Huihui Yang

Qiang Liu

Sylvie Masse

Hao Zhang

Laifeng Li
email: laifengli@mail.ipc.ac.cn

Thibaud Coradin
email: thibaud.coradin@upmc.fr

Prof S Masse

Prof. T Coradin

Hierarchically-organized, well-dispersed hydroxyapatite-coated magnetic carbon with combined organics and inorganics removal properties

Keywords: water treatment, superconducting separation, porous spheres, carbon, hydroxyapatite

Hierarchically-organized, well-dispersed hydroxyapatite-coated magnetic carbon with combined organics and inorganics removal properties

Introduction

Long-term exposure to polluted waters has significant detrimental effects on human health and, in particular, can cause diseases, such as lungs, bladder, kidneys and skin cancer. The development of low-cost and efficient methods for wastewater treatment is therefore an urgent and highly relevant research area [START_REF] Yang | Highcontent, well-dispersed γ-Fe 2 O 3 nanoparticles encapsulated in macroporous silica with superior arsenic removal performance[END_REF][START_REF] Crini | Recent developments in polysaccharide-based materials used as adsorbents in wastewater treatment[END_REF][START_REF] Martin | Activated carbons developed from surplus sewage sludge for the removal of dyes from dilute aqueous solutions[END_REF]. Under normal circumstances, water treatment involves a series of physical, chemical and biological processes [START_REF] Kurniawan | Physicochemical treatment techniques for wastewater laden with heavy metals[END_REF][START_REF] Lee | Effect of coagulation conditions on membrane filtration characteristics in coagulation-microfiltration process for water treatment[END_REF]. A critically timeconsuming step is the coagulation/adsorption process where pollutants are precipitated in a particulate form or adsorb on powders as this often requires a subsequent decantation period [START_REF] Elliott | Content and Fractionation of Heavy Metals in Water Treatment Sludges[END_REF]. Compared to traditional treatment methods, superconducting magnetic separation of sorbents has several advantages, such as the lower investment, smaller area occupied, and shorter period of processing [START_REF] Ohara | Magnetic separation using superconducting magnets[END_REF][START_REF] Igarashi | Study on magnetic separation for decontamination of cesium contaminated soil by using superconducting magnet[END_REF].

Magnetic materials, named as seeds, play a key dual role in the superconducting magnetic separation technology: they need to efficiently immobilize the pollutants and at the same time must be easily separated by application of an external field [START_REF] Ambashta | Water purification using magnetic assistance: A review[END_REF]. Iron and iron oxide nanoparticles are well-suited for this application due to their surface affinity and redox degradation activity towards a wide range of organic and inorganic contaminants [START_REF] Crane | Nanoscale zero-valent iron: future prospects for an emerging water treatment technology[END_REF][START_REF] Xu | Use of iron oxide nanomaterials in wastewater treatment: A review[END_REF]. To avoid issues related to colloidal stability and handling of nanomaterials, 3D hierarchical nanostructures have been developed [START_REF] Fei | Large-scale preparation of 3D self-assembled iron hydroxide and oxide hierarchical nanostructures and their applications for water treatment[END_REF]. As an alternative, it is possible to design (nano)composite powders where the magnetic nanoparticles are associated with a sorbent phase [START_REF] Zhang | Study on industrial wastewater treatment using superconducting magnetic separation[END_REF]. In the field of water remediation, calcium phosphates and carbons represent two important classes of materials with high affinity for inorganic and organic contaminants, respectively [START_REF] Nzihou | Role of phosphate in the remediation and reuse of heavy metal polluted wastes and sites[END_REF][START_REF] Rivera-Utrilla | Activated carbon modifications to enhance its water treatment applications. An overview[END_REF]. HAp and carbon nanoparticles have already been previously individually associated with iron and iron oxide colloids for remediation [START_REF] Oliveira | Activated carbon/iron oxide magnetic composites for the adsorption of contaminants in water[END_REF][START_REF] Zhang | Carbonstabilized iron nanoparticles for environmental remediation[END_REF][START_REF] Dong | Removal of lead from aqueous solution by hydroxyapatite/magnetite composite adsorbent[END_REF][START_REF] Yang | Magnetically recoverable iron oxide-hydroxyapatite nanocomposites for lead removal[END_REF]. However, to our knowledge, the corresponding triphasic system organized within a single composite structure has never been described so far.

Looking for a cheap, easily-scalable source of magnetic carbon, we focused our attention on ultrasonic spray pyrolysis (USP) as a continuous, one-step method to obtain porous iron oxide/carbon microspheres [START_REF] Suh | Magnetic and porous nanospheres from ultrasonic spray pyrolysis[END_REF][START_REF] Skrabalak | Porous carbon powders prepared by ultrasonic spray pyrolysis[END_REF]. The nebulization process is based on the formation of a mist from the aqueous solution under the high-frequency ultrasound radiation. The gas flow carries the droplets into a furnace, where solvent evaporation and precursor decomposition occur, producing the C/Fe 3 O 4 composite. In parallel, in order to achieve the controlled integration of the calcium phosphate phase to these microspheres, we applied a biomimetic approach previously developed for the coating of iron oxide particles with HAp [START_REF] Tang | Biomagnetic of apatite-coated cobalt ferrite: A core-shell particle for protein adsorption and pH-controlled release[END_REF][START_REF] Yang | Surface reactivity of hydroxyapatite nanocoatings deposited on iron oxide magnetic spheres toward toxic metals[END_REF]. This method relies on a pre-coating of a core particle by an acidic aminoacid, followed by addition of calcium and phosphate ions whose precipitation is confined on the particle surface.

Following these approaches, we have prepared here HAp@C/Fe 3 O 4 core-shell three-phasic composite microspheres. We could evidence that the biomimetic interfacial mineralization allows for confinement of the apatite phase on the microsphere surface. The influence of mineralization on the microsphere porosity and its magnetic properties was studied. The adsorption behavior of bare and coated microspheres towards key pollutants, i.e. the antibiotic ampicillin, La 3+ , Hg 2+ and Pb 2+ , was evaluated and compared. This allows for a discussion of the interplay between the different components within the composite architecture, enlightening their potential applications for water treatment.

Material and methods

Preparation and characterization of HAp@C/Fe 3 O 4 microspheres

Magnetic carbon microspheres were prepared according to the literature with some modifications [START_REF] Suh | Magnetic and porous nanospheres from ultrasonic spray pyrolysis[END_REF]. Typically, an aqueous precursor solution was prepared by dissolving glucose (4 g) and ferrous chloride tetrahydrate (3 g) in 100 mL deionized water. The precursor solution was nebulized using a commercially-available household ultrasonic humidifier (1.7 MHz) into a quartz tube placed inside a furnace (700 °C). The flow rate of inert gas (N 2 or Ar) was typically 1 SLPM. The residence time through the furnace was controlled by the gas flow rate and was typically a few seconds. The resulting dark particles were collected into ethanol-filled bubblers and recovered by magnetic separation. These C/Fe 3 O 4 products were washed three times with ethanol.

For hydroxyapatite deposition (Fig. 1), 1 g of the C/Fe 3 O 4 composite powder was first dispersed into 40 mL of a 1 mol L -1 L-aspartic acid solution at 80 °C for 2 h, followed by washing and re-suspension into 40 mL deionized water (sample C/Fe 3 O 4 /A) [START_REF] Yang | Surface reactivity of hydroxyapatite nanocoatings deposited on iron oxide magnetic spheres toward toxic metals[END_REF]. In parallel, Ca(OH) 2 (0.01 mol, 0.748 g) was dissolved in 100 mL of an ethanol-water mixture (50:50 %, v/v) under stirring for 3 h. Then 60 mL of a NH 4 H 2 PO 4 (0.006 mol, 0.67 g) solution was added and the mixture was stirred for 1 h. After this delay, the C/Fe 3 O 4 /A suspension was quickly transferred into the mixture and the solution was aged for 24 h. The powder was recovered by magnetic separation, thoroughly washed with deionized water and dried at 60 °C under vacuum overnight. X-Ray Diffraction (XRD) studies were performed on a Philips PW131 diffractometer, in a 2θ range from 10°-70°. Transmission Electron Microscopy (TEM) imaging was performed on a JEM-2100, operating at 120 kV. Scanning Electron Microscopy (SEM) imaging was conducted on a Hitachi S-4800, operating at 5 kV. N 2 sorption studies were performed at 77 K using a Quantachrome Quadrasorb SI-MP equipment. Thermogravimetric Analysis (TGA) measurements were performed on a Diamond TG/DTA (Perkin Elmer). Magnetic measurements were performed using a vibrating sample magnetometer (VSM) (Lake Shore 7410) in an applied magnetic field of ± 10 kOe at room temperature. X-ray photoelectron spectra (XPS) were recorded on a ESCALAB 250Xi photoelectron spectrometer. The monochromated AlK X-radiation was used as the excitation source.

