Christos Panagiotopoulos
email: christos.panagiotopoulos@univ-amu.fr

Daniel J Repeta

Laura Mathieu

Jean-François Rontani

Richard Sempéré

Molecular level characterization of methyl sugars in marine high molecular weight dissolved organic matter

Keywords:

polysaccharides (APS) Methylated sugars Heptoses Anhydrosugars 3,6-Dideoxysugars Chemical synthesis of methylated sugars GC-MS

Dissolved organic matter (DOM) is the largest active organic carbon reservoir in the ocean (662 GT C), a major fraction (>95%) of which remains chemically uncharacterized. The concentration and isolation of DOM from seawater by ultrafiltration facilitates its chemical characterization by spectroscopic techniques. Using ultrafiltration, silver cation preparative chromatography and gas chromatography coupled with mass spectrometry (GC-MS), we identified 50 novel sugar compounds after hydrolysis of the high molecular weight dissolved organic matter fraction (HMWDOM; the fraction of DOM isolated after ultrafiltration). Sugars were identified by comparison of their mass spectra with those of chemically synthetized standards and with spectra previously described in the literature. Our results showed that mono-and di-methylated hexoses; mono-and di-methylated pentoses; mono-and di-methylated 6-deoxysugars, as well as heptoses, methylated heptoses, 3,6-dideoxysugars and 1,6 anhydrosugars (levoglucosan, mannosan, and galactosan) are components of HMWDOM, which may explain the low apparent yields of sugars recovered by molecular level (HPLC) analyses of HMWDOM after hydrolysis. From three depths spanning the surface (15 m) to bathypelagic (1800 m) ocean in the North Pacific near Hawaii our results showed that mono-and di-methylated hexoses were most abundant in the surface sample (64% of the total identified methylated sugar compounds), while at 1800 m monomethylated 6-deoxysugars were the dominant sugars (42% of the total identified methylated sugar compounds). The high diversity of mono-and di-methylated hexoses in the surface sample most likely suggests an algal and/or bacterial source, while the high abundance of methylated 6-deoxy hexoses in the deep sample points toward an important bacterial contribution because the latter sugars are mostly found in bacterial lipopolysaccharides as well as highly degraded organic material.

Introduction

More than 98% of organic carbon in the ocean is sequestered as dissolved organic carbon (DOC), which is defined as the fraction of carbon that passes through a 0.1-0.7 μm filter and is nonvolatile at pH ≤ 2. Although several studies have acknowledged the important role of DOC in a variety of oceanic processes [START_REF] Carlson | Production and removal processes[END_REF][START_REF] Bronk | Dynamics of DON[END_REF][START_REF] Nelson | Chromophoric DOM in the open ocean[END_REF][START_REF] Mopper | Photochemistry and the cycling of carbon, sulfur, nitrogen and phosphorus[END_REF][START_REF] Wells | Marine colloids and trace metals[END_REF] affecting as such the global carbon cycle [START_REF] Hansell | Dissolved organic matter in the ocean: a controversy stimulates new insights[END_REF], much less is known about its origins and chemical composition. Dissolved organic matter (DOM) includes a wide range of constituents that have different compositions, reactivities and ages [START_REF] Benner | Chemical composition and reactivity[END_REF][START_REF] Repeta | Radiocarbon analysis of neutral sugars in highmolecular-weight dissolved organic carbon: implications for organic carbon cycling[END_REF][START_REF] Eglinton | Marine organic geochemistry[END_REF]. Only a small part of this organic matter is recycled on short time scales, while the largest part (especially the deep-water DOM) is sequestered for long periods of time [START_REF] Williams | Radiocarbon in dissolved organic matter in the central North Pacific Ocean[END_REF][START_REF] Druffel | Cycling of dissolved and particulate organic matter in the open ocean[END_REF][START_REF] Hedges | Global biogeochemical cycles: progress and problems[END_REF][START_REF] Bauer | Carbon isotopic composition of DOM[END_REF]. Currently, only 4-5 GT C of DOC (~10% of DOC) are characterized at the molecular level as a sum of amino acids, lipids and sugars in surface waters [START_REF] Benner | Chemical composition and reactivity[END_REF]. In deep waters the amount of characterized DOC is about 34 GT C (b 5% DOC).

