</td><td>0.24</td><td>0.02</td><td>0.32</td><td>0.04</td><td>0.79</td><td>0.25</td><td>-0.03</td><td>0.05</td></tr><tr><td># 2</td><td>Final GM mg</td><td>25</td><td>0.78</td><td>0.03</td><td>1.16</td><td>0.03</td><td>1.81</td><td>0.32</td><td>0.00</td><td>0.03</td></tr><tr><td># 2</td><td>Final GM mt</td><td>22</td><td>0.84</td><td>0.02</td><td>1.24</td><td>0.02</td><td>2.31</td><td>0.22</td><td>0.00</td><td>0.01</td></tr><tr><td># 2</td><td>Final GM</td><td>37</td><td>0.68</td><td>0.02</td><td>1.01</td><td>0.04</td><td>1.60</td><td>0.24</td><td>0.01</td><td>0.01</td></tr><tr><td colspan="11"><strong>50 μM Fe(II)-ascorbate</strong></td></tr><tr><td></td><td>Initial GM</td><td>42</td><td>-0.09</td><td>0.04</td><td>-0.15</td><td>0.08</td><td>-0.15</td><td>0.66</td><td>-0.02</td><td>0.03</td></tr><tr><td># 1</td><td>Magnetosome (mg)</td><td>49</td><td>-0.29</td><td>0.04</td><td>-0.40</td><td>0.03</td><td>-0.97</td><td>0.33</td><td>0.04</td><td>0.03</td></tr><tr><td># 1</td><td>Magnetite (mt)</td><td>45</td><td>-0.22</td><td>0.03</td><td>-0.29</td><td>0.01</td><td>-0.65</td><td>0.28</td><td>0.03</td><td>0.05</td></tr><tr><td># 1</td><td>Lysate mg</td><td>9</td><td>0.15</td><td>0.01</td><td>0.19</td><td>0.02</td><td>0.51</td><td>0.40</td><td>-0.03</td><td>0.02</td></tr><tr><td># 1</td><td>Lysate mt</td><td>9</td><td>0.26</td><td>0.02</td><td>0.39</td><td>0.03</td><td>0.73</td><td>0.42</td><td>0.02</td><td>0.01</td></tr><tr><td># 1</td><td>Final GM mg</td><td>3</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td># 1</td><td>Final GM mt</td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td># 1</td><td>Final GM</td><td>14</td><td>0.80</td><td>0.01</td><td>1.18</td><td>0.02</td><td>1.47</td><td>0.11</td><td>0.00</td><td>0.02</td></tr><tr><td># 2</td><td>Magnetosome (mg)</td><td>60</td><td>-0.11</td><td>0.01</td><td>-0.17</td><td>0.02</td><td>0.30</td><td>0.32</td><td>0.00</td><td>0.03</td></tr><tr><td># 2</td><td>Magnetite (mt)</td><td>48</td><td>-0.18</td><td>0.02</td><td>-0.28</td><td>0.02</td><td>-0.46</td><td>0.30</td><td>-0.01</td><td>0.03</td></tr><tr><td># 2</td><td>Lysate mg</td><td>9</td><td>0.42</td><td>0.02</td><td>0.62</td><td>0.04</td><td>0.87</td><td>0.23</td><td>0.00</td><td>0.01</td></tr><tr><td># 2</td><td>Lysate mt</td><td>10</td><td>0.09</td><td>0.02</td><td>0.10</td><td>0.04</td><td>0.33</td><td>0.59</td><td>-0.02</td><td>0.02</td></tr><tr><td># 2</td><td>Final GM mg</td><td>3</td><td>1.18</td><td>0.02</td><td>1.79</td><td>0.03</td><td>1.76</td><td>0.34</td><td>0.06</td><td>0.02</td></tr><tr><td># 2</td><td>Final GM mt</td><td>0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><tbody><tr><td># 2</td><td>Final GM</td><td>5</td><td>1.28</td><td>0.06</td><td>1.87</td><td>0.12</td><td>1.95</td><td>0.62</td><td>-0.02</td><td>0.05</td></tr><tr><td colspan="11">100 µM Fe(III)-citrate</td></tr><tr><td></td><td>Initial GM</td><td>108</td><td>0.42</td><td>0.02</td><td>0.63</td><td>0.03</td><td>0.80</td><td>0.16</td><td>0.02</td><td>0.01</td></tr><tr><td># 1</td><td>Magnetosome (mg)</td><td>105</td><td>0.19</td><td>0.01</td><td>0.27</td><td>0.04</td><td>0.29</td><td>0.20</td><td>0.00</td><td>0.04</td></tr><tr><td># 1</td><td>Magnetite (mt)</td><td>96</td><td>0.36</td><td>0.03</td><td>0.51</td><td>0.03</td><td>0.66</td><td>0.28</td><td>-0.02</td><td>0.02</td></tr><tr><td># 1</td><td>Lysate mg</td><td>6</td><td>0.37</td><td>0.04</td><td>0.48</td><td>0.07</td><td>0.74</td><td>0.13</td><td>-0.07</td><td>0.02</td></tr><tr><td># 1</td><td>Lysate mt</td><td>9</td><td>0.49</td><td>0.02</td><td>0.72</td><td>0.05</td><td>0.78</td><td>0.18</td><td>-0.01</td><td>0.03</td></tr><tr><td># 1</td><td>Final GM mg</td><td>35</td><td>1.31</td><td>0.11</td><td>1.92</td><td>0.17</td><td>2.41</td><td>0.24</td><td>-0.01</td><td>0.03</td></tr><tr><td># 1</td><td>Final GM mt</td><td>18</td><td>1.83</td><td>0.01</td><td>2.68</td><td>0.03</td><td>3.49</td><td>0.09</td><td>-0.01</td><td>0.03</td></tr><tr><td># 1</td><td>Final GM</td><td>38</td><td>1.20</td><td>0.04</td><td>1.79</td><td>0.05</td><td>2.36</td><td>0.08</td><td>0.02</td><td>0.01</td></tr><tr><td># 2</td><td>Magnetosome (mg)</td><td>163</td><td>0.41</td><td>0.02</td><td>0.64</td><td>0.03</td><td>0.90</td><td>0.26</td><td>0.03</td><td>0.01</td></tr><tr><td># 2</td><td>Magnetite (mt)</td><td>109</td><td>0.30</td><td>0.02</td><td>0.51</td><td>0.02</td><td>0.75</td><td>0.30</td><td>0.07</td><td>0.01</td></tr><tr><td># 2</td><td>Lysate mg</td><td>7</td><td>0.93</td><td>0.01</td><td>1.40</td><td>0.04</td><td>2.12</td><td>0.34</td><td>0.03</td><td>0.03</td></tr><tr><td># 2</td><td>Lysate mt</td><td>8</td><td>0.62</td><td>0.02</td><td>0.91</td><td>0.03</td><td>1.26</td><td>0.40</td><td>0.00</td><td>0.01</td></tr><tr><td># 2</td><td>Final GM mg</td><td>5</td><td>2.05</td><td>0.12</td><td>3.06</td><td>0.20</td><td>4.28</td><td>0.30</td><td>0.03</td><td>0.02</td></tr><tr><td># 2</td><td>Final GM mt</td><td>26</td><td>0.42</td><td>0.04</td><td>0.65</td><td>0.06</td><td>1.42</td><td>1.13</td><td>0.02</td><td>0.02</td></tr><tr><td># 2</td><td>Final GM</td><td>2</td><td>1.71</td><td>0.02</td><td>2.52</td><td>0.03</td><td>2.32</td><td>1.35</td><td>0.00</td><td>0.05</td></tr><tr><td colspan="11">100 µM Fe(II)-ascorbate</td></tr><tr><td></td><td>Initial GM</td><td>81</td><td>-0.16</td><td>0.03</td><td>-0.23</td><td>0.02</td><td>-0.18</td><td>0.24</td><td>0.01</td><td>0.02</td></tr><tr><td># 1</td><td>Magnetosome (mg)</td><td>68</td><td>-0.61</td><td>0.01</td><td>-0.89</td><td>0.04</td><td>-0.97</td><td>0.64</td><td>0.01</td><td>0.03</td></tr><tr><td># 1</td><td>Magnetite (mt)</td><td>59</td><td>-0.59</td><td>0.02</td><td>-0.87</td><td>0.03</td><td>-0.78</td><td>0.11</td><td>0.01</td><td>0.02</td></tr><tr><td># 1</td><td>Lysate mg</td><td>10</td><td>-0.11</td><td>0.02</td><td>-0.18</td><td>0.01</td><td>-0.03</td><td>0.44</td><td>-0.02</td><td>0.02</td></tr><tr><td># 1</td><td>Lysate mt</td><td>11</td><td>-0.17</td><td>0.02</td><td>-0.25</td><td>0.04</td><td>-0.18</td><td>0.30</td><td>0.00</td><td>0.02</td></tr><tr><td># 1</td><td>Final GM mg</td><td>39</td><td>0.61</td><td>0.03</td><td>0.96</td><td>0.04</td><td>1.24</td><td>0.31</td><td>0.06</td><td>0.03</td></tr><tr><td># 1</td><td>Final GM mt</td><td>39</td><td>0.51</td><td>0.11</td><td>0.70</td><td>0.16</td><td>1.39</td><td>0.65</td><td>-0.05</td><td>0.03</td></tr><tr><td># 1</td><td>Final GM</td><td>30</td><td>0.59</td><td>0.01</td><td>0.88</td><td>0.03</td><td>0.97</td><td>0.30</td><td>0.00</td><td>0.02</td></tr><tr><td># 2</td><td>Magnetosome (mg)</td><td>69</td><td>-0.49</td><td>0.02</td><td>-0.73</td><td>0.02</td><td>-1.42</td><td>0.27</td><td>-0.01</td><td>0.03</td></tr><tr><td># 2</td><td>Magnetite (mt)</td><td>59</td><td>-0.45</td><td>0.02</td><td>-0.64</td><td>0.01</td><td>-0.52</td><td>0.99</td><td>0.02</td><td>0.03</td></tr><tr><td># 2</td><td>Lysate mg</td><td>9</td><td>-0.14</td><td>0.01</td><td>-0.24</td><td>0.05</td><td>-0.50</td><td>0.90</td><td>-0.04</td><td>0.05</td></tr><tr><td># 2</td><td>Lysate mt</td><td>10</td><td>-0.13</td><td>0.02</td><td>-0.21</td><td>0.02</td><td>-0.58</td><td>0.58</td><td>-0.02</td><td>0.02</td></tr><tr><td># 2</td><td>Final GM mg</td><td>33</td><td>0.51</td><td>0.04</td><td>0.72</td><td>0.04</td><td>0.14</td><td>0.60</td><td>-0.03</td><td>0.05</td></tr><tr><td># 2</td><td>Final GM mt</td><td>33</td><td>0.65</td><td>0.01</td><td>0.97</td><td>0.04</td><td>1.10</td><td>0.43</td><td>0.02</td><td>0.03</td></tr><tr><td># 2</td><td>Final GM</td><td>38</td><td>0.43</td><td>0.01</td><td>0.64</td><td>0.03</td><td>0.66</td><td>0.42</td><td>0.01</td><td>0.01</td></tr><tr><td colspan="11">150 µM Fe(III)-citrate</td></tr><tr><td></td><td>Initial GM</td><td>151</td><td>0.41</td><td>0.01</td><td>0.65</td><td>0.02</td><td>0.59</td><td>0.24</td><td>0.05</td><td>0.01</td></tr><tr><td># 1</td><td>Magnetosome (mg)</td><td>26</td><td>-0.11</td><td>0.05</td><td>-0.18</td><td>0.09</td><td>-0.70</td><td>0.21</td><td>-0.02</td><td>0.03</td></tr></tbody></table>
<table><tr><td># 1</td><td>Magnetite (mt)</td><td>23</td><td>-0.10</td><td>0.01</td><td>-0.14</td><td>0.04</td><td>-0.11</td><td>0.17</td><td>0.00</td><td>0.04</td></tr><tr><td># 1</td><td>Lysate mg</td><td>11</td><td>1.24</td><td>0.01</td><td>1.84</td><td>0.06</td><td>2.18</td><td>0.19</td><td>0.01</td><td>0.04</td></tr><tr><td># 1</td><td>Lysate mt</td><td>9</td><td>1.12</td><td>0.01</td><td>1.66</td><td>0.04</td><td>1.95</td><td>0.25</td><td>0.01</td><td>0.02</td></tr><tr><td># 1</td><td>Final GM mg</td><td>121</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td># 1</td><td>Final GM mt</td><td>125</td><td>0.66</td><td>0.05</td><td>0.97</td><td>0.06</td><td>1.44</td><td>0.06</td><td>0.00</td><td>0.02</td></tr><tr><td># 1</td><td>Final GM</td><td>138</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></table>