Adsorption experiments

Solutions with concentrations ranging from 0 to 5 mmol L -1 were prepared from ampicillin (Sigma), La(NO 3 ) 3 , HgCl 2 •6H 2 O and Pb(NO 3 ) 2 •6H 2 O by dissolution in deionized water (pH 6). For kinetics study, 0.2 g of HAp, C/Fe 3 O 4 or HAp@ C/Fe 3 O 4 were added to 100 mL of the solutions and stirred with a orbital shaker at 200 rpm at 25 °C. Sorption isotherms were obtained using 0.1 g of the sorbent powders suspended in 50 mL of the contaminated solution and stirred in the same conditions for 24 h at 25 °C. For metal ion solutions, additional experiments were performed at pH = 4 and 8 by addition of HCl and NaOH, respectively. In all experiments, after magnetic separation, the La 3+ , Hg 2+ , Pb 2+ and Ca 2+ content of the supernatant was determined by inductively coupled plasma spectrometry (ICP) (Varian 710-ES) and residual ampicillin content was analyzed with a UV-Vis spectrophotometer (Varian Cary 5000) at λ= 210 nm.

The Freundlich and Langmuir adsorption isotherm models were used to fit the experimental data. The Langmuir model takes the following form:

q e = q max K l C e 1+ K l C e (1)
where q e is the amount of ampicilin or metal ions adsorbed at equilibrium (mmol g -1 ), q max is the maximum sorption capacity (mmol g -1 ), K l is the Langmuir constant (L mg -1 ), that is related to the binding energy of the species to the active site and C e is the equilibrium concentration (mg L -1 ). The linear form of Freundlich equation can be expressed as follows:

log

q e = 1 n logC e + log K f (2)
where q e is the amount adsorbed at equilibrium (mmol g -1 ), K f (mmol g -1 ) is the Freundlich constant indicating the adsorption capacity and 1/n is indicative of the intensity of the adsorption reaction. When 1/n values are in the range 0.1 < 1/n < 1, the adsorption process is considered favorable.

All experiments in this study were performed in triplicate and the error range was always below 10 %. The results were given as average values, except for values obtained by modeling.

Results and discussion

Preparation and characterization of hydroxyapatite-coated magnetic carbon microspheres

Nebulization and heating of the glucose-FeCl 2 solution led to microspheres with diameter ranging from 400 nm to ca. 1 µm as visualized by SEM in Fig. 2(a). In most cases, the spheres were intact but some fractured particles revealed their hollow structure (inset of Fig.

2(c))

. TEM images were consistent with SEM evidencing that nanoparticles are mostly located on the edge of the microspheres (Fig. 2(e)). XRD patterns of the materials indicated that these nanoparticles consist of magnetite Fe 3 O 4 or maghemite γ-Fe 2 O 3 (Fig. 3(a)). The Fe 2p peak of the XPS spectra showed two main signals at ca. 711 eV and 724 eV, characteristics of the Fe 3 O 4 phase (Fig. 3(b)) [START_REF] Grosvenor | Investigation of multiplet splitting of Fe 2p XPS spectra and bonding in iron compounds[END_REF]. TGA measurements indicated that the microspheres contain 51 ± 1 wt% of carbon (Fig. 4(b)).

After deposition of L-aspartic acid, the addition of the microspheres to a solution of calcium and phosphate salts led to the formation of rod-like crystals protruding from the particle surface (Fig. 2

(b),2(d))

. TEM allowed a more detailed observation of the newly-formed mineral outer layer (thickness ca. 50-100 nm), with rods apparently growing on the external surface of C/Fe 3 O 4 (Fig. 2(f)). XRD patterns of the recovered powders showed new peaks corresponding to the hydroxyapatite (HAp, Ca 10 (PO 4 ) 6 (OH) 2 ) structure (Fig. 3(a)). The XPS spectra confirmed the presence of Ca and P elements on the particle surface (Fig. 3(b)).

Focusing on the Fe 2p region, a shake-up satellite of weak intensity was evidenced at ca. 720 eV. This is indicative of partial oxidation of the magnetite phase leading to the formation of small amounts of maghemite [START_REF] Grosvenor | Investigation of multiplet splitting of Fe 2p XPS spectra and bonding in iron compounds[END_REF]. ICP and EDX indicated a Ca:P molar ratio of 1. Noticeably, such morphologies were obtained only for optimal L-aspartic acid and Ca 2+ /PO 4 3relative concentrations of 1 mol L -1 and 0.5/0.03 mol L -1 respectively. A smaller or larger amount of L-aspartic acid led to the presence of excess HAp in the solution or dissolution of Fe 3 O 4 , respectively. An insufficient or excess amount of calcium/phosphate led to composites with ill-defined morphologies. This underlines the key role of the aspartate pre-coating on favoring HAp formation on the microsphere surface. The biomimetic approach used here is inspired from mineralization processes found in several organisms, and especially described for the formation of aragonite (CaCO 3 ) nacreous shells [START_REF] Falini | Control of Aragonite or Calcite Polymorphism by Mollusk Shell Macromolecules[END_REF]. It relies on the presence of proteins rich in aspartic acid moieties deposited on a rigid organic matrix. When calcium ions are present in the medium, they have a strong tendency to adsorb on the acidic layer where they are chelated by the carboxylate groups. This provides a reactive surface for further deposition of incoming carbonate ions, favoring calcium carbonate nucleation. Such experiments could be replicated in vitro and extended to other rigid substrates and other mineral phases [START_REF] Xu | Biomimetic mineralization[END_REF]. If the density of acidic groups on the surface is not sufficient and/or calcium ions are in excess, then mineral precipitation can occur in solution, independently of the templating substrate. Here it was also observed that an excess of aspartic acid is able to dissolve the iron oxide nanoparticles, plausibly because of its strong chelating properties, as already demonstrated for oxalic acid [START_REF] Panias | Mechanisms of dissolution of iron oxides in aqueous oxalic acid solutions[END_REF]. However we found out that, once optimal concentrations have been determined, the procedure was highly reproducible in terms of mineralization rate and HAp nanocrystal dimensions.

VSM measurements showed that the two composites exhibited a similar hysteresis curve with a coercivity of 235 Oe (Fig. 4(a)). The saturation magnetization (Ms) was is 28 emu g -1 for C/Fe 3 O 4 , and 14 emu g -1 for HAp@C/Fe 3 O 4 , in good agreement with the 1:1 weight ratio for HAp:C/Fe 3 O 4 . In terms of porosity, the two composites exhibited a type IV hysteresis for N 2 sorption, indicating the presence of mesopores (Fig. 4(c)). Application of the BET model indicates that C/Fe 3 O 4 has relatively high specific surface area (ca. 330 m 2 g -1 ) and porous volume (ca. 0.20 cm 3 g -1 ). Interestingly, HAp@C/Fe 3 O 4 exhibits larger specific surface area (ca. 355 m 2 g -1 ) and porous volume (ca. 0.30 cm 3 g -1 ). For C/Fe 3 O 4 , pore size distribution calculated from the BJH model on the desorption branch suggested the preponderance of mesopores with an average diameter of 4 nm and a significant amount of micropores that should mainly originate from the carbonization of glucose (Fig. 4(d)) [START_REF] Suh | Magnetic and porous nanospheres from ultrasonic spray pyrolysis[END_REF]. For HAp@C/Fe 3 O 4 , the mineral layer decreases the accessibility of the mesopores of the microsphere but induces the presence of larger mesopores in the 4-10 nm size range. These mesopores should correspond to inter-particle porosity inside the HAp coating, as previously reported [START_REF] Yang | Surface reactivity of hydroxyapatite nanocoatings deposited on iron oxide magnetic spheres toward toxic metals[END_REF].

Removal studies

To test the adsorption capacities of the composite microspheres, ampicillin, La 3+ , Hg 2+ and Pb 2+ were selected as representative organic and inorganic contaminants (Fig. 5). Based on preliminary kinetics experiments (see Supplementary material), the sorption isotherms were obtained after 24 h of contact between the sorbent powders and the solutions.

Ampicillin is a widespread drug that accumulates in the environment due to its low biodegradability, leading to the proliferation of antibiotic-resistant bacteria [START_REF] Baquero | Antibiotics and antibiotic resistance in water environments[END_REF]. It is mainly in a zwitterionic molecular form between pH 3 and pH 7.2. HAp showed no ampicillin removal capacity whereas C/Fe 3 O 4 exhibited a maximum removal capacity of ca. 0.04 mmol g -1 (Fig. 5(a)). The HAp@C/Fe 3 O 4 composites partially inherited the ampicillin adsorption capacity from C/Fe 3 O 4 and showed a maximum removal capacity of ca. 0.01 mmol g -1 .

Analyses of the experimental data using the Langmuir and Freundlich models are presented in Table 1 and Table 2 and the calculated curves are shown in Fig. 5(a). Correlation coefficients (R 2 ) were similar for the two equations but the low value of the 1/n parameters as well as the apparent underestimation of the maximal sorption capacity by the Freundlich model suggest that the Langmuir model is more adapted to reproduce the experimental data.