Seawater concentrations of dissolved amino acids, simple sugars, and lipids are in the low nanomolar range and are very close to the detection limits of the analytical techniques employed for their characterization. Most DOM is thought to be more complex than the simple biomolecules measured by direct analysis. To access and characterize this fraction of DOM, particularly in the presence of high concentrations of sea salt, a number of isolation techniques including tangential flow ultrafiltration, solid-phase extraction, dialysis, size-exclusion chromatography, and electrodialysis combined with reverse osmosis [START_REF] Tedetti | Determination of low molecular weight dicarboxylic and ketocarboxylic acids in seawater samples[END_REF]Mopper et al., 2007 and references therein;[START_REF] Vetter | Combining reverse osmosis and electrodialysis for more complete recovery of dissolved organic matter from seawater[END_REF] have been explored. Organic matter isolates have been characterized by very powerful analytical tools such as direct temperature-resolved chemical ionization (DT-MS), Fourier transform ion cyclotron mass spectrometry (FT-ICR-MS), high field nuclear magnetic resonance (NMR) spectroscopy, as well as high performance liquid chromatography (HPLC) coupled to Fourier transform infrared spectroscopy (HPLC-FT-IR) thereby providing important insights into DOM chemical composition [START_REF] Minor | Estuarine/ marine UDOM as characterized by size-exclusion chromatography and organic mass spectroscopy[END_REF][START_REF] Panagiotopoulos | Analytical methods for the determination of sugars in marine samples: a historical perspective and future directions[END_REF] and references therein; [START_REF] Kujawinski | Automated analysis of electrospray ionization Fourier transform ion cyclotron resonance mass spectra of natural organic matter[END_REF][START_REF] Koch | Advanced characterization of marine dissolved organic matter by combining reversed-phase liquid chromatography and FT-ICR-MS[END_REF][START_REF] Reemtsma | Molecular and structural characterization of dissolved organic matter from deep ocean by FTICR-MS, including hydrophilic nitrogenous organic molecules[END_REF][START_REF] Hockadey | Electrospray and photoionization mass spectrometry for the characterization of organic matter in natural waters: a qualitative assessment[END_REF][START_REF] Bhatia | Molecular characterization of dissolved organic matter associated with the Greenland ice sheet[END_REF][START_REF] Landry | Compositional differences between size classes of dissolved organic matter from freshwater and seawater revealed by an HPLC-FTIR System[END_REF][START_REF] Minor | Characterization of dissolved organic matter in lake superior and its watershed using ultrahigh resolution mass spectrometry[END_REF]. For example, [START_REF] Boon | Molecular characterization of microgram amounts of oceanic colloid organic matter by direct temperature-resolved ammonia chemical ionization mass spectrometry[END_REF] used DT-MS, to show the presence of amino sugars, furfurals, and methylated sugars in marine ultrafiltrated high molecular weight dissolved organic matter (HMWDOM). Other studies using solid state NMR and FT-ICR-MS showed the presence of carboxylic rich alicyclic molecules (CRAM) as important refractory components of the DOM pool [START_REF] Hertkorn | Characterization of a major refractory component of marine dissolved organic matter[END_REF][START_REF] Woods | Online high-performance size exclusion chromatography-nuclear magnetic resonance for the characterization of dissolved organic matter[END_REF].

All of these studies reported general structural characteristics of complex DOM. Detailed information of component molecules can complement this approach, and provide insight into subtle chemical transformations that may help to explain and to understand organic matter characteristics (e.g. complexity, apparent age, interactions with organisms etc.) [START_REF] Repeta | Radiocarbon analysis of neutral sugars in highmolecular-weight dissolved organic carbon: implications for organic carbon cycling[END_REF][START_REF] Goldberg | Systematic removal of neutral sugars within dissolved organic matter across ocean basins[END_REF][START_REF] Hansell | Net removal of major marine dissolved organic carbon fractions in the subsurface ocean[END_REF].

