Maxime Suroy
email: maxime.suroy@univ-amu.fr.

Brivaëla Moriceau

Boutorh Julia

Madeleine Goutx

Fatty acids associated with the frustule of diatoms and their fate during degradation -a case study in Thalassiosira weissflogii

Keywords: Frustule, Diatoms, Organic Carbon, Lipids, Carbon Export

Diatoms are major actors in the export of organic carbon out of the euphotic zone. Yet, the processes linking biogenic silica and carbon sedimentation fluxes to deep oceanic layers remain unclear. Analysing organic fractions in biominerals is challenging because efficient cleaning often led to structural alteration of organic molecules. Hence, although lipids are widely used as biogeochemical markers in ocean flux study, few studies have dealt with the lipids that are associated with frustules. In the present study, a protocol was set up to extract and quantify the fatty acids associated to the frustule of the diatom species Thalassiosira weissflogii. The protocol involves solvent extraction of diatom external lipids, followed by clean frustule dissolution by 4% NaOH during 1h at 95°C and subsequent solvent reextraction of frustule-associated lipids. Results confirmed that this protocol was efficient first, to isolate the frustule from the rest of the cellular organic carbon and second to extract and quantify fatty acids (FA) associated to frustules of this species. FA composition of the frustules was significantly different from that of the whole cells consisting primarily of 14:0, 16:0 and 18:0 FA, as well as a smaller portion of 16:1 and 18:1 unsaturated FA. Frustuleassociated FA constituted 7 % of the total FA and 1.8 % of the total POC. The 30 days T.

weissflogii degradation/dissolution experiment suggested that frustule FA 14:0 and 16:0 were mainly associated with the bSiO 2 phase dissolving slowly as no degradation of this pool was measured despite 78 % frustule dissolution. At the end of the degradation experiment, this pool constituted 5.8 % of the remaining total POC suggesting an effective protection by the frustule through strong interaction with the organic matrix which is consistent with the correlation observed at depth between Si and POC sedimentation fluxes.

Introduction

Diatoms are characterised by a silicified cell wall, commonly known as the frustule.

This frustule partially consists of amorphous silica that forms mineral structures by precipitation of orthosilicic acid [START_REF] Martin-Jézéquel | Silicon metabolism in diatoms : implications for growth[END_REF]. Such a structure presents great evolutionary advantages to cells, such as turgor resistance and protection against predation [START_REF] Raven | The evolution of silicification in diatoms: inescapable sinking and sinking as escape?[END_REF] and reference therein), or optical properties [START_REF] Kucki | Biological Photonic Crystals: Diatoms[END_REF], partially explaining the success of diatoms and the presumed number of diatom species (more than 200 000, [START_REF] Mann | Biodiversity, biogeography and conservation of diatoms[END_REF]. This success is illustrated by diatom's predominant role in primary production in the ocean [START_REF] Falkowski | Biogeochemical controls and feedbacks on ocean primary production[END_REF] but also in the export of organic matter (OM) to the ocean depths [START_REF] Nelson | Production and dissolution of biogenic silica in the ocean : Revised global estimates , comparison with regional data and relationship to biogenic sedimentation[END_REF][START_REF] Volk | Ocean carbon pumps: Analysis of relative strengths and efficiencies in ocean-driven atmospheric CO2 changes[END_REF]. Diatom frustule-bound organic compounds are diverse. The formation of the frustule starts within silicon deposition vesicles and requires a peculiar association between organic compounds and silicon [START_REF] Hildebrand | Diatoms, biomineralization processes, and genomics[END_REF]. Many studies have demonstrated the role of proteins and polyamines during the precipitation of silica in diatoms [START_REF] Brunner | Analytical studies of silica biomineralization: towards an understanding of silica processing by diatoms[END_REF]. The presence of sugars closely associated with frustule is also confirmed in recent studies, but their role in the biomineralisation process remains unclear [START_REF] Chiovitti | Variations in the substituted 3-linked mannans closely associated with the silicified walls of diatoms[END_REF][START_REF] Tesson | Characterization and localization of insoluble organic matrices associated with diatom cell walls: Insight into their roles during cell wall formation[END_REF]. In contrast, because they are not assumed to be functionally critical to the formation of the biogenic silica, little is known about the potential association of lipids in frustules.

Diatoms [START_REF] Fileman | Vertical profiles of pigments, fatty acids and amino acids: Evidence for undegraded diatomaceous material sedimenting to the deep ocean in the Bellingshausen Sea, Antarctica[END_REF] and frustule-bound organic compounds [START_REF] Bridoux | Structural identification of long-chain polyamines associated with diatom biosilica in a Southern Ocean sediment core[END_REF][START_REF] Bridoux | Widespread distribution and molecular diversity of diatom frustule bound aliphatic long chain polyamines (LCPAs) in marine sediments[END_REF][START_REF] Hedges | Evidence for non-selective preservation of organic matter in sinking marine particles[END_REF][START_REF] Ingalls | Tinted windows: The presence of the UV absorbing compounds called mycosporine-like amino acids embedded in the frustules of marine diatoms[END_REF] provide organic matter to the sediment. Compilation of deep sedimentation fluxes of POC and minerals [START_REF] Armstrong | A new, mechanistic model for organic carbon fluxes in the ocean based on the quantitative association of POC with ballast minerals[END_REF][START_REF] Buesseler | The decoupling of production and particulate export in the surface ocean[END_REF][START_REF] De La Rocha | Factors influencing the sinking of POC and the efficiency of the biological carbon pump[END_REF] show correlations between those fluxes. More specifically, deep sedimentation fluxes of particulate organic carbon (POC) and biogenic silica (bSiO 2 ) are also correlated when expressed basin by basin [START_REF] Ragueneau | Si and C interactions in the world ocean: Importance of ecological processes and implications for the role of diatoms in the biological pump[END_REF]. Using an independent data base, Ragueneau and co-workers (2002) showed that the evolution of the Si/C in all areas of the ocean can be predicted using an empirical relationship. In other words, the sinking of bSiO 2 and POC are linked in such a manner that knowing Si/C in the surface layer, one can theoretically extrapolate the flux of POC from the flux of bSiO 2 . But what processes could explain such a link between the preserved fraction of organic matter and the bSiO 2 ?