<table><tr><td># 2</td><td>Magnetosome (mg)</td><td>7</td><td>0.03</td><td>0.02</td><td>0.06</td><td>0.02</td><td>-0.01</td><td>0.08</td><td>0.02</td><td>0.02</td></tr><tr><td># 2</td><td>Magnetite (mt)</td><td>10</td><td>0.05</td><td>0.02</td><td>0.08</td><td>0.05</td><td>0.12</td><td>0.30</td><td>0.01</td><td>0.02</td></tr><tr><td># 2</td><td>Lysate mg</td><td>16</td><td>0.95</td><td>0.01</td><td>1.39</td><td>0.02</td><td>2.01</td><td>0.23</td><td>-0.02</td><td>0.01</td></tr><tr><td># 2</td><td>Lysate mt</td><td>15</td><td>1.07</td><td>0.02</td><td>1.60</td><td>0.03</td><td>2.21</td><td>0.57</td><td>0.02</td><td>0.03</td></tr><tr><td># 2</td><td>Final GM mg</td><td>99</td><td>0.53</td><td>0.03</td><td>0.76</td><td>0.05</td><td>0.71</td><td>0.59</td><td>-0.02</td><td>0.03</td></tr><tr><td># 2</td><td>Final GM mt</td><td>102</td><td>0.54</td><td>0.01</td><td>0.77</td><td>0.02</td><td>0.74</td><td>0.25</td><td>-0.02</td><td>0.02</td></tr><tr><td># 2</td><td>Final GM</td><td>102</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></table>

