Jules Marcone

Wajdi Chaâbani

Claire Goldmann

Marianne Impéror-Clerc

Doru Constantin
email: constantin@unistra.fr

Cyrille Hamon
email: cyrille.hamon@universite-paris-saclay.fr

Polymorphous packing of pentagonal nanoprisms

Keywords: nanoparticles, pentagonal packing, silver nanorods, superlattices, self-assembly

Packing solid shapes into regular lattices can yield very complex assemblies, not all of which achieve the highest packing fraction. In two dimensions, the regular pentagon is paradigmatic, being the simplest shape that does not pave the plane completely.

In this work, we demonstrate the packing of plasmonic nanoprisms with pentagonal cross section, which form extended supercrystals. We do encounter the long-predicted ice-ray and Dürer packings (with packing fractions of 0.921 and 0.854, respectively), but also a variety of novel polymorphs that can be obtained from these two configurations by a continuous sliding transformation and exhibit an intermediate packing fraction. Beyond the fundamental interest of this result, fine control over the density and symmetry of such plasmonic assemblies opens the perspective of tuning their optical properties, with potential applications in metamaterial fabrication, catalysis or molecular detection.

Covering the plane with regular shapes is a complex and intriguing question that has seen sustained interest since antiquity. Among these shapes, regular pentagons are special because unlike triangles, squares and hexagons, they do not tile the plane without gaps. This made them attractive for use in decorative patterns, such as the traditional Chinese ice-ray motif inspired by the growth of ice crystals into 'rays', [START_REF] Dye | A Grammar of Chinese Lattice[END_REF] which is a periodic striped phase consisting of alternate rows of pentagons in two different orientations. European scientists, philosophers and artists, such as Albrecht Dürer [START_REF] Dürer | Underweysung der Messung, mit dem Zirckel und Richtscheyt[END_REF] or Johannes Kepler, 3 also studied regular arrangements of pentagons, like the so-called Dürer packing. Hales and Kusner have recently demonstrated by a computer-assisted proof [START_REF] Hales | Packings of Regular Pentagons in the Plane[END_REF] that the ice-ray configuration has the largest possible packing fraction η defined as the ratio of the area covered by the objects to the total area (η = 0.921). Ice-ray packing was first demonstrated in numerical simulations in 2005, by the group of Frenkel. [START_REF] Schilling | Monte Carlo Study of Hard Pentagons[END_REF] At lower packing fraction a rotator/plastic phase was found, in which the orientational order for the pentagons was lost while the positional order adopted a hexagonal arrangement. Mathematicians have predicted the existence of other crystalline polymorphs with a slightly lower packing fraction, [START_REF] Hales | Packings of Regular Pentagons in the Plane[END_REF][START_REF] Henley | Sphere Packings and Local Environments in Penrose Tilings[END_REF][START_REF] Kuperberg | Double-Lattice Packings of Convex Bodies in the Plane[END_REF][START_REF] Quiquandon | Merohedral twins revisited: quinary twins and beyond[END_REF] but these lattice polymorphs have yet to be encountered in experiments.

The first experiments about pentagonal packing were done at the macroscopic scale, on an air table, and provided insight into such arrangements. [START_REF] Duparcmeur | Dense Periodic Packings of Regular Polygons[END_REF][START_REF] Duparcmeur | Crystallization of Pentagon Packings[END_REF] More recently, micron-sized pentagonal polymeric platelets were fabricated and assembled into colloidal monolayers: they exhibited a rotator phase, but no crystalline structures. [START_REF] Zhao | Frustrated Rotator Crystals and Glasses of Brownian Pentagons[END_REF] At the molecular length scale, a few examples of pentagonal clusters assembled on a substrate have been demonstrated. [START_REF] Bauert | Building 2D Crystals from 5-Fold-Symmetric Molecules[END_REF][START_REF] Ren | Crystallographic Realization of the Mathematically Predicted Densest All-Pentagon Packing Lattice by C5-Symmetric "Sticky" Fluoropentamers[END_REF][14][START_REF] Wasio | Self-assembly of hydrogen-bonded two-dimensional quasicrystals[END_REF] Finally, at the atomic length scale, phosphorus pentamers deposited on a Ag(111) substrate can pack into a dense monolayer, consisting of alternate rows of pentagons in two different orientations, resembling the ice-ray arrangement. [START_REF] Zhang | Phosphorus Pentamers: Floating Nanoflowers Form a 2D Network[END_REF] Considering the imaging difficulties at the atomic scale and the equilibration issues at the macroscale, a promising alternative for obtaining pentagonal packing is the use of nano-objects. In this respect, metallic nanoparticles are excellent candidates due to their monodispersity, large size compared to molecules and high electron density. For instance, we have recently obtained three-dimensional supercrystals (SCs) of truncated pentatwinned gold bipyramids that show a double-lattice packing related to the pentagonal ice-ray. [START_REF] Lyu | Double-Lattice Packing of Pentagonal Gold Bipyramids in Supercrystals with Triclinic Symmetry[END_REF] Pentatwinned anisotropic metallic nanoparticles have been assembled into standing monolayers on a substrate, [START_REF] Guerrero-Martínez | Gemini-Surfactant-Directed Self-Assembly of Monodisperse Gold Nanorods into Standing Superlattices[END_REF][START_REF] Scarabelli | Design and Fabrication of Plasmonic Nanomaterials Based on Gold Nanorod Supercrystals[END_REF] but so far only plastic crystals [START_REF] Pietrobon | Synthesis of Size-Controlled Faceted Pentagonal Silver Nanorods with Tunable Plasmonic Properties and Self-Assembly of These Nanorods[END_REF] or an apparent hexagonal order 21 have been reported. The discrepancy with the predictions above might be due to a large polydispersity in terms of size and shape or to their small width, which renders difficult the identification of the pentagonal cross section with a scanning electron microscope (SEM).

We therefore hypothesized that noble metal nanoprisms (NPs) with a large enough regular pentagonal cross-section and low size dispersity would be ideal building blocks to observe 2D pentagonal packing.

Here we show that monodisperse pentatwinned gold/silver NPs assemble spontaneously into extended SCs with a characteristic trapezoidal shape. Careful analysis of the intralayer organization reveals that the NPs pack into very regular structures, mostly corresponding to the pentagonal ice-ray. Other less dense crystalline polymorphs are often observed, including the Dürer packing as well as intermediate configurations obtained via a continuous sliding transformation. [START_REF] Hales | Packings of Regular Pentagons in the Plane[END_REF] The experimental system consists in elongated pentagonal silver NPs prepared from gold decahedron seeds, [START_REF] Yang | Controlled Growth of Ag/Au Bimetallic Nanorods through Kinetics Control[END_REF] which have a nearly constant length of about 140 nm and a width spanning from 27.5 to 81.7 nm. Byproducts corresponding to triangular right bipyramids, spheroids, and cubes were discarded by selective precipitation of the nanoprisms via depletion-induced self-assembly. [START_REF] Park | Depletion-Induced Shape and Size Selection of Gold Nanoparticles[END_REF] This step is important, as the presence of byproducts would otherwise affect the extension of the NP layers. Similar SCs were obtained for all NPs with different widths, showing an increasing orientational ordering for the larger NPs (see Section 2 in the Supporting Information for a complete discussion about the optimization of the self-assembly conditions). [START_REF] Agarwal | Mesophase Behaviour of Polyhedral Particles[END_REF][START_REF] Damasceno | Predictive Self-Assembly of Polyhedra into Complex Structures[END_REF][START_REF] Boles | Self-Assembly of Colloidal Nanocrystals: From Intricate Structures to Functional Materials[END_REF][START_REF] Zhou | Chiral Assemblies of Pinwheel Superlattices on Substrates[END_REF][START_REF] Wang | Correlating Superlattice Polymorphs to Internanoparticle Distance, Packing Density, and Surface Lattice in Assemblies of PbS Nanoparticles[END_REF][START_REF] Quan | Solvent-Mediated Self-Assembly of Nanocube Superlattices[END_REF] In the following, we present the results for only one width, for gold/silver NPs of 143.7 ± 9.2 nm in length and 74.8 ± 3.2 nm in width. Scanning electron microscopy (SEM) images show that, after drying on a Si wafer, the NPs solutions leave behind micron-sized SCs with a trapezoidal shape (Figure 1(a-b)). At higher magnification, a multi-layered structure of closely packed nanoprisms in a standing orientation is observed (Figure 1(c-d) and Section 3 in the Supporting Information). The shape of these SCs is different from that usually expected for a SC of standing nanorods in a hexagonal arrangement, which tend to adopt a circular or hexagonal macroscopic shape. [START_REF] Scarabelli | Design and Fabrication of Plasmonic Nanomaterials Based on Gold Nanorod Supercrystals[END_REF][START_REF] Hamon | Controlling the Symmetry of Supercrystals Formed by Plasmonic Core-Shell Nanorods with Tunable Cross-Section[END_REF] Since the SC shape can be related to the local arrangement of the NPs, the obtention of SCs with a trapezoidal shape is a first indication of a new lattice structure. Close view of the SC reveals the pentagonal section of the nanoprisms, often arranged in alternating stripes in an ice-ray fashion. The trapezoidal shape of the SCs can be recovered by combining three Delaunay triangles of the ice-ray unit cell (Figure 1b,f). To assess the symmetry of the lattice inside the SCs, fast Fourier Transforms (FFT) were performed on the SEM experimental images and on model images for comparison (see Figure 2). Although the FFTs are close to a hexagonal pattern, the spots are not arranged along a regular hexagon, and they display the a * (10) and b * (01) spots characteristic of the ice-ray packing with a rectangular symmetry (see Figure 2(a-b)). FFT analysis on a large SC area (see Section 2 in the Supporting Information) confirms the extension of the ice-ray packing to tens of micrometers (i.e. the SC size). In order to confirm that the rectangular symmetry is linked to the pentagonal crosssection, we conducted the self-assembly of monocrystalline gold nanorods for comparison. In this case, the nanorods are arranged in an hexagonal lattice, as confirmed by the FFT and the corresponding model images (Figure 2(c-d)). Such hexagonal ordering has already been reported for monocrystalline nanorods having an octogonal cross section, which is very close to a circular one. [START_REF] Guerrero-Martínez | Gemini-Surfactant-Directed Self-Assembly of Monodisperse Gold Nanorods into Standing Superlattices[END_REF][START_REF] Scarabelli | Design and Fabrication of Plasmonic Nanomaterials Based on Gold Nanorod Supercrystals[END_REF][START_REF] Hamon | Hierarchical Self-Assembly of Gold Nanoparticles into Patterned Plasmonic Nanostructures[END_REF][START_REF] Hamon | Controlling the Symmetry of Supercrystals Formed by Plasmonic Core-Shell Nanorods with Tunable Cross-Section[END_REF][START_REF] Wei | Fabrication of Large-Area Arrays of Vertically Aligned Gold Nanorods[END_REF] Taken together, this set of experiments shows that the rectangular lattice arises from the asymmetric cross section of the pentagonal nanorods, resulting in lower symmetry of the nanostructure.