The preservation of a fraction of organic matter during the sedimentation through the water column to the sediment depends on the balance between sinking and degradation processes. Biominerals in general and bSiO 2 in particular could impact both processes. As suggested by the ballast ratio hypothesis [START_REF] Armstrong | Settling velocity spectra and the ballast ratio hypothesis[END_REF][START_REF] Armstrong | A new, mechanistic model for organic carbon fluxes in the ocean based on the quantitative association of POC with ballast minerals[END_REF][START_REF] Klaas | Association of sinking organic matter with various types of mineral ballast in the deep sea: Implications for the rain ratio[END_REF], the sinking of organic matter ballasted by dense biominerals be it as shells or once incorporated to the same large particles like aggregates or fecal pellets [START_REF] Thornton | Diatom aggregation in the sea: mechanisms and ecological implications[END_REF][START_REF] Turner | Zooplankton fecal pellets, marine snow and sinking phytoplankton blooms[END_REF] should be faster than the sinking of non-ballasted organic matter. Moreover bSiO 2 may also slow down the degradation as some studies hypothesized that the diatom frustule surrounding the cellular organic matter could protect the internal pool against bacterial degradation [START_REF] Goutx | Composition and degradation of marine particles with different settling velocities in the northwestern Mediterranean Sea[END_REF][START_REF] Moriceau | Si-C interactions during degradation of the diatom Skeletonema marinoi[END_REF]. But these studies were undertaken on living diatoms and this hypothesis is not confirmed by degradation studies made on dead diatoms [START_REF] Soler | Impact of nutrient starvation on the biochemical composition of the marine diatom Thalassiosira weissflogii: from the whole cell to the frustule fraction[END_REF].

Other studies have evidenced the presence of another organic pool intimately linked to the frustule that could potentially explain the correlation between organic carbon and bSiO 2 . This fraction of organic matter preserved at great depths is proportional to the flux of biominerals [START_REF] Armstrong | Settling velocity spectra and the ballast ratio hypothesis[END_REF][START_REF] Armstrong | A new, mechanistic model for organic carbon fluxes in the ocean based on the quantitative association of POC with ballast minerals[END_REF][START_REF] François | Factors controlling the flux of organic carbon to the bathypelagic zone of the ocean[END_REF][START_REF] Klaas | Association of sinking organic matter with various types of mineral ballast in the deep sea: Implications for the rain ratio[END_REF][START_REF] Ragueneau | Si and C interactions in the world ocean: Importance of ecological processes and implications for the role of diatoms in the biological pump[END_REF][START_REF] Ragueneau | Si/C decoupling in the world ocean: is the Southern Ocean different? Deep Sea Research Part II[END_REF]. In our study, we hypothesized that the preserved organic matter can be partially formed by the pool of lipids associated to or embedded in the silica matrix.

The presence of an organic coating has been so far mainly deduced from bSiO 2 dissolution experiments on whole versus cleaned frustule [START_REF] Bidle | Accelerated dissolution of diatom silica by marine bacterial assemblages[END_REF][START_REF] Kamatani | Rate of dissolution of diatom silica walls in seawater[END_REF][START_REF] Patrick | The effect of bacteria on the solubilization of silica in diatom frustules[END_REF]. From the experimental results described in the study by [START_REF] Bidle | Accelerated dissolution of diatom silica by marine bacterial assemblages[END_REF], it seems that this pool is removed by bacteria within hours or few days after the death of diatoms. Only few diatoms can reach the sediment with an intact organic coating as shown by a minimum of 50 % dissolution of the bSiO 2 observed in most oceanic sites. The frustule-embedded OM is more difficult to assess as the chemical reagents known to dissolve the silica (NaOH, Na 2 CO 3 and HF) generally react with the targeted polymer organic compounds. The inclusion of proteins in the silica matrix and their implication in silicification have been well-investigated [START_REF] Hildebrand | Biological processing of nanostructured silica in diatoms[END_REF][START_REF] Hildebrand | Diverse and conserved nano-and mesoscale structures of diatom silica revealed by atomic force microscopy[END_REF][START_REF] Kröger | Self-assembly of highly phosphorylated silaffins and their function in biosilica morphogenesis[END_REF][START_REF] Kröger | Polycationic peptides from diatom biosilica that direct silica nanosphere F formation[END_REF][START_REF] Kröger | Characterization of a 200-kDa diatom protein that is specifically associated with a silica-based substructure of the cell wall[END_REF][START_REF] Kröger | A new calcium binding glycoprotein family constitutes a major diatom cell wall component[END_REF]. The presence of other molecules less directly involved on silicification processes, such as lipids or sugars, has been much less studied. [START_REF] Kates | Studies on the biochemistry and fine structure of silica shell formation in diatoms. Lipid components of the cell walls[END_REF] described the lipid composition of the cell wall for the first time after breaking and washing the cells. To our knowledge, this study remains the only one dealing at the molecular level with lipids associated to diatom frustules. Recent works studied this organosilicon structure and noted the importance of lipid-like compounds embedded in the frustule of diatoms using spectroscopic methods [START_REF] Soler | Impact of nutrient starvation on the biochemical composition of the marine diatom Thalassiosira weissflogii: from the whole cell to the frustule fraction[END_REF][START_REF] Tesson | Contribution of multi-nuclear solid state NMR to the characterization of the Thalassiosira pseudonana diatom cell wall[END_REF].

These global methods that avoid dissolution procedures and the potential denaturation of most organic molecules, confirm the presence of OM associated with the frustule but did not reveal details about the structure and amounts of the classes of compounds detected.

In the present work, our approach combined the procedures of [START_REF] Ragueneau | Determination of biogenic silica in coastal waters: applicability and limits of the alkaline digestion method[END_REF] and [START_REF] Chiovitti | Variations in the substituted 3-linked mannans closely associated with the silicified walls of diatoms[END_REF] and enabled the complete dissolution of the frustules and the study of released FA. The protocol was developed on the diatom Thalassiosira weissflogii and a 30-day dissolution/degradation experiment was carried out first to validate our protocol and second to understand the fate of frustule-associated lipids during biodegradation.

Material and Methods

Culture conditions and sampling

The strain Thalassiosira weissflogii (CCMP n°1049) was provided by the National Centre for Marine Algae and Microbiota. Three cultures (A, B and C cultures) were grown in f/2 medium under a 14/10 h light/dark cycle at 18 °C in 2 l glass flasks [START_REF] Guillard | Studies of marine planktonic diatoms : I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran[END_REF] in sterile conditions. Culture C was grown in another growth chamber under similar growth conditions (temperature, light intensity and cycles). Differences in the carbon content of cultured cells were observed (15.6 ± 0.2, 15.6 ± 0.1 and 10.9 ± 0.1 pmol.cell -1 in cultures A, B and C, respectively). The cell concentration was determined by flow cytometry (FACS Calibur, BD biosciences®).

Samples were collected as soon as cultures reached the stationary phase (~10 5 cell.ml -1 and 15 days of growth). Triplicate of 10 ml of culture were sampled in cultures B and C for the 'whole cells' analysis and filtered on pre-combusted GF/F filters (0.7-µm mesh size) as generally done in other studies (Table 1). Lipids from whole cells were extracted according to the Bligh and Dyer protocol (1959) and thus do not include the frustule-associated fraction.

Culture A was not sampled for lipid composition analysis in whole cells. The carbon content of cells in batch A was similar to that of cells in batch B, suggesting composition similarity between A and B cultures. Except the 20:5(n-3) FA (see Fig. 4 below), this latter assumption was supported by the low SD of average percentages of individual FA in total FA, derived from the analysis of FA in triplicate samples from both B and C cultures. To obtain the 'pellet' samples, triplicates of 50 ml of the three cultures were centrifuged at 5400 g (20 min).

