Keywords: Superalloys, SIMS, High temperature Corrosion, Oxidation, Reactor conditions

Corrosion of a chromia-forming nickel base alloy, Haynes 230_, has been investigated under impure helium containing a few Pa of CO and H2O at 900 °C. It has been found that this alloy reacts simultaneously with CO and H2O. Oxidation by CO has been revealed to occur mainly in the first hours. CO diffuses through the scale via short-circuit pathways and oxidizes Al, Cr and Si at the oxide/metal interface. Kinetics of CO oxidation has been investigated and several rate limiting steps are proposed. In the long term, H2O is the major oxidant of chromia-forming nickel base alloys in impure helium. K Ke ey yw

Introduction

High temperature oxidation behaviour of alloys in mixed gas environments has been one of the major scientific and engineering interests in the large field of environmentally induced degradation of materials over the past 15 years [START_REF] Zheng | High temperature corrosion of Cr2O3-forming alloys in CO-CO2-N2 atmospheres[END_REF][START_REF] Young | High-temperature corrosion in mixed gas environments[END_REF][START_REF] Young | Simultaneous oxidation and carburisation of chromia formingalloys[END_REF]. High Temperature gas cooled Reactor (HTR) is a nuclear system with an all graphite core where a mixed gas environment can be found. The helium coolant contains small amounts of impurities typically H2, H2O, CO, and CH4. The challenge here is that the atmosphere is highly diluted [START_REF] Graham | Environmental conditions in HTRs and the selection and developmentof primary circuit materials[END_REF]. Even if their partial pressures are only between a few tens to hundreds of Pa, the gas impurities can react at high temperature with metallic materials, in particular with the chromia-forming alloys for heat exchanger. Gas-metal interactions involved in HTR have been discussed in numerous papers published in the past three decades [START_REF] Brenner | Ternary diagrams for the characterization of metallic corrosion in high-temperature reactors, Gas Cooled Today[END_REF][START_REF] Brenner | The development and application of a unified corrosion model for high temperature gas-cooled reactor systems[END_REF][START_REF] Christ | High-temperature corrosion of the nickel-based alloy Inconel-617 in helium containing small amounts of impurities[END_REF][START_REF] Shindo | Evaporation behavior of hastelloy-X alloys in simulated very high-temperature reactor environments[END_REF][START_REF] Shindo | Corrosion behavior of hightemperature alloys in impure helium environments[END_REF][START_REF] Quadakkers | Thermodynamic ad kinetic aspects of the corrosion of high-temperature alloys in high-temperature gas-cooled reactor helium[END_REF][START_REF] Quadakkers | Corrosion of high-temperature alloys in the primary circuit helium of high-temperature gas-cooled reactors. 1. Theoritical background[END_REF][START_REF] Quadakkers | Corrosion of high-temperature alloys in the primary circuit helium of high-temperature gas-cooled reactors. 1. Experimental results[END_REF] and more recently in France [START_REF] Rouillard | High temperature corrosion of a nickel base alloy by helium impurities[END_REF][START_REF] Rouillard | Oxide-layer formation and stability on a nickel-base alloy in impure helium at high temperature[END_REF][START_REF] Cabet | High temperature reactivity of two chromium-containing alloys in impure helium[END_REF][START_REF] Chapovaloff | Influence of Al on the high temperature corrosion behaviour of Inconel 617 in VHTR primary coolant atmosphere[END_REF]. These works have demonstrated that depending on the composition of the chromia-forming alloy, on the working temperature (850-1000 °C) and on the partial pressures of gas species in helium, two main behaviours could be distinguished: either a passive corrosion based on the formation of a stable, slow-growing, Cr rich oxide layer on the surface or an active corrosion with the destruction of the surface oxide layer and the subsequent decarburization or carburization of the alloy [START_REF] Rouillard | Oxide-layer formation and stability on a nickel-base alloy in impure helium at high temperature[END_REF]. Haynes 230 ® is a good candidate alloy for HTR heat exchanger.It has been shown [START_REF] Rouillard | High temperature corrosion of a nickel base alloy by helium impurities[END_REF][START_REF] Rouillard | Oxide-layer formation and stability on a nickel-base alloy in impure helium at high temperature[END_REF] that Haynes 230 ® forms a Mn-Cr rich oxide layer at 900 °C under a helium atmosphere with 20 Pa H2, 2 Pa CH4, 5 Pa CO and 0.15 Pa H2O. The purpose of the present work is to describe the oxide growth at 900°C under this mixed oxidizing environment with CO and H2O. The composition and morphology of the oxide layer have been accurately analysed. The influence of the water vapour content in helium and the influence of the alloy composition on CO reactivity have been investigated using an evolved gas analysis technique. Moreover two-stage experiments under helium mixture without then with 13 C 18 O have been carried out to precise where is localized the interface of CO reaction. These results combined with accurate analyses of the nature and the morphology of the oxide layer has allowed a better understanding of the kinetics of CO consumption in the HTR conditions. Experimental M Ma at te er ri ia al ls s Haynes 230 ® was provided by Haynes Int. ® in the plate form, annealed at 1230 °C for 10 min then water quenched. A model alloy Ni-22Cr-14W-0.1C was provided by the SMS Centre of the Ecole Nationale Supérieure des Mines de St Etienne. Its chemical composition is based on Haynes 230 ® but it only contains the major elements.It was prepared by electromagnetic melting in cold crucible of a Ni-30Cr ingot with W rods, Ni billets and carbon powder to yield the nominal composition. After casting, the ingot was hot worked at 1260 °C to obtain a rod with a diameter of 18 mm. A 1.5 mm-thick layer was machined off the surface to remove the decarburized zone. Then, the rod was annealed under argon at 1350 °C for 10 h then at 1100 °C for 14 h in order to obtain a homogeneous precipitation of M23C6 carbides. Finally, it was water quenched. Haynes 230 ® and model alloy were cut into rectangular specimens of approximate dimensions 25 × 12 × 2 mm. They were polished down to a 1 µm finish, ultrasonically degreased in an acetone-ethanol mixture and weighed. The chemical composition of both alloys is presented in Oxidation tests were carried out under atmospheric pressure using the gas mixtures described in Table 2. Two specimens were exposed to the flowing gas in a quartz tube reactor inserted in a high temperature furnace. They were heated at a rate of 1 °C/min then maintained at 900 °C (850 °C for test E). After exposure, the samples were cooled at a rate of 2.5 °C/min under pure helium. At the quartz tube outlet, the gas was analysed by Gas Phase Chromatography (GPC) with a helium detector. The time between two analyses was 10 min. In order to check if the relative change in gas concentrations is representative of gas/metal reactions, a blank test was carried out without any specimen. Fig. 1 shows that no gas/gas reaction occurred below 950 °C whereas thermodynamic allows the reaction (1) to occur at high temperature:

4 2 CH + H O CO + 3H → (1)
The results are shown in Fig. 1. It is probably due to the very low partial pressures of gas impurities in helium which prevents the molecules to interact perfectly and consequently the thermodynamic equilibrium to be established. 2 2. .3 3. . M Me et th ho od ds s o of f s su ur rf fa ac ce e a an na al ly ys si is s The corrosion products were analysed by Field Emission Scanning Electron Microscopy, microprobe, Transmission Electron Microscopy (TEM), X-Ray Diffraction (XRD), X-Ray Photoelectron Spectroscopy (XPS) and Glow Discharge Optical Emission Spectroscopy (GDOES). Details on the different apparatus and analysis conditions are given in [START_REF] Rouillard | Oxide-layer formation and stability on a nickel-base alloy in impure helium at high temperature[END_REF].