We next performed SEM image analysis in order to quantify the orientation distribution of the pentagonal cross-section of the NPs within the SCs. Particle outlines were extracted The ice-ray and the Dürer packings are linked to each other by a simple geometrical transformation. [START_REF] Hales | Packings of Regular Pentagons in the Plane[END_REF][START_REF] Kuperberg | Double-Lattice Packings of Convex Bodies in the Plane[END_REF] Let us return to the rectangular unit cell of the ice-ray packing: it contains two pentagons in two different orientations, depicted in red and blue (Figure 1(f)). These two adjacent pentagons can slide along their common edge, resulting in a shift of the position of the inversion center (see Figure 3(e)). This local motion is the starting point of a global sliding transformation of the whole packing. As a result, a continuous family of crystalline polymorphs is obtained, all having a slightly lower packing density than the ice-ray packing.

Note that the relative angle of π/5 between the two orientations for pentagons is preserved upon sliding. Calculations are detailed in Section 3 in the Supporting Information, introducing a sliding parameter u that corresponds to the shift of the inversion center along the edge. This sliding transformation leads continuously from the ice-ray packing (u = 0, η = 0.921) to a Dürer packing (u = 0.5, η = 0.854) (see Figure 3(e)). [START_REF] Hales | Packings of Regular Pentagons in the Plane[END_REF] Doing so, the alternate rows in two different orientations slide along each other and the pentagons tilt with respect to the row direction. Sliding induces the unit cell (a, b) to become oblique instead of rectangular and the space group changes from pm to p2 when u is different from zero.

The arrangements we observe experimentally indeed reside along the continuum between the ideal ice-ray and Dürer packings, as shown by the analysis of the SEM image in Figure 4, corresponding to a polymorph with an intermediate sliding deformation (u = 0.1, η = 0.918). We first determine by image analysis both the distribution for the orientation of the pentagons (angle α in Figure 4(b,c)) and for the bonds between the centers of neighboring particles (angle ϕ in Figure 4(d,e)). Five different orientations of bonds are found and are plotted in different colors. We plot the bond length vs. orientation in Figure 4(e) and compare it to the predictions of the sliding transformation (see Section 4 in the Supporting Information) for the two limiting cases (u = 0 and u = 0.5) and for the configuration with u = 0.1 that best fits the data shown in Figure 4(a). This polymorph is close to the ice-ray but definitely different, with a unit cell that is not rectangular but oblique.

Finally, crystal twinning between domains is also encountered as illustrated in Figure 5.

The SEM image shows an area where two domains are separated by a twinning line shown as a white dashed line. These domains correspond to two twin crystals of the ice-ray packing (Figure 5 Indeed, on the SEM image without the orientation overlay (Figure 5(a)), the twinning line is indiscernible by eye. In contrast to other superlattices, [START_REF] Boles | Self-Assembly of Colloidal Nanocrystals: From Intricate Structures to Functional Materials[END_REF][START_REF] Wang | Interplay between Spherical Confinement and Particle Shape on the Self-Assembly of Rounded Cubes[END_REF][START_REF] Zhao | Twinning of Rhombic Colloidal Crystals[END_REF] such as rhombic platelet lattices, [START_REF] Zhao | Twinning of Rhombic Colloidal Crystals[END_REF] we find that crystal twinning in pentagonal SCs only induces positional disorder while the orientational order is preserved.

So far, only a few examples of the ice-ray pentagonal packing had been encountered, either at the macroscale or at the molecular/atomic scale. Here we obtain a first experimental realisation at the mesoscale with silver nanoprisms self-assembled into extended micronsized supercrystals (Figure 1). In contrast with plastic/rotator phases, the NPs display high positional and orientational ordering related by a π/5 rotation. Extended crystalline order is present and its symmetry is not hexagonal but rectangular, which is a clear signature of the ice-ray pentagonal packing, as proven by the Fourier transform images (Figure 2). The trapezoidal shape of the SCs is related to the symmetry of the unit cell (via its Delaunay triangles), confirming both the very long-range order of the lattices (extending over hundreds of particle rows) and, indirectly, the excellent size and shape monodispersity of the samples.

The presence of polymorphs for crystalline pentagonal packing gives rise to a rich phase behavior. Along with the ice-ray packing, other crystalline structured are evidenced, like the Dürer packing (Figure 3) and intermediate polymorphs (Figure 4). Crystal twinning is also observed (Figure 5). A sliding transformation is introduced to explain such polymorphism (Figure 3(e)). Our analysis reveals a subtle interplay between orientational and positional order. On the one hand, pentagon orientation is conserved even across the boundary between two twin ice-ray crystals or between two types of packing (e.g. ice-ray and Dürer). On the other hand, as the particle's width gets smaller, orientational order decreases while the positional one, given by the length and orientation of the bonds between neighboring NPs, remains fairly constant. Thus, the lattice persists even as the disorientation increases, supporting the presence of the rotator phase detected in numerical simulations. [START_REF] Schilling | Monte Carlo Study of Hard Pentagons[END_REF] The presence of polymorphs can be rationalized by a compromise between the structure that reaches the densest packing (i.e. ice-ray) and the structure that maximizes face to face contact (i.e. Dürer). Although the Dürer structure is less dense than the ice-ray, it allows the formation of face-to-face interparticle contacts, which are favored by strong and localized interactions such as the Van der Waals attraction. This results in the formation of many lattice polymorphs that can be described by the continuous sliding transformation between the two structures. In this sense, the nanoprisms we study in this work are different from other polyhedral nanocrystals such as octahedra, which assemble in a few discrete lattice polymorphs. [START_REF] Boles | Self-Assembly of Colloidal Nanocrystals: From Intricate Structures to Functional Materials[END_REF][START_REF] Gong | Shape-Dependent Ordering of Gold Nanocrystals into Large-Scale Superlattices[END_REF][START_REF] García-Lojo | Structure and Formation Kinetics of Millimeter-Size Single Domain Supercrystals[END_REF][START_REF] Quan | Superlattices with Non-Spherical Building Blocks[END_REF] Compared to earlier macroscopic assemblies [START_REF] Duparcmeur | Dense Periodic Packings of Regular Polygons[END_REF] and numerical simulation studies of hard pentagons, [START_REF] Wang | Structural Characterization of the Packings of Granular Regular Polygons[END_REF][START_REF] Shen | Symmetries in Hard Polygon Systems Determine Plastic Colloidal Crystal Mesophases in Two Dimensions[END_REF] [START_REF] Cersonsky | Relevance of Packing to Colloidal Self-Assembly[END_REF] minimising the packing fraction is not the only parameter relevant for hard colloid crystallization. In this respect, packing pentagonal nanoprisms appears as a promising model system to study crystallization at the mesoscale. The Dürer packing is promising for material design due to the formation of nanochannels between the NPs that would enhance molecular diffusion. Note that an infinity of Dürer tilings exist (with the same packing fraction), but we only observed the one obtained by sliding from the ice-ray configuration. However, a version where all pentagons are in the edge-to-edge configuration would have twice the nanochannel cross-section compared to the Dürer lattices described in this work. Numerical simulations (see the supplementary videos) show that the electric field distribution is different for the various lattice polymorphs, a result that is relevant for plasmon-enhanced spectroscopy and that opens the perspective of using these assemblies for the development of sensing applications.