Frustule isolation

Pellet samples were resuspended in 40 ml milliQ water and were hot-sonicated at 70 °C for 5 min to fragment the cells (Fig. 1). Tubes were subsequently centrifuged at 5400 g for 40 min at 4 °C. The supernatants were removed, and this step was repeated once at lower centrifugation speed (3800 g for 20 min) to eliminate any non-frustule material. After removing the supernatant, the pellets were again resuspended in 40 ml milliQ water and centrifuged at 3200 g for 20 min. This treatment cleared the frustules from most cell content.

However, fluorescence of chlorophyll molecules was noticeable on flow cytometer analysis of the washed pellets indicating external organic material still present on frustule fragments (Fig. 2).

In a second step, this external organic material probably more intimately linked to the frustules than the previous cell content, was separated from fragmented frustules using a modified protocol of Bligh and Dyer. Briefly, the washed pellets were transferred to glass tubes. A monophasic mixture of dichloromethane/methanol/1M salt water (1/2/0.8, v/v/v) (9.5 ml) was added for the lipid extraction. After the addition of an internal standard (nonadecanoic acid methyl ester, Me19:0, Sigma Aldrich®) and sonication (10 min), tube contents were transferred to separatory funnels. Tubes were rinsed with 9.5 ml of the monophasic mixture. The separatory funnel contents were made biphasic by adding 5 ml dichloromethane (DCM) and 5 ml milliQ water. After vigorous shaking and settling, the organic phases were collected, and the aqueous phases were re-extracted with 10 ml DCM.

The organic phases were combined and concentrated under a gentle flux of nitrogen. The lipid extracts such obtained from the pellets of isolated frustules constituted our lipid 'external' fractions.

Extraction of frustule-associated lipids

Frustules cleaned from their 'external' fraction were visualised by scanning electronic microscopy during the cleaning procedure to check for their integrity and the absence of 'external' organic matter at the end of this step. Frustule fragments formed a white layer at the basis of the aqueous phase (methanol/water mixture) remaining in the separatory funnel after the Bligh and Dyer extraction (Fig. 1). Scanning electron microscopy analysis of this white layer samples showed an amalgam of clean silica frustules (Fig. 3). Hydrophilic compounds that may be present in the aqueous phase were not analysed further. The white layer and the top aqueous phase were transferred into glass bottle in which sodium hydroxide (1 mol.l -1 final concentration) was added. These bottles were later transferred to a water bath at 95 °C for 1 h to promote the entire dissolution of frustules. The basic reactant not only dissolves the silica matrix but can cleave covalent associations between FA and polymers. After a rapid cooling on ice, the bottle contents were acidified using HCl and re-extracted in a liquid-liquid phase using 50 ml DCM (the aqueous phase was approximately 70 ml). This last extraction step was repeated three times to ensure the complete extraction of the remaining lipids in the aqueous phase. Extracts thus obtained were called the 'frustule-associated' fractions. The internal standard (Me19:0) was not added before dissolved frustule extraction in order to control the absence of contamination in the 'frustule-associated' extract from the 'external' fraction. Consequently, the same extraction recovery was used for the quantification of both 'external' and 'frustule-associated' lipids.

Biodegradation experiment

For the degradation experiment, the T. weissflogii strain was supplied and grown in a large volume culture by the Experimental Station of Argenton (Ifremer, France). Cells were pelleted and killed by freezing at -20 °C for 4 days. Pellets were resuspended in a Nalgene® flask filled with 10 l of 0.7-µm filtered seawater from Brest Bay (France). The 0.7-µm filtered water contained the natural prokaryotic community required to degrade and utilize the algal material as growth substrate. The flask was incubated on an orbital shaker table at 70 rpm in the dark at 18 °C. The degradation experiment lasted 30 days. Samples (50 ml) were collected from the flask every 6 days, in duplicate or triplicate, and subjected to centrifugation for frustule isolation and lipid extraction as described in the previous section. Briefly, cells were sonicated, pelleted to remove medium and extracted according to Bligh and Dyer for analysis of external FA composition. Cleaned frustules were submitted to NaOH dissolution and reextracted for analysis of frustule-associated FA fraction.

Bulk parameters: bSiO 2 , POC and prokaryotic concentrations

bSiO 2 and POC concentrations were followed during the whole degradation experiment by filtering 10 ml of the batch content on polycarbonate (0.4-µm mesh size) and GF/F (0.7-µm mesh size) filters, respectively. bSiO 2 concentrations were measured using a variation of the method of [START_REF] Ragueneau | Determination of biogenic silica in coastal waters: applicability and limits of the alkaline digestion method[END_REF] described in [START_REF] Moriceau | Si-C interactions during degradation of the diatom Skeletonema marinoi[END_REF]. Filters were digested in 20 mL of 0.2 M NaOH for 3 hours at 95°C and stirred regularly to ensure the dissolution of all bSiO 2 . As no lithogenic silica was present in the diatom culture, only one longer digestion step was done. The solution was then acidified with 5mL of HCl 1M, centrifuged to remove solids and analysed for silicic acid concentrations (dSi). dSi was measured using the molybdate blue spectrophotometric method of [START_REF] Mullin | The colorimetric determination of silicate with special reference to sea and natural waters[END_REF], as adapted by [START_REF] Tréguer | Manuel d'analyse des sels nutritifs dans l'eau de mer[END_REF] and modified by [START_REF] Gordon | A suggested protocol for continuous flow automated analysis of seawater nutrients (phosphate, nitrate, nitrite and silicic acid) in the WOCE Hydrographic Program and the Joint Global Ocean Fluxes Study. WHP Operations and Methods[END_REF] for use in segmented flow colorimetry. We used a Bran and Luebbe Technicon Autoanalyser (<1% precision).POC filters were desiccated overnight in an oven at 50 °C, and POC was quantified by using a Carlo Erba NA 2100 CN analyser coupled to a Finnigan Delta S mass spectrometer.

Prokaryotes were counted over time in 10 ml samples to assess their growth. Free and attached prokaryotes were separated by filtration (3-µm mesh size) and counted on black 0.2µm filters after staining with DAPI (4',6-diamidino-2-phenylindole).

Prokaryotic carbon in degradation flask samples was calculated by using conversion factors (20 and 50 x 10 -15 molC.cell -1 for free and attached prokaryotes, respectively) according to Turley and Mackie (1994) for the north-eastern Atlantic Ocean.

Lipid analysis

Derivatisation and purification.

Lipid extracts were trans-esterified using BF 3 -Methanol and toluene for 1 h at 70 °C.

Fatty acid methyl esters (FAME) were extracted from the mixture with 3 x 2 ml of Hexane/Ethyl Ether (98/2, v/v) and evaporated to dryness under nitrogen. FAME were purified onto a bond elute microcolumn with a mix of Hexane-Ethylacetate (99/1, v/v).

Columns were conditioned with 9 ml Hexane-Ethyl Acetate (99/1, v/v) followed by 3 ml Hexane. Columns were rinsed with 1.4 ml Hexane (to remove hydrocarbons), and FAMEs were eluted by 1.6 ml Hexane-Ethyl Acetate (99/1, v/v).