After test E (see Table 2), the distribution of the 13 C isotope was analysed in the surface scale by Glow Discharge Mass Spectroscopy (GDMS) using a VG9000 spectrometer. The oxide composition as well as the distribution of the oxygen isotope were determined by Ar + -SIMS using a CAMECA Riber MIQ256 secondary ion mass spectrometer. In both experiments, the rate of consumption increased with increasing temperature and reached its maximum at 900 °C. Then, at constant temperature, the reaction rate decreased with time. After roughly 15 h, the reaction rate fell down below the detection limit of the GPC which is about 10 -12 mol/s. The area under the peak in Fig. 2 gives information about the total quantity of CO having reacted. The methane partial pressure difference between the inlet and the outlet of the furnace was too low to be measured. As a consequence, methane reactivity with the specimens was considered to be negligible at 900 °C. Fig. 2 5 are given in Table 3. The oxide layer can be decomposed in two main parts: an upper continuous film and inner nodules. The outer part of the continuous film (spots 1 and 2) was analysed to be Mn/Cr rich oxide (JCPDS No.: 00-006-0504) whereas the inner part (spot 3) was only pure chromia (JCPDS No.: 01-071-0982). The nodules (spot 6) were mainly Cr and Al rich oxides. An aluminium and silicon enrichment was also revealed in the area between the oxide layer and the nodules (spots 4 and 5). Finally, intergranular Al rich oxides were formed deeper in the alloys. In Fig. 3, the white inclusion was a M6-type carbide -with M being mainly W and Crinitially present in the alloy. It can be assumed that this particle has been cut during the initial preparation of the sample. Thus, its flat top should roughly materialize the original alloy surface. Considering this assumption, the interface between the oxide layer and the inner nodules should approximately correspond to the original surface. This is in good agreement with the presence of aluminium and silicon at this interface (see Section 4) since silicon and aluminium oxidized on the surface during the very first stages of oxidation as shown by XPS analysis of a Haynes 230 ® specimen heated at 950 °C (1°C/ min) under helium with a reduced content of oxidizing species. 3 3. . [START_REF] Young | Simultaneous oxidation and carburisation of chromia formingalloys[END_REF] In Fig. 6 the interface between the oxide and the alloy could be located thanks to the inflexion point of the O profile (not represented). The profile indicates that carbon accumulated underneath the oxide surface and progressive decreased down to the asreceived level in the alloy. By considering a sputtering rate equal to 7 µm/min in the prevailing conditions of the GDOES analysis, carbon may have diffused into the alloy bulk over approximately 25 *m. In Fig. 6, the area delimited by the oxide/alloy interface (the vertical dotted line), the initial carbon content (the horizontal dotted line) and the carbon profile gives the carbon content transferred into the alloy during the test. On the other hand, the area under the peak in Fig. 2 corresponds to the amount of CO which concurrently reacted. These two data are reported in Table 4. The amount of carbon found under the oxide layer is in good agreement with the amount of CO which has been consumed (±6%) as expected from Fig. 5 presents the mean thickness of the oxide layer formed after test A (Fig. 3) and test B (Fig. 4) as estimated by FESEM imaging. Besides, GPC analyses (cf. Fig. 2) allowed calculating the total consumption of CO during the tests. These data correspond to a given amount of oxygen transferred to the oxide that eventually can be used to estimate the thickness of oxide produced only by CO -with the assumption that the entire oxide layer was Cr2O3 (molar volume = 28.1 cm 3 ) (see Table 5). Finally, the last column compares the two results in terms of fraction of the oxide layer due to oxidation by CO. It underlines an important result: the higher the water vapour content in helium, the thicker the oxide layer and the lower the CO percentage that reacts with the alloy. Thus CO and H2O were in competition with respect to the oxidation of the alloy. Besides, it is worth noticing that the oxide layer morphology shown in Figs. 3 and4 is different: after test B which contained the lower water vapour content (0.16 Pa), the upper continuous layer was thinner than after test A richer in water vapour (0.4 Pa). Moreover, the inner rough oxide was more pronounced after test B. 3). Its upper part is enriched with manganese, forming a spinel MnxCr(3-x)O4 phase (see Fig. 5 and Table 3). The chemical composition of this oxide layer is identical to that of the oxide formed on Haynes 230 ® at high temperature in air [START_REF] Tawancy | High-temperature oxidation behavior of a wrought Ni-Cr-W-Si-La alloy[END_REF] and is similar to that of other chromium rich nickel base alloys containing a small quantity of manganese when exposed in oxidizing atmospheres [START_REF] Shindo | Evaporation behavior of hastelloy-X alloys in simulated very high-temperature reactor environments[END_REF]. The strong tendency for Mn to concentrate in the outer area of the scale is said to be related to a diffusion coefficient of Mn ions hundred times larger than that of Cr ions in chromia at 900 °C [START_REF] Lobnig | Diffusion of cations in chromia layers grown on iron-base alloys[END_REF]. At the very first steps of exposition, it has been shown that Al and Si start to oxidize and concentrate discontinuously at the original alloy surface. This observation has already been noted on Haynes 230 ® oxidized in air at high temperature [START_REF] Tawancy | High-temperature oxidation behavior of a wrought Ni-Cr-W-Si-La alloy[END_REF]. The presence of these oxidized elements on the surface could be explained by two main reasons: first, the segregation energy is favoured on Haynes 230 ® [START_REF] Pop | Surface segregation in Haynes 230 alloy[END_REF] and secondly, the driving force for their oxidation is higher than for the other elements (Cr and Mn particularly). However, because their content is very low in Haynes 230 ® (0.3-0.4 wt%), the growth of Al and Si rich oxides is limited and Mn/Cr rich oxide starts to grow over them. At longer times, Al rich oxides can still be formed deeper in the alloy, particularly at grain boundaries, where the chemical potential of oxygen set by the dissociation pressure of Cr2O3 is high enough to oxidize aluminium. [START_REF] Brenner | Ternary diagrams for the characterization of metallic corrosion in high-temperature reactors, Gas Cooled Today[END_REF][START_REF] Quadakkers | Corrosion of high-temperature alloys in the primary circuit helium of high-temperature gas-cooled reactors. 1. Theoritical background[END_REF][START_REF] Rouillard | Oxide-layer formation and stability on a nickel-base alloy in impure helium at high temperature[END_REF][START_REF] Lupton | Report JUL-1639 Kernforschungsanlage Jülich GmbH IRW[END_REF], Table 5 shows that the oxide layer formation is due to reactions with H2O and CO according to (2) and (3): χM + yH 2 O=MχOy + yH 2 (g)

Experimental

(2) χM + yCO=MχOy + yC solution [START_REF] Young | Simultaneous oxidation and carburisation of chromia formingalloys[END_REF] where M = Al, Cr, Mn, Si and Csolution represents carbon dissolved in the alloy matrix.

A competition between H2O and CO has been evidenced in Table 4: the higher the water vapour in helium, the lower the probability for CO molecules to react on the surface. Moreover, differences in the reactivity of CO and H2O may be explained by considering the thermodynamic and kinetic of the reactions. Fig. 7 shows that the standard free Gibbs energy of oxidation of Cr by CO strongly depends on Cr activity whereas oxidation by H2O depends a little on aCr: at 900 °C, Cr did not react with CO below an activity of about 0.4 but still react with H2O even when chromium activity is as low as to 0.01. Consequently, Cr2O3 may be formed in the beginning by reactions with both CO and H2O. Then as far as the Cr activity near the oxide/alloy interface decreases, oxidation by CO is less and less possible.

As carbon from CO was transferred beneath the oxide scale (see Fig. 6 and Table 4), it is very likely that CO reacted at the oxide/alloy interface. It is assumed that diffusion occurred via short-circuit pathways. CO molecules then should be dissociated at the oxide/alloy interface leading simultaneously to oxide growth and deposition of carbon. Moreover the industrial alloy containing minor elements like Al and Si consumed more CO than the model alloy as shown in Fig. 2. Fig. 8 shows that from a thermodynamic point of view, CO preferentially oxidizes Al and Si over Cr in Haynes 230 ® even if their activities are much smaller. However, because Al and Si contents are limited in Haynes 230 ® , they do not uniformly oxidize on the surface but rather form small localized nodules. This non uniform precipitation could explain the geometry of the rough inner oxide: Cr cannot be oxidized under areas where Al rich nodules are formed since Al/Al2O3 equilibrium imposes a too low partial pressure of oxygen whereas Cr does oxidize in areas between nodules.

On the other hand, the outer Cr rich oxide layer should mainly be due to oxidation by water vapour according to a mechanism which involves H2O dissociation at the surface of Cr2O3 and bulk Cr 3+ diffusion through the scale. Comparison of the molecule radius of H2O to CO which is respectively 96 pm and 111 pm shows that the water vapour is smaller than carbon monoxide and one can thus expect that it will diffuse faster than CO through the oxide layer and participate actively to the inward part of the oxide layer. It is not what our results reveal. In fact, in VHTR environment, the water vapour partial pressure in helium is always very low (few Pa) and as a consequence most of water molecules reacts at the gas/oxide interface as soon as it is adsorb on the oxide surface. Like CO, some water molecules may also diffuse in high diffusion pathways through the oxide layer but their participation to the inner oxide compared to the one done by CO is negligible because the ratio PCO/PH2O is high (>>10). In the first step, two samples were exposed to a He mixture with 8-10 Pa H2 16 O /200 Pa H 2 . A compact oxide layer should grow only containing 16 O. Then the same samples were exposed to a dry He mixture with 5 Pa 13 C 18 O (H 2 O < 0.01 Pa) without decreasing neither the temperature nor the H2 partial pressure. During this second step, 13 C 18 O is the only species able to react.

The main purpose of test E was to localize the site where CO reacts via the respective positions of 13 C and 18 O profiles versus 16 O profile. After the two-step experiment E, the oxide layer was observed by SEM and its structure and nature revealed to be identical to the one showed in Fig. 3. Fig. 9 shows SIMS profiles of 16 O, 18 O and 52 Cr through the alloy surface after test E and Fig. 10 presents the complementary profiles of 13 C and 16 O obtained by GDMS. The two dotted vertical lines in Figs. 9 and 10 represent the inflexion points of 16 O and 52 Cr signals and should thus materialize the border lines of the oxide/alloy interface. In Figs. 9 and 10, 18 O and 13 C present quite a similar profile from the outside to the alloy bulk: a decreasing slope through the pre-formed continuous oxide layer made of 16 O, then an accumulation near the oxide/alloy interface. Nevertheless, it has to be noted that the intensity level of 18 O signal is hundred times lower than that of 16 O signal meaning that CO did react with the Ni-22Cr-14W-0.1C specimens during the second step but not significantly. The fact that 13 C and 18 O signals follow the same profile through the oxide layer strongly supports that the diffusing species may be CO. This result agrees with our previous assumption that CO should directly react with the metal at the oxide/alloy interface. The two-stage oxidation test E was carried also out for Haynes 230 ® . The SIMS profiles were similar confirming that very few CO molecules reacted at the oxide/alloy interface. According to Basu et al. [START_REF] Basu | Tracer isotope distribution in growing oxide scales[END_REF], the shape of 13 C (and CO) profile is typically representative of a profile of a specie reacting at the oxide/alloy interface and diffusing along shortcircuit pathways such as oxide grain boundaries. Permeability of gas through seemingly compact oxide layers has been discussed in a lot of papers [START_REF] Gilewicz-Wolter | Study of iron oxidation in sulfur dioxide atmospheres by means of the 35S radioisotope[END_REF][START_REF] Singh | Penetration of sulfur through preformed protective oxide scales[END_REF][START_REF] Tomlinson | Hydrogen emission during the steam oxidation of ferritic steels: kinetics and mechanism[END_REF][START_REF] Gibbs | Model for mild-steel oxidation in CO2[END_REF]. It more often calls for gas diffusion into cracks developed in the oxide layer even if such cracks have never been evidenced so far. Recently, Young et al. [START_REF] Zheng | High temperature corrosion of Cr2O3-forming alloys in CO-CO2-N2 atmospheres[END_REF][START_REF] Young | High-temperature corrosion in mixed gas environments[END_REF][START_REF] Young | Simultaneous oxidation and carburisation of chromia formingalloys[END_REF], who have worked on oxidation in mixed gas atmospheres, typically CO/CO2/N2 and CO/CO2/SO2/N2 observed the penetration of carbon bearing molecules through chromia even though any crack was evidenced and though carbon solubility in Cr2O3 is known to be negligible [START_REF] Wolf | Carbon transport through oxide scales on Fe-Cr alloys[END_REF]. They proposed that molecular transport of gas species could occur into the oxide along internal surfaces such as oxide grain boundaries and so called nanochannels or nanopores as surface diffusion at the surface of such nanochannel may be preponderant on the Knudsen diffusion of gas molecules -if the size of the nanochannel is not much higher than that of molecules. According to the conclusions of Young et al. and respect to our SIMS results, CO may also be transferred through the surface chromia layer of NiCrW and Haynes 230 ® via molecular transport through nanochannels. 4 4. .4 4. . K Ki in ne et ti ic cs s o of f C CO O r re ea ac ct ti io on n It was previously evidenced that CO reacts at the inner interface, presumably with Al, Si and Cr to a less extent. Upon edification of the oxide layer, both the available surface for reaction with CO at the oxide/alloy interface and the activity of oxidizable elements decrease. The consumption rate of CO is thus expected to decrease with time as it is actually observed in Fig. 2. After roughly 15 h, the difference between the outlet and the inlet falls below the detection limit of GPC which is 10 -12 mol/cm 2 /s. At this time, the oxide layer appears to be compact on FESEM images (see Fig. 3). A basic approach would argue that after 15 h, CO does not react anymore because of the growth of a perfectly impermeable oxide layer which totally prevents CO from accessing to the oxide/alloy interface. However, SIMS combined to GDMS analyses after test E (see Figs. 9 and 10) discredits this scenario: even after the formation of a continuous compact oxide layer (~500 nm), some CO can still diffuse through the oxide layer and react at the oxide/alloy interface. The reaction rate is not nul but very low (<10 -12 mol/cm 2 /s).