Previous investigations of ampicillin sorption on activated carbons have suggested that the surface interactions were mainly of electrostatic nature [START_REF] Wang | Removal of ampicillin sodium in solution using activated carbon adsorption integrated with H 2 O 2 oxidation[END_REF]. Most carbons have points of zero charge (PZC) of ca. 8 whereas magnetite has a PZC of ca. 6.5 [START_REF] Kosmulski | pH-dependent surface charging and points of zero charge. IV. Update and new approach[END_REF], so that the microspheres are expected to bear a positive charge at pH = 6. In these conditions, ampicillin co-exists in two forms, zwitterionic and cationic. Therefore, sorption via attractive electrostatic interactions is the most plausible mechanism for ampicillin retention by the C/Fe 3 O 4 surface.

HAp particles have PZC values in the 6-7 pH range [START_REF] Narasaraju | Some physico-chemical aspects of hydroxylapatite[END_REF] and their surface is therefore slightly negatively charged or neutral in the conditions of the experiments, with low affinity for ampicillin. In this context, it is interesting to calculate the maximum removal capacity for the Considering the wide importance of rare-earth ions in current technological devices and the related questions in terms of their environmental impact [START_REF] Barry | The acute and chronic toxicity of lanthanum to daphnia carinata[END_REF], the removal of La 3+ by the composite microspheres was investigated. C/Fe 3 O 4 demonstrated no significant La 3+ adsorption capability whereas the maximum removal capacity of HAp towards this species was ca. 2.10 mmol g -1 (Fig. 5(b)). The composite powder exhibited a maximum removal capacity of ca. 0.95 mmol g -1 . Interestingly, when calculated per g of HAp, the HAp@C/Fe 3 O 4 composites has a removal capacity of ca. 1.90 mmol g -1 that is very similar to the pure hydroxyapatite powder. This indicates that the deposition of the HAp nanocrystals on the microsphere surface does not significantly decrease their capacity to interact with La 3+ .

Neither the Langmuir nor Freundlich model could reproduce the experimental data (as indicated by low R 2 values in Table 1 and Table 2), indicating that the depletion in La 3+ from the solution was not a simple adsorption process. Interestingly, XRD data obtained with HAp alone in the presence of La 3+ evidenced the formation of lanthanum phosphate (LaPO 4 ).

Analysis of the supernatant content at the end of the sorption experiments indicated a molar ratio of 0.6 between the retained La 3+ and the released Ca 2+ whereas the P element could not be detected. Considering the HAp and LaPO 4 stoichiometry, the release of 10 calcium ions from hydroxyapatite should be concomitant with the departure of 6 phosphate species, that can precipitate 6 lanthanum ions. Therefore, it is possible to propose that surface dissolution of poorly crystalline hydroxyapatite in contact with water leads to Ca 2+ and PO 4 -release, the latter being able to precipitate LaPO 3 and depleting the solution in La 3+ .

The retention of two widespread highly toxic heavy metals, Hg 2+ and Pb 2+ , was also studied.

Pristine HAp and C/Fe 3 O 4 were able to remove both ions from aqueous solutions, in agreement with the literature [START_REF] Xu | Lead immobilization by hydroxyapatite in aqueous solutions[END_REF][START_REF] Oliva | Removal of cadmium, copper, nickel, cobalt and mercury from water by Apatite II (TM): column experiments[END_REF][START_REF] Kadirvelu | Removal of heavy metals from industrial wastewaters by adsorption onto activated carbon prepared from an agricultural solid waste[END_REF], but to a different extent. For Pb 2+ , HAp alone has a maximal removal capacity of ca. 1.20 mmol g -1 , C/Fe 3 O 4 of ca. 0.05 mmol g -1 and the composite showed a slight improvement compared to HAp (ca. 1.40 mmol g -1 ). Analyses of the experimental data by both Langmuir and Freundlich models showed acceptable R 2 values but the 1/n values were small. In parallel, the XRD diffractograms show distinct peaks that can be attributed to hydroxypyromorphite (Hpy, Pb 10 (PO 4 ) 6 (OH) 2 ). The molar ratio of released Ca 2+ : removed Pb 2+ in the supernatant was almost 1 and no phosphorus could be detected by ICP. Taken together, these data are in favor of a mechanism involving the adsorption of Pb 2+ on the HAp surface. Then two possibilities exist: a cation exchange reaction between Pb 2+ ions in aqueous solution and Ca 2+ ions of HAp or surface dissolution and rapid reprecipitation of HPy.

For Hg 2+ , the maximum removal capacity for HAp and C/Fe 3 O 4 were similar (ca. 0.20 mmol g -1 ) while the composite HAp@C/Fe 3 O 4 showed significantly improved performances (ca. 0.30 mmol g -1 ). On the XRD patterns of the pure HAp powder after contact with Hg 2+ , no obvious crystalline phase was detected although slight modification of the patterns in the 2 = 28-29° domain may indicate the formation of Hg 3 (PO 4 ) 2 (Fig. 3). From the ICP data, the molar ratio between removed Hg 2+ and released Ca 2+ is ca. 0.2. As mentioned earlier, the release of 10 calcium ions upon HAp dissolution is associated with the release of 6 phosphate ions that may precipitate up to 9 Hg 2+ species, a value that is far above the measured ratio.

However, it must be taken into account that the C/Fe 3 O 4 spheres also contribute to the removal of mercury ions. Interestingly, only the Freundlich model was found suitable to reproduce the experimental data, with reasonable R 2 and 1/n values. This reflects the heterogenous nature of the sorbing phase that combines the carbon and the HAp surfaces.

Additional metal removal experiments were performed at pH 4 and pH 8 (Table 3). The evolution of mercury removal with pH indicates that acidic conditions are particularly detrimental to the C/Fe 3 O 4 spheres. This can be attributed to the increased solubility of magnetite with decreasing pH, as confirmed by the detection of high levels of Fe in the supernatant. In contrast, the effect of pH on La 3+ removal suggests that basic conditions decrease the reactivity of HAp. This can be correlated with the decrease solubility of HAp with increasing pH, as confirmed by the measured lower calcium content of the supernatant compared to pH 6. Interestingly, the Pb 2+ removal was almost unaffected by pH variations neither for HAp nor for the composite. Hence, coming back to the proposed mechanisms explaining HPy formation, this result is more in favor of a cation-exchange process than a dissolution/reprecipitation reaction. Indeed, it is not possible to identify what is the relative amount of sorbed Hg 2+ and Pb 2+ on the two components of the composite spheres but the removal capacities of the two individual phases suggest a higher reactivity of the hydroxyapatite layer towards these metals. This is in agreement with the literature showing that similar hydroxyapatite nanolayers can have higher sorption capacity than the HAp powder alone due to lower crystallinity and smaller crystallite size [START_REF] Yang | Surface reactivity of hydroxyapatite nanocoatings deposited on iron oxide magnetic spheres toward toxic metals[END_REF]. Therefore it can be suggested that the increased capacity for Hg 2+ and Pb 2+ binding of the composite materials compared to the two individual phases originates from wellcontrolled architecture of the nanocomposites.

From a practical perspective, the here-described composites appear particularly well-adapted for the simultaneous removal of species with distinct affinities for carbon and hydroxyapatite.

For instance, HAp has a low affinity for antibiotics (ca. 2 mg.g -1 for related compounds [START_REF] Bouyarmane | Parameters influencing ciprofloxacin, ofloxacin, amoxicillin and sulfamethoxazole retention by natural and converted calcium phosphates[END_REF]) while carbons poorly bind La 3+ ions (ca. 0.05 mg.g-1 [START_REF] Murty | Preconcentration of rare earth éléments on activated carbon and its application to groundwater and sea-water analysis[END_REF]). Our experiments suggest the composite approach can be advantageous provided an adjustment of the C:HAp weight ratio as well as a variation of microsphere size or porosity via modulation of the processing parameters. The question is raised about the possible competition between the sorption of organic and inorganic pollutants, a phenomenon that is particularly relevant for the cleaning of natural waters that can contain fulvic and humic acids. Although such a possibility should be taken into account, it is important to point out that the outer layer of the composite consist of the hydroxyapatite phase that has low affinity for organics. Therefore the organic content of natural waters may interfere with the immobilization of organic pollutants but not significantly with the retention of metal ions. In parallel, it is interesting to consider species such as Hg 2+ that have good affinities for both the carbon surface and the hydroxyapatite layer. The retention ability of activated carbons towards mercury in conditions comparable to this work was reported to range between 0.01 mg g -1 and 100 mg g -1 [START_REF] Yardim | Removal of mercury (II) from aqueous solution by activated carbon obtained from furfural[END_REF][START_REF] Zhang | Mercury removal from water using activated carbons derived from organic sewage sludge[END_REF]. However, the highest values are obtained for materials with much higher specific surface area (1100 m 2 g -1 , i.e. three time higher than the microspheres). For hydroxyapatite, reported values vary between 0.5 mg g -1 and 50 mg g -1 (in column experiments) [START_REF] Oliva | Removal of cadmium, copper, nickel, cobalt and mercury from water by Apatite II (TM): column experiments[END_REF]. Therefore the composite phases with removal capacities of ca. 2 mg g -1 are in the lower range of efficiency. Yet, the here-demonstrated increase in removal performance of the composite compared to its individual components suggests that extension of the presented strategy to other carbonaceous supports would be of particular interest.