Extracts from the degradation experiment were first saponified (KOH 1 mol.l -1 for 1 h at 70 °C) and were silylated using a N,O-bis(trimethysilyl)trifluoroacetamide (BSTFA)pyridine mixture according to [START_REF] Rontani | Degradation of particulate organic matter in the equatorial Pacific Ocean: Biotic or abiotic?[END_REF]. Briefly, after saponification, BSTFA and pyridine were added to the dried lipid extract (1/2, v/v). Tubes were placed at 50 °C for 1 h, and then their contents were dried under a gentle stream of nitrogen and resuspended in ethyl acetate and BSTFA. This reaction transforms OH-containing molecules to their trimethylsilyl (TMS) derivatives. Injections of trimethylsilyl ether (TMSE) were made in the 12 hrs following derivatisation due to their unstable nature. This method allowed us to analyse FA, sterols and phytol simultaneously in their TMS ether forms. Indeed, the phytil side chain of chlorophyll is released during saponification (phytol) and transformed to its TMS derivative.

Sterols are also present in diatoms, primarily in a free form (i.e., non-esterified, [START_REF] Volkman | Lipids in marine diatoms of the genus Thalassiosira : Predominance of 24-methylenecholesterol[END_REF]. Four sterols [START_REF] Volkman | Lipids in marine diatoms of the genus Thalassiosira : Predominance of 24-methylenecholesterol[END_REF] were identified, but we combined them in this study. Sterols were quantified by reference to a calibration curve of a cholesterol standard. The quantification of phytol was semi-quantitative using a calibration curve of TMS20:0.

The impact on PUFA of the step involving heating the silica/organic matter complex was tested. The percentage recoveries of arachidonic acid (20:4(n-6)) after incubation for one hour both at 40 °C and 70 °C were in the range of the RSD. Other authors also reported saponification of lipid extracts at 100 °C without noticeable effect on poly-unsaturated FA quantification [START_REF] Chen | Influence of iron on fatty acid and sterol composition of marine phytoplankton and copepod consumers[END_REF][START_REF] Goutx | Composition and degradation of marine particles with different settling velocities in the northwestern Mediterranean Sea[END_REF].

GC/MS analysis.

FAME and TMSE were analysed by gas chromatography/mass spectrometer (GC/MS) (TraceISQ, ThermoElectron) at an ionization energy of 70 eV for m/z range of 50-400 for FAME and 50-600 for TMSE, using hydrogen as carrier gas at a flow rate of 1.2 ml.min -1 .

The injector (used in splitless mode) and detector temperatures were 250 and 320 °C respectively. For FAME analysis, the initial column temperature was held for 2 min at 70 °C then ramped at 12 °C.min -1 (ramp 1) to 140 °C, and then at 5 °C.min -1 (ramp 2) to 200 °C, which was held for 5 min, then finally ramped at 12 °C.min -1 (ramp 3) to 275 °C and held for 10 min. FA, sterols and phytol from the degradation experiment were analysed in their TMS forms using the same conditions as FAME analysis on the same GC/MS. The ramp temperature only was modified as follow: the initial temperature was set at 70 °C and successively increased to 130 °C at 20 °C.min -1 , then to 250 °C at 5 °C.min -1 and finally at 3 °C.min -1 to 300 °C, held for 40 min. The source temperature was set at 200 °C. FAME and TMSE were identified by comparison with retention time and mass spectra of commercial standards (provided by Sigma Aldrich®) and quantified by reference to calibration curves.

The positions of the double bounds were all identified with the exception of that of 16:1, 16:2 and 24:1 for which we did not run standards. All determination coefficients were up to 0.99.

Tricosanoic acid methyl ester standard (Me23:0) was introduced prior to injection to check analytical variability. The mean analytical recovery, taking into account all preparative steps except dissolution, was in the range of the Relative Standard Deviation (4 to 9 % for peak areas and from 0.04 to 0.11 % for the retention time) and was approximately 83 %. As we did not add any standard to the frustule-bound fraction, internal standard (19:0 in its trimethylsilyl or methyl form) was used to check whether any cellular material (that contained the internal standard) may have contaminated the 'frustule-bound' fraction. All concentration values were blank and recovery corrected.

Flow cytometry.

Samples were analysed using the FACSCalibur (BD Biosciences ® ) of the PRECYM flow cytometry platform (http://precym.com.univ-mrs.fr) equipped with a blue (488 nm) aircooled argon laser and a red (634 nm) diode laser. Data were collected using the CellQuest software (BD Biosciences ® ). The analysis was performed a posteriori using SUMMIT v4.3 software (Beckman Coulter). T. weissflogii cells were optically resolved based on their intensities in light scatter and red fluorescence (chlorophyll a related). TruCount beads (BD Biosciences ® ) and 2-µm beads (Fluoresbrite YG, Polyscience ® ) were added to the samples as an internal standard to monitor the instrument stability and to determine the volume analysed by the instrument. Flow cytometry analysis was used to test frustule isolation and to measure prokaryotic presence in samples without additional prokaryotic counting.

Statistical analysis.

All analyses were performed with R (R Core Team, 2012), a freeware for statistical analysis available at http://cran.r-project.org/. A Mann-Whitney U test was also used to test the hypothesis H 0 that there is no difference between FA concentration averages during the degradation experiment. We fixed the significance level at 5 %.

To compare the FA profiles of 'whole cells', 'extrenal' and 'frustule-associated' fractions, a classical multidimensional scaling (MDS) was used on an Euclidean distance matrix of FA standardised concentrations. Using k-means clustering, we partitioned our samples into three groups. The number of clusters in this analysis must be defined by the analyst. We choose three clusters because we have three sample types and also because it was the result of the estimation by optimum average silhouette width using the fpc package in the R software [START_REF] Christian | fpc: Flexible procedures for clustering[END_REF]. Confident ellipses at 50 and 95 % were added to the plot to represent results from cluster analysis and to comfort MDS observations.

Results

FA composition of Thalassiosira weissflogii

Whole cells (WC) fractions from two different cultures (B and C) had similar FA compositions. The average composition (n = 6) was also similar to literature data on the same genus (Table 1) and did not show obvious differences despite various methodologies used in previous studies (Table 1). The composition of WC was dominated by palmitic acid 16:0 (26.4 ± 3.4 %, n = 6) (Table 1). The other dominant FA were those classically found in diatoms: palmitoleic acid (16:1(n-7)) and eicosapentaenoic acid (20:5(n-3) or EPA) (18.3 ± 4.3 % and 22.5 ± 9.6 %, respectively). A characteristic feature was the dominance of 16carbon atom unsaturated FA compared to 18-carbon atom unsaturated FA (38.0 % versus 2.7 %, respectively) (Table 1). Also, 20:0 and 22:0 saturated FA were found in notably low amounts (their sum was < 0.5 %), but their presence was confirmed by the analysis of their mass spectra. Traces of heptadecanoic acid (17:0) and pentadecenoic acid (15:1) represented less than 0.1 % of the total FA in WC. Those FA are generally attributed to prokaryotes (Kaneda, 1991). Considering their low contribution to the total composition, the share of these FA most likely reflected the presence of few bacteria. This scarcity was confirmed by flow cytometry, which showed notably few microorganisms other than diatoms in the cultures (Fig. 2). Therefore, we did not take these bacterial markers into account in the FA profile of T.

weissflogii.