CO P CO P

In the following, the kinetic of CO reaction after the formation of a seemingly compact oxide layer (~15 h) is investigated by comparing the rate of involved processes to the consumption rate: diffusion of CO through the oxide layer, reaction of CO at the oxide/alloy interface or bulk diffusion of oxidizing element to the oxide/alloy interface. On one hand, it has been observed [START_REF] Rouillard | Thermodynamic modelling of the destruction of the surface Cr2O3 on Alloy 230 in the impure helium atmosphere of a gas cooled reactor[END_REF] that CO can be released and transported fast through the scale (8.10 -12 mol/cm 2 /s) because of a specific reaction between oxide and carbon in solution at the oxide/alloy interface. Since the surface diffusion rate of CO through oxide grain boundaries or nanochannels is much too low to explain such a high rate of CO release, faster ways of diffusion such as gas diffusion in nanochannels must exist as well. As discussed in [START_REF] Rouillard | Thermodynamic modelling of the destruction of the surface Cr2O3 on Alloy 230 in the impure helium atmosphere of a gas cooled reactor[END_REF], this nanochannel network may have been constituted during the oxide growth. The rate of CO diffusion through the scale (8.10 -12 mol/cm 2 /s) is thus far higher than the rate of CO reaction and might no be rate limiting. Mention should mades that high diffusion paths with a predominant gas diffusion through the oxide layer usually influences the SIMS/GDMS profiles after two-stage experiments. According to Basu et al. [START_REF] Basu | Tracer isotope distribution in growing oxide scales[END_REF], an accumulation at the oxide/alloy interface and an almost flat profile in the old oxide layer would be expected which is quite different from what has been observed on Figs. 8 and9. A possible reason could be that only very few quantities of CO would have reacted at the oxide/metal interface after the second stage of test E due to the depletion of reactive elements during the first stage. On the other hand, it has been previously evidenced that CO preferentially reacts with Al (see Fig. 8). One could estimate the Al supply at the oxide/alloy interface. Fig. 10 presents the Al profiles obtained by GDOES below the oxide layer formed on Haynes 230 ® after tests C (25 h) and D (6 h). The alloy is depleted in Al near the oxide/alloy interface and Al concentration near this interface decreases with time. This decrease may reflect a mixed kinetic. With the assumption that Al concentration at the oxide/alloy interface is negligible beside the bulk one, it is possible to calculate the upper limit flow of Al to the oxide/alloy interface by the Fick relationship in a semi-infinite solid [START_REF] Philibert | Diffusion and Mass Transport in Solids, Monographie de Physique[END_REF]: 5) [START_REF] Philibert | Diffusion and Mass Transport in Solids, Monographie de Physique[END_REF]:

[ ] 0 Al Al Al Al J D D t π = (
[ ] [ ] [ ] [ ] 0 0 2 i Al Al Al erf D t Al Al χ χ ⎛ ⎞ - = ⎜ ⎜ - ⎝ ⎠ ⎟ ⎟ (5)
in which χ is the depth in cm, [Al] the Al concentration at x in mol/cm 3 and [ ] i Al the Al concentration at the oxide/alloy interface. Eq. ( 5) fits well the two profiles with correlation coefficients equal to 0.99. The diffusion coefficients of Al obtained from Cand D are, respectively, 3.10 -13 cm 2 /s and 5. 10 -13 cm 2 /s. These values are of the same order of magnitude than in pure Ni at 850°C (approximately 6.5 × 10 -13 cm 2 /s [START_REF] Lupton | Report JUL-1639 Kernforschungsanlage Jülich GmbH IRW[END_REF]). and t = 54,000 s (15 h), the relation (4) gives: ≅ 1.8.10 -12 mol/cm 2 /s.

Al J

CO would only react with Al where the molecules can reach the oxide/alloy interface via the previously discussed high diffusion paths. By taking into account one tube-like nanochannel per grain with a cross section A and oxide grains with a mean diameter Φ, the flow of CO consumption would become:

2 3 2 Al CO J A J ≈ Φ (6)
if the diffusion rate of aluminium in the alloy is the rate limiting step of CO consumption, that is to say that J CO is roughly equal to 10 -12 mol/s/cm 2 . After Eq. ( 6) with Φ equal to 60 nm which is themean diameter of the oxide grains assessed by TEM observations, the diameter of the nanochannels has to be equal to 40 nm. This size is too large since the oxide layer appears already compact at this time when observed by TEM. In fact, smaller nanochannels have to be assumed but then, the Al flow to the oxide/alloy interface below them is too low to explain the whole CO reaction. As a consequence Al diffusion to the oxide/alloy interface cannot be the only rate limiting step of CO reaction: Si and Cr may react with CO to a certain extent as well and their diffusion to the oxide/alloy interface may participate to rate limiting step of CO reaction.

Conclusion

At 900 °C under impure helium containing few amounts (ppm) of CO and H2O, chromiaforming nickel based alloy, Haynes 230 ® , develops an upper Mn-Cr rich oxide layer with an inner discontinuous Al oxides. It has been shown by classical then ''double oxidation" experiments that CO reacts mainly during the early time of exposition (a few hours) and that this reaction mainly occurs at the oxide-metal interface. Transport through the scale is likely to occur through oxide grain boundaries and/or nanochannels. After a few hours, H2O becomes the major oxidant. As a consequence, it will control the long term oxidation behaviour of the alloy. In the meantime, CO can always access to the oxide/metal interface but the oxidation by CO becomes very low and the possible rate limiting steps have been quantitevely investigated. It has been proposed that the reaction of CO with the oxidizable elements Al, Si and Cr at the oxide/metal interface actually limits the reaction rate. {1} The test was carried at 850°C because it has been evidenced in [START_REF] Rouillard | Oxide-layer formation and stability on a nickel-base alloy in impure helium at high temperature[END_REF] that the oxide layer suffered from reduction by carbon in solution in the alloy when specimens were exposed at 900°C in helium atmosphere without any CO and containing low partial pressure of water vapour.

Figures captation

F Fi ig gu ur re e 1 1: : O Ou ut tl le et t p pa ar rt ti ia al l p pr re es ss su ur re e o of f C CO O a an nd d C CH H4 4 a as s a a f fu un nc ct ti io on n o of f t ti im me e d du ur ri in ng g a a b bl la an nk k t te es st t -i ie e w wi it th ho ou ut t a an ny y s sa am mp pl le es s -f fo or r a an n i in nl le et t h he el li iu um m m mi ix xt tu ur re e c co on nt ta ai in ni in

results 3 3 .

 3 .1 1. . G Ga as s p ph ha as se e a an na al ly ys se es s Fig. 2 shows the relative change in the CO partial pressure between the outlet and the inlet of the furnace. Due to reaction with the specimens, CO was consumed during the 3 heating up. CO consumption started at around 650 °C in test B with 2 H O P = 0.16 Pa and this temperature increased with the water vapour content in helium up to 850 °C in test A with 2 H O P = 0.4 Pa.

Discussion 4 4 .

 4 .1 1. . O Ox xi id de e n na at tu ur re e a an nd d s st tr ru uc ct tu ur re e Expositions of Haynes 230 ® specimens at 900 °C under impure helium, typically 2-5 Pa CO -0-2 Pa CH4 -20 Pa H2 and 0.01-0.4 Pa H2O, have showed the growth of a continuous chromium rich oxide layer on the alloy surface (see Fig.

4 4 .

 4 .2 2. . O Ox xi id da at ti io on n r re ea ac ct ti io on ns s b by y H H2 2O O a an nd d C CO O As already suggested

4 4 .

 4 .3 3. . S Sp pe ec ci if fi ic c r re ea ac ct ti iv vi it ty y o of f C CO O To verify if the inner oxide nodules could be mainly formed by oxidation with CO, the two-stage oxidation experiment E was carried out on model Ni-22Cr-14W-0.1C alloy at 850 °C {1}

  4) with [Al] 0 the bulk Al concentration, t the holding time at 900 °C and the diffusion coefficient of Al in Haynes 230 ® at 900 °C. Al D Al D can be determined by fitting the Al profiles in Fig. 11 with Eq. (

0

  Al =1.2.10 -3 mol/cm3

a

  By ICP -uncertainty: 3%. b By LECO_. c By weighing -uncertainty: 5%.

Table

  . The carbon content was analysed by combustion coupled with absorption spectroscopy (LECO ® ). Haynes 230 ® composition was obtained by Inductively Coupled Plasma (ICP) after the alloy was dissolved in acid. O Ox xi id da at ti io on n t te es st ts s

	2

  By a capacitive hygrometer with a detection limit of 10 -4 Pa and an accuracy about ± 3°C

