Miguel Cortijo

Vladimir Bulicanu

Kasper S Pedersen

Mathieu Rouzières

Jesper Bendix

Rodolphe Clérac

Elisabeth A Hillard

] Kasper

S Pedersen

Elizabeth A Hillard

Rational self-assembly of tricobalt extended metal atom chains and [MF6]2-building blocks into one-dimensional coordination polymers

Keywords: Coordination polymers, Cobalt Extended Metal Atom Chains, Hexafluoridometallates, Magnetic properties

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Members of the class of compounds coined "Extended Metal Atom Chains (EMACs)" by F. A. Cotton, or metal strings" by S.-M. Peng, consist of a linear arrangement of three to eleven metal ions, wrapped by four polydentate bridging ligands often based on an oligopyridylamine skeleton. [1] Chromium, cobalt, nickel, copper, ruthenium, and rhodium form such structures, which often possess metal-metal bonds. The structure of the first example of this type of complex, [Ni3(dpa)4Cl2], was reported in 1991. [2] Since then, this field has been nourished by notable contributions from the F. A. Cotton, S. M. Peng, and J. F. Berry groups, among others. While Cotton's group performed many of the seminal studies on tri-metallic and penta-metallic EMACs, [3] Peng's group continues to be particularly active in the synthesis and magnetic characterization of long EMACs and the development of new bridging ligands, [4] and Berry's group has especially distinguished itself in the synthesis and characterization of heterometallic EMACs. [5] These materials often display highly complex crystal and electronic structures, which have been elucidated by theoretical work by the M. Bénard/M. Rohmer [6] and J. McGrady [7] groups in particular. The most well-studied EMAC is undoubtedly [Co3(dpa)4Cl2]. [8] This complex crystallizes from dichloromethane and hexane as two solvatomorphs, orthorhombic [Co3(dpa)4Cl2]•CH2Cl2 and tetragonal [Co3(dpa)4Cl2]•2CH2Cl2. The Co-Co distances in the orthorhombic form, 2.3369(4) Å, are crystallographically identical at 298 K because the central cobalt lies on a two-fold axis. On the other hand, in the tetragonal form the cobalt atoms lie on general positions, and the Co-Co distances are quite different, 2.299(1) and 2.471(1) Å at 298 K. Both the "symmetrical" and "unsymmetrical" forms of these compounds have an S = 1/2 ground state and show thermal spin-crossover in the solid state due to population of excited magnetic states. [8] One-dimensional coordination polymers have attracted considerable interest in the molecular magnetism community because of their potential as Single-Chain Magnets (SCMs) [9] and Spin-Crossover materials. [10] An approach to achieve the assembly of such systems consist in the use of paramagnetic building-blocks with two empty or, at least, labile positions, which can be linked by connectors with at least two donor groups. In this regard, it has been shown that the chloride ligands in the axial positions of [Co3(dpa)4Cl2] can be cleanly substituted by more labile ligands such as CH3CN, [11] or BF4 -, [12] and the axial positions can then be subsequently replaced with different ligands, such as CN -, [13] N(CN)2 -, [14] NCS -, [11] and R-C≡C -. [15] It should be mentioned that materials of higher dimensionality using EMACs are still a great synthetic challenge. For example, the [Co3(dpa)4] 2+ moiety (Figure 1) was recently incorporated in twodimensional materials using [M(CN)6] 3-(M = Fe, Co) metalloligands [16] but obtaining crystalline material proved unsuccessful. In the last few years, we became interested in hexafluoridometallates [MF6] 2-(Figure 1) as connectors between {Co3} EMACs. The use of this type of metalloligand for building 1D coordination systems is currently of growing interest, [17,18] as fluoride bridges tend to be highly directional, forming a 180° angle between the metal ions with which they are coordinated, [19] thus favoring the formation of strictly linear chains. A feature that gives this kind of control over the structure is useful in designing materials and predicting their magnetic properties. In this regard, we recently reported three 1D coordination polymers using [ReF6] 2-, [ZrF6] 2-, and[SnF6] 2 3). [20] In the present work, we have selected the paramagnetic [IrF6] 2-(J = 1/2), and the essentially non-magnetic [OsF6] 2-as linkers to apply our synthetic approach to the formation of [Co3(dpa)4IrF6]•2DMF (4) and [Co3(dpa)4OsF6]•2DMF (5). These 5d hexafluoridometallates have been rarely used as metalloligands in molecular magnetic materials and their detailed electronic structures have been only recently elucidated. [18,21]

-linkers as [Co3(dpa)4ReF6]•2DMF (1), [Co3(dpa)4ZrF6]•2DMF (2), [Co3(dpa)4SnF6]•2DMF (

Results and Discussion

Synthesis and spectroscopic characterization. The synthesis of 1-5 was accomplished by slow diffusion of a MeCN solution of the corresponding anhydrous (PPh4)2[MF6] salt into a DMF solution of the in-situ formed [Co3(dpa)4(BF4)2] precursor as described previously. [20] The FT-IR analysis of the [Co3(dpa)4MF6]•2DMF compounds (M = Zr (1), Sn (2), Re (3), Ir (4), and Os ( 5)) in the range of 600-1800 cm -1 yielded, in all cases, spectra similar to that of [Co3(dpa)4Cl2]•CH2Cl2 (Figure S1) with the exception of the bands corresponding to the DMF molecules present in the crystal structure of the coordination polymers. In general, the FT-IR analysis in this range shows very similar features to those of the Hdpa ligand. However, symmetrical and unsymmetrical EMACs give slightly different solid-state IR spectra, as illustrated by the IR spectrum of [Co3(dpa)4Cl2]•xCH2Cl2 obtained as a mixture of symmetrical and unsymmetrical forms. [22] This spectrum shows a splitting of the band around 1153 cm -1 , related to pyridine vibrations, into two peaks at 1153 and 1164 cm -1 , corresponding to the symmetrical and unsymmetrical form, respectively. On the other hand, the IR spectra of 1-5 (Figure S1) do not display a peak at 1164 cm -1 suggesting that there are no unsymmetrical {Co3} moieties in these materials. The decomposition of [Co3(dpa)4Cl2] precursor, when exposed to humid air, occurs within a few weeks resulting in the formation of blue products, characteristic of mononuclear Co(II) products. To test the stability of the 1D assemblies, a sample of 2 was left open to air for 4 months and then an IR spectrum was collected. As seen in Figure S2, the spectrum is identical to that obtained from freshly-filtered crystals. This result thus suggests that connecting the {Co3} clusters into coordination polymers improves the stability of the {Co3} unit. Far IR spectra are more informative for characterizing 1-5, as the M-F bond vibrations are typically found in the 600-150 cm -1 range. [23] The far-IR spectrum of Cs2[Os IV F6] has been reported and consists of one characteristic band at 547 cm -1 for the F-Os(IV) stretching mode and two bands at 259 cm -1 and 231 cm -1 corresponding to F-Os IV -F bending modes. [23a] The far FT-IR spectra of the (PPh4)2[MF6]•2H2O salts (M = Zr, Sn, Re, Ir, and Os) measured in the 600-150 cm -1 range (Table 1 and Figures S3-S7) consist mainly of a broad band that appears in the 180-260 cm -1 range corresponding to the F-M IV -F bending mode, a very strong band at ca. 520 cm -1 corresponding to a P-C stretching mode of the PPh4 + cation, [24] and one (M = Re and Ir) or two (M = Zr, Sn and Os) additional bands in the 450-560 cm -1 region corresponding to F-M IV stretching modes. Considering that the (PPh4)2[MF6]•2H2O compounds are isostructural for M = Sn (ESI), Re, Ir, and Os, [17,18,21] it seems reasonable to consider that the P-C stretching band of the PPh4 + cation masks a F-M IV stretching band for the compounds where M = Re and Ir. After drying the crystals under vacuum, only one M-F stretching band is observed in all cases due to the removal of interstitial water molecules, which are hydrogen bonded to the apical fluoride ions. The far IR spectra of the coordination polymers 1-5 display in all cases a strong band in the 510-570 cm -1 region and a broad band in the 450-500 cm -1 range corresponding to F-M IV stretching modes. A broad and strong band in the 260-220 cm -1 range corresponding to the F-M IV -F bending mode is also observed (Tables 1 and2 andFigure S8-S12). The rest of the bands agree Table 1. Bands related to the M-F bond in the far IR region. [23] 𝜈𝜈̅ /cm -1 well with that reported for [Co3(dpa)4Cl2] in the literature [22] with the exception of (i) a band at 193 cm -1 attributed to the Co-Cl stretch that is absent in the coordination polymers 1-5 and (ii) the presence of bands at ca. 345 cm -1 attributed to Co-F stretching vibrations.

Compound M-F stretching / cm -1 F-M-F bending / cm -1 (PPh4)2[ZrF6]•2H2O (PPh4)2[ZrF6] 1 (PPh4)2[SnF6]•2H2O (PPh4)2[SnF6] 2 (PPh4)2[ReF6]•2H2O (PPh4)2[ReF6] 3 (PPh4)2[IrF6]•2H2O (PPh4)2[IrF6] 4 (PPh4)2[OsF6]•2H2O (PPh4)2[OsF6] 5
[Co3(dpa)4Cl2] [22] 𝜈𝜈̅ /cm -1 5 𝜈𝜈̅ /cm -1 Co-N bend. Co-Cl str. Γ (ring-ring) F-Os-F bend. Co-Co-Co bend. C-N-C bend. Co-N

Description of the crystal structures.

Compounds 1-5 are isostructural, crystallizing in the tetragonal P4/ncc space group. The structures consist of perfectly linear neutral coordination polymers formed of alternating [Co3(dpa)4] 2+ and [MF6] 2-units in which the four-fold axis is coincident with the chain direction and the c axis (Figure 2). In the new compounds 4 and 5, the {Co3} units are slightly unsymmetrical with Co-Co distances of 2.267(1) and 2.274(1) Å in the case of 4 and 2.270(1) and 2.279(1) Å in the case of 5. The [MF6] 2-octahedra are axially elongated in both structures (~2 %) with all the cis-F-M-F bond angles in the range of 90.0 ± 0.6°. Co-F distances are 2.117(3) and 2.178(3) Å for 4 and 2.117(3) and 2.175(3) Å for 5. More detailed crystallographic data can be found in Table 3 andTables S1-S4. The tricobalt units present helicoidal chirality, but the chains themselves are racemic with the [Co3(dpa)4] 2+ units in the ∆ and Λ configurations alternating along the one-dimensional structure (Figure 3). The structures are disordered on the position of the dpa -ligands, such that both enantiomers are found in the same position with a ca. 60 and 40 % occupancy. Two molecules of DMF per structural unit of the [Co3(dpa)4MF6] polymer are found in the crystal structures. These molecules are severely disordered with two molecules of DMF occupying four positions with 1/2 occupancy (Figure S5). Thermogravimetric analysis was performed to confirm the number of solvent molecules in the structures and also to determine their thermal stability (Figure S6). The analysis shows a loss in mass at about 150 °C of ~12% for the two samples, which is the equivalent of two molecules of DMF per structural unit.