Previous studies have shown that most HMWDOM in the upper ocean is comprised of carbohydrates (acylated carbohydrates or APS), but only a minor fraction of the carbohydrate has been identified at the "molecular level" (as monosaccharides after hydrolysis, i.e. fucose, rhamnose, arabinose, galactose, glucose, mannose, and xylose; Fig. 1) [START_REF] Benner | Bulk chemical characteristics of dissolved organic matter in the ocean[END_REF][START_REF] Aluwihare | A major biopolymeric component to dissolved organic carbon in surface water[END_REF][START_REF] Repeta | Chemical characterization of high molecular weight dissolved organic matter in fresh and marine waters[END_REF]. The discrepancy between NMR derived estimates of HMWDOM carbohydrate (50-70% C) and molecular level analyses (10-20% C) has not been explained, and no new sugars have been identified in HMWDOM in about 10-15 years (McCarthy et al., 1996a;[START_REF] Mccarthy | Major bacterial contribution to marine dissolved organic nitrogen[END_REF][START_REF] Aluwihare | A major biopolymeric component to dissolved organic carbon in surface water[END_REF]. The carbohydrate component (APS) of the HMWDOM is water soluble (hydrophilic), exhibits similar Δ 14 C values with the surface dissolved inorganic carbon (DIC), and it is composed from sugar polymers (poly-, oligosaccharides) [START_REF] Repeta | Radiocarbon analysis of neutral sugars in highmolecular-weight dissolved organic carbon: implications for organic carbon cycling[END_REF][START_REF] Panagiotopoulos | Characterization of methyl sugars, 3-deoxysugars and methyl deoxysugars in marine high molecular weight dissolved organic matter[END_REF].

In a recent study, [START_REF] Panagiotopoulos | Characterization of methyl sugars, 3-deoxysugars and methyl deoxysugars in marine high molecular weight dissolved organic matter[END_REF] isolated a methyl sugar fraction from the acid hydrolysis products of HMWDOM (APS-F 3 ; Fig. 1) and identified new methylated sugars by 1-D and 2-D NMR spectroscopy. Because NMR required μmoles of sample at the time, only the most abundant methyl sugars of the samples were identified.

In this study, we further characterized the APS-F 3 component of HMWDOM in terms of methyl sugar composition using gas chromatography mass spectrometry (GC-MS). To do so we first chemically synthetized methylated sugar standards which were then used to identify most of the APS-F 3 methyl sugar components. Here we report the presence of a diverse suite of sugars and methyl sugars within the HMWDOM collected from 3 depths in the North Pacific Ocean. The distributions and the possible sources of these compounds are also discussed.

Materials & methods

HMWDOM sampling

Epipelagic (15 m), mesopelagic (900 m), and bathypelagic (1800 m) seawater samples were collected from the North Pacific Ocean (15 m; 19°43′ N; 156°03′ W, 900 m and 1800 m; 24°25′ N, 156°18′ W) in December 2003 (15 m sample) and June 2002 (900 m and 1800 m samples). The samples were filtered to remove bacteria and small particles using a cleaned (10% HCl) Suporflow dual stage (0.8 μm and 0.2 μm) Gelman polyether sulfone cartridge filter (Pall Corp.) fitted to an Advanta stainless steel housing. HMWDOM samples were collected using a large volume cross flow ultrafiltration system consisting of a stainless steel centripetal pump and membrane housings and a fluorinated high density polyethylene reservoir. The system was plumbed with Teflon tubing and fitted with PVDF valves. We used two ultrafiltration membranes (90 ft 2 each; Separation Engineering, Escondido, CA) that nominally retain organic matter of a molecular weight greater than 1000 Da (>98% rejection of vitamin B 12 ). Membranes were cleaned using isopropanol, detergent (0.01% micro), HCl (0.01 N) and NaOH (0.01 N), stored in sodium azide (0.55 mM), and rinsed with water and seawater immediately before use. The two filters were plumbed in parallel. Between 2763 L (900 m sample) and 8640 L (15 m sample) of seawater was concentrated over several days to approximately 40 L, frozen and returned to Woods Hole for further processing. The HMWDOM samples were concentrated to 2 L using a smaller system fitted with a 10 ft 2 membrane, desalted by repeated (10×) diafiltration with high purity, low carbon, deionized water (Milli-Q water, 2 L), and reduced to 2 L. The final concentrate gave no visible precipitate (AgCl) when an aliquot of permeate was mixed with a 5 mg/50 ml solution of AgNO 3 . After diafiltration was complete, the sample (2 L) was lyophilized to a fluffy off-white powder that was 24-35% by weight carbon with a C/N ratio of 13-16.