	A An na al ly ys se ed d	C Cr r	A Al l	S Si i	N Ni i	M Mn n	O O
	s sp po ot t
	1 1	2 29 9	--	--	3 3	1 11 1	5 57 7
	2 2	2 28 8	--	--	4 4	1 11 1	5 57 7
	3 3	3 34 4	--	--	3 3	--	6 63 3
	4 4	1 18 8	1 17 7	4 4	2 2	--	5 59 9
	5 5	2 27 7	8 8	8 8	2 2	--	5 55 5
	6 6	3 31 1	5 5	--	2 2	--	6 62 2
	T Ta ab bl le e 3 3: : E ED DX XS S a an na al ly ys se es s i in n ( (a at t% %) ). .o of f d di if ff fe er re en nt t s sp po ot ts s i in n t th he e o ox xi id de e l la ay ye er r p pr re es se en nt te ed d i in n F Fi ig gu ur re e 4 4
	Carbon enrichment from Fig.5 (mol)	CO consumption from Fig.1 (mol)
	4.45 × 10 6			4.20 × 10 6
	T Ta ab bl le e 4 4: : C Co om mp pa ar ri is so on n b be et tw we ee en n t th he e c ca ar rb bo on n t tr ra an ns sf fe er rr re ed d i in nt to o H Ha ay yn ne es s 2 23 30 0® ® a an nd d t th he e C CO O c co on ns su um mp pt ti io on n
	d du ur ri in ng g t te es st t C C ( (i in n m mo ol l) ). .
	Test	Water vapour	Mean oxide layer	Oxide layer	% of the oxide
		partial pressure	thickness (µm)	thickness	layer formed by
		(Pa)			estimated by CO	CO reaction
						consumption
						(µm)
	A	4		600		110		~20
	B	1.6	450		190		~55
	T Ta ab bl le e 5 5: : F Fr ra ac ct ti io on n o of f t th he e o ox xi id de e l la ay ye er r f fo or rm me ed d b by y C CO O i in n t te es st t A A a an nd d t te es st t B B; ; m me ea an n o ox xi id de e t th hi ic ck kn ne es ss s f fr ro om m
	T Ta ab bl le e 1 1: : A Al ll lo oy y c co om mp po os si it ti io on n ( F FE ES SE EM M i im ma ag gi in ng g a an nd d t th hi ic ck kn ne es ss s o of f t th he e o ox xi id de e l la ay ye er r e es st ti im ma at te ed d f fr ro om m r re ea ac ct ti io on n w wi it th h C CO O ( (b ba as se ed d o on n F Fi ig g. . (w wt t% %) ). . 1 1) ). .
	Test	Alloy	Input gas composition a (Pa)		Linear	Temp.	Time(h)
							gas flow	(°C)
							rate
							(cm/s)
			H 2	CO	CH 4	H2O b
	A	Haynes	19.5 ±0.4	4.9 ±	2.1 ±	0.4	0.12	900	25
		230®		0.1	0.04
	B	Haynes	1.95 ±0.4	4.9 ±	2.1 ±	0.16	0.12	900	25
		230®		0.1	0.04
	C	Haynes	1.96 ±0.4	2.2 ±	1.9 ±	0.15	0.12	900	6
		230®		0.04	0.04
	D	NicrWC 19.6 ±0.4	4.9 ±	2.1 ±	0.15	0.12	900	25
				0.04	0.04
	E/Step1 NicrWC 20.0 ±0.4	-		8-10	0.14	850	48
	E/Step2 NicrWC 20.0 ±0.4	5.0 ±		<0.01	0.14	850	48
				0.1
				( 13 C 18 O)
	F	NicrWC 20.0 ±0.4	5.0 ±		<0.1	0.14	900	25
				0.1
	a Detection limit: 0.01 Pa and accuracy: 2%.
	T Ta ab bl le e 2 2: : T Te es st ts s g ga as s m mi ix xt tu ur re es s a an nd d c co on nd di it ti io on ns s. .

b

Acknowledgements

The authors are thankful to Mme M.C. Lafont at the Université Paul Sabatier of Toulouse for performing TEM analyses, to Mr P.Bonnaillie for the FESEM pictures performed at the CEA/DEN/DANS/DMN/SRMP, to Pr. S. Chevalier and Dr. O. Heintz for the SIMS analyzes performed at the Institute Carnot of Bourgogne and to Mr. M. Tabarant for the GDOES and the GDMS analyses performed at the CEA/DEN/DANS/DPC/SCP/LRSI.


Rui Zhang
email: rui.zhang@obs-banyuls.fr

Pavla Debeljak
email: pavla.debeljak@gmail.com

Stephane Blain
email: stephane.blain@obs-banyuls.fr

Ingrid Obernosterer
email: ingrid.obernosterer@obs-banyuls.fr

Seasonal shifts in Fe-acquisition strategies in Southern Ocean microbial communities revealed by metagenomics and autonomous sampling

Keywords: marine prokaryotes, iron and organic carbon acquisition, metagenomics, MAGs, Southern Ocean, autonomous sampler

The trace element iron is key for microbial metabolism, as a large number of enzymes involved in carbon-related pathways require iron as a co-factor. The acquisition of both elements is therefore essential for a cell to thrive in a given environment. Despite this key role of iron, the pathways used by diverse prokaryotes to access the different chemical forms of iron remains poorly understood. Understanding the iron acquisition strategies is of particular importance in marine environments where this trace element is present in growthlimiting concentrations. We report here high-resolution seasonal observations from a naturally iron-fertilized region in the Southern Ocean. Applying environmental metagenomics we identified different iron uptake strategies in marine prokaryotic communities and pronounced seasonal changes as an acclimation to varying iron and organic carbon regimes. Using metagenome-assembled genomes we further deciphered the Fe-related gene repertoire of individual taxa assigned to abundant prokaryotic groups. Our study provides novel insights to iron-related ecological strategies of diverse marine prokaryotes and how the acquisition of this element is linked to organic carbon.

Introduction

Organic carbon and iron (Fe) are essential elements for heterotrophic prokaryotes and from a metabolic point of view the two elements are intimately linked. Organic substrates provide building units for the cell and carbon metabolism is a source of energy, but a large number of enzymes involved in carbon-related pathways require Fe as a cofactor. The acquisition of both elements is therefore essential for a cell to thrive in a given environment. The concentration of marine organic matter varies on spatial and temporal scales and a large diversity of substrates with highly variable degrees of bioavailability make up this pool [START_REF] Moran | Deciphering ocean carbon in a changing world[END_REF]. The concentration of inorganic nutrients can regulate the utilization of labile forms of organic matter, and Fe could be a rate-limiting micro-nutrient in High Nutrient Low Chlorophyll (HNLC) regions [START_REF] Obernosterer | Fe and C co-limitation of heterotrophic bacteria in the naturally fertilized region off the Kerguelen Islands[END_REF].

The marine Fe pool is made up by various dissolved and particulate forms originating from biological, lithogenic or authigenic sources [START_REF] Tagliabue | The integral role of iron in ocean biogeochemistry[END_REF], but the availability of these different forms of Fe to prokaryotes remains poorly investigated [START_REF] Debeljak | Homeostasis drives intense microbial trace metal processing on marine particles[END_REF][START_REF] Hogle | Siderophores as an iron source for picocyanobacteria in deep chlorophyll maximum layers of the oligotrophic ocean[END_REF][START_REF] Manck | Petrobactin, a siderophore produced by Alteromonas, mediates community iron acquisition in the global ocean[END_REF]. The presence of multiple chemical forms of Fe is both a challenge and an opportunity for prokaryotes which have evolved a large panel of uptake strategies. Fe requirements and resource acquisition mechanisms vary among taxa [START_REF] Hopkinson | Iron transporters in marine prokaryotic genomes and metagenomes: Iron transporters in marine prokaryotes[END_REF]. Roseobacter genomes contain genes for multiple Fe uptake mechanisms, while SAR11 has a reduced set of transporters [START_REF] Roe | Utilization of Heme as an Iron Source by Marine Alphaproteobacteria in the Roseobacter Clade[END_REF][START_REF] Hogle | Trace Metal Acquisition by Marine Heterotrophic Bacterioplankton with Contrasting Trophic Strategies[END_REF]. Siderophore biosynthesis appears to be restricted to a few prokaryotic groups, and many of these belong to Gammaproteobacteria [START_REF] Hopkinson | Iron transporters in marine prokaryotic genomes and metagenomes: Iron transporters in marine prokaryotes[END_REF][START_REF] Payne | Vibrio Iron Transport: Evolutionary Adaptation to Life in Multiple Environments[END_REF][START_REF] Manck | Petrobactin, a siderophore produced by Alteromonas, mediates community iron acquisition in the global ocean[END_REF]. By contrast, transporters of siderophore-bound Fe are more widespread [START_REF] Hopkinson | Iron transporters in marine prokaryotic genomes and metagenomes: Iron transporters in marine prokaryotes[END_REF][START_REF] Debeljak | Homeostasis drives intense microbial trace metal processing on marine particles[END_REF][START_REF] Hogle | Siderophores as an iron source for picocyanobacteria in deep chlorophyll maximum layers of the oligotrophic ocean[END_REF], a differentiation that can lead to potential cross feeding [START_REF] Cordero | Public good dynamics drive evolution of iron acquisition strategies in natural bacterioplankton populations[END_REF]. The prevalence of specific Fe uptake strategies was shown to be linked to Fe concentrations across large spatial scales [START_REF] Toulza | Analysis of the Global Ocean Sampling (GOS) Project for Trends in Iron Uptake by Surface Ocean Microbes[END_REF]. Together, this results in a complex interplay between the genomic and physiological properties of individual microbes and their interaction among each other and with their micro-environment.

Responses of microbial communities from HNLC waters to additions of Fe and organic carbon in short-term bottle incubation experiments largely depend on location and season [START_REF] Obernosterer | Fe and C co-limitation of heterotrophic bacteria in the naturally fertilized region off the Kerguelen Islands[END_REF]. While these experiments provide evidence that Fe and organic carbon can constrain prokaryotic growth, they do not inform on potential acclimation of microbes to changes in the supply of these elements. The aim of the present study was to better understand the link between microbial Fe uptake strategies, organic matter supply and microbial diversity in the Southern Ocean. Our specific objectives were firstly to determine the seasonal dynamics in the functional repertoire of Fe-and organic substrate acquisition genes and secondly to identify the microbial community members harboring these genes under varying organic matter supply. We carried out our study in the region off Kerguelen Island, a suitable environment because the intense spring and summer phytoplankton blooms induced by natural Fe fertilization [START_REF] Blain | Effect of natural iron fertilization on carbon sequestration in the Southern Ocean[END_REF] result in pronounced seasonal patterns related to Fe and organic carbon availability. To address these objectives in an ocean region that is difficult to access due to logistic constraints, we used an autonomous in situ sampler providing high-resolution observations over an entire seasonal cycle.

Experimental Procedures

Sample collection

We used 12 seawater samples collected with a Remote Autonomous Sampler (RAS-500®, Mac Lane) from 25 October 2016 to 24 February 2017 in surface waters (40 m) of the Kerguelen plateau in the Indian sector of the Southern Ocean (Station A3, 527 m overall depth) (Figure 1). The technical aspects of the RAS and its mooring, and the collection of seawater are described in detail in [START_REF] Blain | Synchronized autonomous sampling reveals coupled pulses of biomass and export of morphologically different diatoms in the Southern Ocean[END_REF]. Briefly, three samples (500 mL each) were collected at each time point; for inorganic nutrient analyses, one sample was filtered in situ through an 0.8 μm polycarbonate filter (PC, 47 mm diameter, Nuclepore, Whatman, Sigma-Aldrich, St Louis, MO), and for phytoplankton and prokaryotic diversity [START_REF] Liu | Seasonal dynamics of prokaryotes and their associations with diatoms in the Southern Ocean as revealed by an autonomous sampler[END_REF][START_REF] Blain | Synchronized autonomous sampling reveals coupled pulses of biomass and export of morphologically different diatoms in the Southern Ocean[END_REF], two samples remained unfiltered. Fixatives (mercuric chloride or glutaraldehyde) were added to the sample bags prior to sampling. Upon recovery of the RAS, seawater for metagenomic analyses (fixed with mercuric chloride) was filtered sequentially through 0.8 and 0.2 µm PC filters (47 mm diameter, Nuclepore, Whatman, Sigma-Aldrich, St Louis, MO) and both filters were kept at -80 °C until DNA extraction.