Conclusions

We demonstrate that multi-phasic materials consisting of hydroxyapatite-coated iron oxidedecorated carbon microspheres prepared from cheap, environment-friendly sources, in solvent-free conditions exhibit the ability to remove both organic and inorganic pollutants from aqueous solutions. Of particular interest is the observation that the composite sorption capacities may exceed that of its individual components, as illustrated here by Hg 2+ and Pb 2+ .

Noticeably thanks to the presence of iron oxide nanoparticles, the composite microspheres can be easily and fully recovered by application of a magnetic field, making them particularly promising as sorbing seeds for superconducting magnetic separation or biological application. Table 1. Adsorption isotherm parameters obtained by Langmuir model.

Langmuir Ampicillin La 3+ Hg 2+ Pb 2+ q max (mmol g -1 ) K l (L mg -1 ) R 2 q max (mmol g -1 ) K l (L mg -1 ) R 2 q max (mmol g -1 ) K l (L mg -1 ) R 2 q max (mmol g -1 ) K l (L mg -1 ) R

  7 ± 0.1, in good agreement with the expected value of 1.67 for HAp. In parallel, TGA measurements yielded to a HAp:C/Fe 3 O 4 weight ratio of 1.1 ± 0.1 (Fig. 4(b)). Note that this ratio closely reflects the reaction conditions where 1 g of the C/Fe 3 O 4 powder was introduced in a solution containing 0.01 mol Ca 2+ and 0.006 mol PO 4 3-that should form 0.001 mol (1 g) of HAp. This implies the HAp formation occurred only on the surface of the C/Fe 3 O 4 spheres.

C/Fe 3 O 4

 34 part of the composites. Considering the 1:1 weight ratio between HAp and C/Fe 3 O 4 in the composite, the adsorption capacity per g of C/Fe 3 O 4 within the composite structure was ca. 0.02 mmol g -1 , i.e. half the value obtained for the bare microspheres. Therefore these results suggest that the antibiotic is mainly adsorbed on the uncoated fraction of the C/Fe 3 O 4 surface.

Fig. 1 .Fig. 2 .

 12 Fig. 1. Overview of the synthetic route to hydroxyapatite-coated magnetic carbon

Fig. 3 .

 3 Fig. 3. (a) XRD patterns of the C/Fe 3 O 4 and HAp@C/Fe 3 O 4 powders before and after La 3+ , Hg 2+ and Pb 2+ removal, with hydroxyapatite powder (HAp) as a reference; (b) XPS spectra of

Fig. 4 .

 4 Fig. 4. (a) VSM measurements of magnetization curves at 298 K of the C/Fe 3 O 4 and

Fig. 5 .

 5 Fig. 5. Removal capacity at equilibrium (q e ) as a function of equilibrium concentration (C e ) of (a) ampicilin, (b) La 3+ , (c) Hg 2+ and (d) Pb 2+ after 24 h of contact between hydroxyapatite

Fig. 1

 1 Fig. 1

Fig. 2 .

 2 Fig. 2.

Fig. 3 26Fig. 4 .Fig. 5 .

 345 Fig. 3

Table 2 .

 2 Adsorption isotherm parameters obtained by Freundlich model.

	2

Table 3 .

 3 Maximal experimental removal capacity (q max,ex ) at various pH Fe 3 O 4 0.90 1.01 0.65 0.26 0.30 0.32 1.39 1.411.43

			La 3+			Hg 2+			Pb 2+
		q max,ex (mmol g -1 )	q max,ex (mmol g -1 )	q max,ex (mmol g -1 )
	pH	4	6	8	4	6	8	4	6	8
	HAp	1.56 2.09 1.42 0.20 0.20 0.22 1.18	1.18 1.19
	C/Fe 3 O 4	----	----	----0.01 0.20 0.27 0.05	0.05 0.05
	HAp@C/

Acknowledgments

Huihui Yang and Laifeng Li acknowledge funding support from the National Natural Science Foundation of China (Grant No. Y4B30471E4).

Supplementary informations

Kinetics of ampicillin, La 3+ , Hg 2+ and Pb 2+ removal by HAp@C/Fe 3 O 4 composites Hierarchically-organized apatite/iron oxide/carbon microspheres were developed

The micro/mesoporous powders combine the sorption capacities of carbon and apatite They demonstrate adsorption capacity towards organic and inorganics pollutants These composites can be useful for superconducting separation wastewater treatment


Anne-Catherine Lehours

Matthew T Cottrell

Océane Dahan

David L Kirchman

Christian Jeanthon
email: jeanthon@sb-roscoff.fr.

Summer distribution and diversity of aerobic anoxygenic phototrophic bacteria in the 4 Mediterranean Sea in relation to environmental variables

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Introduction 56

Aerobic anoxygenic phototrophic bacteria (AAP) represent a functional group that was 57 recently found to account for a significant fraction of the bacterioplankton in marine 58 illuminated environments [START_REF] Kolber | Contribution of aerobic photoheterotrophic 578 Bacteria to the carbon cycle in the ocean[END_REF][START_REF] Cottrell | Aerobic anoxygenic phototrophic bacteria in 512 the mid-Atlantic bight and the north Pacific gyre[END_REF][START_REF] Koblížek | Rapid growth rates of aerobic 574 anoxygenic prototrophs in the Ocean[END_REF]59 Mašín et al., 2006;[START_REF] Sieracki | Distribution of planktonic 643 aerobic anoxygenic photoheterotrophic bacteria in the northwest Atlantic[END_REF][START_REF] Lami | High abundances of aerobic anoxygenic photosynthetic bacteria in the south Pacific 582 Ocean[END_REF][START_REF] Jiao | Distinct distribution 570 pattern of abundance and diversity of aerobic anoxygenic phototrophic bacteria in the global 571 ocean[END_REF]. These 60 bacteriochlorophyll (BChl a)-containing prokaryotes, which can use both light and organic 61 matter for energy production, require oxygen and can use reduced organic compounds as 62 electron donors [START_REF] Yurkov | New light on aerobic anoxygenic phototrophs[END_REF]. Although they are not primary producers, their 63 higher growth rates and efficiency in organic carbon utilization over strict heterotrophs are 64 likely to make them dynamic and significant contributors to the organic carbon production 65 and cycling in the upper ocean [START_REF] Koblížek | Rapid growth rates of aerobic 574 anoxygenic prototrophs in the Ocean[END_REF]. While physiological evidences 66 suggest that they would have a competitive advantage over strict heterotrophs in low-nutrient 67 conditions [START_REF] Yurkov | Impact of light/dark regime on growth rate, biomass 685 formation and bacteriochlorophyll synthesis in Erythromicrobium hydrolyticum[END_REF][START_REF] Suyama | Photosynthetic apparatus in Roseateles depolymerans 61A is transcriptionally induced 657 by carbon limitation[END_REF], the emerging findings 68 indicate that these bacteria may be adapted to a broad range of trophic conditions and are 69 abundant in eutrophic and oligotrophic environments [START_REF] Cottrell | Aerobic anoxygenic phototrophic bacteria in 512 the mid-Atlantic bight and the north Pacific gyre[END_REF]Sieracki et al., 70 2006;[START_REF] Waidner | Aerobic anoxygenic phototrophic bacteria attached to 668 particles in turbid waters of the Delaware and Chesapeake estuaries[END_REF]. 71 Molecular analyses based on the pufM gene encoding the M-subunit of the 72 photosynthetic reaction center have revealed that AAP bacteria belong to different groups of 73 Alpha-, Beta-, and Gammaproteobacteria [START_REF] Béjà | Unsuspected diversity among marine aerobic anoxygenic 485 phototrophs[END_REF][START_REF] Yutin | Assessing 696 diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of 697 the Atlantic and Pacific Oceans using the Global Ocean Sampling expedition metagenomes[END_REF]. Their 74 abundance and distribution have been explored in diverse oceanic regions and have been 75

shown to vary greatly among oceanic regimes [START_REF] Cottrell | Aerobic anoxygenic phototrophic bacteria in 512 the mid-Atlantic bight and the north Pacific gyre[END_REF]2008;[START_REF] Jiao | Distinct distribution 570 pattern of abundance and diversity of aerobic anoxygenic phototrophic bacteria in the global 571 ocean[END_REF], 76 Imhoff, 2001;[START_REF] Yutin | Assessing 696 diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of 697 the Atlantic and Pacific Oceans using the Global Ocean Sampling expedition metagenomes[END_REF][START_REF] Waidner | Diversity and distribution of ecotypes of the aerobic 672 anoxygenic phototrophy gene pufM in the Delaware estuary[END_REF]. However, AAP bacteria 77 remain clearly undersampled in several areas, particularly in oligotrophic environments, that 78 and diversity of these photoheterotrophic bacteria remain yet poorly understood [START_REF] Eiler | Evidence for the ubiquity of mixotrophic bacteria in the upper Ocean: 521 implications and consequences[END_REF]. 80