Magnetic properties

Magnetic susceptibility measurements on polycrystalline samples of 4 and 5 were carried out from 1.9 to 300 K (Figures 4 andS7).

The χT product measured for [Co3(dpa)4IrF6]•2DMF ( 4) is roughly constant (0.91 cm 3 K mol -1 ) in the 20-300 K range of temperatures. This value is in agreement to that expected for an S = 1/2 [Co3(dpa)4] 2--cluster (g = 2.40( 5), C = 0.54 cm 3 K mol -1 ) [9] and an [IrF6] 2-unit with an effective J = 1/2 ground state (g = 2, C = 0.38 cm 3 K mol -1 ). [18] On cooling, the χT product increases giving a value of 1.06 cm 3 K mol -1 at 1.85 K, suggesting the presence of ferromagnetic interactions in the system. The magnetic data were fitted using the Baker model for a S = 1/2 Heisenberg chain model, [25] on the basis of the Hamiltonian 𝐻𝐻 � = -2𝐽𝐽 ∑ 𝑆𝑆 ̂𝑖𝑖 𝑁𝑁 𝑖𝑖=1

• 𝑆𝑆 ̂𝑖𝑖+1 , yielding g = 2.16 (5) and J/kB = +0.13( 6) K. The ferromagnetic interactions between the magnetic [Co3(dpa)4] 2+ and [IrF6] 2- building-blocks along the chain are significantly weaker than those observed in 3 involving the S = 3/2 [ReF6] 2-moiety (J/kB = +9.9(1) K). [Co3(dpa)4OsF6]•2DMF ( 5) shows a temperature-independent χT value between 100 and 300 K of 0.56 cm 3 K mol -1 , close to what is expected for an S = 1/2 system with g = 2.40(5) (0.54 cm 3 K mol -1 ) typical of tricobalt EMACs. Upon cooling, a decrease of χT is observed, suggesting weak antiferromagnetic interactions between the {Co3} magnetic site via the virtually nonmagnetic [OsF6] 2-linkers. [21] Fitting the experimental susceptibility data of 4 using the Bonner-Fisher [26] model for linear S = 1/2 chains, on the basis of the Hamiltonian 𝐻𝐻 � = -2𝐽𝐽 ∑ 𝑆𝑆 ̂𝑖𝑖 𝑁𝑁 𝑖𝑖=1

• 𝑆𝑆 ̂𝑖𝑖+1 , gave values of g = 2.45 (5) and J/kB = -1.9(4) K. In this case, [OsF6] 2-mediates stronger antiferromagnetic interactions than those previously found in 1 or 2 through [ZrF6] 2-(J/kB = -0.82(4) K) or [SnF6] 2-(J/kB ≈ ±0.0(5) K). [20] Unlike [Co3(dpa)4Cl2], but in line with previous findings for 1-3, these coordination polymers do not show any sign of spin crossover behavior up to 300 K confirming that the axial substitution of Cl -by [MF6] 2-stabilizes the S = 1/2 low spin state in tricobalt EMACs.

Conclusions

Two new self-assembled one-dimensional polymers formed by [Co3(dpa)4] 2+ and [MF6] 2-(M = Ir IV (4), and Os IV ( 5)) building blocks have been prepared and studied. Both compounds are isostructural, crystallizing in the same tetragonal space group but ferromagnetic interactions between [Co3(dpa)4] 2+ and [IrF6] 2- magnetic sites are observed (J/kB = +0.13(6) K) whereas [OsF6] 2- mediates significant antiferromagnetic interactions (J/kB = -1.9(4) K). As observed in the Zr, Sn and Re analogues (1-3), no spin crossover behavior is detected below 300 K. In order to clarify the impact of the axial ligand on the T1/2 characteristic temperature of the spin crossover, we are currently studying the effects of various axial halide ligands on the magnetic properties of tricobalt EMACs. The present results further demonstrate the utility of [MF6] 2- anions to rationally build coordination architectures using Extended Metal Atom Chains. Future work in this line will be dedicated to the assembly of other EMACs using hexafluoridometallates. For example, single-molecule magnet properties for [Cr3(dpa)4Cl2] have recently been discovered, and therefore the combination of [Cr3(dpa)4] 2+ with paramagnetic [MF6] 2-are of interest for the design of novel single-chain magnets.

Experimental Section

General Comments. All reactions were carried out under a dry argon or nitrogen atmosphere using Schlenk or glovebox techniques. Tetrahydrofuran (THF), diethyl ether (Et2O), acetonitrile (CH3CN) and dichloromethane (CH2Cl2) were purified using an Innovative Technologies solvent purification system. Anhydrous dimethylformamide (DMF) was purchased from Alfa Aesar and used as received. 2,2'-dipyridylamine (Hdpa) was purchased from Aldrich and recrystallized from hot petroleum ether before use. Anhydrous CoCl2 was purchased from Aldrich and stored in an oven at 130 °C several days before use. [Co3(dpa)4Cl2]•xCH2Cl2 was prepared as previously reported. [8] (PPh4)2[MF6]•2H2O, M = Ir, [18] Os, [21] were prepared according to the respective literature procedures. The crystals of (PPh4)2[MF6]•2H2O were dried under vacuum (10 -3 Torr) at 80 °C overnight to eliminate the water molecules prior to their use.

Physical measurements.

Elemental analyses were carried out by the Service d'Analyse Elémentaire of the University of Lorraine, Nancy. IR spectra were measured on a Nicolet 6700 FT-IR using Smart iTR accessories between 450-4000 cm -1 and 150-600 cm -1 . The magnetic measurements were carried out with the use of an MPMS-XL Quantum Design SQUID magnetometer. This instrument works between 1.8 and 400 K with applied dc fields ranging from -7 to 7 T. Measurements were performed on finely ground polycrystalline samples of 4 and 5 (20.29 and 18.66 mg respectively) sealed in a polypropylene bag (typical sizes and mass: 3 × 0.5 × 0.02 cm; 15-30 mg). Prior to the experiments, the fielddependent magnetization was measured at 100 K in order to detect the presence of any bulk ferromagnetic impurities. Ac susceptibility measurements were made with an oscillating field of 3 Oe with a frequency from 1 to 1500 Hz but no out-of-phase signal was detected above 1.8 K. The magnetic data were corrected for the sample holder, intrinsic diamagnetic contributions, minor ferromagnetic impurities, and temperature-independent paramagnetism. X-ray diffraction measurements were carried out on single crystals selected under immersion oil in ambient conditions and attached to a MiTeGen microloop. The crystals were mounted in a stream of cold nitrogen at 200(2) K and centered in the X-ray beam using a video camera. Data collection was performed on a Bruker APEX II Quasar diffractometer with Mo Kα (λ = 0.71073 Å) radiation. The data were collected using a routine to survey reciprocal space, and were indexed by the APEX II program. [START_REF]Bruker-AXS APEX2, SADABS, and SAINT Software Reference Manuals[END_REF] The structures were solved using direct methods and refined by least-squares refinement on F 2 followed by difference Fourier synthesis. [START_REF] Sheldrick | SHELXL-97: Program for Crystal Structure Refinement[END_REF] All hydrogen atoms were introduced at idealized positions and were allowed to ride on the neighboring atoms with relative isotropic displacement coefficients. CCDC 1573012, 1573013 and 1586059 contain the crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. A summary of crystal and refinement data is shown in Table 3. [a] = 0.0449, wR2 [b] = 0.0608 C46H46Co3F6N14O2Os 1307.98 200(2) P4/ncc 13.6719 (13) 13.6719 (13) 25.558(2) 4777.3(10) 4 1.819 3.754 1.061 R1 [a] = 0.0440, wR2 [b] = 0.0609 [a] R1 = Σ||Fo| -|Fc||/ Σ|Fo|. [b] wR2 = [Σ[w(Fo 2 -Fc 2 ) 2 ] / Σ[w(Fo 2 ) 2 ]] 1/2 , w = 1/σ 2 (Fo 2 ) + (aP) 2 + bP, where P = [max(0 or Fo 2 ) + 2(Fc 2 )]/3.

Figure 1 .

 1 Figure 1. Schematic representation of the building blocks employed in the assembly of the reported 1D coordination polymers.

Figure 2 .

 2 Figure 2. Thermal ellipsoid (50% probability) plot of 4. Only one of the two orientations of the disordered molecule is shown. (C: grey; F: light green; Ir: dark green; N: pale blue; Co: dark blue). H atoms and solvent molecules are omitted for clarity. Selected bond distances for 4: Co-Co: 2.2674(10) and 2.2736(10) Å; Co-F: 2.117(3) and 2.178(3) Å; Ir-Fax: 1.951(3) and 1.965(3) Å; IrFeq: 1.919(2) Å; for 5: Co-Co: 2.2699(9) and 2.2793(10) Å; Co-F: 2.117(3) and 2.175(3) Å; Os-Fax: 1.963(3) and 1.975(3) Å; Os-Feq: 1.917(2) Å.

Figure 3 .

 3 Figure 3. Ball and stick views along (top) c axis and (bottom) b axis of the polymeric species in 4 (red: ∆-{Co3} helix, blue: Λ-{Co3} helix, green: fluoride, and blue: iridium). Only one of the two orientations of the disordered molecule is shown. H atoms and solvent molecules have been omitted for clarity.

Figure 4 .