<table><caption>150 µM Fe(II)-ascorbate</caption><tr><td></td><td>Initial GM</td><td>157</td><td>-0.11</td><td>0.03</td><td>-0.16</td><td>0.01</td><td>-0.18</td><td>0.20</td><td>0.01</td><td>0.03</td></tr><tr><td># 1</td><td>Magnetosome (mg)</td><td>66</td><td>-0.69</td><td>0.00</td><td>-1.02</td><td>0.02</td><td>-1.22</td><td>0.25</td><td>-0.01</td><td>0.02</td></tr><tr><td># 1</td><td>Magnetite (mt)</td><td>53</td><td>-0.58</td><td>0.02</td><td>-0.83</td><td>0.01</td><td>-1.34</td><td>0.31</td><td>0.02</td><td>0.02</td></tr><tr><td># 1</td><td>Lysate mg</td><td>20</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td># 1</td><td>Lysate mt</td><td>17</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td># 1</td><td>Final GM mg</td><td>116</td><td>0.21</td><td>0.03</td><td>0.33</td><td>0.02</td><td>0.38</td><td>0.41</td><td>0.01</td><td>0.04</td></tr><tr><td># 1</td><td>Final GM mt</td><td>104</td><td>0.21</td><td>0.02</td><td>0.32</td><td>0.03</td><td>0.43</td><td>0.57</td><td>0.00</td><td>0.01</td></tr><tr><td># 1</td><td>Final GM</td><td>129</td><td>0.07</td><td>0.05</td><td>0.10</td><td>0.06</td><td>0.09</td><td>0.09</td><td></td><td></td></tr></table>