Figure 1 :

 1 Figure 1: Supercrystals with ice-ray packing. (a-b) SEM images of trapezoidal SCs at different magnifications. (c) Side view showing the 2D packing of the nanoprisms at the surface of the SCs. (d) Close view of an SC surface. The pentagonal cross-section of the NPs is visible in the SEM image. Pentagonal contours are overlaid in the bottom half, color coded to the particles' orientation with respect to the Ox axis, shown as black arrow. (e) Histogram of the orientation angle α for the pentagonal cross-section in (d), with the same color code, and Gaussian fits (solid line). (f) Ice-ray packing annotated with colorcoded Delaunay triangles (each color stands for one distance) as well as vectors a and b of the rectangular unit cell. (g) 2D ice-ray packing of regular pentagons. The blue and red colors indicate the two pentagon orientations.

Figure 2 :

 2 Figure 2: Rectangular versus hexagonal crystalline order. (a) top) SEM image of a rectangular lattice of standing pentagonal gold/silver nanoprisms. bottom) corresponding FFT image (b) top) model of a rectangular lattice of regular pentagons (ice-ray packing). bottom) corresponding FFT image (c) SEM image of a hexagonal lattice of standing monocrystalline gold rods. bottom) corresponding FFT image (d) top) model of a hexagonal lattice of disks. bottom) corresponding FFT image.

Figure 3 :

 3 Figure 3: Grain boundary between ice-ray and Dürer domains. (a) Ideal ice-ray packing. (b) SEM image of an SC surface showing both ice-ray (on the left) and Dürer (on the right) arrangements. The boundary is marked by a dashed white line, as are the limits of the dimer rows in the Dürer domain. (c) Ideal Dürer packing. (d) Histogram of the particle orientation α in the Dürer (top) and ice-ray (bottom) domains, with Gaussian fits. (e) Scheme of the continuous sliding deformation from the ice-ray to the Dürer packing. Values of the corresponding sliding parameter u and packing fraction η are given. The position of the inversion center is depicted by a white circle and slides along the common edge of the blue and red pentagons. Note the deformation of the unit cell from rectangular to oblique and the tilt in orientation of the pentagons with respect to the stripe direction along a. The thin black arrow indicates the direction of the Ox axis used as angle reference for the image analysis (see Figures 1, 3, 4).

Figure 4 :

 4 Figure 4: Analysis of orientational and translational order in a lattice polymorph. (a) Scheme of the polymorph with a sliding parameter of u = 0.1 and a packing fraction of 0.918. The unit cell is oblique. The thin black arrow corresponds to the Ox axis, common reference for the α and ϕ angles. (b) SEM image with overlaid particle contours. (c) Histogram of the particle orientations (angle α). (d) The bonds between the centers of neighboring particles are shown by overlaid links, color coded by length and orientation (angle ϕ). (e) Plot of the bond length vs. bond orientation. Colored points correspond to the experimental image with the same color code as in (d). Symbols are the predictions for the length and orientation of the five different bonds: ice ray-lattice (u = 0, crosses), Dürer lattice (u = 0.5, triangles) and the intermediate value (u = 0.1 solid dots) that best approaches the experimental data points.

Figure 5 :

 5 Figure 5: Twinning line between two domains forming a herringbone texture. (a) SEM image of a defect highlighted with a dashed white line corresponding to a twinning line. (b) Same image where the pentagonal contours are overlaid, color coded to the particles' orientation. The green lines are plotted manually along some pentagon rows to highlight the herringbone texture. On the left, the scheme of the unit cell is shown for each crystal. (c) Histogram of the particle orientations and (d) corresponding height-resolved plot of the orientation. The grey overlay indicates the area covered by the twinning line.

  such supercrystals display a rich phase diagram and open the way to further studies. For instance, in depth investigation of the self-assembly kinetics may reveal the trajectories of materials formation. Varying experimental conditions should help addressing the open and general question of the relative stability of different lattice polymorphs. As pointed out recently,

Acknowledgement

The CNRS is acknowledged for funding and support. This work has benefited from an Investissements d'Avenir grant from Labex PALM (ANR-10-LABX-0039-PALM). J. M. acknowledges financial support by the ANR NIMROD project (ANR-21-CE09-0019) for his PhD. The present work has benefited from the electronic microscopy facility of Imagerie-Gif, (http://www.i2bc.parissaclay.fr), member of IBiSA (http://www.ibisa.net), supported by "France-BioImaging" (ANR10-INBS-04-01), and the Labex "Saclay Plant Science" (ANR-11-IDEX-0003-02). We thank Frederic Kanoufi, Jean-Yves Piquemal and Miguel Comesaña-Hermo for help with the numerical simulations.

Information Available

Synthesis and purification protocols, characterization of the nanoparticles, additional SEM images, image analysis and geometrical relations are available on-line. Numerical simulations showing that the electric field distribution is different for the various lattice polymorphs are provided as four supplementary videos. The Mathematica notebook used to model the sliding transformation is available at: https://www.wolframcloud.com/obj/c70ae9fa-d6a8-4061-9fad-69d93cd42ccf


Nathan Carriot

Raphaëlle Barry-Martinet

Jean-François Briand

Annick Ortalo-Magné
email: annick.ortalo-magne@univ-tln.fr

Gérald Culioli
email: gerald.culioli@univ-avignon.fr

Annick Ortalo

Impact of phosphate concentration on the metabolome of biofilms of the marine bacterium Pseudoalteromonas lipolytica

Keywords: Marine biofilms, LC-MS-based metabolomics, Phosphate depletion, Membrane remodeling, Ornithine lipids

Introduction

Biofilms constitute a mode of life widely distributed in marine environments. These complex communities of marine microorganisms mainly composed of bacteria (but also archaea, diatoms and fungi) can develop on most biotic and abiotic surfaces immersed in seawater [START_REF] Carvalho | Marine biofilms: A successful microbial strategy with economic implications[END_REF][START_REF] Dang | Microbial surface colonization and biofilm development in marine environments[END_REF]. In biofilms, microbial cells are embedded in a threedimensional, highly hydrated matrix of self-produced extracellular polymeric substances (EPS) mainly composed of polysaccharides and proteins, even lipids and extracellular DNA have been also described as matrix constituents [START_REF] Flemming | EPS -Then and now[END_REF][START_REF] Flemming | Biofilms: an emergent form of bacterial life[END_REF]. This matrix promotes adhesion processes on surfaces and maintains the cohesion between biofilm microorganisms. Furthermore, the biofilm matrix acts as a nutrient source by trapping nutrient elements and molecules, or by the degradation of constitutive EPS, and protects the microbial cells against a wide range of environmental stresses [START_REF] Flemming | The biofilm matrix[END_REF]. Thus, the biosynthesis by biofilm cells of biopolymers and metabolites reflects the adaptive capacities of microbial communities to environmental constraints.

Marine biofilms play crucial roles in marine ecosystems through their implication in biogeochemical cycles and trophic interactions [START_REF] Anderson | Marine and estuarine natural microbial biofilms: ecological and biogeochemical dimensions[END_REF]. In such living structures, the phenotypic plasticity of some bacteria results in their quick responses to the highly fluctuating marine conditions [START_REF] Stocker | Marine microbes see a sea of gradients[END_REF]. Similar to hydrodynamic conditions or the nature of the colonized substrate, the availability of nutrients in the marine environment is one of the most important factors influencing biofilm development, as well as its species and biochemical compositions [START_REF] Allan | Effect of nutrient limitation on biofilm formation and phosphatase activity of a Citrobacter sp[END_REF][START_REF] Lawes | Elevated change bacterial community composition and connectivity: high throughput sequencing of young marine biofilms[END_REF]. More particularly, phosphorus (P) is an essential micronutrient for living organisms [START_REF] Karl | Microbially mediated transformations of phosphorus in the sea: New views of an old cycle[END_REF]. Whether in its organic or inorganic forms, P is present in most compartments of both terrestrial and marine ecosystems, such as water, soil or sediment. Phosphate is generally present in low amounts in oceans with concentrations ranging typically from nanomolar values in surface waters increasing with depth to micromolar values [START_REF] Paytan | The oceanic phosphorus cycle[END_REF]. In living microorganisms, P is considered an essential element, as it is involved through adenosine phosphates or NADPH in a wide range of energy-requiring reactions. P also enters, in the form of a phosphate group, the composition of DNA and RNA molecules, phosphoproteins, polyphosphates, as well as phospholipids which are key components of cell membranes [START_REF] Violaki | Organic phosphorus in atmospheric deposition over the Mediterranean Sea: An important missing piece of the phosphorus cycle[END_REF]. The main sinks of organic P are nucleic acids, phosphoproteins and phospholipids whose biosynthesis creates a demand for this nutrient. While nucleic acids and phosphoproteins cannot be synthesized without P, the strategy of polar lipids remodeling is widespread among phytoplankton and diverse marine heterotrophic bacteria [START_REF] Carini | SAR11 lipid renovation in response to phosphate starvation[END_REF][START_REF] Sebastián | Lipid remodelling is a widespread strategy in marine heterotrophic bacteria upon phosphorus deficiency[END_REF][START_REF] Van Mooy | Phytoplankton in the ocean use non-phosphorus lipids in response to phosphorus scarcity[END_REF]. Under P deficiency, these organisms have developed strategies to maintain the membrane integrity by replacing amphiphilic membrane phospholipids with non-P lipids whose variable polar head groups contain sulfur (sulfolipids), mono-or disaccharides (glycolipids), betaines (betaine lipids) or amino acids (aminolipids) [START_REF] Diercks | Accumulation of novel glycolipids and ornithine lipids in Mesorhizobium loti under phosphate deprivation[END_REF][START_REF] Geiger | Amino acidcontaining membrane lipids in bacteria[END_REF]. Therefore, while it is well accepted that the biochemical composition of bacterial membranes varies from one microorganism to another, such a variability has been also demonstrated within a same species depending on environmental factors, and more particularly P limitations [START_REF] Sohlenkamp | Bacterial membrane lipids: diversity in structures and pathways[END_REF].