 4 Figure 4. Temperature dependence of the χT product for 4 (red) and 5 (blue) at 1000 Oe (χ is defined as molar magnetic susceptibility equal to M/H per mole of [Co3(dpa)4MF6]). The solid lines are the fits as discussed in the text.

  dpa)4Cl2]•xCH2Cl2 (50 mg, 0.05 mmol) and AgBF4 (20 mg, 0.10 mmol) in 10 mL of DMF was stirred overnight and filtered to remove AgCl. A solution of the corresponding anhydrous (PPh4)2[MF6] salt (0.05 mmol) in 10 mL of CH3CN was prepared and layered over the DMF solution in a glass tube (200 mm, ∅ 10 mm). The DMF and CH3CN solution were separated by 4 mL of a 1:1 mixture of DMF and CH3CN. Dark green blocks were collected from the walls of the tubes after 3-4 weeks. 4: Yield 37 mg

Table 2 .

 2 Assignment of the bands observed in the far FT-IR spectra of 5•and analogues of the building units.

	456, 496	218
	480	218
	450b, 510	218
	511,556	246
	548	246
	500b, 570	246
	499, 520*	237
	511	236
	500b, 539	238
	521*, 554	256
	546	256
	504b,562	259
	509, 543	244
	539	244
	492b; 549	250

Table 3 .

 3 Crystal and structure refinement data for 4-5.

		4	5
	Empirical formula	C46H46Co3F6N14O2Ir
	Formula weight / g mol -1	1309.97
	Temperature / K	200(2)
	Space group	P4/ncc
	a /Å	13.7071(13)
	b /Å	13.7071(13)
	c /Å	25.505(3)
	Volume / Å 3	4791.9(10)
	Z	4
	Calculated density / g	1.816
	cm 3	3.868
	µ / mm -1	1.106
	Goodness-of-fit on F 2	R1
	R indices
	(all data)

Acknowledgements

This work was supported by the CNRS, the University of Bordeaux, the Conseil Régional de la Nouvelle Aquitaine, the Marie Sklodowska-Curie grant 706556 CHIMMM (Postdoctoral fellowship for MC) and the Erasmus Mundus European program (PhD fellowship for VB). JB acknowledges support from The Danish Research Council under grant 12-125226. KSP thanks The Danish Research Council for a DFF | Sapere Aude: Research Talent award (grant 4090-00201). The authors thank the GdR MCM-2 (Magnétisme et Commutation Moléculaires), the MOLSPIN COST action CA15128, L. Favello (for crystallographic assistance), P. Dagault (for TGA analysis), I. Oyarzabal (for useful scientific discussions), and E. Durand and A. Tressaud (for the synthesis of the [MF6] 2-precursors).


Solange Duhamel
email: sduhamel@ldeo.columbia.edu

France Van Wambeke

Dominique Lefevre

Mar Benavides

Sophie Bonnet

Mixotrophic metabolism by natural communities of unicellular cyanobacteria in the western tropical South Pacific Ocean 1,*

Keywords: cyanobacteria, mixotrophy, photoheterotrophy, carbon cycling, organic molecules

Mixotrophy in natural marine cyanobacteria mixotrophy by marine picocyanobacteria is more likely to be an adaptation to low inorganic nutrient availability as cell specific uptake rates of organic molecules containing nutrients were much higher (35.5±16.5 time for leucine uptake by Prochlorococcus) than for glucose.

These findings are a significant contribution for developing a mechanistic understanding of the diverse cellular physiologies of different bacterioplankton groups, and to improve our understanding of microbial adaptations to light and nutrient availability, but also our knowledge of marine dissolved organic matter cycling, and the role of light in bacterioplankton biogeochemical functions in the ocean.

Originality-Significance Statement: Marine unicellular cyanobacteria of the genus Prochlorococcus and Synechococcus are major contributors to primary production and carbon (C) export in the open ocean. These picocyanobacteria are considered to be photoautotrophic, but recent evidence suggests they may also benefit from assimilation of organic compounds (mixotrophy). Most studies investigating the light-dependent organic C uptake potential of marine cyanobacteria have been performed with cultures, while only one field study has demonstrated glucose uptake by Prochlorococcus in the Atlantic Ocean. Hence, in situ data is lacking to assess the potential mixotrophic nutrition of these globally relevant marine cyanobacteria, how it compares to their autotrophic nutrition mode (CO 2 fixation), and its environmental controls (nutrients, light levels, etc.).

To fill this gap, we used a combination of radiolabeled organic molecules in incubation experiments where both light availability and photosynthetic electron transport were controlled, followed by flow cytometry cell sorting to separate picoplankton groups in natural samples along an oligotrophic to ultraoligotrophic transect in the Western Tropical South Pacific (WTSP) Ocean. Our results demonstrate that natural Prochlorococcus and Synechococcus can incorporate organic molecules, including glucose, and that rates are reduced in the dark or when photosynthesis is inhibited. This mixotrophic metabolism by unicellular cyanobacteria was widespread in the tested trophic gradient in the WTSP Ocean. In comparison, the low-DNAcontent bacteria, a group dominated by photoheterotrophic bacteria from the SAR11 group, also presented large (~35%) light-enhanced cell-specific glucose uptake, but generally lower than for Prochlorococcus and Synechococcus (~50%). Nevertheless, parallel group specific C uptake from 14 C-sodium bicarbonate suggest that the most abundant marine phytoplankton, Prochlorococcus and Synechococcus remain primarily autotrophic. Our results also indicate that

Summary

Cyanobacteria are major contributors to ocean biogeochemical cycling. However, mixotrophic metabolism and the relative importance of inorganic and organic carbon assimilation within the most abundant cyanobacteria are still poorly understood. We explore the ability of Prochlorococcus and Synechococcus to assimilate organic molecules with variable C:N:P composition and its modulation by light availability and photosynthetic impairment. We used a combination of radiolabeled molecules incubations with flow cytometry cell sorting to separate picoplankton groups from the western tropical south Pacific Ocean. Prochlorococcus and Synechococcus assimilated glucose, leucine, and ATP at all stations, but cell-specific assimilation rates of N and P containing molecules were significantly higher than glucose. Incubations in the dark or with an inhibitor of photosystem II resulted in reduced assimilation rates. Light-enhanced cell-specific glucose uptake was generally higher for cyanobacteria (~50%) than for the low nucleic acid fraction of bacterioplankton (LNA, ~35%). Our results confirm previous findings, based mainly on cultures and genomic potentials, showing that Prochlorococcus and Synechococcus have a flexible mixotrophic metabolism, but demonstrate that natural populations remain primarily photoautotrophs. Our findings indicate that mixotrophy by marine cyanobacteria is more likely to be an adaptation to low inorganic nutrient availability rather than a facultative pathway for carbon acquisition.

Introduction

Unicellular marine cyanobacteria (e.g., Prochlorococcus and Synechococcus) are major contributors to primary production and carbon (C) export in the open ocean [START_REF] Li | Autotrophic picoplankton in the tropical ocean[END_REF][START_REF] Richardson | Small phytoplankton and carbon export from the surface ocean[END_REF][START_REF] Johnson | Prochlorococcus: Approved for export[END_REF][START_REF] Martiny | Widespread metabolic potential for nitrite and nitrate assimilation among Prochlorococcus ecotypes[END_REF].

Cyanobacteria are aerobic oxygenic prokaryotes that use a chlorophyll-based lightharvesting complex and CO 2 as a C source. Therefore, marine representatives have been classically considered photoautotrophs [START_REF] Karl | Microbial oceanography: paradigms, processes and promise[END_REF], and Prochlorococcus and Synechococcus have long been considered to be unable to use organic C sources such as glucose [START_REF] Waterbury | Biological and ecological characterization of the marine unicellular cyanobacterium synechococcus[END_REF][START_REF] Béjà | Photoheterotrophic marine prokaryotes[END_REF]. However, the simple picture that marine cyanobacteria are purely photoautotrophic is actually much more complex. Indeed, recent molecular evidences indicate that organic compound uptake genes are ubiquitous within marine picocyanobacteria [START_REF] Yelton | Global genetic capacity for mixotrophy in marine picocyanobacteria[END_REF], suggesting that these photosynthetic microorganisms may present mixotrophic metabolism. Prochlorococcus and Synechococcus have the genetic capacity to use not only organic molecules with key limiting chemical elements such as nitrogen (N) and phosphorus (P), but also molecules devoid of such elements, such as glucose [START_REF] Gao | The Cyanobacterial NAD Kinase Gene sll1415 Is Required for Photoheterotrophic Growth and Cellular Redox Homeostasis in Synechocystis sp Strain PCC 6803[END_REF][START_REF] Gomez-Baena | Glucose uptake and its effect on gene expression in Prochlorococcus[END_REF][START_REF] Muñoz-Marín | Prochlorococcus can use the Pro1404 transporter to take up glucose at nanomolar concentrations in the Atlantic Ocean[END_REF], 2017[START_REF] Yelton | Global genetic capacity for mixotrophy in marine picocyanobacteria[END_REF]. Unicellular marine cyanobacteria are thus potentially capable of degrading and assimilating a wide range of organic molecules, but only a handful of studies have measured assimilation rates of some selected organic molecules such as amino acids, adenosine-5'-triphosphate (ATP) or dimethylsulfoniopropionate [START_REF] Michelou | Light-stimulated bacterial production and amino acid assimilation by cyanobacteria and other microbes in the North Atlantic Ocean[END_REF], Mary et al., 2008a,b Duhamel et al., 2012, Ruiz-Gonzalez et al., 2012b[START_REF] Björkman | Differential assimilation of inorganic carbon and leucine by Prochlorococcus in the oligotrophic North Pacific Subtropical Gyre[END_REF][START_REF] Evans | Photoheterotrophy of bacterioplankton is ubiquitous in the surface oligotrophic ocean[END_REF]. Results suggest that marine picocyanobacteria may obtain nutrients mixotrophically via the uptake of N and/or P-containing organic molecules when facing nutrient limitation [START_REF] Yelton | Global genetic capacity for mixotrophy in marine picocyanobacteria[END_REF].