Only the DNA extracted from the 0.2 µm filters were used for metagenomic analyses.

Temperature and salinity were obtained from sensors mounted on the RAS.

DNA extraction and metagenome sequencing

Total DNA was extracted from 0.2 µm PC filters using the DNeasy PowerWater Kit (Qiagen) according to the manufacturer's instructions with a few modifications. Each filter was cut into small pieces with the scissors, and added solution PW1 and incubated at 65°C for 10 min to promote cell lysis, the complete details of DNA extraction are given in the study by [START_REF] Liu | Seasonal dynamics of prokaryotes and their associations with diatoms in the Southern Ocean as revealed by an autonomous sampler[END_REF]. DNA concentration was measured using quantus fluorometer (Promega) with QuantiFluor® Double stranded DNA (dsDNA) system. Metagenome samples (n=12) were sequenced with an Illumina NovaSeq 6000 system using 2 × 150 bp chemistry at Fasteris SA, Inc. (Switzerland), and yielded 59-83 Mio pairs of metagenomic reads per sample, corresponding to a total of 263.8 GB (Table S1). The data sets are available in the European Nucleotide Archive (ENA) repository at https://www.ebi.ac.uk/ena under the project ID PRJEB56376.

Metagenome assembly

The raw reads were checked with FastQC v0.11.9 and processed with Trimmomatic (v 0.32) [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF]. The short reads that passed the quality control (QC) were individually assembled by MEGAHIT v1.2.9 [START_REF] Li | MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph[END_REF] (parameters used --presets meta-large). We also used metaSPAdes v3.14.1 (Nurk et al., 2017) (parameters used -k 21, 33, 55, 77, 99, 127) to assemble short reads for comparison. MetaQuast with default parameters was used to evaluate metagenomic assembly [START_REF] Mikheenko | MetaQUAST: evaluation of metagenome assemblies[END_REF]. For assembly using MEGAHIT, the total number of contigs varies between 0.1 and 1.6 Mio with an average length of 600 bp (Table S2). For the assembly using metaSPAdes, the total number of contigs varies between 0.3 and 1.8 Mio with an average length of 500 bp (Table S2). Due to the length of N50 we decided to continue the analysis from the MEGAHIT assembly results.

Metagenomic analysis

ORFs (open reading frames) were predicted using Prodigal v2.6.3 [START_REF] Hyatt | Prodigal: prokaryotic gene recognition and translation initiation site identification[END_REF] (parameters used -p meta) (Table S3). CD-HIT v4.8.1 [START_REF] Li | Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences[END_REF]) (parameters used -c 1 -aS 1 -g 1) was used to construct a non-redundant protein database pooling the twelve samples (10 984 076 proteins). Based on the non-redundant protein sequence number, we constructed the non-redundant gene sequence set. Salmon v1.4.0 [START_REF] Patro | Salmon provides fast and bias-aware quantification of transcript expression[END_REF] was used for read recruitment of the short reads to the non-redundant gene sequences collection, and for the quantification of each gene in each sample (parameters used -incompatPrior 0.0 --seqBias --gcBias --biasSpeedSamp 5 -validateMappings). The quantified protein occurrences for each sample were normalized as genes per kilobase million (GPM) using the following formula: 𝐺𝑃𝑀 = 10 ! × 𝑡𝑜𝑡𝑎𝑙 𝑟𝑒𝑎𝑑𝑠 𝑚𝑎𝑝𝑝𝑒𝑑 𝑡𝑜 𝑔𝑒𝑛𝑒𝑠/𝑔𝑒𝑛𝑒 𝑙𝑒𝑛𝑔𝑡ℎ 𝑖𝑛 𝑏𝑝 𝑠𝑢𝑚(𝑡𝑜𝑡𝑎𝑙 𝑟𝑒𝑎𝑑𝑠 𝑚𝑎𝑝𝑝𝑒𝑑 𝑡𝑜 𝑔𝑒𝑛𝑒𝑠/𝑔𝑒𝑛𝑒 𝑙𝑒𝑛𝑔𝑡ℎ 𝑖𝑛 𝑏𝑝)

This value can be applied to the metagenomes to remove the effect of library size and gene length, and GPM are thus comparable among samples.

To detect genes that are involved in Fe-related pathways, we used FeGenie [START_REF] Garber | FeGenie: A Comprehensive Tool for the Identification of Iron Genes and Iron Gene Neighborhoods in Genome and Metagenome Assemblies[END_REF] based on individual contig files for alignment and annotation (parameters used --metat 8 --norm). FeGenie is a bioinformatics tool to annotate iron-related genes by combining a Hidden Markov Model (HMM) and BLAST. HMM is a probabilistic model used for gene finding of sequencing data. The authors of FeGenie have constructed their own database of HMMs for Fe related genes by obtaining HMMs from Pfam/TIGRFAMS or by manually constructing HMMs. The HMMs were calibrated through queries against the NCBI's database. Bit score cutoffs that optimally delineate between true and false positives among hits from the non-redundant protein database were identified by manually inspecting each hmm search result. In the present study we used this comprehensive library to identify proteins that are related to Fe transport and storage, and siderophore biosynthesis and transport (Table S4). The genes identified from the present dataset passed the recommended bit score cutoffs. Different pathways can share homologous genes, as is the case for example in siderophore biosynthesis. HMMs developed in FeGenie are sensitive to the entirety of each gene family. In the metagenomes used here, not all domains of a given siderophore pathway were detected at each time point (see Fig. S3). This is most likely due to the overall lower GPMs of siderophore biosynthesis genes as compared to other Fe-related genes, and thus a limit of detection at our sequencing depth. In order to obtain GPM for the identified FeGenie proteins, each protein sequence related to the Fe pathway in each sample was retrieved in fasta format and mapped against the non-redundant protein database using DIAMOND blastp. Proteins at 100% similarity were retrieved with the according GPM.

For the taxonomic affiliation of Fe-related genes, sequences were obtained using Centrifuge v1.0.4 [START_REF] Kim | Centrifuge: rapid and sensitive classification of metagenomic sequences[END_REF] by searching against the GTDB database [START_REF] Parks | GTDB: an ongoing census of bacterial and archaeal diversity through a phylogenetically consistent, rank normalized and complete genome-based taxonomy[END_REF] and the alignments with highest score were kept. We then calculated the relative contribution of a prokaryotic group assigned to a gene of interest. The limitation of this approach is that taxonomy is assigned based on relatively short sequencing length of the individual genes; for this reason, we considered mainly the family level and used metagenome assembled genomes for a more resolutive description. Therefore, the GPM for each Fe-related gene in each sample were summed separately (corresponding to a), the GPM per taxa associated with each Fe-related gene in each sample were summed (corresponding to b), and the relative contribution of the prokaryotic group was calculated as b/a. Organic substrate transporter annotation was carried out based on the non-redundant protein sequences set against TCDB [START_REF] Saier | TCDB: the Transporter Classification Database for membrane transport protein analyses and information[END_REF] using DIAMOND blastp (parameters used --moresensitive -e 1e-5), and amino acid similarity of ≥ 70% was chosen as threshold for further analysis. CAZymes were annotated based on the non-redundant protein sequences set using hmmsearch against the dbCAN database (e value < 1 × 10-10, coverage > 0.3) [START_REF] Zhang | dbCAN2: a meta server for automated carbohydrate-active enzyme annotation[END_REF]. The domain with the highest coverage was selected for sequences overlapping multiple CAZymes domains. The phylogenetic affiliation of organic substrate transporters and CAZymes sequences and the relative contribution of prokaryotic groups was determined using the same method as described above.

Visualization of data was performed using the packages ggplot2 in the R v4.0.3 and SigmaPlot v14.5 software. The heatmap was generated using the pheatmap package in the R v 4.0.3, with Euclidean distance as the distance function.

Analysis of Metagenome Assembled Genomes (MAG)

One MAG assigned to each SAR11 and Nitrincolaceae, and two MAGs assigned each to Polaribacter and Rhodobacteraceae were downloaded from data previously published by [START_REF] Sun | Microbial iron and carbon metabolism as revealed by taxonomy-specific functional diversity in the Southern Ocean[END_REF]. The seawater samples from which the MAGs were constructed were collected from 3 sites in the Kerguelen region including the study site of the RAS deployment. After sequencing, metagenomes were assembled and binned using the methods described by Sun et al. [START_REF] Sun | Microbial iron and carbon metabolism as revealed by taxonomy-specific functional diversity in the Southern Ocean[END_REF]. MAGs were reassessed using CheckM (v1.1.2) [START_REF] Parks | CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes[END_REF] for completeness, and the completeness of the 6 MAGs used in the present study ranged from 55-94% (Table S5). The ANI values between MAGs, calculated using the OrthoANIu algorithm [START_REF] Yoon | A large-scale evaluation of algorithms to calculate average nucleotide identity[END_REF], were below 95%. ORFs of MAGs were predicted using Prodigal v2.6.3 [START_REF] Hyatt | Prodigal: prokaryotic gene recognition and translation initiation site identification[END_REF] (parameters used -p meta) and functional annotation of MAGs was carried out using the same method as above. To estimate the abundance of MAGs and their Fe-and organic substrate transporter and CAZymes-related genes across the twelve metagenomes, we first used Bowtie2 v2.4.4 [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF] with default parameters to recruit short reads from the twelve metagenomes and samtools [START_REF] Li | The Sequence Alignment/Map format and SAMtools[END_REF] was used to convert the resulting SAM files into sorted and indexed BAM files. We used anvi'o v7.1 [START_REF] Eren | Anvi'o: an advanced analysis and visualization platform for 'omics data[END_REF] to profile short metagenomic reads aligned to MAGs to estimate coverage statistics per metagenome. Briefly, we first used the program 'anvi-gen-contigs-database' turning MAGs into a contigs-db, then used the program 'anvi-profile' to process the BAM file and generate individual anvi'o profile database, and used the program 'anvi-merge' to merge these individual profiles into a final merged profile database which stored the coverage statistics of each MAGs in the 12 metagenomes. Finally, the program 'anvi-summarize' generated mean coverage values of each MAG and each gene within them across the twelve metagenomes and was visualized by the program 'anvi-interactive' in 'gene mode'.