Sample preparation

HMWDOM samples (200-300 mg) were hydrolyzed in 2 M trifluoroacetic acid (TFA) at 120 °C under a helium atmosphere. Approximately 14.5 g of TFA was added to 43 g of water in a 250 mL round bottom flask. The solution was heated to temperature and the HMWDOM was added as a solution in approximately 2-3 mL of water. After 4 h, the dark brown solution was cooled, frozen at -20 °C, and freezedried. The dried sample was dissolved in 2-3 mL of water and applied to a 6-8 mm i.d. column packed with 4 g anion exchange resin (OH -form, Bio-Rex® 5 resin, Bio-Rad Laboratories, Hercules, CA). The column was preconditioned by rinsing with 15 mL of NaOH (0.5 M) followed by Milli-Q water until the pH returned to approximately 7. The column was drained of water and the sample was applied. Carbohydrates were eluted with 20-40 mL of water, collected, concentrated to a small volume (few mL) by rotary evaporation and freeze dried. The recovery of HMWDOM (by weight) after hydrolysis and ion exchange was 85-90%.

Chromatography

Chromatography was performed on a Waters pump (Model 510) and a Shodex RI-71 refractive index detector. Sample injection was performed with a manual Rheodyne injector fitted with a 100 μL sample loop. Carbohydrates were separated by the degree of polymerization on two sulfonated styrene-divinylbenzene gel analytical columns (30 cm × 7.8 mm i.d.; particle size 8 μm; cross linking 8%) in the silver form (Supelcogel™ Ag, Supelco, Inc., Bellefonte PA) connected in series. The columns were placed into a column heater (Tiberline TL-50) set at 80 °C. Sugars were eluted with water at a flow rate of 0.5 mL min -1 . Prior to use, the mobile phase was degassed with He for 30 min. The total weight of the sample injected onto the column did not exceed 10 mg/injection. Retention times for neutral sugars and oligosaccharides were determined from authentic fucose, rhamnose, arabinose, galactose, glucose, mannose, xylose, and maltooligosaccharides (DP 2-7). Fig. 1 depicts a typical chromatogram of a hydrolyzed APS sample. The suite of the seven neutral sugars (fucose, rhamnose, arabinose, galactose, glucose, mannose, and xylose) were identified within the APS-F 4 fraction, while the APS-F 3 fraction contained the methylated-, and the methylated deoxy-sugars [START_REF] Panagiotopoulos | Characterization of methyl sugars, 3-deoxysugars and methyl deoxysugars in marine high molecular weight dissolved organic matter[END_REF]. The different carbohydrate fractions (F 1 -F 5 ) were collected manually for further chemical characterization. The sugars within the APS-F 3 fraction were reduced and acetylated (see below) to produce alditol acetate derivatives which were further analyzed by GC-MS.

Preparation of partially methylated alditol acetates (PMAAs)

2.4.1. Acquisition and preparation of partially methylated methyl glycosides Sugar standards of fucose, rhamnose, arabinose, glucosamine, galactose, mannose, xylose, ribose, quinovose (6-deoxy glucose), 2-deoxy ribose, gluco-heptose, and manno-heptose were obtained from Sigma-Aldrich (St. Louis, MO, USA). Sugars were methylated according to the protocol of [START_REF] Ciucanu | A simple and rapid method for the permethylation of carbohydrates[END_REF] with some important modifications (Fig. 2). Briefly 10 mg of a standard sugar was dissolved in 1 mL of 40 mg mL -1 NaOH/DMSO. The mixture was well vortexed for 1 min. Then 0.1 mL of MeI was added and the solution was centrifuged for 4 min. After 11 min at ambient temperature, 1 mL of Milli-Q water and 1 mL of CH 2 Cl 2 were added to the solution. The organic layer (lower) was transferred in a second vial and the CH 2 Cl 2 was removed under a stream of N 2 . In contrast to the protocol of [START_REF] Ciucanu | A simple and rapid method for the permethylation of carbohydrates[END_REF] that produces fully permethylated sugars which are further recovered in the CH 2 Cl 2 phase, we found that the aqueous phase contained most of the mono-, di-, and tri-methylated sugars. Therefore, the aqueous layer was not discarded but kept and freeze dried to remove the excess of DMSO and the water. The dried residue was extracted with a few mL of ethyl acetate, combined with the CH 2 Cl 2 extract, filtered through quartz wool (precombusted at 500 °C for 3 h), and dried under a stream of N 2 .