Direct evidences that marine picocyanobacteria use organic molecules to obtain energy or C are scarce, particularly under in situ conditions. To date, only one study by [START_REF] Muñoz-Marín | Prochlorococcus can use the Pro1404 transporter to take up glucose at nanomolar concentrations in the Atlantic Ocean[END_REF] demonstrated that Prochlorococcus in the Atlantic Ocean can take up organic C lacking other essential nutrients at nanomolar concentrations in the light (using radiolabeled glucose). A limited number of studies have attempted to quantify the relative contribution of mixotrophy compared to photoautotrophy in C assimilation by natural communities of marine cyanobacteria. First attempts to quantify the contribution of glucose uptake to total C assimilation in Prochlorococcus indicated that it may be very small (<1%; [START_REF] Muñoz-Marín | Prochlorococcus can use the Pro1404 transporter to take up glucose at nanomolar concentrations in the Atlantic Ocean[END_REF]. However, measurements of C assimilation from inorganic and organic substrates were done separately (in the same sampling area but on separate cruises and by different users) and only one taxon was considered, making comparison between fluxes and taxonomic groups difficult [START_REF] Paoli | Ecological advantages from light adaptation and heterotrophic-like behavior in Synechococcus harvested from the Gulf of Trieste (Northern Adriatic Sea)[END_REF][START_REF] Muñoz-Marín | Prochlorococcus can use the Pro1404 transporter to take up glucose at nanomolar concentrations in the Atlantic Ocean[END_REF][START_REF] Benavides | Dissolved organic matter uptake by Trichodesmium in the Southwest Pacific[END_REF]. Thus, it is still unclear if organic C assimilation by marine unicellular cyanobacteria is ubiquitous, and how it contributes to total C uptake to different groups of picocyanobacteria (e.g. high-light and low-light adapted Prochlorococcus and Synechococcus, [START_REF] Partensky | Prochlorococcus: advantages and limits of minimalism[END_REF] in comparison to CO 2 fixation.

Additional critical gaps in our understanding of picocyanobacteria mixotrophic metabolism concern regulating factors. In particular, we still don't know how organic C assimilation by marine unicellular cyanobacteria depends upon light availability and photosynthetic electron transport in natural settings [START_REF] Moore | More mixotrophy in the marine microbial mix[END_REF]. Recent findings showed that light enhances picocyanobacteria uptake rates of amino acids, ATP or dimethylsulfoniopropionate [START_REF] Michelou | Light-stimulated bacterial production and amino acid assimilation by cyanobacteria and other microbes in the North Atlantic Ocean[END_REF], Mary et al., 2008a[START_REF] Duhamel | Light dependence of phosphorus uptake by microorganisms in the North and South Pacific subtropical gyres[END_REF], Ruiz-Gonzalez et al., 2012b[START_REF] Björkman | Differential assimilation of inorganic carbon and leucine by Prochlorococcus in the oligotrophic North Pacific Subtropical Gyre[END_REF][START_REF] Evans | Photoheterotrophy of bacterioplankton is ubiquitous in the surface oligotrophic ocean[END_REF].

Interestingly, organic nutrient assimilation by the low nucleic acid bacterioplankton (LNA), which has been shown to be numerically dominated by the photohererotrophs SAR11 [START_REF] Mary | SAR11 dominance among metabolically active low nucleic acid bacterioplankton in surface waters along an Atlantic meridional transect[END_REF][START_REF] Hill | Differential responses of Prochlorococcus and SAR11-dominated bacterioplankton groups to atmospheric dust inputs in the tropical Northeast Atlantic Ocean[END_REF], Gómez-Pereira et al., 2013, Zubkov et al., 2015), was enhanced to a comparable extent to that by Prochlorococcus (Gómez-Pereira et al., 2013). However, the mechanisms of this light enhancement are not well understood. Recently, [START_REF] Muñoz-Marín | Glucose Uptake in Prochlorococcus: Diversity of Kinetics and Effects on the Metabolism[END_REF] explored the potential role of photosynthetic electron transport in the regulation of glucose uptake by Prochlorococcus SS120 in laboratory cultures. Using different photosynthesis inhibitors, they showed that glucose uptake was significantly reduced or even inhibited. This remains untested with natural populations.

To improve our understanding of marine unicellular cyanobacteria utilization of organic molecules and answer pressing questions about their mixotrophic metabolism in the wild, we conducted a set of experiments in the WTSP during the OUTPACE cruise (Oligotrophy to UlTra-oligotrophy PACific Experiment). Based on the ubiquity of picocyanobacterial organic compound uptake genes [START_REF] Yelton | Global genetic capacity for mixotrophy in marine picocyanobacteria[END_REF], but low glucose uptake rates by Prochlorococcus in the Atlantic Ocean [START_REF] Muñoz-Marín | Prochlorococcus can use the Pro1404 transporter to take up glucose at nanomolar concentrations in the Atlantic Ocean[END_REF], we hypothesize that mixotrophy by Prochlorococcus and Synechococcus is an adaptation to nutrient limitation rather than a facultative pathway for carbon acquisition. To test this hypothesis, we measured group-specific assimilation rates of organic molecules containing C only, C and N, or C, N and P (glucose, leucine, and ATP, respectively) in Prochlorococcus and Synechococcus. We then compared C assimilation from glucose and sodium bicarbonate to test how much C is assimilated via autotrophic or mixotrophic pathways. We also compared the light enhancement of leucine, ATP, and for the first time, glucose, by natural Prochlorococcus, Synechococcus and LNA, and tested the effect of the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU).

We hypothesize that as for organic nutrient assimilation, organic C uptake is enhanced in the light, and that it is partially tied to photosynthetic electron transport.

Results

Description of the study area

Three incubation experiments were carried out in the WTSP at the long duration (LD, 72 h) stations A, B and C (thereafter LDA, LDB, LDC, respectively) chosen for their contrasted biogeochemical conditions (Table 1, [START_REF] Moutin | Preface: The Oligotrophy to the UlTra-oligotrophy PACific Experiment (OUTPACE cruise[END_REF]. At LDA and LDB, seawater was sampled within the well-lit top mixed layer (ML at 54% PAR, 7 and 9 m depths, respectively) and at the deep chlorophyll maximum (DCM, 0.3% PAR, 70 and 90 m, respectively), while LDC was sampled at 60 m depth (10% PAR), to compare results between microbial communities adapted to different light levels and nutrient conditions.

The ML was 14, 21, and 34 m deep, at LDA, LDB, and LDC, respectively [START_REF] Moutin | Nutrient availability and the ultimate control of the biological carbon pump in the Western Tropical South Pacific Ocean[END_REF].

Station LDA was relatively oligotrophic (Table 1), with moderately high chlorophyll a concentrations compared to typical open ocean regional values (0.36±0.05 μg l -1 , [START_REF] Benavides | Dissolved organic matter uptake by Trichodesmium in the Southwest Pacific[END_REF]. Station LDB was sampled in an elevated chlorophyll a patch, about twofold the concentration at LDA in the ML (0.83±0.07 μg l -1 , [START_REF] Benavides | Dissolved organic matter uptake by Trichodesmium in the Southwest Pacific[END_REF]de Verneuil et al., 2018). Compared to LDA, LDB exhibited similar low nutrient concentrations in the ML but depleted inorganic nutrients at the DCM (Table 1).

Prochlorococcus, Synechococcus and LNA abundances were 2.3, 1.4 and 3.6-fold higher in the ML of LDB than at LDA; and respectively were 2.3-6.1, 13.1-31.8 and 1.5-4.5 fold higher in the ML than at the DCM (Table 1). LDC presented characteristics of the oligotrophic south Pacific gyre (lower surface chlorophyll concentrations and deeper DCM, [START_REF] Claustre | Introduction to the special section biooptical and biogeochemical conditions in the South East Pacific in late 2004: the BIOSOPE program[END_REF][START_REF] Moutin | Preface: The Oligotrophy to the UlTra-oligotrophy PACific Experiment (OUTPACE cruise[END_REF]). Glucose concentrations were on average 2.6 to 4.3-fold higher at LDA than at LDB and LDC. Leucine and ATP concentrations were on average 1.4 to 4.5-fold higher at LDA than at LDB and LDC (Table 1).

Assimilation rates of organic molecules in light incubations

The assimilation of radiolabeled organic molecules with C alone, or C with N or P (glucose, leucine and ATP, respectively) was measured in separate incubations. Flow sorting of radiolabeled cells after incubation in the light showed that Prochlorococcus, Synechococcus and LNA assimilated glucose, leucine and ATP at all sampled stations and depths (Fig. 1). Group-specific assimilation rates of glucose followed trophic gradients and were generally higher at LDB>LDA>LDC, and higher in the ML than at the DCM (Fig. 1 a,b). Cell specific rates of glucose assimilation by LNA were 4.8 and 1.7-fold higher than Prochlorococcus in the ML at LDA and LDB, respectively, and 2.3fold higher at LDA-DCM, but Prochlorococcus presented higher cell specific rates than LNA at LDB-DCM and LDC-60m (4.1 and 1.3 times, respectively, Fig. 1a).

Synechococcus presented 1.4±0.2-fold higher cell specific assimilation rates than Prochlorococcus at LDB (p<0.05) and LDC, but about half the rate by Prochlorococcus at LDA, although differences were not significant at LDA and LDC. Because Synechococcus was much less abundant than Prochlorococcus and LNA (on average Prochlorococcus, Synechococcus and LNA represented 46±5, 2±2 and 52±7% of the cumulated abundance (Prochlorococcus+Synechococcus+LNA), respectively; Table 1), the contribution of the Synechococcus group to glucose assimilation rates was at least an order of magnitude lower than that of Prochlorococcus and LNA groups (0.90±1.12, 0.08±0.11 and 1.95±2.52 pmol Glc l -1 h -1 on average, for Prochlorococcus, Synechococcus and LNA groups, respectively, Fig. 1b). Consequently, Synechococcus accounted for < 3% of the total glucose assimilation by the three combined sorted groups (Prochlorococcus+Synechococcus+LNA), while LNA accounted for > 65% at LDA and LDB-ML, and Prochlorococcus contributed to 70 and 54% at LDB-DCM and LDC.

Thus, Prochlorococcus can be a large contributor to glucose assimilation rates in comparison to SAR11-like photoheterotrophic groups. But compared to the total microbial community glucose assimilation (i.e. bulk rates), Prochlorococcus accounted for only 4.9±3.3%, similar to results obtained in the Atlantic Ocean [START_REF] Muñoz-Marín | Prochlorococcus can use the Pro1404 transporter to take up glucose at nanomolar concentrations in the Atlantic Ocean[END_REF].

Group-specific assimilation rates of leucine and ATP were also higher at LDB and LDA than at LDC, and were higher in the ML than at the DCM (p<0.05, Fig. 1). On average, Prochlorococcus and LNA groups accounted for 50±23 and 21±7% of leucine assimilation by the total microbial community, respectively; and 17±12 and 40±17% of ATP assimilation by the total microbial community, respectively. The Synechococcus group contributed to < 1% of the leucine and ATP assimilation by the total microbial community. Cell specific leucine and ATP assimilation rates by Synechococcus were roughly an order of magnitude lower than by Prochlorococcus, except for ATP at LDB and LDC, where assimilation rates by Synechococcus were similar to those by Prochlorococcus.