<table><tr><td># 2</td><td>Magnetosome (mg)</td><td>74</td><td>-0.53</td><td>0.03</td><td>-0.79</td><td>0.03</td><td>-1.36</td><td>0.38</td><td>-0.01</td><td>0.01</td></tr><tr><td># 2</td><td>Magnetite (mt)</td><td>51</td><td>-0.40</td><td>0.02</td><td>-0.64</td><td>0.04</td><td>-1.32</td><td>0.57</td><td>-0.05</td><td>0.01</td></tr><tr><td># 2</td><td>Lysate mg</td><td>12</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td># 2</td><td>Lysate mt</td><td>14</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td># 2</td><td>Final GM mg</td><td>118</td><td>0.19</td><td>0.02</td><td>0.29</td><td>0.11</td><td>0.62</td><td>0.29</td><td>0.01</td><td>0.09</td></tr><tr><td># 2</td><td>Final GM mt</td><td>106</td><td>0.22</td><td>0.02</td><td>0.31</td><td>0.06</td><td>0.28</td><td>0.23</td><td>-0.01</td><td>0.05</td></tr><tr><td># 2</td><td>Final GM</td><td>101</td><td>0.20</td><td>0.01</td><td>0.31</td><td>0.02</td><td>0.66</td><td>0.30</td><td>0.01</td><td>0.03</td></tr></table>

<footnote>* Sample type: GM stands for growth medium (initial or final), mt for magnetosome, mt for magnetite extract. For instance, "Final GM mg" corresponds to the final growth medium of the bottle used for magnetosome extraction.</footnote>

<footnote>** [Fe] corresponds to the iron concentration recovered in one liter of growth medium.</footnote>
Table 2

Fractions of cellular iron in lysate and magnetite, and difference in their Fe isotope compositions, obtained for MV-1 cultures with Fe(II)-ascorbate and Fe(III)-citrate. The mass balance and isotope difference are calculated from either magnetosome or magnetite.

<table><thead><tr><th>[Fe] µM. replicate #</th><th>Calculation from</th><th>Fraction of cellular iron in lysate</th><th>Fraction of cellular iron in magnetite</th><th>Δ<sup>56</sup>Fe<sub>lysate-mag</sub> * ‰</th><th>±2SD ‰</th></tr></thead><tbody><tr><td colspan="6"><strong>Fe(II)-ascorbate</strong></td></tr><tr><td>50µM. repl. #1</td><td>magnetosome</td><td>0.15</td><td>0.85</td><td>0.44</td><td>0.10</td></tr><tr><td>50µM. repl. #1</td><td>magnetite</td><td>0.17</td><td>0.83</td><td>0.47</td><td>0.10</td></tr><tr><td>50µM. repl. #2</td><td>magnetosome</td><td>0.13</td><td>0.87</td><td>0.53</td><td>0.06</td></tr><tr><td>50µM. repl. #2</td><td>magnetite</td><td>0.17</td><td>0.83</td><td>0.27</td><td>0.08</td></tr><tr><td>100µM. repl. #1</td><td>magnetosome</td><td>0.12</td><td>0.88</td><td>0.50</td><td>0.04</td></tr><tr><td>100µM. repl. #1</td><td>magnetite</td><td>0.16</td><td>0.84</td><td>0.42</td><td>0.09</td></tr><tr><td>100µM. repl. #2</td><td>magnetosome</td><td>0.11</td><td>0.89</td><td>0.35</td><td>0.07</td></tr><tr><td>100µM. repl. #2</td><td>magnetite</td><td>0.14</td><td>0.86</td><td>0.31</td><td>0.08</td></tr><tr><td>150µM. repl. #1</td><td>magnetosome</td><td>0.23</td><td>0.77</td><td>n.a.</td><td>n.a.</td></tr><tr><td>150µM. repl. #1</td><td>magnetite</td><td>0.24</td><td>0.76</td><td>n.a.</td><td>n.a.</td></tr><tr><td>150µM. repl. #2</td><td>magnetosome</td><td>0.14</td><td>0.86</td><td>n.a.</td><td>n.a.</td></tr><tr><td>150µM. repl. #2</td><td>magnetite</td><td>0.21</td><td>0.79</td><td>n.a.</td><td>n.a.</td></tr><tr><td colspan="6"><strong>Fe(III)-citrate</strong></td></tr><tr><td>50µM. repl. #1</td><td>magnetosome</td><td>0.21</td><td>0.79</td><td>0.10</td><td>0.09</td></tr><tr><td>50µM. repl. #1</td><td>magnetite</td><td>0.20</td><td>0.80</td><td>0.14</td><td>0.20</td></tr><tr><td>50µM. repl. #2</td><td>magnetosome</td><td>0.31</td><td>0.69</td><td>0.33</td><td>0.17</td></tr><tr><td>50µM. repl. #2</td><td>magnetite</td><td>0.30</td><td>0.70</td><td>0.16</td><td>0.09</td></tr><tr><td>100µM. repl. #1</td><td>magnetosome</td><td>0.06</td><td>0.94</td><td>0.19</td><td>0.10</td></tr><tr><td>100µM. repl. #1</td><td>magnetite</td><td>0.08</td><td>0.92</td><td>0.13</td><td>0.10</td></tr><tr><td>100µM. repl. #2</td><td>magnetosome</td><td>0.04</td><td>0.96</td><td>0.52</td><td>0.06</td></tr><tr><td>100µM. repl. #2</td><td>magnetite</td><td>0.07</td><td>0.93</td><td>0.32</td><td>0.07</td></tr><tr><td>150µM. repl. #1</td><td>magnetosome</td><td>0.29</td><td>0.71</td><td>1.34</td><td>0.12</td></tr><tr><td>150µM. repl. #1</td><td>magnetite</td><td>0.30</td><td>0.70</td><td>1.22</td><td>0.05</td></tr><tr><td>150µM. repl. #2</td><td>magnetosome</td><td>0.71</td><td>0.29</td><td>0.93</td><td>0.05</td></tr><tr><td>150µM. repl. #2</td><td>magnetite</td><td>0.59</td><td>0.41</td><td>1.02</td><td>0.08</td></tr></tbody></table>