Reduction and acetylation of methylated standard sugars

Partially methylated methyl glycosides obtained in the previous step were dissolved in 1 mL of 2 M TFA and hydrolyzed for 2 h at 100 °C [START_REF] Wang | An alternative method for the rapid synthesis of partially O-methylated alditol acetate standards for GC-MS analysis of carbohydrates[END_REF]. This step converts the ketal group of sugars to hemiacetal and thus facilitates the subsequent step of reduction. The methylated standard sugars obtained after treatment with TFA were reduced and acetylated to their corresponding alditol acetate derivatives according to the protocol of [START_REF] Panagiotopoulos | Spectrophotometric and chromatographic analysis of carbohydrates in marine samples[END_REF]. This procedure (reduction and acetylation) was applied as well to our APS-F 3 samples prior to their GC-MS analysis (Fig. 2). Briefly, in a 4-5 mL vial sugar samples/standards were dissolved in 0.25 mL of 1 M NH 4 OH/NaBH 4 (or NaBD 4 ). The solution was flushed with N 2 , and kept at room temperature for a minimum of 1 h. The reaction was quenched with a few drops of glacial acetic acid until bubbling ceased. Acetic acid was removed by adding 0.5 mL methanol and the solution was evaporated under N 2 . This last step was repeated three times. The dry alditols were acetylated with 100 μL of acetic anhydride and 20 μL of 1-methyl imidazole. The sample was mixed thoroughly and let stand for 15 min. Excess acetic anhydride was quenched with 0.5 mL of Milli-Q water. After 10 min at room temperature, 0.5 mL of CH 2 Cl 2 was added and the solution was vortex-mixed. The organic layer containing the alditol acetates was transferred to a 4 mL clean vial and dried over anhydrous sodium sulfate. The solution was then filtered through quartz wool (precombusted at 500 °C for 3 h) and the organic layer was transferred to another clean 4 mL vial. The CH 2 Cl 2 was removed under a stream of N 2 . The alditol acetates were then dissolved in ethyl acetate (1-2 mL) and analyzed by GC-MS. All methylated sugar standards and samples were run in duplicate. Alditol acetate derivatives are stable for 3-4 months when kept at 4 °C in the dark.

Gas chromatography mass spectroscopy (GC-MS)

Standards of PMAA and APS-F 3 samples were analyzed using an Agilent 5975C mass spectrometer connected to a 6850 gas chromatograph operating in electron impact mode. The following operating conditions were employed: 30 m × 0.25 mm (i.d.) fused silica capillary column coated with DB-5 (5% phenyl-methylpolysiloxane; film thickness 0.20 μm). The following conditions were used: oven temperature programmed from 90 to 150 °C at 10 °C min -1 after a delay of 1 min and then from 150 to 230 °C at 2 °C min -1 ; helium was used as carrier gas at 1 ml min -1 ; injector (pulsed splitless) temperature, 250 °C; electron energy, 70 eV; source temperature, 230 °C; and cycle time, 0.2 s. Alditol acetates were eluted between 10 and 30 min (Fig. 3). Data acquisition and processing were performed with Agilent Chem-Station software (Agilent Technologies).

Results and discussion

Identification and interpretation of PMAAs mass spectra

Gas chromatography-mass spectrometry is a powerful tool for the partial characterization of simple sugars. However, epimeric hexoses (glucose, mannose, and galactose) and their derivatives (methyland deoxy-hexoses), which are abundant in HMWDOM, display nearly identical mass spectra, and can only be distinguished by their chromatographic behavior. For example, GC-MS can easily distinguish hexoses methylated at different positions (2-OMe vs 6-OMe) since they exhibit different fragmentation patterns, but cannot distinguish 2-OMe glucose and 2-OMe-mannose without comparing retention times to authentic compounds. We therefore performed partial chemical synthesis of PMAA to generate a chromatographic and spectral database of the methylated derivatives for each of the major monosaccharides found in APS.