The Mediterranean Sea is an ideal environment for these ecological studies as it offers 81 a range of trophic conditions including extreme oligotrophy, particularly in summer when the 82 water column is strongly stratified [START_REF] Berman | Secchi disk depth 488 record: a claim for the eastern Mediterranean[END_REF]. While the N:P ratio is close to the 83 Redfield ratio (16:1) in most oceanic waters, Mediterranean waters have a higher ratio, 84 especially in the eastern Basin, leading to strong phosphorus limitation (Moutin & Raimbault, 85 2002). A complex thermohaline circulation coupled with regional hydrodynamic features also 86 contributes to the establishment of many different oceanic regions throughout the 87 Mediterranean Sea [START_REF] Manca | Physical and 600 biochemical averaged vertical profiles in the Mediterranean regions: an important tool to trace 601 the climatology of water masses and to validate incoming data from operational 602 oceanography[END_REF]. As an example, the exchange of the Atlantic and 88

Mediterranean water masses at the Strait of Gibraltar induces marked salinity and temperature 89 gradients [START_REF] Gascard | Water masses and circulation in the Western Alboran Sea and 544 in the Straits of Gibraltar[END_REF]. Although the different trophic conditions available in the 90 Mediterranean Sea provide a unique context to link nutrient availability, trophic status and 91 functioning of the food web to the dynamics of photoheterotrophic populations, the 92 distribution and diversity patterns of AAP bacteria have been only partially explored (Oz et 93 al., 2005;[START_REF] Yutin | Novel primers reveal wider diversity among marine 693 aerobic anoxygenic phototrophs[END_REF]2008). 94

In the present study, we analyzed the biogeography patterns of AAP populations 95 October 1999 during the PROSOPE cruise aboard the R.V. La Thalassa (Fig. 1). For 111 molecular diversity studies of AAP populations, 1.45-5 l water samples were retrieved using 112 12 l Niskin bottles fitted on a Rosette sampler equipped with conductivity, temperature and 113 depth (CTD) sensors. Seawater was prefiltered through 3 µm pore-size, 47 mm diameter, 114 Nuclepore filters using moderate vacuum in order to separate picoplankton from larger 115 organisms. Picoplanktonic cells were collected by filtration as previously described (Marie et 116 al., 2006;[START_REF] Garczarek | High vertical and low horizontal diversity of 540 Prochlorococcus ecotypes in the Mediterranean Sea in summer[END_REF]. The filtered biomass was transferred into a cryovial 117 containing 3.5 ml of DNA lysis buffer (0.75 M sucrose, 50 mM Tris-HCl, pH 8) and 118 immediately frozen in liquid nitrogen. DNA extraction was performed as previously described 119 [START_REF] Marie | Eukaryotic picoplankton communities 606 of the Mediterranean Sea in summer assessed by molecular approaches (DGGE, TTGE, 607 QPCR)[END_REF]. Ancillary data (nutrients, dissolved oxygen, chlorophyll a, salinity, 120 temperature, etc.) and methods used to analyze them are available from the PROSOPE web 121 site (http://www.obs-vlfr.fr/cd_rom_dmtt/pr_main.htm). 122 123

Environmental pufM gene amplification 124

Multiple combinations of previously designed primer sets [START_REF] Achenbach | Photosynthetic and phylogenetic primers for 476 detection of anoxygenic phototrophs in natural environments[END_REF]125 Béjà et al., 2002;[START_REF] Yutin | Novel primers reveal wider diversity among marine 693 aerobic anoxygenic phototrophs[END_REF] were tested (data not shown). On the basis of specificity 126 and efficiency (e. g. yield) results, we selected PufMF forward (5 '-127 TACGGSAACCTGTWCTAC-3', Béjà et al., 2002) and PufWAW reverse primers (5'-128 (dATP,dCTP,dGTP,134 dTTP; Eurogenetec), 10 pmoles of each oligonucleotide primer, 2.5 U of GoTaq Flexi DNA 135 polymerase (Promega) and 50 to 100 ng of template DNA. Amplifications were carried out in 136 a GeneAmp PCR system 9700 (Applied Biosystems) with the following parameters: 95°C for 137 5 min, followed by 35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s, with a final 138 extension step at 72°C for 10 min. Amplified products were checked by electrophoresis in 139 1.5% agarose in 0.5× Tris-Acetate-EDTA (TAE) buffer and further quantified with a DNA 140 quantitation fluorescence assay kit (Sigma-Aldrich). 141 142

Temporal Temperature gel Gradient Electrophoresis (TTGE) profiling and analyses 143

One hundred nanograms of each amplified product were electrophoresed along an 8% 144 (wt/vol) polyacrylamide gel (ratio acrylamide to bis-acrylamide 37.5:1) containing 7M urea, 145 1.25X TAE, 0.06% of N,N,N',N'-Tetramethylethylenediamine (Temed) and 0.06% 146 ammonium persulfate using the DCode Universal Mutation Detection System (BioRad,147 Hercules, CA). Runs were performed in 1.25x TAE at 68 V for 17 h with a temperature range 148 of 66 to 69.7°C and a ramp rate of 0.2°C h -1 . Standard markers were generated with a mixture 149 of pufM PCR products amplified from Erythrobacter longus strain OCh 101 T , E. litoralis 150 strain T4 T , Roseobacter denitrificans strain OCh 114 T (CIP104266), Dinoroseobacter shibae 151 strain DFL12 T . Gel images were obtained at 100-µm resolution using a Typhoon Trio variable 152 mode imager (Amersham Biosciences, Piscataway, NJ). Typhoon scans were acquired using 153 Belgium). In band assignment, a 1% band position tolerance (relative to total length of the 158 gel) was applied, which indicates the maximal shift allowed for two bands in different TTGE 159 patterns to be considered as identical. The number of bands in a profile was expressed as the 160 phylotype richness and the Shannon-Weiner index (H') was calculated as previously 161 described [START_REF] Hill | Using ecological diversity measures 557 with bacterial communities[END_REF]. The Smith and Wilson evenness index (E var , Smith & Wilson, 162 1996) was calculated using the Ecological Evenness Calculator software 163 (http://www.nateko.lu.se/personal/benjamin.smith/software). 164 165

Clone library construction and analyses 166

Fresh PCR products were cloned using the TOPO TA cloning kit according to the 167 manufacturer's instructions (Invitrogen Corporation, Carlsbad, CA, USA). Recombinant 168 clones were screened for insert-containing plasmids by direct PCR amplification with M13 169 forward and reverse primers. Clones were sequenced using an ABI 3130 POP7 sequencer 170 (Applied Biosystems, Foster City, CA) at the Biogenouest Sequencing-Genotyping Platform 171 (Roscoff site). Clone libraries were screened for chimeric sequences with Chimera_Check 172 program available on the RDP website [START_REF] Cole | The ribosomal database project (RDP-II): 508 previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy[END_REF]. The 388 remaining sequences were 173 subjected to BLAST search against publicly available sequences to determine their 174 approximate phylogenetic affiliations. A conservative value of 94 % nucleic acid sequence 175 similarity [START_REF] Zeng | Genetic diversity assessment of anoxygenic 701 photosynthetic bacteria by distance-based grouping analysis of pufM sequences[END_REF] [START_REF] Mullins | Genetic comparisons reveal the 618 same unknown bacterial lineages in Atlantic and Pacific bacterioplankton communities[END_REF]. The Shannon-Weiner index (H', [START_REF] Hill | Using ecological diversity measures 557 with bacterial communities[END_REF], 179 the richness-estimator S chao1 [START_REF] Hughes | Counting the uncountable: 563 statistical approaches to estimating microbial diversity[END_REF] and the Abundance-base Coverage 180 Estimator (ACE, [START_REF] Chazdon | Statistical methods for 494 estimating species richness of woody regeneration in primary and secondary rain forests of 495 NE Costa Rica[END_REF] were computed using EstimateS software Version 7.5. 181 (K. Colwell, http://purl.oclc.org/estimates). 182

A pufM database containing more than 700 aligned sequences of cultured species and 183 environmental clones retrieved from GenBank database 184 (http://www.ncbi.nlm.nih.gov/Genbank/) and the GOS scaffold nucleotide sequences was 185 constructed using ARB [START_REF] Ludwig | ARB: a software 597 environment for sequence data[END_REF]. Sequences were translated to protein and the 186 resulting alignment was then used to manually realign nucleotide sequences. A neighbor-187 joining tree was first constructed with all the sequences longer than 600 bp and the robustness 188 of inferred tree topologies was tested by bootstrap analysis (1000 resamplings) using PHYLIP 189 [START_REF] Felsenstein | PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the 528 author[END_REF]. Shorter sequences were aligned as above and added to the tree using 190 ADD-BY-PARSIMONY algorithm. Phylogenetic tree display and annotation were performed 191 with iTOL software [START_REF] Letunic | Interactive tree of life (iTOL): an online tool for phylogenetic tree 591 display and annotation[END_REF]. The pufM sequences obtained in this study are 192 deposited in the GenBank database under accession No. GQ468944 to GQ468986. 193 194

Statistical analyses 195

The normality of environmental variables was checked using Shapiro-Wilk (Shapiro, 196 1965) and Anderson-Darling [START_REF] Stephens | EDF Statistics for Goodness of Fit and Some Comparisons[END_REF] tests and variables were transformed when 197 necessary to correct for deviations from normality. Principal component analysis (PCA), 198 performed with XLSTAT version 6.01 (Addinsoft), was used to group samples according to 199 environmental variables using the Pearson correlation coefficient. 200