Light-enhanced uptake of organic molecules and relationship to photosynthesis

Flow sorting of labeled cells incubated in light or dark bottles showed that light enhanced the cell specific uptake of all radiolabeled organic molecules tested here, including glucose, and in most cases to a larger extent in Prochlorococcus and Synechococcus as compared to LNA (Fig. 2). On average, incubations in the light represented an enhancement of 44±18, 57±30 and 35±11% of dark glucose uptake, 73±23, 57±30 and 35±11% of dark leucine uptake and 56±25, 35±24 and 43±30% of dark ATP uptake by Prochlorococcus, Synechococcus and LNA, respectively. In comparison, incubations in the light represented an enhancement of 92±11 and 99±2% of dark 14 C-sodium bicarbonate uptake by Prochlorococcus and Synechococcus, respectively; where dark uptake was negligible (Fig. 2g). Interestingly, the light enhancement of organic molecule uptake rates was higher at the DCM than in the ML for Prochlorococcus and Synechococcus (light to dark ratios of glucose, leucine and ATP uptake were 1.3-2.1 (ML) vs. 2.2-5.5 (DCM), 1.1-4.1 (ML) vs. 2.2-11.4 (DCM), and 1.9-10.5 (ML) vs. 1.5-1.7 (DCM), respectively; Fig. 3,4,5). In comparison, light enhancement of organic molecule uptake by LNA was mostly similar between ML and DCM. In SYBR stained samples, high-DNA-content bacteria (HNA) could be properly distinguished from Prochlorococcus only in samples from the DCM and differences between light and dark incubations were either not significant or higher in the dark (t-test, p<0.05, Fig. 2 b,d,f), and thus this group is not further discussed here. However, these results indicate that the light enhancement of organic molecule assimilation by Prochlorococcus could not be a result of by-sorting HNA bacteria overlapping in size with Prochlorococcus.

Additions of the photosystem II inhibitor DCMU resulted in reduced

Prochlorococcus, Synechococcus and LNA glucose uptake to a level not statistically different from rates in the dark, except for Prochlorococcus and LNA at LDB-ML where uptake was higher in DCMU compared to the dark (Fig. 3). Cell specific leucine uptake was lower when DCMU was added, significantly for Prochlorococcus and LNA at all stations/depths but only in the ML for Synechococcus (Fig. 4). For Prochlorococcus, DCMU resulted in higher or equal rates than in the dark while for LNA, DCMU resulted in lower rates than in the dark. In most cases, DCMU resulted in decreased cell specific ATP uptake for the three groups compared to light samples, but differences between dark and DCMU were not significant (except for Synechococcus at LDC, Fig. 5).

Bacterial production

Because bacterial production is commonly measured using leucine or thymidine assays, the effect of light and DCMU on the incorporation rates of leucine and thymidine into trichloroacetic acid (TCA) insoluble material (Leu inc and Tdr inc , respectively) was estimated in separate incubations. Light affected Leu inc to a larger extent than Tdr inc (Fig. 6a,b,e). Leu inc and Tdr inc rates were 12-57% (40±21%) and 2-27 % (11±14%) lower in the dark than in the light, respectively (Fig 6e). The addition of DCMU resulted in an average decrease of 68±10% and 49±23% in Leu inc and Tdr inc rates in the light, respectively (Fig. 6a,b). The Leu inc to Tdr inc ratio was on average 1.6±0.5 times higher in the light than in the dark, 1.5±0.3 times higher in the light than with DCMU (Fig. 6c).

Discussion

Characterization of Prochlorococcus and Synechococcus mixotrophic metabolism.

Owing to their capability to utilize sunlight and atmospheric CO 2 for growth, Prochlorococcus and Synechococcus are commonly considered photoautotrophs. Yet, recent evidence has shown that the uptake of organic N-(leucine, amino acids) and P-(ATP) molecules by Prochlorococcus and Synechococcus is enhanced in the light [START_REF] Michelou | Light-stimulated bacterial production and amino acid assimilation by cyanobacteria and other microbes in the North Atlantic Ocean[END_REF], Mary et al., 2008b[START_REF] Duhamel | Light dependence of phosphorus uptake by microorganisms in the North and South Pacific subtropical gyres[END_REF][START_REF] Gomez-Pereira | Comparable light stimulation of organic nutrient uptake by SAR11 and Prochlorococcus in the North Atlantic subtropical gyre[END_REF]. Therefore previous studies (e.g. [START_REF] Björkman | Differential assimilation of inorganic carbon and leucine by Prochlorococcus in the oligotrophic North Pacific Subtropical Gyre[END_REF], Gomez-Pereira et al., 2012[START_REF] Michelou | Light-stimulated bacterial production and amino acid assimilation by cyanobacteria and other microbes in the North Atlantic Ocean[END_REF][START_REF] Moore | More mixotrophy in the marine microbial mix[END_REF][START_REF] Muñoz-Marín | Prochlorococcus can use the Pro1404 transporter to take up glucose at nanomolar concentrations in the Atlantic Ocean[END_REF], 2017[START_REF] Zubkov | Photoheterotrophy in marine prokaryotes[END_REF][START_REF] Zubkov | High rate of uptake of organic nitrogen compounds by Prochlorococcus cyanobacteria as a key to their dominance in oligotrophic oceanic waters[END_REF] since the early work of [START_REF] Rippka | Photoheterotrophy and chemoheterotrophy among unicellular bluegreen algae[END_REF] have commonly defined this nutritional plasticity in marine cyanobacteria as photoheterotrophy, although sensus stricto this term defines organisms that use light for energy, but cannot use CO 2 as their sole C source. However, these studies demonstrating light-enhancement of N or Pcontaining organic molecules uptake did not directly verify if marine cyanobacteria could also use organic molecules containing only C (e.g. glucose) and if light also enhances organic C assimilation. To the best of our knowledge, only [START_REF] Muñoz-Marín | Prochlorococcus can use the Pro1404 transporter to take up glucose at nanomolar concentrations in the Atlantic Ocean[END_REF] demonstrated that Prochlorococcus could assimilate glucose, a molecule devoid of heteroatoms (N or P), in natural seawater. Our results geographically expand these findings from the Atlantic Ocean and demonstrate that not only Prochlorococcus but also Synechococcus assimilate glucose in biogeochemically distinct marine environments of the WTSP. Interestingly, cell-and group-specific assimilation rates of glucose appeared to follow trophic gradients, similar to organic C uptake by Trichodesmium [START_REF] Benavides | Dissolved organic matter uptake by Trichodesmium in the Southwest Pacific[END_REF]. Further, we demonstrate that light enhanced cell specific glucose uptake by nearly 50% for Prochlorococcus and Synechococcus, suggesting that variability in light availability (e.g. changes in light intensity due to euphotic layer PAR gradient, diel sunlight rhythm, cloud coverage) could largely impact organic C assimilation by these cyanobacteria. Similar results were also found in cultures of the low-light Prochlorococcus SS120 strain in which 24h incubation in the dark induced a 40% decrease in glucose uptake [START_REF] Gomez-Baena | Glucose uptake and its effect on gene expression in Prochlorococcus[END_REF]. Thus, marine cyanobacteria may be a significant competitor of heterotrophic bacteria for this labile molecule, especially during the day, potentially creating temporal patterns between strictly heterotrophic and mixotrophic bacterioplankton. Expanding this finding to all labile C molecules, such temporal patterns could affect dissolved organic matter (DOM) remineralization and C sequestration via the microbial C pump.

We also explored the participation of photosynthetic electron transport in the regulation of organic molecules assimilation using the photosynthesis inhibitor DCMU [START_REF] Rippka | Photoheterotrophy and chemoheterotrophy among unicellular bluegreen algae[END_REF][START_REF] Stanier | Autotrophy and heterotrophy in unicellular blue-green algae[END_REF][START_REF] Neilson | The uptake and utilization of organic carbon by algae: an essay in comparative biochemistry[END_REF][START_REF] Paerl | Ecophysiological and trophic implications of light-stimulated amino acid utilization in marine picoplankton[END_REF][START_REF] Moore | More mixotrophy in the marine microbial mix[END_REF]. In chlorophyll and bacteriochlorophyll containing organisms, DCMU blocks electron flow between photosystem II and plastoquinone, resulting in no O 2 and NADPH production, but allowing ATP synthesis through cyclic electron flow around photosystem I. Our results show that DCMU completely inhibited CO 2 fixation by Prochlorococcus and Synechococcus but only partially inhibited their assimilation of glucose, leucine and ATP. For most samples and molecules examined, the assimilation rates were significantly higher or not statistically different in the light with DCMU compared to the dark, suggesting that organic molecules incorporation is partially tied to photosynthetic production of energy in the light. The light harvested by Prochlorococcus and Synechococcus photosynthetic apparatus may thus transfer energy into ATP that can be used in the active transport of organic molecules. Similarly, [START_REF] Muñoz-Marín | Glucose Uptake in Prochlorococcus: Diversity of Kinetics and Effects on the Metabolism[END_REF] found different inhibitory effects on Prochlorococcus SS120 glucose uptake using the quinone analogue inhibitor of cytochrome b6f complex in photosystem I -DBMIB (~100%), or the inhibitor of photosystem II -DCMU (~50%), and argued that the ATP generated by photosystem I could maintain up to 50% of the glucose uptake. Seawater used in our experiments was sampled in the early morning and thus cells were likely light energy depleted, assuring that continuing uptake in our dark incubations was not a result of stored energy during the light phase. Therefore, our results demonstrate that natural populations of Prochlorococcus and Synechococcus are nutritionally and metabolically flexible. We confirm that in situ, picocyanobacteria can assimilate selected organic molecules including glucose, a molecule devoid of heteroatoms, and show that assimilation rates are reduced but continue even in the dark or when photosynthesis is impaired. This mixotrophic metabolism could explain the recent findings of the substantial presence of Prochlorococcus in the aphotic ocean [START_REF] Jiao | Presence of Prochlorococcus in the aphotic waters of the western Pacific Ocean[END_REF] and the survival of specific strains of Prochlorococcus in extended darkness [START_REF] Coe | Survival of Prochlorococcus in extended darkness[END_REF].