The elution order of PMAAs depends generally on their degree of methylation (Table 1; Fig. 3). Under the chromatographic conditions used in our analysis, permethylated sugars (which are less polar) eluted first followed by tetra-, tri-, di-, and mono-methylated sugars [START_REF] Sassaki | Rapid synthesis of partially O-methylated alditol acetate standards for GC-MS: some relative activities of hydroxyl groups of methyl glycopyranosides on Purdie methylation[END_REF][START_REF] Wang | An alternative method for the rapid synthesis of partially O-methylated alditol acetate standards for GC-MS analysis of carbohydrates[END_REF]. Fig. 3 depicts a typical TIC (Total Ion Current) chromatogram of the partially methylated galactose. Previous studies indicated that PMAAs mass spectra are characterized by primary and secondary fragments [START_REF] Björndal | Mass spectrometry of partially methylated alditol acetates[END_REF]. Primary fragments result from the fragmentation of the parent alditol, while secondary fragments arise from primary fragments after the elimination of acetic acid (m/z 60), ketene (m/z 42), MeOH (m/z 32), CH 2 O (m/z 30) or acetic anhydride (m/z 102). It is worth noting that PMAAs mass spectra do not display the molecular ion (Fig. 4). The identification of standard PMAAs was made by the comparison of mass spectra with literature data [START_REF] Sassaki | Rapid synthesis of partially O-methylated alditol acetate standards for GC-MS: some relative activities of hydroxyl groups of methyl glycopyranosides on Purdie methylation[END_REF][START_REF] Wang | An alternative method for the rapid synthesis of partially O-methylated alditol acetate standards for GC-MS analysis of carbohydrates[END_REF]. To confirm PMAAs structure attributions, the reduction step was also performed using sodium borodeuteride (NaBD 4 ). This reagent induces an asymmetry in the alditol acetates through the introduction of deuterium at the reducing carbon and allows the discrimination of symmetrical derivatives such as 2,3-OMe 2 glucose and 4,5-OMe 2 glucose; 3-OMe glucose and 4-OMe glucose [START_REF] Sassaki | Rapid synthesis of partially O-methylated alditol acetate standards for GC-MS: some relative activities of hydroxyl groups of methyl glycopyranosides on Purdie methylation[END_REF]. In general, for each monosaccharide 10-14 methylated derivatives were identified (Fig. 3).

Identification and distribution of sugars in the APS-F 3 samples

Sugars in our APS-F 3 samples were identified by comparing their respective retention time and mass spectra to the authentic standards of PMAA. Likewise, we identified most of the major peaks within the 12-30 min chromatographic window, while several unknown peaks appeared at >30 min (see below; Fig. 5). In total, we identified 38 methylated monosaccharides (3 methylated heptoses, 19 methylated hexoses, 6 methylated pentoses, and 10 methylated 6-deoxy hexoses), one heptose, two 2-amino 6-deoxy hexoses (fucosamine and quinovosamine), three 1-6 anhydro hexoses (galactosan, levoglucosan, and mannosan), one 2-deoxy pentose (xylose or arabinose), two 3,6dideoxy hexoses (3-deoxy fucose and 3-deoxy rhamnose) and two tetroses (Table 2; Fig. 6). Due to the imperfect separation between fractions F 3 and F 4 , we also found within the APS-F 3 fraction the seven major neutral sugars identified in the APS-F 4 fraction, where they prevailed [START_REF] Repeta | Radiocarbon analysis of neutral sugars in highmolecular-weight dissolved organic carbon: implications for organic carbon cycling[END_REF]. Unidentified compounds eluting between 10 and 40 min represented 32, 45, and 24% of the total chromatogram (integrated area) for the 15, 900, and 1800 m samples, respectively (Fig. 5). Unidentified peaks at >30 min most likely correspond to mono-and di-methylated heptoses as well as monomethylated amino sugars. Based on their characteristic fragments however, their structures could not be fully resolved.