The 'external' fractions showed of the three cultures (A, B, and C), as for whole cells, a dominance of 16:0, 16:1(n-7) and 20:5(n-3) FA (18.0 ± 4.4 %, 18.2 ± 3.2 % and 28.6 ± 6.8 %, respectively, n = 9) (Fig. 4). Taking into account the 16:2 and 16:3(n-4) FA, these FA represented more than 85.9 % of the total FA for both WC and 'external' fractions.

The 'frustule-associated' fraction was dominated by saturated 14:0 and 16:0 FA (17.3 ± 5.9 % and 57.0 ± 4.9 %, respectively) (Fig. 4). This predominance of saturated FA led to a lower contribution of other FA. Contrarily to the WC and 'external' fraction, unsaturated FA with 16-carbon atoms were much less abundant in frustule-associated fraction (38.0 %, 41 % and 10.8 % in WC, 'external' and 'frustule-associated' fractions, respectively) (Fig. 4).

Moreover, no branched nor 17:0 FA prokaryote markers were found in these fractions emphasizing the non-contamination of 'frustule-associated' fraction. The major difference with FA composition of the whole cell was the low degree of unsaturation of the FA from the frustule.

Using k-means clustering method and MDS, comparison of FA profiles enabled to identify three "groups": WC and 'external' fractions from culture A and B on the one hand, WC and 'external' fractions from culture C on the other hand, and a third group composed of 'frustule-associated' fractions from all cultures (Fig. 5). The fact that A, B and C 'frustuleassociated' fractions were gathered together emphasized the specificity of frustule FA profiles regardless of the culture conditions. The difference between culture C and the two other cultures may be explained by a higher contribution and variability of 20:5(n-3) FA in culture C in both 'external' and whole cells samples (Fig. 4), which were most probably related to the different growth chamber (see section 2.1) or to a lag time for the sampling regarding the growth curve.

Biotic degradation of diatom cells

Diatom material for the degradation experiment was obtained from the continuous reactor culture from Argenton. The overall degradation of the diatom material was reflected in the decrease of both POC and bSiO 2 concentrations over the 30-day incubation period (from 1480.2 to 496.6 ± 14.5 µmol.l -1 and from 342.9 µmol.l -1 to 76.4 ± 2.4 µmol.l -1 for POC and bSiO 2 , respectively) (Fig. 6). As expected, prokaryotes largely contributed to diatom decomposition, as shown by the increase in prokaryotic carbon during the first 4 days (Fig. 6).

A total of 22 % of the initial bSiO 2 still remained in the batch at the end of the incubation experiment.

During the course of the degradation experiment, we focused on the kinetics of 'frustule-associated' FA and compared them with the kinetics of external lipids to highlight differences in degradation trends of the two fractions. External phytol and sterols represented 10.5 ± 3.2 % of the total external lipid pool (FA + Sterols + Phytol) at day 0. Their decrease over time from 17.7 ± 5.9 µmol C.l -1 to below the detection limit at day 18 for phytol and from 18.0 ± 6.3 µmol C.l -1 to 5.6 ± 0.8 µmolC.l -1 for sterols, well reflected the degradation of external algal material (Fig. 7). Total sterol concentrations exhibited an increase at day 6, mainly because of the analytical variability that was quite high between replicates, for the 24methylene cholesterol in particular. We were not able to attribute this increase to any process.

Interestingly, neither phytol nor sterols were detected in the 'frustule-associated' fraction.

This observation and the fact that the internal standard (19:0) added to the pellet extraction medium has never been found in the 'frustule-associated' fraction confirmed that our protocol accurately separated these two fractions.

The saturated FA proportion (especially 18:0) of the 'external' fraction during degradation was higher than in cultures used to set up the protocol. Several factors may increase saturated FA in pellets. The culture used for degradation was more mature than culture used to set up the protocol and proportions of unsaturated FA more susceptible to oxidative attack than saturated ones, decrease in mature cells. Moreover, prokaryotes enriched in saturated FA, were present in the culture medium at initial time of the degradation. In addition to this, centrifugation used to sample the pellet for the degradation experiment concentrates OM in general and not only diatoms. Thus, there was no possible interpretation of the kinetics of degradation of external saturated FA. On the opposite, unsaturated FA that dominated the FA in the diatom Thalassiosira weissflogii (50.7±6.5 % of Total FA ) (Table 1) are often used as markers of fresh diatom material [START_REF] Balzano | Changes in fatty acid composition in degrading algal aggregates[END_REF][START_REF] Najdek | Fatty acid and phytoplankton compositions of different types of mucilaginous aggregates in thenorthern Adriatic[END_REF].

They were proportionally less concentrated in the "degradation culture" (Table 2). However, their decay over time (from 68.5 ± 27.5 µmol C.l -1 to 26.7 ± 10.2 µmol C.l -1 between days 0 and 30) (Fig. 7) and that of their individual components (Fig. 8) that characterized the algae in both the WC and external fractions, namely C16:1n7, C16:2n4, C16:3n4, C18:1n7, C18:1n9, C20:5n3 and C22:6n3 (Fig. 4), also reflected the prokaryotic degradation of the algal lipids.

At the beginning of the degradation experiment, saturated FA, 14:0, 16:0 and 18:0, constituted the major part of the 'frustule-associated' FA (92.5 % of the total FA), as seen in the diatom cultures (Fig. 3). The concentration of the frustule-associated saturated FA 14:0 and 16:0 did not exhibit significant differences over time (Fig. 9), whereas the concentration of 18:0 decreased significantly after day 12 (Mann-Whitney U test, p < 0.05). This finding suggested a different association with silica phases of the 18:0 compared to the 14:0 and 16:0.

Possibly, 18:0 was associated with a more soluble silica phase, the dissolution of this phase being then necessary for 18:0 to be bioavailable. Unsaturated FA such as hexadecenoic acid 16:1 and oleic acid (18:1(n-9)) were still present in the frustule of T. weissflogii at the end of incubation. More generally, these results were in line with a protective effect of the frustule on its associated lipid fraction.

Discussion

Evidence for frustule-associated fatty acids in Thalassiosira weissflogii

The FA composition of Thalassiosira weissflogii was similar to that reported in the literature for diatoms, with a predominance of 16:0, 16:1(n-7) and 20:5(n-3), which are often used as diatom markers [START_REF] Dalsgaard | Fatty acid trophic markers in the pelagic marine environment[END_REF] (Table 1). The proportions of saturated FA (approximately 35 %) were comparable to those found in the same species. Only few composition differences existed compared to results on the same species from Klein Breteler et al. ( 2005), with respect to unsaturated FA, especially the proportions of 16:1(n-7) and 20:5(n-3). This may come from differences in growth conditions (detailed in Table 1) known to impact physiological status and biochemical pools [START_REF] Coombs | Studies on the biochemistry and fine structure of silica shell formation in diatoms. Chemical composition of Navicula pelliculosa during silicon-starvation synchrony[END_REF][START_REF] Vrieling | Growth and silica content of the diatoms Thalassiosira weissflogii and Navicula salinarum at different salinities and enrichments with aluminium[END_REF].