n.a.: values not available (due to a loss of a few lysate samples)

* isotopic difference between lysate and magnetite or magnetosome, calculated as:

$$ \Delta^{56}\text{Fe}_{\text{lysate-mag}} = \delta^{56}\text{Fe}_{\text{lysate}} - \delta^{56}\text{Fe}_{\text{magnetite/magnetosome}} $$
<table><caption>Table 3
Iron isotope compositions measured in the growth medium and calculated for initial dissolved Fe(II) and Fe(III).</caption><thead><tr><th>Iron source</th><th>Fe<sub>tot</sub></th><th>Fe(II)/Fe<sub>tot</sub></th><th>Fe(III)/Fe<sub>tot</sub></th><th>δ<sup>56</sup>Fe<sub>Fe tot</sub></th><th>δ<sup>56</sup>Fe<sub>Fe(II)</sub><sup>*</sup></th><th>δ<sup>56</sup>Fe<sub>Fe(III)</sub><sup>*</sup></th></tr></thead><tbody><tr><td>50 µM Fe(II)-ascorbate</td><td>41.0</td><td>0.99</td><td>0.01</td><td>-0.09</td><td>-0.12</td><td>2.68</td></tr><tr><td>100 µM Fe(II)-ascorbate</td><td>79.1</td><td>0.98</td><td>0.02</td><td>-0.16</td><td>-0.21</td><td>2.59</td></tr><tr><td>150 µM Fe(II)-ascorbate</td><td>163.3</td><td>0.93</td><td>0.07</td><td>-0.11</td><td>-0.34</td><td>2.48</td></tr><tr><td>50 µM Fe(III)-citrate</td><td>56.0</td><td>0.74</td><td>0.26</td><td>0.45</td><td>-0.32</td><td>2.53</td></tr><tr><td>100 µM Fe(III)-citrate</td><td>101.0</td><td>0.31</td><td>0.69</td><td>0.42</td><td>-1.65</td><td>1.28</td></tr><tr><td>150 µM Fe(III)-citrate</td><td>152.7</td><td>0.39</td><td>0.61</td><td>0.41</td><td>-1.41</td><td>1.51</td></tr></tbody></table>

<footnote>* Values calculated for Fe(II) and Fe(III) assuming an isotope equilibrium of 2.8‰ (Welch et al., 2003).</footnote>
<figure><img src="image_2.png" /><figcaption>Fig. 1. Transmission electron microscopy (TEM) images representative of MV-1 cells and their magnetosomes observed from bottle cultures to purified single nanocrystals of magnetite. (A) Single MV-1 cell showing a chain of magnetosomes (i.e. dark chain of nanocrystals). (B) Extracted magnetosomes chains illustrating that magnetite crystals preserved their alignment when the membrane was still present. (C) Zoom on a single magnetosome, with the black arrow pointing to the biological membrane surrounding magnetite. The dashed line highlights the external surface of the membrane. (D) Zoom on a magnetite nanocrystal for which the organic membrane has been removed.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Fig 2. Iron distribution in the final reservoirs (i.e. after harvesting) for all MV-1 cultures, with either Fe(II)-ascorbate (A, B, C) or Fe(III)-citrate (D, E, F). The values indicated are expressed as percentage of the total Fe recovered at the end of the experiment, and correspond to the mean values calculated from replicate analyses. The standard deviation calculated from replicate experiments does not exceed ± 3% of each value except for the final growth medium (GM) and magnetosome in the Fe(III)-citrate condition at 100 µM, for which standard deviation between replicates reaches ±14% and ±12%, respectively.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Fig 3. Average iron isotope compositions of the growth medium (GM) before and after culture as well as those of the cellular compartments of MV-1 (magnetite, magnetosome, bacterial lysate) in the final state. (A) Results obtained in Fe(II)-ascorbate experiments; the gray vertical bar indicates the range of variation measured for the initial GM for the three Fe(II) conditions at 50 (light green), 100 (green), and 150 (dark green) µM. (B) Results obtained in Fe(III)-citrate experiments; the gray vertical bar represents the range of variation measured for the initial GM for the three Fe(III) conditions at 50 (light orange), 100 (orange) and 150 (dark orange) µM. Squares: growth medium after culture; diamonds: cell lysate; circles bordered in black: magnetosomes; circles bordered in white: magnetite. Uncertainties are contained within the symbols.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Fig 4. Iron isotope anomaly expressed as $\Delta^{57}\text{Fe}$, and plotted versus $\delta^{56}\text{Fe}$ values for magnetite (circles bordered in white) and magnetosome (circles bordered in black) precipitated from an initial growth medium containing (A) Fe(II)-ascorbate and (B) Fe(III)-citrate. The different colors correspond to initial Fe concentrations of 50, 100 150 µM. The error bars are expressed as $\pm$2SD. The horizontal gray zone represents the external reproducibility based on laboratory (internal) and international standards ($\pm$ 0.06‰, 2SD, n = 61).</figcaption></figure>
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