The Mediterranean Sea is overall one of the most oligotrophic marine environments in the world, with the exception of specific coastal locations with anthropogenic inputs, where mineral nutrients, and particularly P, are present in limited quantity [START_REF] Van Wambeke | Longitudinal and vertical trends of bacterial limitation by phosphorus and carbon in the Mediterranean Sea[END_REF]. P concentrations are so low in such locations that this nutrient is often considered to be the major limiting element for bacterial growth in Mediterranean marine environments [START_REF] Dyhrman | Microbes and the marine phosphorus cycle[END_REF][START_REF] Pinhassi | Seasonal changes in bacterioplankton nutrient limitation and their effects on bacterial community composition in the NW Mediterranean Sea[END_REF]). Among different groups from the heterotrophic bacterial community of the eastern Mediterranean Sea tested for their response to P availability, previous results have shown contrasting levels of bacterial metabolic activity (i.e. assimilation of leucine determined using micro-autoradiography combined with fluorescence in situ hybridization), with proteobacteria activity being the most stimulated in P-rich conditions [START_REF] Sebastián | Heterogeneity in the nutrient limitation of different bacterioplankton groups in the Eastern Mediterranean Sea[END_REF].

For the present study, the impact of P starvation was evaluated on a marine bacterial strain belonging to the Pseudoalteromonas genus (-proteobacteria class), which is commonly represented in marine environments. Moreover, bacteria species of the genus Pseudoalteromonas show adaptative advantages such as the ability to exude a large number of EPS [START_REF] Poli | Bacterial exopolysaccharides from extreme marine habitats: Production, characterization and biological activities[END_REF]) and to produce a wide range of bioactive chemicals [START_REF] Offret | Spotlight on antimicrobial metabolites from the marine bacteria Pseudoalteromonas: Chemodiversity and ecological significance[END_REF]).

The strain selected for this study, Pseudoalteromonas lipolytica TC8, had previously been isolated from a natural biofilm formed on a silicon coupon immersed in the bay of Toulon, France (NW Mediterranean Sea) [START_REF] Brian-Jaisson | Identification of bacterial strains isolated from the Mediterranean Sea exhibiting different abilities of biofilm formation[END_REF][START_REF] Camps | Antifouling activity of commercial biocides vs. natural and natural-derived products assessed by marine bacteria adhesion bioassay[END_REF]. P. lipolytica TC8 was chosen as a suitable candidate to study the impact of P starvation on marine biofilms formed in vitro due to its capacity to easily grow in vitro in biofilm mode. Moreover, previous investigations of P. lipolytica TC8 biochemical production have shown the ability of this bacterial strain to biosynthesize, in addition to phospholipids, non-P lipids such as aminolipids [START_REF] Favre | Discrimination of four marine biofilm-forming bacteria by LC-MS metabolomics and influence of culture parameters[END_REF][START_REF] Favre | Metabolome and proteome changes between biofilm and planktonic phenotypes of the marine bacterium Pseudoalteromonas lipolytica TC8[END_REF][START_REF] Favre | Metabolomic and proteomic changes induced by growth inhibitory concentrations of copper in the biofilm-forming marine bacterium Pseudoalteromonas lipolytica[END_REF].

In this context, LC-MS-based untargeted metabolomics appears as a well-suited approach to assess the effect of an environmental factor on organisms and has already proven effective for the analysis of marine biofilms [START_REF] Bauermeister | Intra-clade metabolomic profiling of MAR4 Streptomyces from the Macaronesia Atlantic region reveals a source of anti-biofilm metabolites[END_REF][START_REF] Favre | Discrimination of four marine biofilm-forming bacteria by LC-MS metabolomics and influence of culture parameters[END_REF][START_REF] Favre | Metabolome and proteome changes between biofilm and planktonic phenotypes of the marine bacterium Pseudoalteromonas lipolytica TC8[END_REF][START_REF] Favre | Metabolomic and proteomic changes induced by growth inhibitory concentrations of copper in the biofilm-forming marine bacterium Pseudoalteromonas lipolytica[END_REF]. Over the past decades, technological progress in chromatography and mass spectrometry has revolutionized this field of metabolomics. For example, these advances allow for the comparison of the metabolism of living organisms under different conditions and, more precisely, for the identification of metabolites which were differently expressed [START_REF] Chang | A practical guide to metabolomics software development[END_REF]. For this last point, statistical analyses combined with the powerful molecular networking approach [START_REF] Aron | Reproducible molecular networking of untargeted mass spectrometry data using GNPS[END_REF][START_REF] Nothias | Bioactivity-based molecular networking for the discovery of drug leads in natural product bioassay-guided fractionation[END_REF][START_REF] Wang | Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking[END_REF] help in the annotation of specific chemomarkers.

The annotation of metabolites of interest is always a delicate step in LC-MS-based metabolomics, as many of the LC-MS signals are inherent to samples collection and treatment (e.g. contaminants, degradation products), or related to a single molecule (e.g. adducts, fragments). At the beginning of the annotation process, a m/z feature can be defined by: (i) its retention time, (ii) its accurate mass and then its molecular formula, and eventually (iii) its MS/MS fragmentation pattern (fragment ions, neutral losses). If a chemical standard is available, the comparison of these three criteria (or at least of the first two) with those of the unknown feature leads easily to a robust and efficient annotation. If this is not the case, the search for accurate mass/molecular formula with or without MS/MS data in the literature regarding the studied organism and/or in specialized databases (e.g. PubChem, HMDB, MetLin, Lipidmaps) can also allow for the annotation of a specific m/z feature, however with a lower level of confidence [START_REF] Chaleckis | Challenges, progress and promises of metabolite annotation for LC-MS-based metabolomics[END_REF]. More recently, a bioinformatic tool known as molecular networking (MN) has been developed and allows, on the theoretical basis that similar chemical structures tend to give closely-related MS/MS data, for the facilitation of the annotation step of the LC-MS-based metabolomics workflow [START_REF] Watrous | Mass spectral molecular networking of living microbial colonies[END_REF]. Such a MN provides a map of the chemical space of a set of samples where nodes can be annotated not only by spectral comparison with spectral libraries and commercial/in-house compound databases [START_REF] Fox Ramos | CANPA: Computer-Assisted Natural Products Anticipation[END_REF]), but also by comparing their MS/MS data with those of other nodes found in a common cluster (e.g. Carriot et al. 2021).

In this study, the aim was to investigate the impact of P starvation on the metabolism of a model biofilm-forming marine bacterium, P. lipolytica TC8, by mimicking relevant environmental conditions. For that purpose, five minimal culture media, in which phosphate concentration was modified in an ecologically-relevant manner to correspond to different ocean and sea conditions over the world, were used to evaluate the resulting changes on the biofilm-forming capacity and the metabolome of P. lipolytica TC8. Moreover, the metabolomics workflow used in this study focused on the annotation of key discriminating and statistically robust metabolites depending on the P concentrations.

2

Materials and methods

Preparation of bacterial cultures

Pseudoalteromonas lipolytica TC8 is a -proteobacterium isolated from an artificial substratum immersed in the bay of Toulon (NW Mediterranean Sea, France; 43°06′23′′N, 5°57′17′′E) [START_REF] Brian-Jaisson | Identification of bacterial strains isolated from the Mediterranean Sea exhibiting different abilities of biofilm formation[END_REF][START_REF] Camps | Antifouling activity of commercial biocides vs. natural and natural-derived products assessed by marine bacteria adhesion bioassay[END_REF]). Cells of P. lipolytica TC8, stored at -80 °C, were placed in a sterile flask containing 30 mL of Väätänen nine salt solution (VNSS) [START_REF] Mårdén | Physiological and morphological changes during short term starvation of marine bacterial islates[END_REF]. Precultures were made in triplicate and the flasks were placed for 48 h at 20 °C in an incubator shaker (120 rpm). Bacterial precultures were subsequently diluted in VNSS in three Erlenmeyer flasks to reach an optical density at 600 nm (OD600) of 0.1. The resulting precultures were incubated at 20 °C with stirring at 120 rpm. The optical density was monitored every hour until the end of the exponential phase (OD600 0.6) with a spectrophotometer (Genesys 20 spectrophotometer; Thermo Fisher Scientific, Waltham, MA, USA). Medium blanks were also prepared in parallel with only VNSS. Once the precultures reached an OD600 equal to 0.6, 30 mL of the bacterial cultures were placed in 50 mL Falcon tubes in triplicate for each culture medium. The tubes were centrifugated (Z 200 A; Hermle LaborTechnik GmbH, NJ, USA) at 6000 rpm for 10 min at room temperature and the supernatants were removed. The resulting pellets were suspended in 15 mL of nine salt solution (NSS) [START_REF] Dahlbäck | The hydrophobicity of bacteria -An important factor in their initial adhesion at the air-water inteface[END_REF] and then vortexed. The tubes were centrifuged at 4000 rpm for 15 min at room temperature.