In addition to these cellular lipids, our study showed that a significant pool of FA inaccessible to conventional lipid extraction technics was associated to the frustule of the diatom Thalassiosira weissflogii. It is now well established that existing interactions between the silica frustule and the organic matter depends on the diatom freshness or at the opposite of their burial period up to their fossilization in sediments. Breaking these interactions requires the use of either strong acids or oxidizing agents, either solvent. Depending on objectives, some protocols are adapted to the study of the silica content of the frustule and aim at denaturing and removing all organic material that it contains [START_REF] Kamatani | Physical and chemical characteristics of biogenous silica[END_REF][START_REF] Loucaides | Dissolution of biogenic silica from land to ocean: Role of salinity and pH[END_REF], others require the removal of silica to release organic matter [START_REF] Bridoux | Structural identification of long-chain polyamines associated with diatom biosilica in a Southern Ocean sediment core[END_REF]Ingalls et al 2003;[START_REF] Kröger | A new calcium binding glycoprotein family constitutes a major diatom cell wall component[END_REF]. In the latter case, the difficulty lies in preserving the integrity of the molecular structures after treatment of the sample. Recently, Morales et al.

2013 proposed a successful protocol to clean fresh diatoms for isotopic analysis of diatom frustule-bound nitrogen. However, the harsh oxidative treatment transformed all organic nitrogen into nitrates preventing analysis of biochemical groups and molecular structures.

To our knowledge the first characterisation of a lipid pool embedded into the frustules of T. weissflogii was done by using nuclear magnetic resonance on SDS/EDTA/H 2 O 2 extracted frustules of T. pseudonana by [START_REF] Tesson | Contribution of multi-nuclear solid state NMR to the characterization of the Thalassiosira pseudonana diatom cell wall[END_REF]. Their results indicated the presence of C=C from double bonds, -(CH n )-from aliphatic chains and C=O from carbonyl groups of FA. Previously, [START_REF] Kates | Studies on the biochemistry and fine structure of silica shell formation in diatoms. Lipid components of the cell walls[END_REF] were the first to strongly demonstrate a lipid/frustule association in seven marine diatom species and to analyse FA. These authors washed frustules using distilled water and they added, after a Bligh and Dyer lipid extraction, a methanolic-HCl step to cleave bound-lipids. As such, the cell wall lipid composition was likely dominated by cellular material together with externally bound-lipids as no silica dissolution step was included in the protocol, as shown by our experiment in which fatty acid composition of external fraction was mainly identical to the fatty acid composition of the whole cell.

In our study, we pushed a step further the protocol of Kate and Volcani by adding an hot NaOH silica dissolution step after the first B&D extraction of washed fragmented diatom pellets. Briefly, in our protocol, frustule pellets were isolated from the cellular material by sequential washings and centrifugation of fragmented diatoms in pure water followed by a B&D extraction of external lipids. Then, silica frustules were dissolved with NaOH (1mol. l -1 ) and re-extracted at acidic pH by dichloromethane solvent to obtain the frustule-associated lipids.

Long chain carboxylic acids are major constituents of diatom lipids. They are free or esterified to glycerol, to other alcohols, or to more complex glycosidic and phosphatidic moieties. After sonication and water washing treatments, a fraction of the cellular lipids remained attached to the silica frustule through either hydrogen bonding, or electrostatic and hydrophobic interactions with other molecules constitutive of the cell wall. This was confirmed by the flow cytometry analysis of frustule pellets showing chla after cells fragmentation and washings.

The B&D protocol was particularly suitable to remove the lipid external fraction with preservation of intact molecular structures. The monophasic solvent extraction mixture (chloroform/methanol/0.1M NaCl in water) is a powerful extracting agent for both disruption of these bonding's and lipid dissolution (Kates, 1986), while the second step through addition of proper amounts of chloroform and water, achieves an efficient separation of hydrophobic and hydrophilic compounds by changing the monophasic mixture into biphasic. The NaOH treatment of frustules free from the external organic material gives access to the organic matter associated to the silica matrix. Not only NaOH increases silica dissolution but it is widely use to cleave hydrogen and covalent bonds, which favour separation of FA from the silica phase and most probably from the organic matrix that may be present. We did not use oxidizing agent such as perchloric acid suitable for the extraction of aqueous metabolites (Ingalls et al. 2009) as it has a deleterious effect on lipid components. We tested the use of HF to dissolve silica but precautions required for its use (plastic or polypropylene containers) makes its use incompatible with the analysis of lipids.

The efficiency of our protocol for isolating and extracting frustule-associated lipids was demonstrated first by the absence of cellular compounds (i. e. phytol and sterols) in frustule-associated lipids, secondly by the fact that the internal standard (19:0) added to the cellular fraction was not recovered with the frustule-associated lipid fraction (see section 2.4) and finally, because FA markers of prokaryotes that contributed for a few percent of the pellets total carbon were also absent from the frustule-associated lipids. In addition, we used our isolation/extraction protocol at different stages of dissolution of the silica phases during a bacterial degradation experiment of these diatoms. Frustule-associated FA thus extracted were found similar to that of frustule-associated FA in fresh Thalassiosira weissflogii diatom culture validating our protocole and showing little accessibility of these FA to degradation.

External FA were identified as cellular lipids because of their composition similar to that of FA in whole cells. While results did not show any specific FA marker of the frustule, frustule-associated FA were characterised by a very low degree of unsaturation with predominance of14:0, 16:0 and 18:0 FA. Although high pH (NaOH 1 mol.l -1 ) and the absence of chlorophyll favour neither autoxidation nor photooxidation [START_REF] Petit | Photochemical production and behavior of hydroperoxyacids in heterotrophic bacteria attached to senescent phytoplanktonic cells[END_REF][START_REF] Rontani | Visible light-dependent degradation of lipidic phytoplanktonic components during senescence: a review[END_REF], the reactivity of unsaturated FA to oxidation could have led to a decrease of unsaturation degree due to experimental conditions and extraction process. However, tests to determine the effect of temperature (70 °C) on polyunsaturated FA (20:4(n-6)), and previous observations that report unsaturated FA conservation during hot saponification (> 100 °C, see section 2.7.1), suggest that our protocol had little effect on FA unsaturation. Oleic acid (18:1(n-9)) and the unidentified 16:1 FA were present in the 'frustule-associated' fraction isolated from T. weissflogii cultures, even after 30 days of degradation. Thus, 16:1 and 18:1 FA may likely be associated to a resistant to degradation/dissolution phase of organic or silica matrices.