Fig. 5. Rayleigh distillation models compared to results obtained on MV-1 cultures with Fe(II)-ascorbate (green circles) or Fe(III)-citrate (orange circles). Evolution of the $\delta^{56}\text{Fe}$ value of the growth medium (A) and magnetite/magnetosomes (B) versus fraction of iron remaining in the solution (F in Eq. 2 and 4). The isotope fractionation factors ($\alpha$) are given for each curve. The isotope compositions of magnetite/magnetosome are modelled as cumulated products using a set of $\alpha$ values very close to those of the growth medium. Data measured in magnetosomes are circled in black.
<figure><img src="image_8.png" /><figcaption>Fig. 6. Comparison of Fe isotope compositions of magnetite and magnetosome fractions extracted from the same culture conditions. Cultures with Fe(III)-citrate and Fe(II)-ascorbate correspond to orange and green circles respectively. The concentrations of initial Fe sources (50, 100 or 150 µM) are indicated by the various light to dark colors. The 1:1 line is represented as well as an extended region of ±0.2‰ (dotted lines). Overall, magnetite and magnetosome cultivated under similar conditions show identical values.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>Fig. 7. Iron isotope difference between lysate and magnetite/magnetosome ($\Delta^{56}\text{Fe}_{\text{lysate-mag}} = \delta^{56}\text{Fe}_{\text{lysate}} - \delta^{56}\text{Fe}_{\text{mag}}$) reported as a function of the fraction of cellular Fe in the lysate (lower X-axis) and magnetite (higher X-axis) for the various MV-1 culture conditions. Cultures with Fe(III)-citrate and Fe(II)-ascorbate correspond to orange and green circles respectively. The concentrations of initial Fe sources (50, 100 or 150 µM) are indicated by the various light to dark colors. Data calculated from magnetosomes are circled in black. The culture with Fe(II)-ascorbate at 150 µM are not plotted because the lysate samples were lost (and not measured) and the difference between lysate and magnetite/magnetosome can thus not be calculated.</figcaption></figure>
# Supplementary Material

## Iron isotope fractionation in magnetite from the marine magnetotactic bacterium *Magnetovibrio blakemorei*

Vincent Busigny<fnref n="1" />, François P. Mathon<fnref n="1" />,<fnref n="2" />, Matthieu Amor<fnref n="3" />,
François Guyot<fnref n="4" />, Nicolas Menguy<fnref n="4" />, Christopher T. Lefevre<fnref n="2" />

<footnote n="1">¹ Université de Paris, Institut de Physique du Globe de Paris, CNRS, F-75005, Paris, France</footnote>

<footnote n="2">² Aix-Marseille Université, CEA, CNRS, BIAM, 13115 Saint-Paul-lez-Durance, France</footnote>

<footnote n="3">³ Univ. Lyon, ENSL, UCBL, UJM, CNRS, LGL-TPE, F-69007 Lyon, France</footnote>

<footnote n="4">⁴ Muséum National d'Histoire Naturelle, Sorbonne Université, UMR CNRS 7590. Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie (IMPMC), Paris, France</footnote>
<figure><img src="image_10.png" /></figure>

Fig. S1. Monitoring of Fe concentration and speciation in culture media containing (A) Fe(II) ascorbate or (B) Fe(III) citrate. The data correspond to the mean values obtained for each culture condition (3 triplicates were measured for all conditions). Three experiments were performed with initial Fe concentration target near 50, 100 and 150 µM. Total Fe is the sum of Fe(II) and Fe(III) concentrations. The cultures with Fe(II) ascorbate show that almost all Fe occurs as Fe(II). In contrast, the cultures with Fe(III) citrate show that Fe occurs dominantly as Fe(II) and then as Fe(III). This indicates that part of the Fe(III) is reduced in the media.

Comparison of δ<sup>56</sup>Fe of the whole cell with Rayleigh distillation models
The Fe isotope compositions of the whole cells were calculated from data obtained on lysate and magnetite/magnetosome as:

$$ \delta^{56}Fe_{cell} = f_{lys} \times \delta^{56}Fe_{lys} + f_{mag} \times \delta^{56}Fe_{mag} \quad (\text{Eq. S1}) $$

where $\delta^{56}Fe_{cell}$, $\delta^{56}Fe_{lys}$ and $\delta^{56}Fe_{mag}$ are the isotope compositions of the cell, lysate and magnetite or magnetosome, and $f_{lys}$ and $f_{mag}$ are the fraction of cellular Fe in the lysate and magnetite or magnetosome, respectively. Therefore, $f_{lys} = 1 - f_{mag}$ for every condition.