The supernatants were removed and the pellets were diluted in marine minimal medium (MMM) [START_REF] Ostling | Behaviour of IncP-1 plasmids and a miniMu transposon in a marine Vibrio sp.: Isolation of starvation inducible lac operon fusions[END_REF] with different phosphate concentrations (Table 1 and see Part 3.1) chosen to mimic different realistic environmental conditions. The optical densities were adjusted (OD600 0.5) at that time, corresponding to a bacterial density of about 5 × 10 6 CFU/mL.

For the following experiments, several types of culture containers were used. However, they are all made of the same material (polystyrene) and, in all experiments, they were inoculated with the same precultures at an identical optical density.

2.2

Characterization of the biofilms of P. lipolytica TC8

Biofilm quantification

Biofilm forming ability and monitoring of the evolution of bacterial biofilms according to phosphate concentration were carried out through crystal violet (CV) staining. CV allows the quantification of biofilm biomass, exopolymeric matrix and bacterial (living and dead) cells being stained [START_REF] Kragh | Into the well -A close look at the complex structures of a microtiter biofilm and the crystal violet assay[END_REF]). More precisely, 5 mL of a bacterial preculture were placed in each well of a 6-well polystyrene plate (Greiner Bio One, Frickenhausen, Germany).

Biofilms and medium blanks were made in triplicate. The plates were then incubated at 20 °C.

After 24 h, the medium was removed, and the wells were rinsed three times with 6 mL of NSS.

The plates were subsequently dried in an oven (50 °C, 30 min) and 5 mL of a CV solution (0.01 %) were deposited in each well. After 10 min, the plates were rinsed three times with 5 mL of Milli-Q water (Waters-Millipore, Milford, MA, USA), dried at room temperature for 10 min, and 5 mL of 95 % ethanol (VWR, Fontenay-sous-Bois, France) were added directly in the wells. An incubation time of 10 min at 20 °C under shaking (120 rpm) was performed.

Finally, 200 μL of the solution of each well were transferred to a 96-well plate (Greiner Bio-One) and analyzed with a microplate reader (Infinite® M200 pro; TECAN, Switzerland) at 595 nm (Fig. 1A).

Biofilm viability and cellular organization

The monitoring of cell evolution was performed using the "live/dead" kit (Bacterial Viability Kit; Thermo Fisher Scientific, Eugene, OR, USA) which consists of two fluorescent markers:

SYTO® 9 (labelling all the bacterial cells) and propidium iodide (PI) (labelling only dead cells).

The staining was done directly in 96-well black polystyrene microplates with a clear bottom (Corning Incorporated, NY, USA). Bacterial samples and medium blanks were tested in triplicate. 200 μL of preculture were placed in each well and incubated at 20 °C. After 24 h, the wells were rinsed three times with 200 μL of a NaCl solution (36 g/L). 0.3 μL of the staining solution (PI at 20 mM/SYTO® 9 at 3.34 mM; 1/1, v/v) were then introduced in each well.

Incubation was done at room temperature in the dark for 15 min. Each well was rinsed three times (NaCl at 36 g/L, 200 μL/well) in order to remove the excess of fluorescent markers.

200 μL of NaCl (36 g/L) were put in the wells which were then analyzed by a microplate reader.

The absorbance wavelengths used were those of PI (538/617 nm) and SYTO® 9 (470/515 nm).

A confocal microscope reading (Zeiss LSM 510 meta, Göttingen, Germany) was also performed to highlight the viability and the cellular organization of the biofilms (Fig1B).

Gross biochemical composition

For these assays, biofilms were obtained from 200 µL of a bacterial preculture placed in each well of 96-well polystyrene plates (Greiner Bio One). These plates were then incubated at 20 °C. After 24 h, the plates were removed and rinsed with 150 µL of NSS. The protein content of the biofilm samples was quantified by the Bradford-based Uptima coo protein assay (Interchim, Montluçon, France). The carbohydrate content was determined by a colorimetric method with glucose as a standard [START_REF] Dubois | Colorimetric method for determination of sugars and related substances[END_REF]. In this method, the use of concentrated sulfuric acid allows the obtention of monosaccharides through acidic hydrolysis of biofilm samples and leads to furfural derivatives which form colored complexes with phenol (maximum absorbance wavelength at 490 nm). Each quantification experiment was performed in triplicate (Supplemental Fig. S1).

2.3

Metabolomic analysis

Sample preparation

The biofilm cultures were done in polystyrene Petri dishes in which 25 mL of preculture (OD600 0.5) were inoculated (24 h, 20 °C) and each medium was tested in triplicate. The supernatants of the Petri dishes were removed to keep only the biofilms and to eliminate the bacterial planktonic cells. Each Petri dish was rinsed two times with 3 mL of the corresponding culture medium. The resulting solution containing the adherent biofilm was placed in a tube.

For each sample, the quenching and the extraction were carried out simultaneously by adding 5 mL of ethyl acetate (analytical grade; Carlo Erba Reagents, Peypin, France) in each tube and by vortexing the mixture [START_REF] Favre | Discrimination of four marine biofilm-forming bacteria by LC-MS metabolomics and influence of culture parameters[END_REF]). The tubes were then placed in an ultrasonic bath (Bransonic 2150E-DTH; Danbury, CT, USA) for 30 min at a temperature of 20 °C. The upper phase containing the organic extract was placed in 8 mL vials and dried under N2. The vials were then filled with argon and kept at -20 °C. Medium blanks (in triplicate for each culture condition) were also prepared using the same protocol, however without bacterial cells.

UHPLC-MS data acquisition

The bacterial dried extracts (mass values ranging from 8 to 10 mg) and the medium blanks The elution gradient started at 5 % B and was maintained for 2 min, then increased to 100 % B (linear ramp) in 8 min and was maintained for 4 min. The composition of the mobile phase was restored at 5 % B (linear ramp) over 0.01 min and was maintained for 1.99 min, for a total run time of 16 min.

The capillary voltage of the MS spectrometer was set at 4500 V (positive mode), and the nebulizing parameters were set as follows: nebulizing gas (N2) pressure at 0.4 bar, drying gas (N2) flow at 4 L/min, and drying temperature at 180 °C. Mass spectra were recorded from m/z 50 to 1200 at a mass resolving power of 25,000 full width at half-maximum (FWHM, m/z = 200) and a frequency of 2 Hz. Tandem mass spectrometry analyses were performed thanks to a collision induced dissociation (CID), fragmenting the two most intense precursor ions by scan, with a collision energy selected on the basis of the ion mass (ramp from 15 to sample for internal mass calibration, and the mass spectrometer was calibrated with the same solution at the beginning of the injection sequence.

Data processing and statistical analysis

LC-MS raw data were respectively converted into ".netCDF" files using DataAnalysis (version 4.3; Bruker Daltonics) and processed with MZmine-2.51 [START_REF] Pluskal | MZmine 2: Modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data[END_REF]). The data matrix was submitted to three filtering steps using an in-house script on R. Each step of the script consisted of removing successively experimental and analytical bias according to signal/noise ratio (using blanks; minimal S/N ratio: 6), coefficient of variation (using QCs; minimal CV: 20 %), and coefficient of correlation (using biofilm samples; minimal CC: 80 %). For multivariate analyses, the resulting data matrix was log10-transformed, mean-centered and analyzed using MetaboAnalyst 5.0 online webtool (https://www.metaboanalyst.ca/ ; [START_REF] Chong | Using MetaboAnalyst 4.0 for comprehensive and integrative metabolomics data analysis[END_REF]. Data were analyzed using principal component analysis (PCA), followed by partial least squares discriminant analysis (PLS-DA) to reveal differences between biofilms of P. lipolytica TC8 grown with various phosphate concentrations. The VIPs (variable importance in the projection) representing the most discriminating m/z features between the different culture conditions were classified according to their VIP score (the most discriminating metabolite having the highest VIP score) (Table 2 and Supplemental Table S1).

Feature annotation via molecular networking

Raw UPLC-MS/MS data were converted into ".mzXML" files using DataAnalysis. For each culture condition, the MS/MS data of one biofilm sample and one medium blank were used to Data from GNPS platform were imported into Cytoscape (version 3.7.0), using nodes to represent each spectrum and edges to represent a similarity of MS/MS fragmentation between two connected nodes (Fig. 3).