The very strong similarity between FA profiles of frustules of the different cultures (Fig. 5), although clear differences appeared for others fractions, indicated that 'frustuleassociated' FA were less sensitive to growth conditions than cellular FA. Nevertheless, the study of [START_REF] Soler | Impact of nutrient starvation on the biochemical composition of the marine diatom Thalassiosira weissflogii: from the whole cell to the frustule fraction[END_REF] shows an effect of nutrient starvations, especially phosphorus starvation, on the FTIR-analysed biochemical composition of T. weissflogii frustule. These authors suggested that biochemical changes lead to frustule structure changes leading in fine to changes in bSiO 2 dissolution. Therefore, although frustule-associated FA in sediments may not be used as markers of nutrient regime during paleoproduction, changes in other biochemical fractions should be considered when studying frustules in deep sediments. [START_REF] Kates | Studies on the biochemistry and fine structure of silica shell formation in diatoms. Lipid components of the cell walls[END_REF] suggested that lipids could be distributed according to their unsaturation level in the frustule. The degradation of the 18:0 and some unsaturated FA (16:1(n-7), 18:2(n-6), 20:4(n-6) and 20:5(n-3)) of the frustule while 14:0 and 16:0 were almost not degraded at all is in agreement with a non-homogenous association of FA with the frustule. These results also confirm the existence of different organic pools in the silica matrix of the frustules [START_REF] Abramson | The use of soft X-ray spectromicroscopy to investigate the distribution and composition of organic matter in a diatom frustule and a biomimetic analog[END_REF][START_REF] Tesson | Contribution of multi-nuclear solid state NMR to the characterization of the Thalassiosira pseudonana diatom cell wall[END_REF][START_REF] Tesson | Characterization and localization of insoluble organic matrices associated with diatom cell walls: Insight into their roles during cell wall formation[END_REF] and of different silica phases associated with different pool of organic matter [START_REF] Moriceau | Si-C interactions during degradation of the diatom Skeletonema marinoi[END_REF]. Using a spectroscopic method, [START_REF] Abramson | The use of soft X-ray spectromicroscopy to investigate the distribution and composition of organic matter in a diatom frustule and a biomimetic analog[END_REF] concluded that the OM encased within the structure of the frustule, which is resistant to different chemical attacks, may be protected from decomposition until the release of this matter into the surrounding environment occurs. This last method advantageously allowed locating the organic pools in Cylindrotheca fusiformis because of its thin frustule. However it is not valid to accurately quantify organic pools and it can hardly be adapted to other species as thicker frustules absorb X-rays too strongly. In contrast, our methodology may be used to quantify the pool of FA embedded in the frustule of a large choice of diatom species.

Implications for the export of organic matter

In some cases, huge numbers of fresh diatom cells can sink out of the euphotic layer [START_REF] Martin | Export and mesopelagic particle flux during a North Atlantic spring diatom bloom[END_REF], carrying with them different pool of organic matter; extra-, intra-cellular and intra-frustule. On the contrary to what was previously thought, the fraction of organic matter embedded or associated to the frustule could be an important fraction of the total POC (1.8 % of the diatom POC in surface water and up to 5.8 % of the diatom POC after 30 days of degradation).

To better understand the implication of this result in term of export, we compared our experimental data to the in situ data compiled by [START_REF] Ragueneau | Si/C decoupling in the world ocean: is the Southern Ocean different? Deep Sea Research Part II[END_REF]. More precisely, data from the Northern Antarctic Circumpolar Current site (NACC) were used for comparison because bSiO 2 dissolution at this site (82 % of bSiO 2 dissolution at 4224 m depth) was comparable to the final dissolution measured in our degradation/dissolution experiment (78 % of our initial bSiO 2 after 30 days incubation). The 'frustule-associated' FA represented 5.8 % of the total POC at the end of our experiment. The amount of these 'frustule-associated' FA for each mol of bSiO 2 after 78 % of dissolution was 0.23 molC FA.mol bSiO 2 -1 . Assuming that 14:0 and 16:0 FA are associated to the less soluble phase of the frustule without excluding possible association with the organic matrix, the flux of the 'frustule-associated' FA at 4224 m may be estimated from the bSiO 2 flux (0.14 mol.m -2 .yr -1 ) found at this site. This calculated 'frustule-associated' FA flux amounted to 0.0322 molC.m -².yr -1 which represents 64 % of the POC flux at 4224 m at NACC site. When N in frustules is on the order of 15 µmol N/g opal and the C associated with amino acids is also reported in units of µmol C/g [START_REF] Ingalls | Tinted windows: The presence of the UV absorbing compounds called mycosporine-like amino acids embedded in the frustules of marine diatoms[END_REF], this calculation suggests that our protocol extracts frustule-bound fatty acids and another FA fraction not embedded but strongly associated to the frustule and the organic matrix.

Keeping in mind the limitation of this comparison, especially in terms of the species studied (low latitude widespread species) and experimental conditions that did not consider in situ conditions (temperature, pressure, grazing) our study still raises the possible importance of the contribution of 'frustule-associated' FA to the organic carbon flux. According to our simple calculation, these 'frustule-associated' FA could represent an important part of the POC reaching the sediment. Interestingly, this assessment is in line with the correlation observed at depth between the sedimentation flux of POC and the sedimentation flux of bSiO 2 as revealed by the study of [START_REF] Armstrong | Settling velocity spectra and the ballast ratio hypothesis[END_REF], 2002), Francois et al. (2002), [START_REF] Klaas | Association of sinking organic matter with various types of mineral ballast in the deep sea: Implications for the rain ratio[END_REF] and [START_REF] Ragueneau | Si and C interactions in the world ocean: Importance of ecological processes and implications for the role of diatoms in the biological pump[END_REF].

In further studies of sinking particles and sediments, a more systematic hydrolysis/dissolution of deep samples should be used to consider this intra-frustule organic matter and to correctly estimate the sedimentation of the different organic pools. Until now, the different protocol used showed that this fraction is taken into account when measuring total POC but not when characterizing the different organic pools. As such, this fraction could be part of the large uncharacterized organic matter pool at depths [START_REF] Wakeham | Lipid biomarkers for heterotrophic alteration of suspended particulate organic matter in oxygenated and anoxic water columns of the ocean[END_REF].

As shown by our degradation/dissolution results, the 'frustule-associated' organic matter is not available to prokaryotes before the complete frustule dissolution. As a result and because of the saturation in dissolved silicon in interstitial water in bottom sediments [START_REF] Van Cappellen | Biogenic silica dissolution in the oceans: Reconciling experimental and field-based dissolution rates[END_REF], its release in sediments should be slower than previously thought and could not be a valuable source of energy for the organisms feeding on the seafloor. Another point of interest is the better preservation of lipids in sediments and their usefulness as biomarkers in paleoceanographic studies [START_REF] Wakeham | Lipid biomarkers for heterotrophic alteration of suspended particulate organic matter in oxygenated and anoxic water columns of the ocean[END_REF]. The fact that sterols are not found embedded in the frustule of T. weissflogii, although many deep sediment analysis reveal their presence [START_REF] Wakeham | Molecular indicators of diagenetic status in marine organic matter[END_REF], could be used to estimate the proportion of intracellular versus 'frustule-associated' organic carbon in opal dominated sediments.