Results

Environmental context

The site of the RAS deployment is located southeast of Kerguelen Island in the Indian Sector of the Southern Ocean (Figure 1). This naturally Fe-fertilized region has been extensively studied during previous oceanographic cruises focusing on distinct time periods, that are early spring, mid and late summer (projects KEOPS1&2 and MOBYDICK). The deployment of the RAS from 25 October 2016 to 24 February 2017 has for the first time provided a full seasonal picture of the physical context [START_REF] Pellichero | Relaxation of Wind Stress Drives the Abrupt Onset of Biological Carbon Uptake in the Kerguelen Bloom: A Multisensor Approach[END_REF], inorganic nutrient concentrations and characteristics of the consecutive phytoplankton blooms and their carbon and trace element export [START_REF] Blain | Synchronized autonomous sampling reveals coupled pulses of biomass and export of morphologically different diatoms in the Southern Ocean[END_REF][START_REF] Blain | Vertical Flux of Trace Elements Associated with Lithogenic and Biogenic Carrier Phases in the Southern Ocean[END_REF]. Briefly, the spring phytoplankton bloom started in late October and peaked by mid-November and the summer bloom occurred in early January (Figure S1A). Diatoms dominated both blooms but varied considerably in their composition between seasons [START_REF] Blain | Synchronized autonomous sampling reveals coupled pulses of biomass and export of morphologically different diatoms in the Southern Ocean[END_REF]. Surface water temperature steadily increased from 1.70°C to 4.17°C over the course of the sampling period. Concentrations of nitrate (range 21.95 to 27.69 μM) and silicic acid (range 2.45-8.24 μM) decreased during the observation period, while ammonium concentrations (range 0.3 to 2 μM) reached a maximum in late December (Figure S1B) [START_REF] Blain | Synchronized autonomous sampling reveals coupled pulses of biomass and export of morphologically different diatoms in the Southern Ocean[END_REF].

Seasonal trends in Fe and C transporters

In total 28 662 Fe-related genes were annotated in the FeGenie database, 66 005 genes related to substrate transport in the TCDB database and 136 460 genes related to enzymatic activity in the CAZymes database. Fe-related genes exhibited lowest normalized gene abundances (genes per kilobase million, GPM) (10-600 GPM), followed by organic carbon membrane transporters (600 to 1400 GPM) and CAZymes (7000 to 12000 GPM).

Normalized gene abundances (GPM) of each of these categories revealed pronounced seasonal patterns (Figure 2). Transporters of Fe ions (Fe 3+ , Fe 2+ ) (Figure 2A), siderophores and heme (Figure 2B) had the highest GPM after the blooms. Increases in GPM occurred rapidly after the spring bloom and with a time lag of about one month after the summer bloom. Siderophore biosynthesis genes had one peak during the spring bloom, and a second peak after the summer bloom in parallel with various Fe transporters (Figure 2B). Organic substrate transporters revealed the highest GPM during the spring bloom and a smaller peak was observed one month after the summer bloom (Figure 2C). Organic substrate transporters increased also during the final spring bloom decline, following the seasonal peak in CAZymes.

The most abundant transporters of Fe 3+ ions were fbpABC, futA1/A2 and fbpB-futB, and the dominant Fe 2+ ion transporter genes were feoAB and efeUOB (Figure 3A) (Table S4).

Siderophore uptake genes were dominated by the exbB-exbD-tonB complex and among the genes identified for the transport of specific siderophores were those of pyoverdin (fpv), aerobactin (hat), ferric enterobactin (pir), ferripyochelin (fpt), and vibrioferrin (pvu) (Figure 3B). We further identified four siderophore biosynthesis clusters related to pyochelin (pch), pyoverdin (pvd), rhizobactin (rhb) and vanchrobactin (vab) (Figures S2 andS3). Among other Fe-related genes identified, a seasonal trend driven by the two phytoplankton blooms was also detectable for Fe regulation (fur, dtxR, fecR), while genes for iron storage (ftn) remained stable over time (Figure S2). Transporters of organic substrates were dominated by those for amino acids and proteins, followed by sugars, glycerol and organic acids, and transporters for vitamins were also present throughout the season (Figure 3C). During the sampling period, 379 families of CAZymes were identified, among which 191 glycoside hydrolase (GH) families, 82 glycosyltransferase (GT) families, 41 polysaccharide lyase (PL) families, 34 carbohydrate-binding module (CBM) class families, 16 carbohydrate esterase (CE) families and 13 auxiliary activities (AA) families (Figure 3D).

To obtain a more detailed picture of the seasonal patterns of genes involved in Fe and organic substrate acquisition, we determined the similarity in the temporal trend among individual genes (Figure 4). This analysis highlights 4 periods. The first period, corresponding to the spring bloom (6 Nov and 17 Nov), was characterized by seasonally highest GPM of genes coding for siderophore synthesis (e.g., Pyochelin (pch), Pyoverdin (pvd), Vanchrobactin (vab)) and transport (e.g., catecholate receptor (piu), ferripyochelin (fpt), Vibriobactin (viuB)), concurrently with several organic substance transporters, such as amino acids, sugars, spermidine/putrescine, and sulfonates. During the second period, that corresponds to the immediately following decline of the spring bloom (28 Nov), concurrent peaks in transporters of Fe 3+ ions (fbp, fut), heme (hmu) and siderophores (e.g., Vibrioferrin (pvu), Aerobactin (hat), Pyoverdin (fpv)) were observed. This period further revealed high GPM of several CAZymes (GT, CE, GH). The third period extends from the final declining phase of the spring bloom to the summer bloom (9, 20 and 31 Dec) and was characterized by enhanced GPM of transporters of Fe 3+ ions (fbpB-futB, futA1/A2, yfe). The fourth period is represented by two sampling dates one month after the summer bloom (22 Jan and 2 Feb).

Siderophore synthesis genes present already during the first phase re-emerged (e.g., Pch, Pvd, Vab) in combination with a new group of siderophore synthesis (e.g., Rhizobactin (rhb)) and transport (Ferric Enterobactin (pir), Legiobactin (lbt)) genes. During the fourth period, transporters of Fe peaked in parallel with those of organic compounds and siderophore synthesis genes, a pattern that contrasted our observations during the spring bloom.

Linking function to taxonomy at the family level

These temporal changes in the functional repertoire were paralleled by shifts in the prokaryotic community composition [START_REF] Liu | Seasonal dynamics of prokaryotes and their associations with diatoms in the Southern Ocean as revealed by an autonomous sampler[END_REF] raising the question of the link between microbial diversity and Fe and organic substrate acquisition. To investigate whether the genes of interest were harbored by specific prokaryotic groups we assigned the taxonomy of the individual genes belonging to the different gene categories. We illustrate here the taxonomic assignments of 4 genes, each representing the transport of a distinct form of Fe and each characterized by a marked seasonal trend; these are transport of Fe 3+ ions (fbp) and Fe 2+ ions (feo), of heme (hmu) and the siderophore ferric enterobactin receptor (pir) (Figure 5). The taxonomic assignments of other Fe-related genes are illustrated in Figure S4. The transporter fbp (Fe 3+ ions) was assigned to several prokaryotic groups and their respective relative contributions changed considerably over time (Figure 5). During the spring bloom (period 1) when GPM were low, transport of Fe 3+ ions was assigned to Pseudomonadaceae, Pelagibacteraceae and Rhodobacteraceae, while Nitrincolaceae dominated the taxonomic assignments (75 % of all fbp genes) when GPM peaked during the bloom decline (period 2).

Rhodobacteraceae (in particular the genus Amylibacter, Figure S5) were the most abundant contributors to fbp (19-51%) during the remaining season and Pseudomonadaceae had an increasing share towards the end of the season (4-35%). A contrasting picture was obtained for feo (transport of Fe 2+ ions) that was assigned to mainly two groups, Sphingomonadaceae in early spring (period 1) (28-39%) and Flavobacteriaceae throughout the remaining season (16-89%). Flavobacteriaceae were dominated by the genus Polaribacter (Figure S5) during the spring bloom decline (period 2). Heme uptake (hmu) revealed a seasonal abundance pattern similar to that of fbp, but had distinct taxonomic assignments. Porticoccaceae (7-43%), Flavobacteriaceae (2-76%) and Nostocaceae (2-14%) were the main contributors to heme uptake throughout the season. During the bloom decline (phase 2) when hmu peaked Polaribacter irgensii (Flavobacteriaceae) had a dominant contribution, and Cellulophaga, hydrogenovibrio and Kordia (Flavobacteriaceae) (Figure S5) prevailed over the remaining season. The ferric enterobactin receptor pir had a seasonal abundance pattern different to that of heme, with peaks during period 1 and 4, but overall similar taxonomic assignments at the family level. Flavobacteriaceae (6-66%), with substantial contributions of Polaribacter, Pseudomonadaceae and Porticoccaceae (1-13%) were the dominant contributors to pir, and Sphingomonadaceae (0-15%) and Cellvibrionacaea (0-30%) had seasonally variable contributions. The taxonomic assignments of genes encoding for organic substrate transporters and CAZymes revealed overall small differences in the prokaryotic groups contributing to each category, but marked temporal changes in their relative contributions were observed, in particular during period 2 (Figures S6 andS7).

Genome-resolved functional potential

To obtain insights on the functional potential for the acquisition of both Fe and organic substrates, we used previously published metagenome assembled genomes (MAGs) obtained from samples collected in the Kerguelen region, including our study site [START_REF] Sun | Microbial iron and carbon metabolism as revealed by taxonomy-specific functional diversity in the Southern Ocean[END_REF].

We choose MAGs belonging to SAR11, Nitrincolaceae, Rhodobacteracea, and the genus Polaribacter (Flavobacteriaceae) (Figure 6 and Table S5). These MAGs belong to prokaryotic groups with substantial contributions to the different gene categories (Figure 5 and Figures S5-S7). Further, ASVs belonging to each of these groups were shown to be abundant and have pronounced seasonal relative abundance patterns, based on 16S rRNA gene sequencing from the same RAS deployment [START_REF] Liu | Seasonal dynamics of prokaryotes and their associations with diatoms in the Southern Ocean as revealed by an autonomous sampler[END_REF]. Mapping our metagenomic short reads to these MAGs revealed that they largely varied in their respective coverages and had each a distinct seasonal trend. SAR11 MAG133 had slightly higher coverage in early spring as compared to the remaining season. By contrast, Nitrincolaceae MAG115 revealed one sharp peak during period 2. The 2 Rhodobacteraceae MAGs had both higher coverage during the transition between the spring and summer blooms (period 3). By contrast, the 2 Polaribacter MAGs differed in their overall coverage and their seasonal dynamics. The inventories of the Fe-and organic substrate-acquisition genes provided some insights on the metabolic versatility of these MAGs. SAR11 MAG133 harbored a reduced set of Fe (fut, fbpB-futB, Fe 3+ ions) and organic substrate (sugars and amino acids) transporters.