Then, considering the isotope mass balance of our experimental system, we can write an equation similar to Equation 3 (main text) but with no simplification, so that:

$$ \delta^{56}Fe_{GM initial} = F \times \delta^{56}Fe_{GM} + (1 - F) \times \delta^{56}Fe_{cell} \quad (\text{Eq. S2}) $$

where F is the fraction of iron remaining in the growth medium, and the subscripts GM and cell stand for the growth medium and whole cell (including lysate and magnetite/magnetosome), respectively. Equation S2 can be modified to express the isotope composition of whole cell as:

$$ \delta^{56}Fe_{cell} = \frac{1}{1-F} \times (\delta^{56}Fe_{GM initial} - \delta^{56}Fe_{GM} \times F) \quad (\text{Eq. S3}) $$

In the model, $\delta^{56}Fe_{GM}$ is calculated from Equation 2 (see main text) and includes the fractionation factor $\alpha$ between growth medium and cell as a free parameter. Rayleigh distillation curves calculated from Equation S3 are illustrated in Fig. S2 with corresponding $\alpha$ values and compared with the experimental results on MV-1 (calculated from Eq. S1 above). Cultures with Fe(II)-ascorbate and Fe(III)-citrate are compatible with $\alpha$ values between growth medium and cell ranging from 0.9991 to 0.9997, and from 0.9993 to 0.9999, respectively (Fig. S2). These values are very close to those obtained from fits of the magnetite/magnetosome data (Fig. 5b), indicating the limited effect of lysate fractions on global mass balance approach. This is due to the fact that most of the cellular Fe in MV-1 is transferred to and stored in magnetosome.
<figure><img src="image_11.png" /><figcaption>Fig. S2. Rayleigh distillation models compared to results obtained on MV-1 cultures with Fe(II)-ascorbate (green circles) or Fe(III)-citrate (orange circles). Evolution of the δ<sup>56</sup>Fe value of the whole cell (i.e. including lysate and magnetosome, see Eq. S1) *versus* fraction of iron remaining in the solution (F in Eq. S2 and S3). The isotope fractionation factors (α) of the Rayleigh models are given for each curve. Data calculated from magnetosomes are circled in black, the others being calculated from magnetite.</figcaption></figure>
Table S1