Conclusion

Our study suggests an interesting and effective protocol to approach the FA composition of the organic matter embedded in or strongly associated to the frustule. Clear differences exist between the FA composition of the whole cell and the FA composition of the OM embedded and associated to the frustule which are characterised by a very low degree of unsaturation. Moreover, FA composition of the whole cell seem a lot more sensitive to small differences in the growth conditions than FA associated to the frustule. When T. weissflogii underwent a 30 days degradation, the FA associated to the frustule represented 5.8 % of the remaining POC suggesting the importance of their role in carbon export. With our study we infer that organic matter associated to the frustule or embedded in the frustule may be consider when investigating carbon export. This pool could partially explain the correlation observed at depth between Si and C sedimentation fluxes despite the decoupling between bSiO 2 dissolution and POC degradation processes.

indicated for three FA: 16:1, 16:1 and 16:3. For a convenient presentation, we assume their identity according to their contribution. θ Two FA, 20:4(n-6) and 20:5(n-3), are pooled in their study. 4 From this study (average of two independent cultures). φ In our study, the position of some double bonds are not localized.

Tr = Trace<0.05% as we assume in [START_REF] Volkman | Lipids in marine diatoms of the genus Thalassiosira : Predominance of 24-methylenecholesterol[END_REF]. For non detected FA, a -is indicated. UNS = unsaturated.

Figure captions

Figure 1. Schematic representation showing the different steps of the protocol. Cells are broken using hot sonication and centrifuged three times with milliQ water to remove intracellular material. Pellets of fragmented frustules are then extracted using organic solvents following the Bligh and Dyer protocol (B&D). The organic phase contains the external fraction. Frustules accumulated at the interface form a "white layer" composed of washed frustules (see photo of scanning electron microscopy). Frustules are dissolved with NaOH 1M at 95°C to release frustule-associated fraction that is further extracted with CH 2 Cl 2 at pH<2.

Figure 2. Cytograms of whole cells (A) and isolated frustules after three centrifugations with 40 ml of MilliQ water (B). Cytograms represent the chlorophyll fluorescence of particles function of their granularity (side scatter). Some frustules still show fluorescence of chlorophyll molecules indicating presence of external material.

Figure 3. Frustules fragments of Thalassiosira weissflogii freed from their "external fraction" observed in scanning electron microscopy.

Figure 4. Relative FA compositions of whole cells, external and frustule-associated fractions. All samples are gathered under each bar by averaging the FA contribution in each culture. n = 6 for whole cells (culture B and C) and n = 9 for the two other fractions (cultures A, B and C). Error bars represent the standard deviation.

Figure 5. Graphical representation of the classical multidimensional scaling analysis of whole cells samples (black diamonds), external fractions (grey dots) and frustule-associated fractions (white triangles). Confident ellipses containing 50 and 95% of the data used to build clusters obtained from k-means analysis are represented.

Figure 6. Evolution of concentrations of total POC (black dots), bacterial OC (black squares), and biogenic silica (bSiO 2 , grey triangles) during the course of the degradation experiment. Solid and dashed lines represent averaged value for each parameter. OC (particulate and bacterial) and bSiO 2 concentrations are expressed as µmol.l -1 . For a convenient representation, the y axis for bSiO 2 concentration is on the right of the graph.

Figure 7 .

 7 Figure1. Schematic representation showing the different steps of the protocol. Cells are broken using hot sonication and centrifuged three times with milliQ water to remove intracellular material. Pellets of fragmented frustules are then extracted using organic solvents following the Bligh and Dyer protocol (B&D). The organic phase contains the external fraction. Frustules accumulated at the interface form a "white layer" composed of washed frustules (see photo of scanning electron microscopy). Frustules are dissolved with NaOH 1M at 95°C to release frustule-associated fraction that is further extracted with CH 2 Cl 2 at pH<2.Figure2. Cytograms of whole cells (A) and isolated frustules after three centrifugations with 40 ml of MilliQ water (B). Cytograms represent the chlorophyll fluorescence of particles function of their granularity (side scatter). Some frustules still show fluorescence of chlorophyll molecules indicating presence of external material.Figure3. Frustules fragments of Thalassiosira weissflogii freed from their "external fraction" observed in scanning electron microscopy.Figure4. Relative FA compositions of whole cells, external and frustule-associated fractions. All samples are gathered under each bar by averaging the FA contribution in each culture. n = 6 for whole cells (culture B and C) and n = 9 for the two other fractions (cultures A, B and C). Error bars represent the standard deviation.Figure5. Graphical representation of the classical multidimensional scaling analysis of whole cells samples (black diamonds), external fractions (grey dots) and frustule-associated fractions (white triangles). Confident ellipses containing 50 and 95% of the data used to build clusters obtained from k-means analysis are represented.Figure6. Evolution of concentrations of total POC (black dots), bacterial OC (black squares), and biogenic silica (bSiO 2 , grey triangles) during the course of the degradation experiment. Solid and dashed lines represent averaged value for each parameter. OC (particulate and bacterial) and bSiO 2 concentrations are expressed as µmol.l -1 . For a convenient representation, the y axis for bSiO 2 concentration is on the right of the graph.Figure7. Concentrations (µmolC.l -1 ) of total unsaturated FA (black dots and solid

Figure 8 .

 8 Figure 8. Concentrations (µmolC.l -1 ) of individual unsaturated FA characteristic of external fractions at each sampling day during the course of the degradation experiment. Error bars represent standard deviation (n = 3). At day 18 and 30, n = 1.Figure9. Mean concentrations (µmolC.l -1 ) of total FA (dots and solid line), saturated

Figure 9 .

 9 Figure 8. Concentrations (µmolC.l -1 ) of individual unsaturated FA characteristic of external fractions at each sampling day during the course of the degradation experiment. Error bars represent standard deviation (n = 3). At day 18 and 30, n = 1.Figure9. Mean concentrations (µmolC.l -1 ) of total FA (dots and solid line), saturated

  Figure 1

  Figure 3. 815

  Figure 7.

Acknowledgments

We thank Daniel Delmas from Ifremer-Brest for prokaryote counting during the degradation experiment. We also thank Chantal Bezac for SEM micrography. The authors are grateful to two anonymous reviewers who greatly help improving the manuscript. Flow cytometry analyses were performed with the PRECYM platform (http://www.com.univmrs.fr/PRECYM/) with the help of Aude Barani. This work was supported by the UTIL project of the LEFE/CYBER program of CNRS/INSU. MS was supported by a Ph.D grant from Aix-Marseille University.

* Some FA bacterial markers were found in lipid extracts from degradation experiment samples (15:0iso, 15:0anteiso and 17:0). We choose to not take them into account and keep the composition found during growth of T.

weissflogii. However, it is clear that other FA such as palmitic and stearic acids are shared by diatoms and bacteria.

φ Another 16:1 FA was found in FA profiles but the position of its double bond was not localized.