Methylated sugars

The methylated sugar fraction APS-F 3 accounted between 2 and 3% of the total integrated area of the sample chromatograms (Fig. 1). Relative area provides a semi-quantitative estimate of the contribution of methyl sugars to APS as it does not account for likely differences in Table 1. Peaks were identified as a function of their mass spectra and comparison with literature data [START_REF] Sassaki | Rapid synthesis of partially O-methylated alditol acetate standards for GC-MS: some relative activities of hydroxyl groups of methyl glycopyranosides on Purdie methylation[END_REF][START_REF] Wang | An alternative method for the rapid synthesis of partially O-methylated alditol acetate standards for GC-MS analysis of carbohydrates[END_REF]. the refractive index of major neutral sugars (Section 2.3), methyl sugars and polysaccharides. Interestingly enough, methylated 6-deoxysugars also occur in the unhydrolyzed APS-F 1 polymer [START_REF] Panagiotopoulos | Characterization of methyl sugars, 3-deoxysugars and methyl deoxysugars in marine high molecular weight dissolved organic matter[END_REF]. [START_REF] Quan | Periodate oxidation of marine high molecular weight dissolved organic matter: Evidence for a major contribution from 6-deoxy-and methyl sugars[END_REF] using periodate oxidation also showed the presence of methylated sugars within the APS pool, however as MeOH (end product of -OMe group after periodate oxidation) was not measured, the total amount of methylated sugars in APS was not estimated. Currently, we do not know the exact contribution of the methylated sugar component in the whole APS pool and the HMWDOM in general.

The most abundant methylated sugars in the surface sample were the 2-OMe glucose (10% of the total APS-F 3 sugars), 3-OMe rhamnose (6%), 2,4 di-OMe galactose (6%), 4-OMe glucose (5%), and 2-OMe rhamnose (5%) in agreement with previous results [START_REF] Panagiotopoulos | Characterization of methyl sugars, 3-deoxysugars and methyl deoxysugars in marine high molecular weight dissolved organic matter[END_REF]. At 1800 m depth, the major methylated sugars were the 2-OMe fucose (24%), 2-OMe galactose (18%), 2-OMe rhamnose (5%), 2-OMe glucose (4%), and 2,4 di-OMe galactose (4%). Trimethylated and permethylated sugars were not detected in our samples indicating that mono-, and di-methylated sugars prevail in APS pool.

Previous investigations have indicated changes in the relative amounts of dissolved, hydrolyzable neutral sugars with depth in different oceanic basins [START_REF] Benner | Bulk chemical characteristics of dissolved organic matter in the ocean[END_REF]McCarthy et al., 1996a;[START_REF] Mccarthy | Major bacterial contribution to marine dissolved organic nitrogen[END_REF][START_REF] Skoog | Aldoses in various size fractions of marine organic matter. Implications for carbon cycling[END_REF][START_REF] Sempéré | Distribution and bacterial availability of dissolved neutral sugars in the south east Pacific[END_REF][START_REF] Kaiser | Biochemical composition and size distribution of organic matter at the Pacific and Atlantic time-series stations[END_REF][START_REF] Goldberg | Systematic removal of neutral sugars within dissolved organic matter across ocean basins[END_REF] and we find similar trends in unidentified and methyl sugars within the APS-F 3 fraction. A large body of work on carbohydrate concentrations in seawater indicates that combined neutral sugar concentrations, total sugar yields (total sugar-C/DOC%), and mol % glucose, galactose and mannose + xylose are higher in surface vs deep waters pointing to a recent accumulation of DOM [START_REF] Skoog | Aldoses in various size fractions of marine organic matter. Implications for carbon cycling[END_REF][START_REF] Sempéré | Distribution and bacterial availability of dissolved neutral sugars in the south east Pacific[END_REF][START_REF] Kaiser | Biochemical composition and size distribution of organic matter at the Pacific and Atlantic time-series stations[END_REF][START_REF] Goldberg | Systematic removal of neutral sugars within dissolved organic matter across ocean basins[END_REF]. Our results on methylated sugar composition are in agreement with these observations indicating that surface APS-F 3 sugars are a less diagenetically altered material. Finally, our findings on methylated sugar composition may explain in part the discrepancy between the amount of total carbohydrate measured colorimetrically and that of neutral carbohydrates measured by HPLC since methylated sugars were never included in HPLC or GC measurements [START_REF] Borch | Concentration and compositionof dissolved combined neutral sugars (polysaccharides) inseawater determined by HPLC-PAD[END_REF][START_REF] Benner | Chemical composition and reactivity[END_REF].