For TTGE patterns, pairwise similarity matrices were calculated using the Dice and the Bray-201

Curtis equations for presence/absence and relative peak height data, respectively. 202 Dendrograms were generated from the Dice matrix using the method described by Ward 203 Analysis of similarity (ANOSIM, [START_REF] Clarke | Change in marine communities: an approach to statistical 504 analysis and interpretation, 2 nd edition[END_REF] was used to test the hypothesis 207 that communities within each cluster were more similar to each other than to communities in 208 other clusters. Correlations between similarity matrices were calculated using a Mantel test 209 [START_REF] Mantel | The detection of disease clustering and a generalized regression approach[END_REF] with 10000 permutations and were performed with XLSTAT version 6.01. 210

Relationships among samples were visualized using the ordination technique 211 multidimensional scaling (MDS) using a standardized stress with 1000 iterations computed 212 with XLSTAT version 6.01. Canonical correspondence analyses (Legendre & Legendre, 213 1998) used to determine the extent to which selected environmental variables explained 214 patterns of similarity in community composition were performed using PAST 1.81 (Hammer 215 et al., 2001) The main biological and physico-chemical parameters of the waters were analyzed 239 using a principal component analysis (PCA) including nineteen variables (salinity, 240 temperature, dissolved oxygen, light, pH, depth, phosphate, nitrate, nitrite, particulate organic 241 carbon, nitrogen, phosphorus, total chlorophyll a, accessory pigments, bacterial production, 242 abundances of Synechococcus, Prochlorococcus, non photosynthetic bacteria and 243 picoeukaryotes) (Fig. S2). This analysis allowed separation of the Mediterranean Sea euphotic 244 zone into two distinct layers above and in/or below the DCM, whose discrimination was 245 mainly explained by biological variables and nutrient levels (NO 3 , NO 2 , PO 4 ) respectively. 246

All surface waters except that of the Morocco upwelling and the Gibraltar Strait (St. 1) were 247 nutrient depleted (Figs. S1 andS2). Covariation of biological parameters with particulate 248 organic compounds, oxygen, pH and photosynthetically available radiation (PAR) are 249 consistent with the characteristics of the upper layer where photosynthesis supports the 250 formation of organic matter [START_REF] Lucea | Nutrient (N, P and Si) and carbon 594 partitioning in the stratified NW Mediterranean[END_REF] allowing heterotrophic activities as indicated 251 by the inter-correlation of these parameters with bacterial production and cell densities (Fig. 252 S1 and S2). Deep euphotic waters located in or below the DCM were relatively similar as they 253 were significantly more similar to each other than to those in or below the DCM (R=0.419, 281 p<0.001). Dimension 1 from the TTGE/MDS analyses was negatively correlated to nutrient 282 variables (NO 3 and NO 2 ). In contrast to dimension 1, dimension 2 co-varied with numerous 283 variables characterizing biological activity (e.g. oxygen, bacterial production, cell densities). 284

Variables significantly related to dimensions 1 and 2 were integrated in a Canonical 285Correspondence Analysis (CCA) performed from relative intensity of TTGE bands. CCA 286 revealed that more than 50% of the variability of AAP communities was described by the 11 287 selected variables (Fig. 3). The four clusters (A to D) previously detected by HCA analysis 288 were retrieved. Cluster D was mainly separated according to NO 3 and NO 2 variables, while 289 clusters A and C were discriminated by pH, PAR, total organic carbon, biogenic activity 290 variables and particulate nitrogen and phosphorus. None of the selected variables clearly 291 explained pattern of similarity within cluster B. 292 293

Phylogenetic analyses of pufM genes 294 295

Ten out of the 29 samples were selected for phylogenetic analyses on the basis of their 296 location and the diversity of AAP populations. Of a total of 388 clones analyzed, 44 distinct 297 OTUs were identified after grouping the sequences at 94% nucleic acid sequence similarity 298 (Fig. 4, Table S1). Coverage values (Table 1) and rarefaction curves (data not shown) 299 indicated that most of the diversity was detected in most libraries (> 71%). Differences in 300 AAP population diversity were not significant between samples collected above and below 301 the DCM. 302 MDS for AAP populations from the 10 selected samples were performed from Dice 303 similarity matrices for both TTGE and clone library analyses (Fig. S4). As also expressed by 304 the significant correlation between Dice similarity matrices for TTGE and clone libraries 305 13 (R=0.39, p=0.05), both methods gave reliable information. The discrepancy observed for St.9-306 65m could be due to the low coverage of the clone library (Table 1). 307 About half of the pufM sequences were less than 94% identical to known sequences 308 (Table S1). Most sequences were related to clones retrieved from the Mediterranean Sea 309 (29%) and coastal environments (52%) including the Delaware and Monterey Bays. A unique 310 sequence (PROSOPE-7) was affiliated with a clone from the Atlantic Ocean. The phylogenetic 311 analysis demonstrated that the sequences were distributed into 7 of the 12 phylogroups 312 previously defined by [START_REF] Yutin | Assessing 696 diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of 697 the Atlantic and Pacific Oceans using the Global Ocean Sampling expedition metagenomes[END_REF]. None of the sequences was affiliated with the A, D, 313 H, J and L groups (Fig. 4, Table S1) that are mainly found in anoxic environments (Yutin et 314 al., 2007). 315

Only two sequences, belonging to PROSOPE-48 and PROSOPE-52 phylotypes, were 316 highly similar to that of cultured representatives (Methylobacterium radiotolerans and 317

Erythrobacter longus, respectively). Among the 7 AAP groups recovered from this study, 318 only groups B and K were present in all samples (Fig. 5). PROSOPE-6 which shared 99% 319 similarity with a clone from the Monterey Bay (Béja et al., 2002) was the main phylotype of 320 group B. Representing 11% of the pufM sequences, PROSOPE-6 was prevalent (> 80%) at 321 coastal and mesotrophic stations (Fig. 4, Table S1). Most of our sequences (~80%) fell into 322 Group K which contained Gammaproteobacteria representatives including few isolates such 323 as Congregibacter litoralis KT71 [START_REF] Eilers | Isolation of 524 novel pelagic bacteria from the german bight and their seasonal contributions to surface 525 picoplankton[END_REF] and strains NOR5-3 and NOR51B and 324 HTCC2080 [START_REF] Cho | Polyphyletic photosynthetic reaction centre genes in oligotrophic 501 marine Gammaproteobacteria[END_REF] and BAC clones EBAC65D09 and EBAC29C02 (Béjà et al., 325 2002), all related to the OM60 clade [START_REF] Rappé | Phylogenetic diversity of marine coastal 636 picoplankton 16S rRNA genes cloned from the continental shelf off Cape Hatteras, North 637 Carolina[END_REF]. Among the 30 OTUs affiliated to 326 Group K, 10 (i.e. PROSOPE -10, -11, -38) and 9 (i.e. PROSOPE -12, -15, -45) phylotypes were 327 only detected at the coastal Atlantic (St. UPW and St. 1) and at Mediterranean stations, 328 respectively. Among gammaproteobacterial sequences, PROSOPE-34, which represented up to 329 20% of the total pufM sequences, dominated at meso-and eutrophic stations while other 330 in freshwater and estuarine environments [START_REF] Page | Representative freshwater bacterioplankton 630 isolated from Crater Lake, Oregon[END_REF][START_REF] Yutin | Assessing 696 diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of 697 the Atlantic and Pacific Oceans using the Global Ocean Sampling expedition metagenomes[END_REF]. This could 338 suggest that closely related organisms also thrive in oceanic surface waters. 339