Relative importance of mixotrophy for picocyanobacterial C assimilation.

Cell-specific glucose uptake in natural Prochlorococcus was on average 0.00021±0.00011 fg C cell -1 h -1 , similar to results by [START_REF] Muñoz-Marín | Prochlorococcus can use the Pro1404 transporter to take up glucose at nanomolar concentrations in the Atlantic Ocean[END_REF] in the Atlantic Ocean (0.00010±0.00008 fg C cell -1 h -1 , where added and ambient glucose concentrations were comparable to those in our study). Synechococcus had higher cell specific glucose assimilation rates than Prochlorococcus, but using an average biovolume of 0.17 and 0.33 µm 3 for Prochlorococcus and Synechococcus, respectively [START_REF] Grob | Contribution of picoplankton to the total particulate organic carbon concentration in the eastern South Pacific[END_REF], we calculated that glucose uptake by Prochlorococcus and Synechococcus was similar (0.0013±0.0007 and 0.0009±0.0005 fg C µm -3 h -1 , respectively). Compared to carbon uptake from 14 C-sodium bicarbonate (4.4±1.7 and 44±36 fg C cell -1 d -1 for Prochlorococcus and Synechococcus, respectively), glucose uptake represented a small fraction (<1%) of total (inorganic + organic) C uptake, similar to the values calculated using results in [START_REF] Muñoz-Marín | Prochlorococcus can use the Pro1404 transporter to take up glucose at nanomolar concentrations in the Atlantic Ocean[END_REF]. This implies that mixotrophy may represent a marginal fraction of Prochlorococcus and Synechococcus C uptake in the Atlantic and Pacific Oceans, and confirms culture-based studies where Prochlorococcus actively takes up glucose when available, but remains primarily autotrophic [START_REF] Muñoz-Marín | Glucose Uptake in Prochlorococcus: Diversity of Kinetics and Effects on the Metabolism[END_REF]). Yet considering that glucose is only one of the greatly diverse dissolved organic C molecules present in the ocean [START_REF] Moran | Deciphering ocean carbon in a changing world[END_REF], the low assimilation rates may be the result of glucose uptake competition with other sugars. Thus, other organic C compounds need to be tested as substrates for mixotrophic growth by marine cyanobacteria.

However, mixotrophy by marine picocyanobacteria may represent a greater advantage in response to inorganic nutrient limitation than to access an alternative form of C. Indeed, we found that cell specific uptake of leucine and ATP by Prochlorococcus were on average 35.5±16.5 and 3.4±2.3 times greater than cell specific uptake rates of glucose (mol:mol). Still, [START_REF] Muñoz-Marín | Prochlorococcus can use the Pro1404 transporter to take up glucose at nanomolar concentrations in the Atlantic Ocean[END_REF] argued that the bioenergetic advantage of glucose uptake vs. glucose synthesis de novo may save Prochlorococcus significant energy for other metabolic uses.

Photoheterotrophy by LNA bacteria.

The LNA group has been previously characterized using molecular tools and results consistently show that it is largely dominated by SAR11 [START_REF] Mary | SAR11 dominance among metabolically active low nucleic acid bacterioplankton in surface waters along an Atlantic meridional transect[END_REF], 2008b[START_REF] Gomez-Pereira | Comparable light stimulation of organic nutrient uptake by SAR11 and Prochlorococcus in the North Atlantic subtropical gyre[END_REF][START_REF] Morán | More, smaller bacteria in response to ocean's warming[END_REF], a highly abundant group in the subtropical Pacific [START_REF] West | Distinct spatial patterns of SAR11, SAR86, and Actinobacteria diversity along a transect in the ultra-oligotrophic south Pacific Ocean[END_REF]. SAR11 is a clade of proteorhodopsin-based photoheterotrophic bacteria, which are characterized by light-controlled growth and proteorhodopsin expression [START_REF] Lami | Light-dependent growth and proteorhodopsin expression by Flavobacteria and SAR11 in experiments with Delaware coastal waters[END_REF]. Like in other studies (Mary et al., 2008b[START_REF] Zubkov | Photoheterotrophy in marine prokaryotes[END_REF][START_REF] Gomez-Pereira | Comparable light stimulation of organic nutrient uptake by SAR11 and Prochlorococcus in the North Atlantic subtropical gyre[END_REF][START_REF] Evans | Photoheterotrophy of bacterioplankton is ubiquitous in the surface oligotrophic ocean[END_REF] we showed that light enhances the assimilation of leucine and ATP by the LNA group. In our experiments, Tdr inc was an excellent proxy of strict heterotrophic bacterial production as the Tdr inc rates were similar in light and dark incubations, unlike Leu inc rates (Fig. 6). This confirms that increased Leu inc rates in the light were mostly due to photoheterotrophy and mixotrophic capacities of Prochlorococcus, but not to an indirect effect related to enhanced phytoplankton excretion/exudation in the light. Light-enhanced cell-specific glucose uptake by LNA bacteria was large (~35%), albeit lower than for Prochlorococcus and Synechococcus, implying that photons can supply a significant part of the energy demand during daytime and the requirement for organic molecules as energy sources is significantly decreased. Surprisingly, the addition of DCMU reduced organic molecules incorporation by LNA. DCMU has been widely used to study phytoplankton metabolism [START_REF] Jeanjean | The effect of metabolic poisons on ATP level and on active phosphate uptake in Chlorella pyrenoidosa[END_REF][START_REF] Lewis | Relationships between vertical mixing and photoadaptation of phytoplankton: similarity criteria[END_REF][START_REF] Garrigue | The use of photosynthesis inhibitor (DCMU) for in situ metabolic and primary production studies on soft bottom benthos[END_REF][START_REF] Ikeya | Study on phosphate uptake of the marine cyanophyte Synechococcus sp. NIBB 1071 in relation to oligotrophic environments in the open ocean[END_REF][START_REF] Delorenzo | Use of metabolic inhibitors to characterize ecological interactions in an estuarine microbial food web[END_REF][START_REF] Laurent | The influence of photosynthesis on host intracellular pH in scleractinian corals[END_REF][START_REF] Halsey | Contrasting strategies of photosynthetic energy utilization drive lifestyle strategies in ecologically important picoeukaryotes[END_REF], including photoheterotrophy [START_REF] Estep | Stable hydrogen isotope fractionations during autotrophic and mixotrophic growth of microalgae[END_REF][START_REF] Paerl | Ecophysiological and trophic implications of light-stimulated amino acid utilization in marine picoplankton[END_REF][START_REF] Johnson | Interaction between starch breakdown, acetate assimilation, and photosynthetic cyclic electron flow in Chlamydomonas reinhardtii[END_REF][START_REF] Knoop | Flux balance analysis of cyanobacterial metabolism: the metabolic network of Synechocystis sp. PCC 6803[END_REF][START_REF] You | Photoheterotrophic fluxome in Synechocystis sp. strain PCC 6803 and its implications for cyanobacterial bioenergetics[END_REF][START_REF] Oren | Expression and functioning of retinal-based proton pumps in a saltern crystallizer brine[END_REF][START_REF] Muñoz-Marín | Glucose Uptake in Prochlorococcus: Diversity of Kinetics and Effects on the Metabolism[END_REF]. These studies indicate that DMCU is not lethal, that its inhibitory effect is reversible and that it does not affect heterotrophic processes, even in autotroph-heterotroph symbiotic associations [START_REF] Vandermeulen | The effect of inhibitors of photosynthesis on zooxanthellae in corals and other marine invertebrates[END_REF][START_REF] Mühlbauer | Light-dependent formation of the photosynthetic proton gradient regulates translation elongation in chloroplasts[END_REF][START_REF] Francoeur | Evaluation of the efficacy of the photosystem II inhibitor DCMU in periphyton and its effects on nontarget microorganisms and extracellular enzymatic reactions[END_REF]. Combined with good efficiency against algal photosynthesis (Fig. 6d), and because it was also chosen to study photoheterotrophic metabolism in culture isolates of Prochlorococcus [START_REF] Muñoz-Marín | Glucose Uptake in Prochlorococcus: Diversity of Kinetics and Effects on the Metabolism[END_REF], we used DCMU as selective inhibitor of the photosystem II in cyanobacteria. However, we observed a reduction in organic molecules incorporation in LNA which suggests that DCMU affected LNA directly or indirectly. A direct effect could be either toxicity on heterotrophic metabolism of strict heterotrophs or an action on the light-driven proton pump, proteorhodopsin. Yet, at the end of incubation, LNA cell abundances were similar between treatments, indicating that DCMU may not be lethal to LNA. Moreover, DCMU did not inhibit the light-driven proton pump of the cyanobacteria Gloeobacter violaceus which has two types of lightdriven proton pumps, chlorophyll-based photosystems and rhodopsin [START_REF] Choi | Cyanobacterial Light-Driven Proton Pump, Gloeobacter Rhodopsin: Complementarity between Rhodopsin-Based Energy Production and Photosynthesis[END_REF].

Alternatively, the reduction of activity by LNA in DCMU samples may be indirect, resulting from the inhibition of photosynthate production by phytoplankton (photosynthesis was inhibited, Fig. 6d) on which bacteria rely greatly as a source of labile organic substrates for growth [START_REF] Church | Light dependence of [ 3 H]Leucine incorporation in the oligotrophic North Pacific Ocean[END_REF], Ruiz-Gonzalez et al., 2012a[START_REF] Moore | More mixotrophy in the marine microbial mix[END_REF].

Implication of picocyanobacterial uptake of leucine for bacterial production measurements in the ocean.