The MN thus created was used to annotate metabolites of P. lipolytica TC8, and more specifically the VIPs. Various methods were used to annotate a metabolite: (i) during the computational process for generating the network, GNPS platform performed a fragment retention times were compared with those of molecules of our in-house database, and (iv) MS data and MS/MS fragmentation patterns of nodes belonging to a same cluster of the MN were manually interpreted using their similarity and the related bibliographic data.

Results and discussion

The general monitoring of biofilm formation was carried out through staining with CV, confocal laser scanning microscopy, and gross biochemical characterization, while untargeted LC-MS metabolomics coupled to molecular networking allowed for observation of main changes at the metabolome level.

Selection of ecologically relevant phosphate concentrations for the culture of biofilms of P. lipolytica TC8

The aim of this study was to evaluate the effect of a gradient of relevant environmental P concentrations on biofilms of P. lipolytica TC8 (Table 1). The lowest concentration chosen corresponded to the phosphate concentration experienced by this bacterial strain in the environment from which it has been isolated (i.e. bay of Toulon, NW Mediterranean Sea). This geographical area is typically defined as nutrient limited, mostly due to a lack of phosphate [START_REF] Lazzari | Spatial variability of phosphate and nitrate in the Mediterranean Sea: A modeling approach[END_REF]), but its coastal bay waters contain higher levels of phosphate (10 -4 mM).

The second concentration (10 -3 mM) corresponded to maximum phosphate concentrations observed in coastal locations from the NE Atlantic Ocean [START_REF] Michaels | Inputs, losses and transformations of nitrogen and phosphorus in the pelagic North Atlantic Ocean[END_REF])) and mean phosphate concentrations observed in the Baltic Sea [START_REF] Frumin | Total phosphorus distribution over the Baltic Sea sub-regions[END_REF]. The third concentration (10 -2 mM) tended to mimic phosphate levels reached in estuarine ecosystems with higher river discharges [START_REF] Lochet | Transfers between a eutrophic ecosystem, the river Rhône, and an oligotrophic ecosystem, the north-western Mediterranean Sea[END_REF]. The last concentration corresponded to the concentration of the marine minimal medium (1.32 mM), often used to isolate and grow marine bacteria in "realistic" conditions [START_REF] Patrauchan | Calcium influences cellular and extracellular product formation during biofilm-associated growth of a marine Pseudoalteromonas sp[END_REF]. Finally, an intermediate concentration of 10 - 1 mM between 10 -2 and 1.32 mM was selected in order to avoid big gaps between phosphate concentration values along the selected gradient.

Impact of phosphate concentration on the amount and the structure of biofilms of

P. lipolytica TC8

Growing bacteria in minimum and well-defined culture media has several advantages, such as promoting biofilm formation (due to the limited availability of certain nutrients) and allowing for the observation of the specific effects of some nutrients (e.g. phosphate in this study) on the biofilm growth and properties. In addition, the use of these carbon-limited culture media tends to avoid interactions between nutrients and organic matter that could drastically alter nutrient bioavailability. Through a simple crystal violet (CV) staining assay, the influence of medium composition on the amount of biofilm formed can be investigated [START_REF] Haney | Critical assessment of methods to quantify biofilm growth and evaluate antibiofilm activity of host defence peptides[END_REF]. Such a monitoring is frequently used as it allows for the evaluation of the total biofilm biomass, including the microbial cells and the surrounding exopolymeric matrix [START_REF] Barraud | Methods for studying biofilm dispersal in Pseudomonas aeruginosa[END_REF].

Regarding the biofilm cultures of P. lipolytica TC8, a decrease in their amount was observed in dependency with the decrease of phosphate concentration (Fig. 1A). With phosphate being the only modified nutrient, a direct correlation between the decrease in the P content of the culture medium and the biomass of biofilms formed was shown. More precisely, significant shifts of the quantity of biofilm were observed between the three following groups of samples: i) 1.32/10 -1 mM, ii) 10 -2 and 10 -3 mM, and iii) 10 -4 mM. These results echoed previous works which have also observed a decrease in the biofilm formation under P-limiting conditions [START_REF] Ghosh | Phosphate deficiency induced biofilm formation of Burkholderia on insoluble phosphate granules plays a pivotal role for maximum release of soluble phosphate[END_REF][START_REF] Lehtola | Biofilm formation in drinking water affected by low concentrations of phosphorus[END_REF][START_REF] Rinaudi | Effects of nutritional and environmental conditions on Sinorhizobium meliloti biofilm formation[END_REF], even if the impact of phosphate starvation on cultured marine bacterial biofilms has never been studied to date.

Confocal imaging was used to quantify living/dead bacterial cells, as well as to examine the structure of biofilms of P. lipolytica TC8 grown under various phosphate concentrations. No differences were observed in the ratio between living and dead cells in the different culture media (data not shown). The reduction of the biofilm amounts observed by CV staining did not only come from a cell mortality induced by P starvation, but also from a reduction of the living biomass. Regardless of the culture conditions, biofilms formed by P. lipolytica TC8 always displayed a highly compact layer covering the entire surface. Differences, however, can be noted in the biofilm structure (Fig. 1B). While CLSM images revealed a carpet-like appearance of the biofilm formed at the lowest phosphate concentration (10 -4 mM), three dimensional clusters were observed at at a phosphate concentration of 10 -3 mM. Given that an established biofilm is structured by microbial cells and EPS, the differences in the defined architecture might be due to growth rates and EPS production. Among EPS, polysaccharides participate to the structuration of biofilms by providing aggregative properties [START_REF] Karygianni | Biofilm matrixome: Extracellular components in structured microbial communities[END_REF]) . Here, an increased secretion of such exopolymers might be correlated with the P content of the culture medium and would be responsible of clusters formation. This hypothesis was supported by results of protein and carbohydrate measurements by colorimetric methods which revealed a significant decrease (~40 %) of carbohydrates when the P concentration goes from 1.32 mM to 10 -4 mM while no statistically significant variations (HSD Tukey's test) were observed for the protein content along the selected gradient (Supplemental Fig. S1).

These results revealed that the quantity and the structure of biofilms of P. lipolytica TC8 were impacted by P starvation at the lowest phosphate concentration used in this study (10 -4 mM).

Discrimination of biofilms of P. lipolytica TC8 depending on the phosphate concentration by LC-MS-based metabolomics

The untargeted LC-(+)-ESI-MS metabolomics approach designed for the analysis of biofilms of P. lipolytica TC8 cultured with different phosphate concentrations produced a first dataset with 728 m/z features. Three drastic filtering steps were applied in order to facilitate the detection and the selection of relevant ions, corresponding to the most abundant and statistically robust metabolites, and to focus the annotation effort on these specific metabolites. At the end of this treatment, only 62 m/z features were kept in the final data matrix (Supplemental Table S1). A first PCA analysis including blanks and QCs was implemented (Supplemental Fig. S2).

First, a clear discrimination between blanks and the other samples was observed on the corresponding PCA score plot. Moreover, QCs were centered near the origin of the PCA score plot and were sparsely scattered, relative to the observed dispersion of biofilm samples, which is in accordance with high quality data [START_REF] Broadhurst | Guidelines and considerations for the use of system suitability and quality control samples in mass spectrometry assays applied in untargeted clinical metabolomic studies[END_REF].

When blank samples were removed from the PCA analysis and only biofilms were considered and plotted (Fig. 2A), the resulting PCA score plot showed a clear distinction between the biofilm samples according to the phosphate concentration used for each bacterial culture. Thus, these samples were discriminated along the first component, which accounted for 32.6 % of the total variance. In accordance with the effect of the lowest phosphate conditions (10 -4 mM) on the amount and the structure of biofilms of P. lipolytica TC8, the samples obtained at this phosphate concentration showed a more marked difference from all other samples along the first component of the PCA.

In order to find chemomarkers linked with the P starvation, a PLS-DA five-class model was built (one class for each phosphate concentration). The corresponding PLS-DA score plot showed a similar trend to the one observed for the PCA score plot (Fig. 2B). The different biofilm samples were distinguished due to the first component (32.3 % of the total variance) from the richest (1.32 mM) phosphate-containing culture medium to the lowest (10 -4 mM). The second component (10.1 % of the total variance) accentuated the difference between biofilms obtained with a phosphate concentration of 10 -3 mM and those from other culture conditions.

The overall quality of the model was evaluated as good through a 10-fold internal crossvalidation giving R 2 , Q 2 and Q 2 /R 2 values greater than 0.5 (R 2 1 comp. = 0.86, Q 2 1 comp. = 0.69 and Q 2 1 comp./R 2 1 comp. = 0.80) while a permutation test (n = 100, p = 0.01) allowed for its validation [START_REF] Triba | PLS/OPLS models in metabolomics: the impact of permutation of dataset rows on the K-fold cross-validation quality parameters[END_REF].

So, the fluctuations of the metabolome of biofilms of P. lipolytica TC8 through the progressive variation of phosphate concentration in the culture medium indicated a progressive and correlated biosynthetic response of bacterial cells.

Annotation of discriminant metabolites

The next step of this study was to annotate the metabolites which were the most differentially expressed by biofilms of P. lipolytica TC8 according to the five different culture conditions.

Through the predictive PLS-DA five-model, the most discriminant m/z features were determined among the whole dataset using their VIP score (Table 2 and Supplemental Table S1).