<table><caption>Iron concentrations (µmol/L) and redox states of the growth media monitored during MV-1 cultures.</caption><thead><tr><th>Day</th><th>Fe(II)</th><th>± 1SD</th><th>Fe(III)</th><th>± 1SD</th><th>Fe<sub>tot</sub></th><th>± 1SD</th><th>Fe(II)/Fe<sub>tot</sub></th><th>Fe(III)/Fe<sub>tot</sub></th></tr></thead><tbody><tr><td colspan="9"><strong>50 µM Fe(II)-ascorbate</strong></td></tr><tr><td>0</td><td>40.7</td><td>0.9</td><td>0.4</td><td>0.2</td><td>41.0</td><td>1.1</td><td>0.99</td><td>0.01</td></tr><tr><td>1</td><td>38.1</td><td>0.9</td><td>1.5</td><td>0.3</td><td>39.6</td><td>1.3</td><td>0.96</td><td>0.04</td></tr><tr><td>2</td><td>29.8</td><td>2.1</td><td>0.7</td><td>0.8</td><td>30.5</td><td>2.9</td><td>0.98</td><td>0.02</td></tr><tr><td>3</td><td>8.6</td><td>5.7</td><td>0.3</td><td>0.1</td><td>8.9</td><td>5.8</td><td>0.97</td><td>0.03</td></tr><tr><td colspan="9"><strong>100 µM Fe(II)-ascorbate</strong></td></tr><tr><td>0</td><td>77.8</td><td>0.8</td><td>1.3</td><td>0.7</td><td>79.1</td><td>1.5</td><td>0.98</td><td>0.02</td></tr><tr><td>1</td><td>78.4</td><td>0.4</td><td>0.8</td><td>0.4</td><td>79.3</td><td>0.8</td><td>0.99</td><td>0.01</td></tr><tr><td>2</td><td>74.1</td><td>1.5</td><td>0.9</td><td>0.2</td><td>75.0</td><td>1.7</td><td>0.99</td><td>0.01</td></tr><tr><td>3</td><td>56.7</td><td>7.1</td><td>1.0</td><td>0.5</td><td>57.7</td><td>7.6</td><td>0.98</td><td>0.02</td></tr><tr><td>4</td><td>34.1</td><td>3.3</td><td>0.7</td><td>0.6</td><td>34.8</td><td>3.9</td><td>0.98</td><td>0.02</td></tr><tr><td colspan="9"><strong>150 µM Fe(II)-ascorbate</strong></td></tr><tr><td>0</td><td>151.2</td><td>4.8</td><td>12.1</td><td>1.2</td><td>163.3</td><td>6.0</td><td>0.93</td><td>0.07</td></tr><tr><td>1</td><td>151.7</td><td>3.1</td><td>13.2</td><td>1.6</td><td>164.8</td><td>4.7</td><td>0.92</td><td>0.08</td></tr><tr><td>2</td><td>145.6</td><td>1.3</td><td>10.1</td><td>1.2</td><td>155.7</td><td>2.5</td><td>0.94</td><td>0.06</td></tr><tr><td>3</td><td>130.4</td><td>6.9</td><td>11.8</td><td>0.5</td><td>142.2</td><td>7.4</td><td>0.92</td><td>0.08</td></tr><tr><td>4</td><td>110.1</td><td>5.9</td><td>8.5</td><td>2.2</td><td>118.5</td><td>8.1</td><td>0.93</td><td>0.07</td></tr><tr><td colspan="9"><strong>50 µM Fe(III)-citrate</strong></td></tr><tr><td>0</td><td>41.5</td><td>2.8</td><td>14.4</td><td>3.8</td><td>56.0</td><td>6.6</td><td>0.74</td><td>0.26</td></tr><tr><td>3</td><td>21.3</td><td>4.8</td><td>9.8</td><td>4.3</td><td>31.1</td><td>9.0</td><td>0.68</td><td>0.32</td></tr><tr><td colspan="9"><strong>100 µM Fe(III)-citrate</strong></td></tr><tr><td>0</td><td>31.3</td><td>6.8</td><td>69.7</td><td>9.5</td><td>101.0</td><td>16.3</td><td>0.31</td><td>0.69</td></tr><tr><td>3</td><td>18.1</td><td>7.9</td><td>42.2</td><td>14.5</td><td>60.2</td><td>22.4</td><td>0.30</td><td>0.70</td></tr><tr><td>4</td><td>10.0</td><td>4.7</td><td>18.5</td><td>9.1</td><td>28.6</td><td>13.8</td><td>0.35</td><td>0.65</td></tr><tr><td colspan="9"><strong>150 µM Fe(III)-citrate</strong></td></tr><tr><td>0</td><td>60.3</td><td>19.4</td><td>92.5</td><td>4.9</td><td>152.7</td><td>24.3</td><td>0.39</td><td>0.61</td></tr><tr><td>1</td><td>34.6</td><td>11.1</td><td>116.2</td><td>6.3</td><td>150.9</td><td>17.4</td><td>0.23</td><td>0.77</td></tr><tr><td>2</td><td>45.4</td><td>15.3</td><td>106.7</td><td>7.7</td><td>152.2</td><td>23.0</td><td>0.30</td><td>0.70</td></tr><tr><td>3</td><td>36.9</td><td>11.9</td><td>114.2</td><td>4.6</td><td>151.1</td><td>16.5</td><td>0.24</td><td>0.76</td></tr><tr><td>4</td><td>36.8</td><td>12.1</td><td>109.4</td><td>5.8</td><td>146.2</td><td>18.0</td><td>0.25</td><td>0.75</td></tr><tr><td>7</td><td>30.3</td><td>9.8</td><td>98.0</td><td>9.9</td><td>128.2</td><td>19.7</td><td>0.24</td><td>0.76</td></tr></tbody></table>

<footnote>* Fe concentration determined by the ferrozine method (Viollier et al., 2000).</footnote>
Table S2

Iron distribution between final growth medium, lysate, magnetosome and magnetite fractions for all culture conditions.

<table><thead><tr><th>Iron source</th><th>[Fe]<br>µM</th><th>Replicate<br>analysis</th><th>Final growth<br>medium</th><th>Lysate</th><th>Magnetosome</th><th>Magnetite</th></tr></thead><tbody><tr><td rowspan="6">Fe(II)-ascorbate</td><td rowspan="2">50</td><td># 1</td><td>0.62</td><td>0.08</td><td>0.15</td><td>0.15</td></tr><tr><td># 2</td><td>0.62</td><td>0.12</td><td>0.14</td><td>0.13</td></tr><tr><td rowspan="2">100</td><td># 1</td><td>0.30</td><td>0.05</td><td>0.34</td><td>0.31</td></tr><tr><td># 2</td><td>0.10</td><td>0.05</td><td>0.51</td><td>0.34</td></tr><tr><td rowspan="2">150</td><td># 1</td><td>0.85</td><td>0.05</td><td>0.06</td><td>0.05</td></tr><tr><td># 2</td><td>0.89</td><td>0.07</td><td>0.01</td><td>0.02</td></tr><tr><td rowspan="6">Fe(III)-citrate</td><td rowspan="2">50</td><td># 1</td><td>0.14</td><td>0.14</td><td>0.38</td><td>0.35</td></tr><tr><td># 2</td><td>0.08</td><td>0.13</td><td>0.43</td><td>0.35</td></tr><tr><td rowspan="2">100</td><td># 1</td><td>0.42</td><td>0.08</td><td>0.27</td><td>0.23</td></tr><tr><td># 2</td><td>0.41</td><td>0.07</td><td>0.27</td><td>0.24</td></tr><tr><td rowspan="2">150</td><td># 1</td><td>0.69</td><td>0.07</td><td>0.13</td><td>0.11</td></tr><tr><td># 2</td><td>0.68</td><td>0.06</td><td>0.16</td><td>0.11</td></tr></tbody></table>