Despite being important for our understanding of biological productivity in the ocean, light enhanced bacterial production and uptake of leucine by the most abundant marine microbes, Prochlorococcus, Synechococcus and LNA, have been the subject of a limited number of studies [START_REF] Church | Light dependence of [ 3 H]Leucine incorporation in the oligotrophic North Pacific Ocean[END_REF][START_REF] Michelou | Light-stimulated bacterial production and amino acid assimilation by cyanobacteria and other microbes in the North Atlantic Ocean[END_REF], Mary et al., 2008b[START_REF] Ruiz-Gonzalez | Away from darkness: a review on the effects of solar radiation on heterotrophic bacterioplankton activity[END_REF][START_REF] Björkman | Differential assimilation of inorganic carbon and leucine by Prochlorococcus in the oligotrophic North Pacific Subtropical Gyre[END_REF]. In incubations with saturating concentrations of leucine (20 nM), previous reports found that Prochlorococcus contributes significantly to bacterial production estimates in the North Pacific [START_REF] Björkman | Differential assimilation of inorganic carbon and leucine by Prochlorococcus in the oligotrophic North Pacific Subtropical Gyre[END_REF], North Atlantic [START_REF] Michelou | Light-stimulated bacterial production and amino acid assimilation by cyanobacteria and other microbes in the North Atlantic Ocean[END_REF] and Mediterranean Sea (Talarmin et al., 2011a), both in dark or in the light. Here we show that light enhanced bulk Leu inc rates (incorporation rates of leucine into TCA insoluble material), as well as cell-specific leucine uptake by Prochlorococcus, Synechococcus and LNA in the tropical southwest Pacific Ocean. However, light significantly enhanced bulk Tdr inc rates to a much lesser extent (dark to light ratio was 73-100 %, mean 89%). This may be due to thymidine being preferentially used by heterotrophic bacteria than leucine which can be used by mixotrophic phytoplankton [START_REF] Michelou | Light-stimulated bacterial production and amino acid assimilation by cyanobacteria and other microbes in the North Atlantic Ocean[END_REF][START_REF] Björkman | Differential assimilation of inorganic carbon and leucine by Prochlorococcus in the oligotrophic North Pacific Subtropical Gyre[END_REF]. Indeed, in our samples, over a third of the total leucine uptake was attributable to the Prochlorococcus group. Unfortunately, due to low thymidine specific activity, we were not able to measure its group-specific uptake. This should be verified in future studies as cyanobacteria tested so far do not incorporate Tdr in culture, which was related to a probable lack of thymidine kinase [START_REF] Pollard | Validity of the tritiated thymidine method for estimating bacterial growth rates: measurement of isotope dilution during DNA synthesis[END_REF]. The contribution of picocyanobacteria to bacterial production estimates, particularly using 3 H-leucine, should thus be considered when measuring bacterial production in marine environments, even in dark incubations (Talarmin et al., 2011a[START_REF] Björkman | Differential assimilation of inorganic carbon and leucine by Prochlorococcus in the oligotrophic North Pacific Subtropical Gyre[END_REF]. [START_REF] Longnecker | Variation in cell-specific rates of leucine and thymidine incorporation by marine bacteria with high and with low nucleic acid content off the Oregon coast[END_REF] found higher Leu inc /Tdr inc ratio in dark incubated HNA than in the LNA group, and also higher in surface samples than below, particularly in the open-sea station. Besides the general hypothesis of higher rates of protein synthesis relative to DNA synthesis in larger cells, this result could also be partially due to the difficulty to separate Prochlorococcus from HNA during cell sorting after SYBR green DNA staining, particularly in sub-surface waters. As seen from seawater cultures, the Leu inc /Tdr inc ratio can be representative of decoupling between cell division and biomass production (Chin-Leo & Kirchman 1990).

In situ, Leu inc rates has been shown also to vary more than Tdr inc rates along diel cycles [START_REF] Riemann | Advances in estimating bacterial production and growth in aquatic systems[END_REF]). Consequently, the use of Leu inc /Tdr inc ratio as a proxy of unbalanced growth should be misleading when samples are incubated in the light as we demonstrated that it was also affected by photoheterotrophic processes (higher in the light than in the dark or with DCMU).

Conclusion

We present several lines of evidence that natural Prochlorococcus and Synechococcus can assimilate organic molecules with variable C:N:P composition, as well as organic molecules devoid of heteroatoms (i.e. glucose). Prochlorococcus and Synechococcus assimilated organic molecules in the light but also in the dark or when photosynthesis was altered by DCMU, albeit at significantly reduced rates, verifying previous findings in culture indicating that cyanobacteria are nutritionally versatile. Yet Prochlorococcus and Synechococcus C uptake from glucose was small compared to CO 2 uptake, indicating that they obtain carbon primarily through an autotrophic metabolism.

Nevertheless, mixotrophy by these unicellular cyanobacteria was widespread in biogeochemically distinct regions of the WTSP Ocean and cell and group-specific assimilation rates were generally higher in surface than at the DCM. However, cellspecific assimilation rates of the N-and P-containing molecules (leucine and ATP) were significantly higher than that of glucose. Thus, mixotrophy by marine cyanobacteria is more likely to be an adaptation to low inorganic nutrient availability. Many details of marine cyanobacteria mixotrophic metabolism remain to be elucidated. In particular, additional experiments will be necessary to evaluate the global importance of organic vs. inorganic C uptake by marine cyanobacteria and assess the diel variability in these processes, in order to improve C fluxes models [START_REF] Gasol | Towards a better understanding of microbial carbon flux in the sea[END_REF][START_REF] Zubkov | Photoheterotrophy in marine prokaryotes[END_REF].

Further study of mixotrophic metabolism is likely to contribute not only to our understanding of microbial adaptations to light and nutrient availability but also to our knowledge of marine DOM cycling, and the role of light in modulating bacteria and cyanobacteria biogeochemical functions in the ocean.

Experimental Procedures

Field sampling

This study was conducted in the WTSP along trophic gradients during the OUTPACE cruise (DOI: http://dx.doi.org/10.17600/15000900, RV L'Atalante, February-April 2015) between New Caledonia and Tahiti [START_REF] Moutin | Preface: The Oligotrophy to the UlTra-oligotrophy PACific Experiment (OUTPACE cruise[END_REF]. Three incubation experiments were carried out at the long duration stations LDA, LDB, and LDC, selected for their contrasted biogeochemical conditions (Table 1). At LDA and LDB, seawater was sampled within the well-lit top mixed layer (ML at 54% PAR, 7 and 9 m depths, respectively) and at the DCM (0.3% PAR, 70 and 90 m, respectively). LDC was sampled at 60 m depth (10% PAR).

Bacterioplankton enumeration

Bacterioplankton groups were enumerated from untreated samples using a BD Influx flow cytometer (BD Biosciences, San Jose, CA, USA). Prochlorococcus and Synechococcus were enumerated in unstained samples while the low-DNA-content (LNA) and high-DNA-content (HNA) bacteria groups were discriminated in a sample aliquot stained with SYBR Green I DNA dye (0.01% final), following published protocols [START_REF] Gasol | Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria[END_REF][START_REF] Duhamel | Microbial response to enhanced phosphorus cycling in the North Pacific Subtropical Gyre[END_REF]. Using a forward scatter detector with small particle option and focusing a 488 plus a 457 nm (200 and 300 mW solid state, respectively) laser into the same pinhole greatly improved the resolution of dim surface Prochlorococcus population from background noise in unstained samples. However, in stained samples from the ML, Prochlorococcus overlapped with HNA bacteria and HNA abundances were calculated by subtracting Prochlorococcus enumerated from unstained samples. Calibration and alignment were done using 1-μm yellow-green microspheres (Polysciences, USA).

Incubation experiments

Seawater collected at the LD stations was distributed into acid-washed and sample rinsed transparent polycarbonate bottles for separate incubations with different radioactive-labeled molecules and under different treatment conditions. For each radioactive molecule tested, a killed control was prepared by adding paraformaldehyde (0.5 % final w/v) for 30 min before adding the radioisotope. Bottles were incubated in on-deck blue-shielded incubators to mimic the amount of transmitted light at the corresponding sampled depth and cooled with surface seawater. Samples were treated with or without addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, at 20 µM final, for 15 min before adding the radioisotope). For dark incubations, the bottles were masked using multi-layers of matte black aluminum foil (Rosco Matte Black Cinefoil).

For each treatment, D-[6-3H(N)]-glucose (45.7 Ci mmol -1 ), L-[3,4,5-3H(N)]-Leucine (112 Ci mmol -1 ) and [2,5',8-3H]-Adenosine-5'-triphosphate (52 Ci mmol -1 ), were used in separate incubations to measure assimilation rates of organic C alone (glucose, Glc), and N-(leucine, Leu) and P-(Adenosine-5'-triphosphate, ATP) -enriched organic compounds, respectively (Perkin Elmer, Waltham, MA, USA). Isotope additions were kept as low as possible considering the specific activity and sensitivity of the cell sorting procedure described below: 3 H-glucose, 3 H-leucine and 3 H-ATP were added at a final concentration of 2, 7 and 1 nmol l -1 , respectively. Samples were incubated for 4 to 6 h and uptake linearity was checked before each experiment. Additional incubations were done using [ 14 C]-sodium bicarbonate (43.3 mCi mmol -1 ) at 3.3 µCi ml -1 final concentration from dawn to dusk (~8 h). At the end of incubation, samples were fixed with paraformaldehyde (0.5 % final, for 15-min in the dark), 20 µl were sampled to measure total activity (dpm l -1 , with β-phenylethylamine for 14 C samples) and determine the concentration of added molecules (S*, nmol l -1 ), 4-ml were filtered onto 0.2-µm polycarbonate membranes to monitor incorporation by the total microbial community (total activity -dpm l -1 , and total microbial assimilation rate -nmol l -1 h -1 ) and 30 to 50 ml was gently concentrated to 4 ml and preserved at -80°C for flow cytometry cell sorting. Radioactivity was measured using scintillation cocktail with low background and high 3 H counting efficiency (Ultima Gold LLT, Perkin Elmer) and a Packard Tri-Carb 3110 TR liquid scintillation counter with ultra-low-level option kit. The turnover time (h) was calculated by dividing the total activity (dpm l -1 ) by the activity on the 0.2-µm filter (dpm l -1 h -1 ). The total microbial assimilation rate (nmol l -1 h -1 ) was calculated by dividing the substrate concentration (ambient concentration (S a ) plus S*) by the turnover time (h). The ambient concentrations of glucose, leucine and ATP were estimated using a concentration series bioassay of untreated live samples as described by [START_REF] Wright | Use of glucose and acetate by bacteria and algae in aquatic ecosystems[END_REF] and modified by [START_REF] Zubkov | Amino acid uptake of Prochlorococcus spp. in surface waters across the South Atlantic Subtropical Front[END_REF], which represents an upper estimate of ambient concentrations. Thus, calculated assimilation rates represent an upper estimate of assimilation rates at in situ concentrations.