From the LC-MS/MS data obtained with the biofilm samples of P. lipolytica TC8, a molecular network (MN) was built (Fig. 3). It was constituted by 109 nodes, most gathered in seven main clusters (labelled from A to I), from which 36 were also found in the filtered MS data matrix used for the building of the five-class PLS-DA model (58 % of the metabolites of the matrix appeared in the MN). The differences between the number of nodes detected in the MN and the number of m/z features of the filtered data matrix were due to: (i) the data dependent acquisition parameters used here which did not allow for the weaker ions in a first stage of tandem MS to be analyzed in MS/MS, and (ii) the filtering steps used for the MS data matrix (but not for the MS/MS data from which the MN was built).

When focusing on the ten main VIPs (Table 2) of the five-class PLS-DA model, seven of them were detected in the MN. VIPs n°1, 2, and 4 were found in cluster B, together with two other nodes (Fig. 3). The MS/MS fragmentation pattern of these five m/z features has already been observed in previous studies of this same bacterial strain [START_REF] Favre | Discrimination of four marine biofilm-forming bacteria by LC-MS metabolomics and influence of culture parameters[END_REF][START_REF] Favre | Metabolome and proteome changes between biofilm and planktonic phenotypes of the marine bacterium Pseudoalteromonas lipolytica TC8[END_REF][START_REF] Favre | Metabolomic and proteomic changes induced by growth inhibitory concentrations of copper in the biofilm-forming marine bacterium Pseudoalteromonas lipolytica[END_REF]. These compounds all showed an intense diagnostic fragment ion at m/z 115.09 (C5H11N2O2), as well as other ions at m/z 133 (C5H13N2O2) and 70 (C4H8N) (Supplemental Fig. S3), which are characteristic of amino-acid lipids known as ornithine lipids (OLs) [START_REF] Moore | Elucidation and identification of amino acid containing membrane lipids using liquid chromatography/high-resolution mass spectrometry[END_REF]). The characteristic MS/MS fragmentation of OLs included the sequential loss of (i) H2O from the ornithine part, (ii) the fatty acid chain esterified to the hydroxyl group of the ornithine-linked fatty acid, and (iii) the amide-linked fatty acid [START_REF] Zhang | Characterization of ornithine and glutamine lipids extracted from cell membranes of Rhodobacter sphaeroides[END_REF]). On the basis of this MS/MS fragmentation pattern, VIPs n°1, 2 and 4 were annotated as OL (18:1/18:1), OL (16:0, 16:0) and OL (18:1/16:0), respectively. In the same way, the two other nodes of cluster B were annotated as lyso-OL (18:1) and lyso-OL (16:0). For VIP n°3, a specific tandem MS/MS run specifically targeted on the corresponding ion allowed for an interpretable MS/MS spectrum and the annotation of this VIP as OL (14:0, 18:0).

VIP n°5 was detected in cluster C and revealed a molecular formula (C19H36O4) and a MS/MS fragmentation pattern in accordance with those of a monoacylglycerol bearing a C16:1 fatty acyl chain (Fig. 3, Table 2 and Supplemental Fig. S3) [START_REF] Gao | Monoacylglycerol analysis using MS/MS ALL quadruple time of flight mass spectrometry[END_REF] ). The same detailed analysis of MS/MS data of the other nodes of this cluster allowed for the annotation of another monoacylglycerol derivative and three fatty acids (Fig. 3).

VIPs n° 6 and 7 were included in the largest cluster of the MN (cluster A, Fig. 3). A large part of the nodes constituting this cluster were characterized by a MS/MS spectrum showing the most intense fragment ion obtained following a neutral loss of m/z 141.02 (C2H8NO4P) from the precursor ion (Supplemental Fig. S3). Such a fragmentation pattern is typical of phosphatidylethanolamines (PEs) and corresponds to the loss of the phosphoethanolamine moiety [START_REF] Godzien | Rapid and reliable identification of phospholipids for untargeted metabolomics with LC-ESI-QTOF-MS/MS[END_REF][START_REF] Suárez-García | Development and validation of a UHPLC-ESI-MS/MS method for the simultaneous quantification of mammal lysophosphatidylcholines and lysophosphatidylethanolamines in serum[END_REF]). Thus, VIP n°6 (m/z 624.3879,

[C30H59NO10P] + ) was putatively annotated as the oxidized PE (16:0/9:0, O2) using fragment ([M -C2H8NO4 -C16:0 + H] + ) for the characterization of the fatty acid groups (Table 2). Using the same procedure, the VIP n°7 was annotated as the oxidized PE (16:0/9:1; O), while 12 nodes of the same cluster were annotated as PEs and 10 others as lyso-PEs. Moreover, the six remaining nodes observed in cluster A were annotated as phosphatidylglycerol lipids (PGs) on the basis of a neutral loss of m/z 172.01 from the precursor ion, giving intense fragment ions observed on their MS/MS data [START_REF] Murphy | Mass spectrometric analysis of long-chain lipids[END_REF]. Three additional lyso-PEs were also detected in the MN and constituted cluster I. The fact that these three nodes were not included into the cluster A can be explain by the occurrence of highly unsaturated acyl chains in their chemical structure, which should give specific MS/MS fragment ions. As for VIP n°3, a specific LC-MS/MS run allowed for the fragmentation of the ion corresponding to VIP n°8

and the annotation of this metabolite as the oxidized PE (16:1/9:1; O).

VIP n°10 was observed in cluster G (Fig. 3) and, as with the two other nodes of this cluster, showed a molecular formula and a MS/MS fragmentation pattern in accordance with those of ceramide lipids (Table 2 and Supplemental Fig. S3) [START_REF] Hsu | Complete structural characterization of ceramides as [M-H]ions by multiple-stage linear ion trap mass spectrometry[END_REF].

Other compounds, such as dipeptides or phenylalanine, were also annotated in the MN leading to the final annotation of 53 metabolites belonging to eight chemical families.

Thus, using MN and analysis of MS/MS fragmentation pattern, nine from the ten first VIPs and 32 from the 62 m/z features of the final data matrix were putatively annotated (Table 2, Supplemental Table S1 and Fig. S3).

Membrane lipid adaptation of P. lipolytica TC8 under phosphate-limiting conditions

Depending on the phosphate concentration, the amounts of the most representative VIPs were given as boxplots in Fig. 4. These results revealed that several OLs (VIPs n°1 to 4) and a monoacylglycerol (VIP n°5) were increasingly biosynthesized by the bacterial cells as soon as the phosphate concentration decreased in the culture medium. About 50 % of the sequenced bacteria (mainly Gram-negative bacteria) can biosynthesize OLs and these non-phosphorous glycolipids are found more abundantly in the outer part of bacterial membranes [START_REF] Sohlenkamp | Bacterial membrane lipids: diversity in structures and pathways[END_REF] . In several studies, the occurrence of OLs has been demonstrated to be related to abiotic stresses such as nutrient-limiting conditions (in most cases, phosphate-limiting conditions; e.g. [START_REF] Lewenza | The olsA gene mediates the synthesis of an ornithine lipid in Pseudomonas aeruginosa during growth under phosphate-limiting conditions, but is not involved in antimicrobial peptide susceptibility[END_REF], osmotic stress (e.g. [START_REF] Lopalco | Adjusting membrane lipids under salt stress: the case of the moderate halophilic organism Halobacillus halophilus[END_REF], increased temperatures or acidic conditions [START_REF] Vences-Guzmán | Ornithine lipids and their structural modifications: from A to E and beyond[END_REF]. The role of these aminolipids in non-stress related phenotypes has been also addressed (See in Sohlenkamp 2019). Our findings were thus in accordance with previous studies dealing with the effect of P limitations on bacterial membranes through an increase in the production of non-phosphorous lipids (e.g. [START_REF] Barbosa | Accumulation of ornithine lipids in Vibrio cholerae under phosphate deprivation is dependent on VC0489 (OlsF) and PhoBR system[END_REF][START_REF] Benning | Accumulation of a novel glycolipid and a betaine lipid in cells of Rhodobacter sphaeroides grown under phosphate limitation[END_REF][START_REF] Geiger | Amino acidcontaining membrane lipids in bacteria[END_REF][START_REF] Lewenza | The olsA gene mediates the synthesis of an ornithine lipid in Pseudomonas aeruginosa during growth under phosphate-limiting conditions, but is not involved in antimicrobial peptide susceptibility[END_REF].

Consistent with these first findings, three PEs (VIPs n°6 to 8) were under-expressed when the phosphate concentration decreased. Surprisingly, these three metabolites are oxidized PEs, while all the other annotated "non-oxidized" phospholipids (i.e. PEs, lyso-PEs, PGs and lyso-PGs) of the final dataset were not differentially expressed depending on the different phosphate concentrations. These results agreed with a previous study dealing on P. lipolytica TC8 cultures in non-limited phosphate conditions which has highlighted that PEs were mainly produced in biofilms rather than in planktonic cultures [START_REF] Favre | Metabolome and proteome changes between biofilm and planktonic phenotypes of the marine bacterium Pseudoalteromonas lipolytica TC8[END_REF]. They differed from several other studies which have described an important shift in the bacterial membrane composition, leading to a decrease production of phospholipids, such as PEs, and their subsequent substitution with non-phosphorous lipids, such as OLs, under P-limiting conditions (i.e. [START_REF] Sandoval-Calderón | Plasticity of Streptomyces coelicolor membrane composition under different growth conditions and during development[END_REF].