Discussion

340

The Mediterranean Sea is semi-enclosed allowing the study, on a reduced scale, of 341 processes typical of the world's oceans. It constitutes a unique environment for ecological 342 studies as it offers a range of trophic conditions, including extreme oligotrophy, particularly in 343 summer when the water column is strongly stratified. The PROSOPE cruise was organized a 344 few years before the discovery of the ecological significance of AAP bacteria in oceanic 345 waters [START_REF] Kolber | Contribution of aerobic photoheterotrophic 578 Bacteria to the carbon cycle in the ocean[END_REF]. Therefore, the sampling strategy was not intended to allow the 346 examination of their abundance and their contribution to the microbial community structure 347 using infrared microscopy and fluorometry methods. One of the main objectives of the 348 present study was to investigate environmental factors influencing biogeography of AAPs by 349 analyzing the pufM gene diversity in archived bacterial community DNA samples. We agree 350 that top-down regulation factors probably contribute to the distribution of AAP populations 351 [START_REF] Koblížek | Rapid growth rates of aerobic 574 anoxygenic prototrophs in the Ocean[END_REF][START_REF] Zhang | Roseophage RDJL 1, infecting the aerobic anoxygenic 705 phototrophic bacterium Roseobacter denitrificans OCh114[END_REF] The diversity of AAP populations as evident in pufM sequences was relatively 359 constant along both transects and was similar to that reported by [START_REF] Jiao | Distinct distribution 570 pattern of abundance and diversity of aerobic anoxygenic phototrophic bacteria in the global 571 ocean[END_REF] in 360 oligotrophic oceanic provinces. We found a positive correlation between the PAR and 361 diversity (H'/E var ) indices, suggesting that AAP populations are more diverse in the upper 362 surface layer and that light affected their community structure. In contrast to Jiao et al. 363 (2007), who reported that the AAP diversity decreased with increasing Chla concentrations, 364 we did not find a significant correlation between these variables. Our study found, however, 365 an inverse relationship between AAP diversity and nitrate levels. This result together with the 366 highest richness observed for the highly oligotrophic station MIO also supports the hypothesis 367 that inorganic nitrogen concentrations may affect their distribution (Waidner & Kirchman, 368 2007) and that AAP diversity decreases with increasing trophic status [START_REF] Jiao | Distinct distribution 570 pattern of abundance and diversity of aerobic anoxygenic phototrophic bacteria in the global 571 ocean[END_REF]. This 369 suggests that the scarcity of inorganic nutrients and dissolved organic matter in oligotrophic 370 environments leads to a higher specialization of AAP assemblages and increases the 371 functional redundancy (Curtis & Sloan, 2004) for this group, as it has been observed 372 elsewhere for other bacterial populations [START_REF] Wohl | Functional redundancy supports biodiversity and 679 ecosystem function in a closed and constant environment[END_REF]. This hypothesis is consistent 373 with the contrasting variation of abundance and diversity of AAP assemblages previously 374 suggested by a large-scale survey analysis of their distribution patterns [START_REF] Jiao | Distinct distribution 570 pattern of abundance and diversity of aerobic anoxygenic phototrophic bacteria in the global 571 ocean[END_REF]. 375

Variation in the composition of AAP assemblages and environmental context 376

The HCA and MDS analyses suggest that AAP populations follow nutrient status and 377 that oceanic Mediterranean populations differed from those of the Atlantic coast. This 378 clustering deduced from TTGE analyses was consistent with the clone library results with 379 reported for the whole bacterial community structure in the Mediterranean Sea (Acinas et al., 384 1997;[START_REF] Ghiglione | Role of environmental factors for the vertical distribution (0-1000 m) of 548 marine bacterial communities in the NW Mediterranean Sea[END_REF] and in other oligotrophic waters, including the Pacific and 385 Atlantic oceans, the Caribbean Sea (Lee & Fuhrman, 1991), and Antarctic environments 386 [START_REF] Murray | 622 Seasonal and spatial variability of bacterial and archaeal assemblages in the coastal waters off 623 Anvers Island, Antarctica[END_REF]. Multivariate analyses of environmental parameters and molecular 387 fingerprinting profiles revealed that the variation in AAP populations with depth is due to 388 synergetic driving forces similar to those involved in the vertical stratification of bacterial 389 communities at the DYF station [START_REF] Ghiglione | Role of environmental factors for the vertical distribution (0-1000 m) of 548 marine bacterial communities in the NW Mediterranean Sea[END_REF]. Fifty percent of the variability of 390 AAP composition was explained by the selected environmental parameters. This suggests that 391 the composition of AAP assemblages may be additionally influenced by other factors such as 392 specific and multiple interactions with other organisms of their environment such as their 393 bacterial counterparts, protists, viruses, and metazoans [START_REF] Fuhrman | Microbial community structure and its functional implications[END_REF] as well as intrinsic 394 photoheterotrophic capabilities and physiological peculiarities of each species. Indeed, AAP 395 bacteria have diverse metabolisms, ranging from generalists (e.g. Erythrobacter, Roseobacter) 396 to specialized species (e.g. Citromicrobium) able to grow on a broad and a narrow spectrum 397 of carbon sources [START_REF] Yurkov | New light on aerobic anoxygenic phototrophs[END_REF]. Further in-depth studies on the physiology 398 and metabolism of environmentally significant organisms and on their biological interactions 399 with other planktonic counterparts are essential to better understand the ecology of marine 400

AAP. 401

Phylogenetic composition of populations 402

Most pufM sequences retrieved from our samples belonged to clades previously 403 identified in a global metagenomic survey [START_REF] Yutin | Assessing 696 diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of 697 the Atlantic and Pacific Oceans using the Global Ocean Sampling expedition metagenomes[END_REF]. As previously observed in 404 environmental surveys of these genes (e.g. [START_REF] Béjà | Unsuspected diversity among marine aerobic anoxygenic 485 phototrophs[END_REF], most of the OTUs retrieved in 405 this study were distantly related to known anoxygenic phototrophs. More than 80% of our 406 pufM sequences had their best matches with clone sequences obtained from the Mediterranean 407 Sea and from coastal areas suggesting that AAP populations in this sea are different from 408 those in open oceans. We acknowledge, however, that this observation may be biased by the 409 low number of offshore pufM sequences available in the databases. 410

In contrast to previous PCR-based studies [START_REF] Oz | Roseobacter like bacteria in Red 626 and Mediterranean Sea aerobic anoxygenic photosynthetic populations[END_REF][START_REF] Yutin | Novel primers reveal wider diversity among marine 693 aerobic anoxygenic phototrophs[END_REF], 411

Alphaproteobacteria constitute a minor fraction of the Mediterranean AAP community. 412 Members of the Roseobacter-related group (group G), often a key player in diverse marine 413 systems [START_REF] Buchan | Overview of the marine Roseobacter lineage[END_REF][START_REF] Yutin | Assessing 696 diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of 697 the Atlantic and Pacific Oceans using the Global Ocean Sampling expedition metagenomes[END_REF], were only detected at Atlantic coastal 414 stations. While this finding supports the notion that Roseobacter-like bacteria are favored by 415 nutrient-rich conditions, in agreement with the common association of Roseobacter with 416 phytoplankton blooms [START_REF] González | Bacterial community structure associated with a dimethylsulfoniopropionate-553 producing North Atlantic algal bloom[END_REF][START_REF] Suzuki | Quantitative mapping of 660 bacterioplankton populations in seawater: field tests across an upwelling plume in Monterey 661 Bay[END_REF], it seems to contradict their 417 suspected important role and dominance in the Mediterranean Sea [START_REF] Oz | Roseobacter like bacteria in Red 626 and Mediterranean Sea aerobic anoxygenic photosynthetic populations[END_REF]. However, 418 since Roseobacter species can be more abundant in winter than in summer, this discrepancy 419 with previous studies [START_REF] Oz | Roseobacter like bacteria in Red 626 and Mediterranean Sea aerobic anoxygenic photosynthetic populations[END_REF][START_REF] Yutin | Novel primers reveal wider diversity among marine 693 aerobic anoxygenic phototrophs[END_REF] could be explained by the different 420 sampling period. 421

Previous studies have revealed a high contribution of Gammaproteobacteria to AAP 422 populations [START_REF] Béjà | Unsuspected diversity among marine aerobic anoxygenic 485 phototrophs[END_REF][START_REF] Hu | Abundant presence of the gamma-like proteobacterial 560 pufM gene in oxic seawater[END_REF][START_REF] Yutin | Assessing 696 diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of 697 the Atlantic and Pacific Oceans using the Global Ocean Sampling expedition metagenomes[END_REF]. However, the large 423 proportions observed in this study (~ 80%) have never been reported from any environments. 424

Possible PCR biases cannot be definitively ruled out to explain the large dominance of 425 Gammaproteobacteria in our samples. However, by using the same primer combination to 426 study the diversity of AAP in arctic waters and in coastal systems of the English Channel, we 427 did not find such high percentages of gammaproteobacterial sequences (Lehours et al.,Dahan 428 et al.,unpublished results). Consistent with previous observations, gammaproteobacterial 429 [START_REF] Hu | Abundant presence of the gamma-like proteobacterial 560 pufM gene in oxic seawater[END_REF]Jiao et al. 430 2007). Athough some gammaproteobacterial phylotypes were obtained from both nutrient 431 levels, the distribution of most suggests an adaptation to specific trophic conditions. For 432 example, some of our cloned pufM sequences from the Atlantic coast were similar to the 433 sequences belonging to the OM60 clade abundant in coastal oceans [START_REF] Béjà | Unsuspected diversity among marine aerobic anoxygenic 485 phototrophs[END_REF]Yutin 434 et al., 2007). Although prevalent at coastal stations, phylogroup B also showed a ubiquitous 435 distribution across the different trophic regimes [START_REF] Yutin | Assessing 696 diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of 697 the Atlantic and Pacific Oceans using the Global Ocean Sampling expedition metagenomes[END_REF]. Onboard enrichment 436 experiments performed along the PROSOPE transect demonstrated a switch from a 437 phosphorus limitation in the surface layer to organic carbon limitation in the deep chlorophyll 438 maximum [START_REF] Van Wambeke | 664 Longitudinal and vertical trends of bacterial limitation by phosphorus and carbon in the 665 Mediterranean Sea[END_REF]. Specific adaptation capabilities to extreme 439 oligotrophy are likely to explain the success of phylogroups B and K in typical Mediterranean 440 waters. In line with this hypothesis, genes coding for several storage compounds were 441 identified in the genome of the gammaproteobacterium Congregibacter litoralis (Fuchs et al., 442 2007). Among them, the storage compounds cyanophycin and polyphosphate probably reflect 443 an adaptation to fluctuating carbon and phosphorus availability. 444 445