In Nitrincolaceae MAG115 we identified transporters for siderophore-bound Fe (exbB-exbD complex and fpv for pyoverdin) and for several organic substrates (sugars, amino acids, peptides) and vitamins. Both Rhodobacteraceae MAGs harbored transporters for Fe 3+ ions (yfe, fbp), but appear to differ in their potential of organic substrate acquisition. We identified transporters of several substrates in Rhodobacteraceae MAG20 while only amino acid transporters were detected in Rhodobacteraceae MAG52. Among the MAGs considered here, Polaribacter MAG88 had the most diverse Fe-gene inventory, including heme and bacterioferritin, absent from MAG64. The 2 Polaribacter MAGs harbored both transporters of Fe 2+ ions (feo), proteins and carboxylic acids.

Discussion

We observed pronounced seasonal patterns in the prevalence of genes for the transport of Fe and organic substrates by prokaryotic communities in the Southern Ocean. The acquisition of Fe was temporally decoupled from that of organic carbon over the course of the spring phytoplankton bloom, while a concerted access to both elements occurred after the summer bloom. These dynamics on the community level likely result from seasonal changes in the requirements of Fe and organic carbon and the functional capabilities to acquire the different chemical forms in which these elements are present by diverse microbial taxa. Using metagenome assembled genomes, we illustrate differences in the Fe-and organic substrate acquisition repertoires among abundant prokaryotic taxa. These observations combined with marked seasonal changes in microbial community composition [START_REF] Liu | Seasonal dynamics of prokaryotes and their associations with diatoms in the Southern Ocean as revealed by an autonomous sampler[END_REF] provide new insights on the potential ecological niches of prokaryotes in the Fe-and organic carbonconstrained Southern Ocean.

Seasonal microbial response to phytoplankton blooms induced by natural iron fertilization

Natural Fe-fertilization in the region off Kerguelen Island leads to annually occurring phytoplankton blooms that impact ecosystem structure and functioning [START_REF] Blain | Effect of natural iron fertilization on carbon sequestration in the Southern Ocean[END_REF].

Heterotrophic prokaryotes respond markedly to these blooms in terms of growth, biomass production and respiration and thereby contribute to the processing of a substantial fraction of primary production during different seasons [START_REF] Christaki | Seasonal microbial food web dynamics in contrasting Southern Ocean productivity regimes[END_REF]. The microbial communities present and active in these naturally Fe-fertilized waters vary over the course of the bloom [START_REF] Liu | Seasonal dynamics of prokaryotes and their associations with diatoms in the Southern Ocean as revealed by an autonomous sampler[END_REF] and are distinct to communities in surrounding HNLC waters during different seasons [START_REF] West | Major differences of bacterial diversity and activity inside and outside of a natural iron-fertilized phytoplankton bloom in the Southern Ocean[END_REF][START_REF] Landa | Shifts in bacterial community composition associated with increased carbon cycling in a mosaic of phytoplankton blooms[END_REF][START_REF] Hernandez-Magana | Prokaryotic diversity and activity in contrasting productivity regimes in late summer in the Kerguelen region (Southern Ocean)[END_REF]. Differences among prokaryotic taxa in Fe requirements and acquisition strategies as well as in the metabolic potential for the uptake of diverse phytoplankton-derived organic substrates could be one underlying mechanism [START_REF] Teeling | Substrate-Controlled Succession of Marine Bacterioplankton Populations Induced by a Phytoplankton Bloom[END_REF][START_REF] Debeljak | Microbial iron metabolism as revealed by gene expression profiles in contrasted Southern Ocean regimes[END_REF].

Niche differentiation in relation to Fe and organic carbon was recently described on a spatial scale [START_REF] Sun | Microbial iron and carbon metabolism as revealed by taxonomy-specific functional diversity in the Southern Ocean[END_REF], but a temporal perspective is thus far lacking.

Microbial strategies and Fe sources during the spring bloom

The prevalence of organic substrate transporters during the spring bloom (period 1) points to a community that rapidly responds to the supply of labile phytoplankton-derived substrates.

Organic carbon has been identified as a growth-limiting resource for the winter community in the Kerguelen region [START_REF] Obernosterer | Fe and C co-limitation of heterotrophic bacteria in the naturally fertilized region off the Kerguelen Islands[END_REF][START_REF] Landa | Shifts in bacterial community composition associated with increased carbon cycling in a mosaic of phytoplankton blooms[END_REF]. Our data suggest that amino acids, organic acids, sugars and sulfonates are the most prominent substances utilized, confirmed by metatranscriptomics and -proteomics data at the same study site (Debeljak et al., unpublished) and as reported previously from phytoplankton blooms in other marine environments [START_REF] Teeling | Substrate-Controlled Succession of Marine Bacterioplankton Populations Induced by a Phytoplankton Bloom[END_REF][START_REF] Francis | North Sea spring bloom-associated Gammaproteobacteria fill diverse heterotrophic niches[END_REF]. The seasonal pattern of organic substrate transporters was, however, decoupled from that of Fe-transporters during the spring bloom, raising the question of how prokaryotes meet their Fe-requirements.

Dissolved Fe concentrations are high in surface waters in early spring (0.16 nM) [START_REF] Quéroué | High variability in dissolved iron concentrations in the vicinity of the Kerguelen Islands (Southern Ocean)[END_REF], but heterotrophic prokaryotes compete with fast growing small phytoplankton for this Fe source [START_REF] Fourquez | Microbial iron uptake in the naturally fertilized waters in the vicinity of the Kerguelen Islands: phytoplankton-bacteria interactions[END_REF]. As reported for diverse members of this clade [START_REF] Liu | Seasonal dynamics of prokaryotes and their associations with diatoms in the Southern Ocean as revealed by an autonomous sampler[END_REF][START_REF] Dinasquet | SAR11 clade microdiversity and activity during the early spring blooms off Kerguelen Island, Southern Ocean[END_REF] SAR11 MAG133 was abundant in early spring. The small streamlined genomes of SAR11 [START_REF] Giovannoni | SAR11 Bacteria: The Most Abundant Plankton in the Oceans[END_REF] have reduced Fe-uptake repertoires [START_REF] Hogle | Trace Metal Acquisition by Marine Heterotrophic Bacterioplankton with Contrasting Trophic Strategies[END_REF], as observed also for SAR11 MAG133 (Fe 3+ ion transporters fut, fbpB-futB). Using MICRO-CARD-FISH, SAR11 accounted for about 25% of community Fe uptake, while the contribution to leucine uptake was 50% in early spring in fertilized and HNLC waters [START_REF] Fourquez | Uptake of Leucine, Chitin, and Iron by Prokaryotic Groups during Spring Phytoplankton Blooms Induced by Natural Iron Fertilization off Kerguelen Island (Southern Ocean)[END_REF], indicating an efficient organic substrate utilization even under conditions when access to Fe is constrained. Low Fe requirements and an efficient transport of Fe and organic substrates could be a strategy of SAR11 to take advantage of the pool of labile substrates supplied by phytoplankton.

Lithogenic particles present a potentially important source of Fe in early spring above the Kerguelen plateau [START_REF] Blain | Vertical Flux of Trace Elements Associated with Lithogenic and Biogenic Carrier Phases in the Southern Ocean[END_REF]. This Fe source is geochemically complex and needs to be rendered biologically available via dissolution as for example through the binding to high affinity ligands [START_REF] Kalinowski | Rates of bacteriapromoted solubilization of Fe from minerals: a review of problems and approaches[END_REF]. Among the strongest binding ligands are catecholate-type siderophores and particle-associated prokaryotes were shown to express the genes implicated in the respective biosynthesis pathways [START_REF] Debeljak | Homeostasis drives intense microbial trace metal processing on marine particles[END_REF]. The prevalence of a catecholate-type siderophore receptor gene (piu) during spring indicates that this Fe source could in part account for the prokaryotic demand. Taxonomic assignments revealed that Pseudomonadaceae were the most prominent group harboring this receptor, an observation that supports previous findings on the role of this group in siderophore biosynthesis and uptake [START_REF] Schalk | An overview of siderophore biosynthesis among fluorescent Pseudomonads and new insights into their complex cellular organization[END_REF]. An alternative strategy could be the utilization of an internal reservoir, such as Fe stored in specific proteins (bacterioferritin, Ftn) [START_REF] Andrews | Bacterial iron homeostasis[END_REF]. This capacity was shown for strains belonging to Pseudoalteromonas [START_REF] Mazzotta | Characterization of the Fe metalloproteome of a ubiquitous marine heterotroph, Pseudoalteromonas (BB2-AT2): multiple bacterioferritin copies enable significant Fe storage[END_REF] and several prokaryotic groups, in particular Flavobacteriaceae, Halieaceae and Altermonadaceae contributed to the expression of the respective genes above the Kerguelen plateau in early spring [START_REF] Debeljak | Microbial iron metabolism as revealed by gene expression profiles in contrasted Southern Ocean regimes[END_REF]. We identified the gene coding for ferritin in Polaribacter MAG88, which had increased coverage during spring, in contrast to Polaribacter MAG64 that lacked this gene and revealed enhanced coverage later in the season. These observations illustrate potential niche differentiation among closely related taxa. The utilization of internal Fe stored as ferritin by some taxa could be a possible mechanism for the apparent temporal decoupling in requirements of organic substrates and Fe.

Fe acquisition strategies during the spring bloom decline

Microbial gene inventories related to Fe and organic carbon transport drastically changed during the decline of the spring bloom (period 2). The prevalence of all CAZyme families indicates the presence of a chemically different pool of organic matter and the need to cleave polysaccharides to access organic carbon [START_REF] Teeling | Substrate-Controlled Succession of Marine Bacterioplankton Populations Induced by a Phytoplankton Bloom[END_REF]. The synchronized enhancement in the GPM of different types of Fe-transporters, including those for Fe 3+ and Fe 2+ ions, Fe bound to siderophores (Vibrioferrin (pvu), Aerobactin (hat), Pyoverdin (fpv)) and heme suggest an increased prokaryotic Fe demand in response to the shift in the organic carbon regime during the phytoplankton bloom decline. In contrast to early spring, remineralization is a key process providing different sources of Fe to the system. Our observations allow us to provide insights into possible Fe and organic carbon related strategies of three bacterial groups with distinct abundance patterns.