The fact that only oxidized PEs were impacted by phosphate concentration was quite interesting given that oxidized phospholipids have been attracting more and more interest over the past few years, especially in the medical field [START_REF] Nie | The role of oxidized phospholipids in the development of disease[END_REF]). In the case of membranes, the occurrence of oxidized phospholipids can drastically affect their properties and their organization [START_REF] Tyurina | Redox (phospho)lipidomics of signaling in inflammation and programmed cell death[END_REF].

Conclusion

The objective of this work was to evaluate the impact of phosphate-limiting conditions on biofilms of the bacterial strain P. lipolytica TC8 which had already shown its ability to biosynthesize alternative non-P analogues to phospholipids [START_REF] Favre | Discrimination of four marine biofilm-forming bacteria by LC-MS metabolomics and influence of culture parameters[END_REF][START_REF] Favre | Metabolome and proteome changes between biofilm and planktonic phenotypes of the marine bacterium Pseudoalteromonas lipolytica TC8[END_REF]. Despite a moderate effect of various environmentally-relevant phosphate concentrations on the biomass, the gross polymeric composition and the structure of the biofilms of P. lipolytica TC8 formed in vitro, an untargeted LC-MS-based metabolomics analysis revealed clear differences at the metabolome level. Combining drastic filtering steps and annotation through MN, this metabolomics approach focusing on a few abundant and statistically robust annotated metabolites demonstrated that phosphate concentration generated significant changes in the chemical composition of membranes with higher amounts of the non-phosphorous OLs at low phosphate concentrations. Conversely, a decrease of oxidized PEs was observed in the same culture conditions, while the biosynthesis of non-oxidized PEs remained globally unchanged.

Together with and in addition to previous works [START_REF] Favre | Discrimination of four marine biofilm-forming bacteria by LC-MS metabolomics and influence of culture parameters[END_REF][START_REF] Favre | Metabolome and proteome changes between biofilm and planktonic phenotypes of the marine bacterium Pseudoalteromonas lipolytica TC8[END_REF][START_REF] Favre | Metabolomic and proteomic changes induced by growth inhibitory concentrations of copper in the biofilm-forming marine bacterium Pseudoalteromonas lipolytica[END_REF], all results agreed with the fact that P. lipolytica TC8 testified to a certain plasticity of membrane lipid composition in response to phenotypic and environmental changes. From a taxonomic perspective, such results are not particularly obvious because Pseudoalteromonas spp, and more broadly -proteobacteria, are not known for their ability to produce aminolipids such as ornithine lipids [START_REF] Sohlenkamp | Bacterial membrane lipids: diversity in structures and pathways[END_REF]. The analysis of

In the marine environment, bacteria must be able to adapt their membranes to maintain their biological functions. As environmental conditions are multiple and changing, associating a modification of the cytoplasmic membrane with an environmental condition is complicated in marine environment. Here, the fact of studying a single fluctuation allowed us to describe the impact of phosphate deficiency on the adaptation of the membrane of a marine bacterial strain cultivated in biofilm. This mechanism might be a selective advantage for bacteria present in environments experiencing rapid and massive P concentration changes, such as those found in estuaries [START_REF] Li | Spatiotemporal variability in phosphorus species in the Pearl River estuary: Influence of the river discharge[END_REF][START_REF] Lochet | Transfers between a eutrophic ecosystem, the river Rhône, and an oligotrophic ecosystem, the north-western Mediterranean Sea[END_REF]. Previous works have shown that bacterial OLs biosynthesis could increase under various stress conditions (e.g. temperature, pH) [START_REF] Vences-Guzmán | Ornithine lipids and their structural modifications: from A to E and beyond[END_REF]). So, it cannot be excluded that the lipid remodelling observed in this study was a response of P. lipolytica TC8 common to all types of stress and this question could be an avenue for further work.

Further research should also focus on the specific roles of the oxidized PEs described here and on the full characterization of their chemical structure through targeted natural products chemistry and annotation using MN (Carriot et al. 2021). Moreover, the coupling of such a metabolomics approach with other omics and their application to natural biofilms sampled in marine environments with P-contrasted conditions might be useful to better understand the adaptative strategies developed by marine bacteria upon P deficiency.

(

  mass values ranging from 3 to 7 mg) were solubilized in 1 mL of LC-MS grade methanol (Fisher, Loughborough, UK). A pool sample solution was prepared taking 100 μL of each sample (biofilms and medium blanks). The resulting solution was divided into several HPLC vials that were used as quality-control samples (QCs). At the beginning of the injection sequence, two analytical blanks (methanol only) and a QC sample (×10) were injected prior to the samples. Biofilms and medium blanks were then randomly injected to avoid any possible time-dependent changes in LC-MS chromatographic fingerprints while a QC sample was injected every five samples. Two analytical blanks (methanol only) and a QC sample were then injected at the end of the sequence run.Sample were analyzed by UHPLC-ESI-QToF-MS in positive mode. The UHPLC-MS instrumentation consisted of a Dionex Ultimate 3000 Rapid Separation (Thermo Fisher Scientific, Sunnyvale, CA, USA) chromatographic system equipped with a RS pump, a temperature-controlled autosampler, a thermostated column compartment and an UV-Vis diode array detector. This system was coupled via an ESI interface to a QToF Impact II mass spectrometer (Bruker Daltonics, Champs-sur-Marne, France). The separations were carried out on a reverse phase column (150  2.1 mm, 1.7 μm, Kinetex Phenyl-Hexyl; Phenomenex, Le Pecq, France) equipped with a pre-column (SecurityGuard cartridge; Phenomenex). The column oven temperature was maintained at 40 °C and the flow rate was set at 0.5 mL/min. The autosampler temperature was set at 4 °C and the injection volume was 5 μL. Mobile phases were: (A) water and (B) acetonitrile (LC-MS grade; Carlo Erba Reagents) containing each 0.1 % (v/v) of formic acid (Ultra grade Fluka; Sigma-Aldrich, St Quentin-Fallavier, France).

  build a molecular network (MN). The Global Natural Products Social Molecular Networking online workflow (GNPS web platform; http://gnps.ucsd.edu) was used to create a MN of related compounds based on the similarity of their tandem MS fragmentation patterns[START_REF] Wang | Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking[END_REF]. The principle of MN was based on the fact that molecules with similar chemical structures share similar MS/MS fragmentation pathways leading to common fragment ions and neutral losses. From MS/MS spectra, the creation of a MN allowed for: (i) the identification of clusters of related compounds, (ii) the detection of a set of structural analogues of a reference molecule, and (iii) the comparison of the metabolic production of different samples. The parameters used are: precursor ion mass tolerance (Da): 0.02, fragment ion mass tolerance (Da): 0.02, minimum cosine score: 0.70, minimum matched fragment ions: 6 and network TopK: 10.

  ions at m/z 313.2732 [C19H37O3] + ([M -C2H8NO4 -C9:0(2O) + H] + ) and 245.1381 [C12H21O5] +

Table 2 .

 2 List of the first ten biomarkers (VIPs) putatively annotated by LC-MS for the chemical discrimination of biofilm extracts of P. lipolytica TC8 according to the phosphate concentration.

Figure 1 .

 1 Figure 1. Effect of phosphate concentrations on biofilms of P. lipolytica TC8 in terms of

Figure 2 .

 2 Figure 2. PCA (2A) and PLS-DA (2B) score plots obtained from (+)-ESI-LC-MS profiles of

Figure 3 .

 3 Figure 3. Molecular network built with (+)-ESI-LC-MS/MS data of extracts of biofilms of P.

Figure 4 .

 4 Figure 4. Normalized concentrations of the eight metabolites with the highest VIP scores

Acknowledgements

The authors would like to acknowledge Dr S. Greff (Aix-Marseille University, IMBE, France) for the acquisition of LC-MS profiles and for the helpful discussions, and J. Dechaux (Université de Toulon, MAPIEM) for her help during the culture of biofilms and their subsequent extraction and analysis. The authors are also grateful to S.

Ortalo-Magné for her careful English language editing of the manuscript.

Funding

This work was funded by the French Ministry of Higher Education, Research and Innovation and the University of Toulon (PhD. grant of N. Carriot). LC-MS experiments were conducted on the regional metabolomics platform MALLABAR funded by the Institute of Ecology and Environment (INEE) of the French National Centre for Scientific Research (CNRS), the Total Foundation and the French Sud-PACA regional council.

Data availability

The metabolomics and metadata reported in this paper are available as supplementary material (Table S1). Raw data for LC-ESI-(+)-MS/MS experiments were deposited and are publicly available on the MassIVE platform under the IDs MSV000083860.

Author contributions

JFB, AOM and GC conceived and designed research. NC and RBM conducted experiments. NC, AOM and GC analyzed data. NC, JFB, AOM and GC wrote the manuscript. All authors read and approved the manuscript.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.