Conclusion 446

The molecular analyses of AAP diversity conclusively demonstrated that typical 447 Mediterranean populations varied greatly along depth profiles, with strong and opposite 448 gradients of light and nutrient availability. This variation with depth was much greater than 449 seen across both transects. During stratification, the vertical distribution of AAP bacteria 450 seems to be governed by the same environmental variables as that of the whole bacterial 451 community [START_REF] Ghiglione | Role of environmental factors for the vertical distribution (0-1000 m) of 548 marine bacterial communities in the NW Mediterranean Sea[END_REF]. This result, however, does not imply that they may act as 452 strict heterotophs in situ and that phototrophy has a minor impact on their distribution and 453 lifestyle. Since evidence from other studies indicate that AAP use their phototrophic 454 [START_REF] Cho | Polyphyletic photosynthetic reaction centre genes in oligotrophic 501 marine Gammaproteobacteria[END_REF][START_REF] Spring | The Photosynthetic Apparatus and Its 649 Regulation in the Aerobic Gammaproteobacterium Congregibacter litoralis gen. nov., sp. 650 nov[END_REF], additional variables 455 may influence their populations in the extreme oligotrophic conditions prevailing in the 456 Mediterranean Sea. 457 This study was the first to reveal such a high abundance of gammaproteobacterial 458 AAP in the environment. An ecotypic differentiation was suggested by both TTGE and 459 cloning approaches. Further culture efforts are therefore needed to expand the diversity of 460 gammaproteobacterial isolates and to delineate the environmental parameters that govern the 461 found to be dominant (>80%) at meso-eutrophic and oligotrophic conditions, respectively. 802 the "biological activity": PROC (Prochlorococcus cell concentration, cell.ml -1 ), SYN 21 (Synecococcus cell concentration, cell.ml -1 ), BACT (bacteria cell concentration, cell.ml -1 ), 22 EUK (eukaryotes cell concentration, cell.ml -1 ), BP (bacterial production, ng C.l -1 .h -1 ), Chla 23 (chlorophyll a, µg. l -1 ), PIGM (accessory pigments, µg.l -1 ). Black dashed arrows represent 24 variables characterizing the "trophic status":TOC (total organic carbon, µM), NO

activity

  the oceans. The environmental factors controlling the population structure 79

  collected in late summer along two transect during the PROSOPE (PROductivité des 96 Systèmes Océaniques PElagiques) cruise. To explore what environmental factors control 97 structure and diversity of AAP populations in the Mediterranean Sea, we monitored their 98 longitudinal and vertical changes using a pufM-based PCR-Temporal Temperature Gel 99 Electrophoresis (TTGE) survey and analyzed the pufM clone libraries from selected stations collected from seven stations along two transects in the 108 Mediterranean Sea (from Gibraltar to the Ionian Sea off the north-east coast of Lybia and 109 north-west to the French coast through the Tyrrhenian and Ligurian Seas) in September and 110

  ,[START_REF] Yutin | Novel primers reveal wider diversity among marine 693 aerobic anoxygenic phototrophs[END_REF] to amplify partial sequences of the 129 pufM gene (245 bp fragments). For TTGE analyses, a 5'-Cy5-labeled PufMF primer and a added at the 5'-end 132 were used. Reaction mixture (50 µL) contained the following components: 5X buffer (10µl), 133 2 mM MgCl 2 , 10 pmoles of each deoxyribonucleotide triphosphate

  photomultiplier tube voltages to maximize signal without saturating fingerprint bands. Band 156

  . The consistence of a cluster was expressed by the cophenetic correlation which 204 calculates the correlation between the dendrogram-derived similarities and the matrix 205 similarities. A distance of 35 was used to separate clusters in the hierarchical classification. 206

  available at http://palaeo-electronica.org/2001_1/past/issue1_01.sampled the highly eutrophic Morocco upwelling off Agadir 220 (St. UPW, Fig. 1) and then proceeded to a first Mediterranean west-east (W-E) transect, 221 through a markedly decreasing gradient of surface chlorophyll a concentrations, from the 222 mesotrophic St. 1 at the Strait of Gibraltar to St. MIO located in a highly oligotrophic area in 223 the center of the Ionian Sea. A second transect sampled the Tyrrhenian and Ligurian Seas (St. 224 9, St. DYF). 225 Plots of the depth variations of environmental variables analyzed along both these 226 transect revealed the complexity of the studied zones in which several physical and chemical 227 gradients were superimposed (Figs. S1a to S1l). Temperature and salinity transects reflected 228 low-salinity, low-temperature Atlantic waters penetrating through the 229 Gibraltar Strait and flowing along the African coast (Figs. S1a-b). Thus a strong halocline was 230 observed between St. 3 and St. 5 identifying a partitioning of surface waters into westwards 231 and typical high salinity eastwards (21.6°C and 36.7‰ for St. 1 to 26°C and 38.9‰ for St. 232 MIO, respectively). The upwelling at St. UPW supplied abundant dissolved nutrients to the 233 surface layer, which supported phytoplankton growth as indicated by the surface maximum in 234 chlorophyll a (Fig. S1c). A progressive W-E deepening of the nitracline and phosphacline 235 down to 98 and 124 m depth at St.1 and St. MIO, respectively, was noted (Figs. S1d-e). 236 Along the second transect (from St. MIO to St. DYF), concentrations of nitrates and 237 phosphates increased below the deep chlorophyll maximum (DCM). 238

  enriched in nutrients and depleted in oxygen (Fig.S1). Although a strong halocline 254 partitioned W-E surface waters, salinity did not explain variability among samples. This 255 might result from the superimposed longitudinal and vertical variation trends of salinity 256 concentrations (Fig.S1b). Discrimination of surface and deep euphotic layers in the 257 Mediterranean Sea mainly according to their nutrient status suggests that the vertical gradient 258 may prevail over the longitudinal gradient during summer stratification. 259 Spatial variability, diversity and structure of AAP populations 260 PCR amplified pufM genes from different depths (Fig.S3) were analyzed by temporal 261 temperature gradient gel electrophoresis (TTGE) which yielded a total of 79 unique bands 262 with an average of 34± 6 bands in each sample (Fig.2). A Mantel test also showed that Bray-263 Curtis and Dice similarity matrices calculated from TTGE profiles were significantly 264 correlated (r=0.748, p<0.05). The hierarchical clustering analysis (HCA) identified four 265 clusters of AAP bacteria (Fig. 2) confirmed by ANOSIM statistics performed from both 266 presence/absence and relative intensity of TTGE bands (data not shown). Cluster A contained 267 populations from the coastal stations UPW and St. 1, together with those of the surface waters 268 of the Algerian Basin (St. 3-5m). Cluster B represented the deep euphotic zone of the 269 Algerian Basin (St. 3) and the Strait of Sicily (St. 5) whereas Cluster C grouped TTGE 270 profiles of samples collected above the DCM in the Ionian, Tyrrhenian, Ligurian Seas and the 271 Strait of Sicily. AAP bacteria from deep euphotic waters of stations St. 9 and St. DYF 272 clustered together in Cluster D. Richness and diversity (E var and H') values were not 273 significantly different between clusters (data not shown). E var , H' and richness were positively 274 correlated to PAR and biological activity indicators and were negatively correlated with NO 3 275 and NO 2 concentrations. 276 To obtain two-dimensional-coordinates for samples and to confirm HCA groupings, 277 ordination of Bray-Curtis similarities among sample profiles was performed by non-metric 278 MDS). With the exception of samples St1-80m and St. 3-5m, the 279 four clusters recovered by HCA were defined (data not shown). Populations above the DCM 280

  .e. prevailed (>80%) at oligotrophic stations. The 331 distribution of the other groups was sporadic. A few clones recovered at MIO and UPW 332 stations clustered in the group C. The Roseobacter-like (group G) and Rhodobacter-like 333 (groups E and F) clones were only distributed in the nutrient rich coastal waters of the 334 Morocco upwelling (st. UPW) and the Strait of Gibraltar (UPW and St. 1) (Fig. 5). The 335 phylotype PROSOPE-7 consisting of a few clones retrieved at station MIO grouped into the 336 group I. The closest cultured relatives of this group are Betaproteobacteria widely distributed 337

  , however, this study provides one of the first look 352 on bottom-up factors that contribute to longitudinal and vertical distribution and diversity of

  that stratification is a critical factor determining the vertical 382 distribution of AAP species in the Mediterranean Sea. Similar vertical stratification has been 383

  in eutrophic and oligotrophic waters

782Figure 1 .Figure 2 .Figure 3 .Figure 4 .

 1234 Figure 1. Track of the PROSOPE cruise superimposed on the composite SeaWiFS image of 783

Figure 5 .Figure S2 .

 5S2 Figure 5. Distribution of the AAP phylogroups along the PROSOPE transects based on their 803

  Figure S.1

Acknowledgements

Supplementary material