Nitrincolaceae, represented by MAG115, rapidly responded to the bloom decline. The marked seasonal pattern of MAG115, reaching highest coverages of all MAGs considered here, matched the relative abundances of the respective ASVs as determined from 16S rRNA gene sequences [START_REF] Liu | Seasonal dynamics of prokaryotes and their associations with diatoms in the Southern Ocean as revealed by an autonomous sampler[END_REF]. Nitrincolaceae dominated the taxonomic assignments of transporters of Fe 3+ ions (fbp) during the bloom decline and MAG115 harbored several siderophore uptake genes, such as pyoverdine (fpv), a siderophore containing catecholate and hydroxamate groups. MAG115 also contained a large repertoire of organic substrate transporters indicating its capacity in the uptake of various labile compounds (B. Francis et al., 2021). The rapid response to a pulsed supply of organic carbon by taking advantage of its potential to utilize different forms of Fe suggests a copiotrophic metabolic strategy, and idea that is further supported by the concurrent expression both at the transcriptomic and proteomic level of genes related to Fe-and organic carbon transport by Nitrincolaceae MAG115 (Debeljak et al., unpublished).

Polaribacter MAG88 (Flavobacteriaceae) also responded to the bloom decline, but its coverage remained comparatively low. This MAG harbored a large repertoire of transporters for organic substrates and Fe, and among the latter we identified the heme transporter hmu.

Heme is a porphyrin-bound source of Fe that can account for a large fraction of cellular Fe concentrations in phytoplankton and can become a source to heterotrophs when cells are destroyed such as by viral lysis or grazing [START_REF] Honey | Heme b in marine phytoplankton and particulate material from the North Atlantic Ocean[END_REF]. Utilization of heme was demonstrated experimentally for strains belonging to the Roseobacter clade [START_REF] Roe | Utilization of Heme as an Iron Source by Marine Alphaproteobacteria in the Roseobacter Clade[END_REF][START_REF] Hogle | Direct Heme Uptake by Phytoplankton-Associated Roseobacter Bacteria[END_REF], but many other abundant microbial groups appear to lack heme transporters [START_REF] Hogle | Direct Heme Uptake by Phytoplankton-Associated Roseobacter Bacteria[END_REF]. In the present study, Flavobacteriaceae had a substantial share of the taxonomic assignments of heme transporters (hmu), in particular during the bloom decline. Our data suggest that members belonging to Flavobacteriaceae could use this source of Fe, to account for its requirements during the degradation of phytoplankton derived organic substances. This potential niche specialization could play an important role in large particles where access to this source of Fe is facilitated.

Both Roseobacter MAGs had increased coverage during the transition between the spring decline and summer bloom, matching observations of members of this clade at the ASVs level [START_REF] Liu | Seasonal dynamics of prokaryotes and their associations with diatoms in the Southern Ocean as revealed by an autonomous sampler[END_REF]. While functional Fe-profiles were overall similar for both MAGs, MAG20 appears to have a larger organic substrate repertoire than MAG52. An analysis of Roseobacter genomes revealed the presence of multiple and diverse pathways for the acquisition of inorganic and organically bound Fe and several copies of ABC transporters [START_REF] Hogle | Trace Metal Acquisition by Marine Heterotrophic Bacterioplankton with Contrasting Trophic Strategies[END_REF]. This indicates that this group can make use of a variety of chemical forms of Fe, an idea that is supported by observations on the transcriptomic [START_REF] Debeljak | Microbial iron metabolism as revealed by gene expression profiles in contrasted Southern Ocean regimes[END_REF][START_REF] Debeljak | Homeostasis drives intense microbial trace metal processing on marine particles[END_REF] level. This versatility with respect to Fe transport let us hypothesize that specialization of organic substrate utilization could be the underlying mechanism for the similar seasonal patterns of the two Roseobacter MAGs.

Parallel Fe and C acquisition following the productive season

Despite the smaller magnitude of the summer phytoplankton bloom as compared to spring, the community functional repertoire markedly changed with a time lag of about 1 month, contrasting the rapid response to the spring bloom. The late summer period is characterized by slightly higher concentrations of dissolved organic carbon and seasonally highest prokaryotic abundance [START_REF] Christaki | Seasonal microbial food web dynamics in contrasting Southern Ocean productivity regimes[END_REF][START_REF] Hernandez-Magana | Prokaryotic diversity and activity in contrasting productivity regimes in late summer in the Kerguelen region (Southern Ocean)[END_REF] and remineralization presents the main source of Fe [START_REF] Blain | Distribution of dissolved iron during the natural iron-fertilization experiment KEOPS (Kerguelen Plateau, Southern Ocean)[END_REF][START_REF] Sarthou | The fate of biogenic iron during a phytoplankton bloom induced by natural fertilisation: Impact of copepod grazing[END_REF].

These environmental conditions could be favorable for the energetically costly biosynthesis of siderophores, supported by our observation of the diverse assemblage of genes for the acquisition of siderophore-bound Fe at the end of the season. Among these the gene of the siderophore receptor pir (ferric enterobactin receptor) was assigned to Flavobacteriaceae, and the two Polaribacter MAGs harbored both this gene. Many members of Flavobacteriaceae, including Polaribacter, are specialized in the degradation of complex compounds, such as polysaccharides [START_REF] Xing | Niches of two polysaccharide-degrading Polaribacter isolates from the North Sea during a spring diatom bloom[END_REF][START_REF] Kappelmann | Polysaccharide utilization loci of North Sea Flavobacteriia as basis for using SusC/D-protein expression for predicting major phytoplankton glycans[END_REF]. Our results indicate that the concurrent access to organic substrates and siderophore-bound Fe could be a strategy that renders these taxa efficient degraders of organic matter in the Southern Ocean.

By considering transporters for different forms of Fe and organic carbon, two elements that were shown to constrain prokaryotic growth in the Southern Ocean, we provide insights on temporal dynamics in nutrient acquisition at the community level. The distinct seasonal patterns in Fe transporters with respect to those of organic substrates suggest that there could be a switch in nutrient requirements along the season. Our results extend the use of biomarker genes that have allowed to identify nutrient stress of specific microbial groups on spatial scales [START_REF] Saito | Multiple nutrient stresses at intersecting Pacific Ocean biomes detected by protein biomarkers[END_REF][START_REF] Garcia | Linking regional shifts in microbial genome adaptation with surface ocean biogeochemistry[END_REF][START_REF] Ustick | Metagenomic analysis reveals global-scale patterns of ocean nutrient limitation[END_REF]. The taxon-specific genomic capacities to access the different chemical forms of Fe could be the basis for ecological niches and drive the observed changes in prokaryotic community composition [START_REF] Liu | Seasonal dynamics of prokaryotes and their associations with diatoms in the Southern Ocean as revealed by an autonomous sampler[END_REF] as an acclimation to the supply of phytoplankton-derived organic matter in this naturally fertilized region of the Southern Ocean.

Figure 1 .

 1 Figure 1. Map of the region around Kerguelen Island and location of the deployment of the remote autonomous sampler (white dot). Insert shows global position of Kerguelen Island as indicated by a square. Monthly composite of chlorophyll for November 2016. Modified from Blain et al.[START_REF] Blain | Vertical Flux of Trace Elements Associated with Lithogenic and Biogenic Carrier Phases in the Southern Ocean[END_REF].

Figure 2 .

 2 Figure 2. A. Temporal changes of Chlorophyll a (green shaded area) and abundance of genes for Fe 3+ and Fe 2+ transporters (A), siderophore-bound Fe and heme transporters, and siderophore biosynthesis (B) and carbon substrate transporters and carbohydrate-active enzymes (CAZymes) (C). Normalized gene abundances are given in genes per kilobase million (GPM) (see Material and Methods for details).

Figure 3 .

 3 Figure 3. Abundance of individual genes contributing to Fe 3+ and Fe 2+ transport (A), heme and siderophore transport (B), organic substrate transport (C) and carbohydrate-active enzymes (CAZymes, D) at the 12 time points. Transporters of fatty acids, lipoproteins, ammonia, exopolysaccharides, and urea are pooled as 'others'. CAZyme classes include auxiliary activities (AA), carbohydrate-binding modules (CBM), carbohydrate esterases (CE), cohesin, glycoside hydrolases (GH), glycosyltransferases (GT), polysaccharide lyases (PL), and S-layer homology domain (SLH). Normalized gene abundances are given in genes per kilobase million (GPM) (see Material and Methods for details).

Figure 4 .

 4 Figure 4. Heatmap illustrates the seasonal patterns of genes involved in Fe and organic substrate transporters and related processes. Similarity between genes is based on Euclidian distance cluster analysis. Color intensity is determined by Z-score transformed normalized gene abundances (GPM). The vertical lines separate the four periods.

Figure 5 .

 5 Figure 5. Temporal changes of normalized gene abundance (GPM) and relative contribution of prokaryotic groups to specific genes associated with Fe 3+ , Fe 2+ , heme and siderophore transport. Prokaryotic groups are based on the family level. Prokaryotic groups discussed in the text are marked in red.

Figure 6 .

 6 Figure 6. Mean coverage of MAGs in the twelve samples and inventories of genes related to Fe and organic substrate transport and CAZymes. Mean coverage on the upper panel represents the average depth of coverage across contig (the coverage of each nucleotide in a contig divided by the length of the contig). Mean coverage on the lower panel represents the Σ coverage of each bp in a gene / gene length.

Acknowledgements

We thank the team of the Technical Division of the Institute of the Sciences of the Universe (DT-INSU) for the design and construction of the mooring for the RAS. We thank the captains and the crews of the R/V Marion Dufresne for their support during the deployment and the recovery of the RAS. The project SOCLIM (Southern Ocean and Climate) was supported by the Climate Initiative of the BNP Paribas Foundation, the French Polar Institute (Institut Polaire Paul Emile Victor), and the French program LEFE-CYBER of the CNRS-INSU. We thank Yan Liu who carried out the DNA extraction. We thank the French Institute of Bioinformatics (IFB; https://www.france-bioinformatique.fr) for providing computing resources. We thank to the reviewers for their insightful comments on a previous version of this manuscript. This work is part of the PhD thesis of R.Z. supported by the China Scholarship Council (CSC; No. 202006220057).

Data availability statement

The data sets generated and analysed during the current study are available in the European Nucleotide Archive (ENA) repository at https://www.ebi.ac.uk/ena under the project ID PRJEB56376.

Competing of interests

The authors declare no competing interests.

Author contributions

Rui Zhang, Pavla Debeljak, Stephane Blain and Ingrid Obernosterer designed the research.

Bioinformatic analyses were performed by Rui Zhang. Rui Zhang and Ingrid Obernosterer wrote the first draft of the manuscript. All authors contributed to editing the manuscript.

Competing of interests

The authors declare no competing interests.