Flow cytometry cell sorting

Bacterioplankton groups were characterized as described above. Note that because of the overlap in Prochlorococcus with HNA in stained ML samples, we did not systematically sort the HNA population for which results were biased by the contribution of Prochlorococcus activity. The Influx flow cytometer was set at the highest sorting purity (1.0 drop single mode) and potential attached cells were discarded using a pulse width vs. forward scatter plot. The drop delay was calibrated using Accudrop Beads (BD Biosciences, USA) and sorting efficiency was verified manually by sorting a specified number of 1-μm yellow-green microspheres (Polysciences, USA) onto a glass slide and counting the beads under an epifluorescence microscope. Using 1.0 drop single mode we systematically recovered 100% of the targeted beads. Performance was validated by sorting Prochlorococcus and Synechococcus from natural samples and reanalysing the sorted cells flow cytometrically to confirm sort purity, which exceeded 96 %, a result similar to [START_REF] Baer | Stoichiometry of Prochlorococcus, Synechococcus, and small eukaryotic populations in the western North Atlantic Ocean[END_REF]. Increasing numbers of cells from the same incubation sample were sorted (20,000-600,000 LNA and 10,000-300,000 Prochlorococcus and Synechococcus). Sorted cells were assessed by liquid scintillation following Talarmin et al. (2011b). The 14 C-labeled samples were acidified with 0.5 ml of 1N HCl for 24 h to remove any unincorporated 14 C-sodium bicarbonate. For each group, at least three samples were sorted and regression analysis between the number of cells sorted and the radioactivity taken up by the sorted cells was used to calculate the per cell activity (dpm cell -1 ). Radioactivity in the killed control sorted samples (dpm cell -1 ) was deduced from radiaoactivity in the respective sorted samples. The cell-specific assimilation rate (nmol cell -1 h -1 ) was calculated by dividing the radioactivity per cell (dpm cell -1 ) by the total microbial activity (dpm l -1 ) measured in the same treatment, and then multiplied by the total microbial assimilation rate at ambient plus added organic substrate concentration (S a +S*, nmol l -1 h -1 ). Statistical tests (one-way ANOVA, Tukey's multiple comparisons test) were carried out to assess significant differences between treatments (P < 0.05) using Prism 6 (GraphPad software, La Jolla, CA, USA).

Bulk bacterial production

Samples were incubated with [methyl-3 H]-thymidine (Tdr, 20 nM, 48.8 Ci mmol - 1 ) or with L-[3,4,5-3 H(N)]-leucine (Leu, 6 nM, 112 Ci mmol -1 ) for 5 to 6 h. Killed controls and incubation terminations were fixed with formalin 1% final concentration.

Tdr samples were treated by the filtration technique [START_REF] Bell | Estimating production of heterotrophic bacterioplankton via incorporation of tritiated thymidine[END_REF], ice cold-trichloroacetic acid (TCA) extract) and Leu samples by the centrifuge technique [START_REF] Smith | A simple, economical method for measuring bacterial protein synthesis rates in seawater using 3 H-leucine[END_REF].

In both methods, an ethanol rinse was included. Note that bulk bacterial production experiments could not be done at LDA but were done at the short duration station 8 instead (hereafter SD8, in the ML, 12m). We occasionally checked that we were working at saturating concentration of Tdr and Leu by testing activities using a range of concentrations of Tdr varying from 5 to 60 nM and of Leu varying from 2 to 45 nM. We confirmed that the Tdr concentration was saturating at all stations and that Leu concentration was saturating at all stations, excepted at SD8 where measured rates were about half the maximum velocities. Latitude (Lat.), longitude (Long.), fraction of surface photosynthetically active radiation (PAR, %) received at the corresponding depth (m), temperature (T, C), Prochlorococcus (Pro), Synechococcus (Syn), and LNA cell abundances (10 3 cell ml -1 ), phosphate and nitrate concentrations (PO 4 3-and NO 3 -nmol l -1 ), and ambient concentrations of glucose (Glc), leucine (Leu) and ATP (nmol l -1 ).

Fig. 1 :

 1 Fig. 1: Cell specific (a, c, e, amol cell -1 h -1 ) and group specific (b, d, f, pmol l -1 h -1 ) assimilation rates of glucose (a, b), leucine (c, d) and ATP (e, f) by Prochlorococcus (Pro, black bars), Synechococcus (Syn, white bars) and LNA bacteria (LNA, grey bars) in incubations in the light. Error bars represent standard deviation on triplicate samples. * indicate non-measurable rates (killed control corrected rates ≤ killed control).

Fig. 2 :

 2 Fig.2: Scatter plots comparing cell specific uptake (10 -3 dpm cell -1 ) in the light (ordinate) and in the dark (abscissa) by picocyanobacteria (Pro: black filled circles, Syn: white filled circles; a, c, e, g) and bacteria (LNA: black filled squares, HNA: grey filled squares; b, d, f), for 3 H radiolabeled glucose ( 3 H-Glc, a, b), leucine ( 3 H-Leu, c, d) and ATP ( 3 H-ATP, e, f), and for 14 C radiolabeled sodium bicarbonate ( 14 C-PP, g). Error bars represent standard deviation on triplicate samples. The dotted lines represent the 1:1 ratio.

Fig. 4 :

 4 Fig. 4: Cell specific leucine assimilation rate (amol Leu cell -1 h -1 ) at LDA -ML (a), LDA -DCM (b), LDB -ML (c), LDB -DCM (d) and LDC -60m (e), in incubations in the light (white bars), in the dark (black bars), and in the light with DCMU (checker board pattern) for Prochlorococcus (Pro), Synechococcus (Syn) and LNA bacteria (LNA). Error bars represent standard deviation on triplicate samples. One-way ANOVA multiple treatment comparison results are represented by white or black circles when values are significantly (P < 0.05) different from the light or the dark treatments, respectively. * indicate non-measurable rates (killed control corrected rates ≤ killed control).

Fig. 5 :

 5 Fig. 5: Cell specific ATP assimilation rate (amol ATP cell -1 h -1 ) at LDA -ML (a), LDA -DCM (b), LDB -ML (c), LDB -DCM (d) and LDC -60m (e), in incubations in the light (white bars), in the dark (black bars), and in the light with DCMU (checker board pattern) for Prochlorococcus (Pro), Synechococcus (Syn) and LNA bacteria (LNA). Error bars represent standard deviation on triplicate samples. One-way ANOVA multiple treatment comparison results are represented by white or black circles when values are significantly (P<0.05) different from the light or the dark treatments, respectively. * indicate non-measurable rates (killed control corrected rates ≤ killed control).

Figure 6 .

 6 Figure6. Bacterial production rates measured using leucine (a, Leu inc , pmol Leu l -1 h -1 ) or thymidine (b, Tdr inc , pmol Tdr l -1 h -1 ) incorporation into TCA insoluble material; leucine to thymidine incorporation ratio (c, Leu inc to Tdr inc ratio); primary production rates (d, nmol C l -1 d -1 ) in incubations in the light (white bars), in the dark (black bars), and in the light with DCMU (white and grey checker board pattern); dark to light ratio (e, %) for Leu inc (grey bars, Leu) and Tdr inc (light grey bars, Tdr). Error bars represent standard deviation on triplicate samples (a) or absolute difference between duplicate samples (b, thymidine in the light and light with DCMU). One-way ANOVA multiple treatment comparison results are represented by white or black circles when values are significantly (P < 0.05) different from the light or the dark treatments, respectively.

Table and Figure Legends:Table 1 .

 and1 Ancillary data Characteristics of the seawater samples collected at stations LDA, LDB and LDC used in experiments.

Acknowledgments

We thank T. Moutin, chief scientist of the OUTPACE cruise, O.R. Anderson, A. Martiny and an anonymous reviewer for their insightful comments to previous versions of this manuscript. We are indebted to O. Grosso and S. Helias-Nunige for nutrient measurements and to G. Rougier and M. Picheral for their help in CTD rosette management and data processing. We are grateful to the crew of the R/V L'Atalante for outstanding shipboard operation. SD was funded by the National Science Foundation (OCE-1434916). MB was funded by the People Program (Marie Skłodowska-Curie Actions) of the European Union's Seventh Framework Program (FP7/2007-2013) under REA grant agreement number 625185. This is a contribution of the OUTPACE project (https://outpace.mio.univ-amu.fr/) funded by the French research national agency (ANR-14-CE01-0007-01), the LEFE-CyBER program (CNRS-INSU), the GOPS program

(IRD) and the CNES.


Djedjiga Benouioua

Denis Candusso

Fabien Harel

Pierre Picard

Voltage Singularity-Based Diagnostic for PEM Fuel Cell Stack designed for Operation in OECHP Units

come   L'archive ouverte pluridisciplinaire

Voltage Singularity-Based Diagnostic for PEM Fuel Cell Stack designed for Operation in µCHP Units

D. Benouioua 1,2,4 , D. Candusso 1,2,4 , F. Harel 3,4 , and P. Picard 1,5 1 ITE EFFICACITY, 14-20 boulevard Newton, Champs-sur-Marne, F-77447 Marne la Vallée, France. A non-intrusive and without disturbance diagnostic strategy for a low temperature Proton Exchange Membrane Fuel Cell (PEMFC) designed to operate in µCHP units is proposed. The developed diagnosis strategy consists of two phases:  In the first phase, an experimental campaign is conducted on a 12 cell PEMFC stack dedicated to stationary application. It is designed and marketed by Riesaer Brennstoffzellentechnik GmbH and Inhouse Engineering GmbH, Germany. Thereby, a database is built; it includes many physical signals measured from the stack and the testbench ancilliaries such as: gas pressures, flows, temperatures… and the voltage stack signal recorded with a sampling frequency of 3kHz for nominal and faulty operating conditions.  In the second phase, the research of relevant indicators for the PEMFC State-of-Helath (SoH) is made by analyzing the stamped singularities in the stack voltage signal according to the various operating conditions.

The approved approach implements two data analysis steps: o A data pre-processing step based on the filtering of a voltage signal (acquired during 1 hour) to achieve the separating of its low and high frequencies components. Then, a Useful Voltage (UV) signal was obtained by computing the Root Mean Square 'RMS' on the windowed filtered voltage signal (with a window size of 0.066 s). o A data processing step that consists of computing a punctual singularity strength in the UV signals and summarizing all the founded singularities strength in the form of a singularity spectrum (SS). The signal processing method adopted in this study, namely the Wavelet Leaders-Based Multifractal Analysis (WLMA), has been published in previous works [1,2] and showed high-performance results on a PEMFC designed for transport application. The results of the SS features classification enable the identification of a wide range of operating failures for the studied PEMFC stack dedicated to µCHP application.