The number of methylated sugars decreased in our samples from 36 in the surface sample, to 26 at 900 m and 25 at 1800 m. Mono-, and di-methylated hexoses in particular accounted for 41% and 24%, respectively of the total methyl sugars in the surface sample, whereas these percentages dropped to 35% and 12% for the 1800 m sample (Fig. 7). These results indicate that there is a net loss of diversity and relative mass of mono-and di-methylated hexoses with depth. Gal. 30.69 85, 115, 145, 170, 187, 217, 259, 289, 361 2 3-OMe Gal. b 29.24 85, 87, 99, 127, 129, 159, 189, 261, 333 3 2-OMe Gal. 28.05 87, 97, 117, 129, 139, 159, 231, 259, 333 4 2,4-OMe 2 Gal. 26.48 87,101,117,129,139,159,189,233,305 5 6-OMe Gal. 25.93 87,97,115,129,139,157,184,259,333 6 2,3-OMe 2 Gal. 25.50 85,87,99,101,117,127,159,187,201,261 7 3,6-OMe 2 Gal. 24.07 87,99,113,129,189,233 8 2,6-OMe 2 Gal. 23.36 58,87,101,117,129,143,185,231,305 9 2,3,5-OMe 3 Gal. 22.58 85,101,117,127,143,159,201,233 10 3,4,6-OMe 3 Gal. 21.20 74,87,101,117,129,143,161,189,233 11 2,3,6-OMe 3 Gal. 20.66 87,99,101,117,129,131,161,173,233 12 2,5,6-OMe 3 Gal. 20.41 59,87,89,97,117,129,143,185,231,305 13 2,3,4,6-OMe 4 Gal. 17.90 71,87,101,117,129,145,161,205 14 2,[START_REF] Rha | OMe 2 Gal.; 2,6-OMe 2 Gal.; 3,6 dideoxy Gal. Rhodella maculata Polysaccharide Extracellular mucilage of red alga Evans et al., 1974 6-OMe GlcN. Photosynthetic bacteria LPS of bacterial cell walls (Gram-negative) Bacterial strain Mayer et al., 1974 3-OMe Man. 3-OMe L-Rha. 3-OMe Rib. 2-OMe Gal. 2,3-OMe 2 Gal. Photosynthetic bacteria, cyanobacteria LPS of bacterial cell walls (Gram-negative) Bacterial strain Tharanathan et al., 1978 6-OMe D,L Man. 2,3-OMe 2 L-Rha. 3-OMe Xyl. Emiliania huxleyi Acidic polysaccharide Coccoliths culture Fichtinger-Schepman et al., 1979 3,6 dideoxy hexose; 3-OMe Man.; 4-OMe Man.; ManN; 2,3,4-OMe 3 Ara. Cyanobacterium Synechococcus LPS of bacterial cell walls (Gram-negative) Bacterial strain Schmidt et al., 1980 Mono-, di-, and tri-methylated 6-deoxysugars; di-, tri-, tetra-methylated hexoses and pentoses Humic and fulvic acids Soil Ogner, 1980 3,4-OMe 2 Rha. 3-OMe Glc. 2-OMe Rha. 2,3-OMe 2 Fuc. Mycobacterium Glycopeptidolipid antigens Serovar Brennan et al., 1981 Mono, di, and tri-methylated 6-deoxysugars; 3,6 -dideoxy hexose; monomethylated pentoses; mono, di-methylated hexoses; heptose, 2-OMe heptose[END_REF]5,6-OMe 4 Gal. 17.51 59,89,101,117,129,143,161,205 15 2,[START_REF] Rha | OMe 2 Gal.; 2,6-OMe 2 Gal.; 3,6 dideoxy Gal. Rhodella maculata Polysaccharide Extracellular mucilage of red alga Evans et al., 1974 6-OMe GlcN. Photosynthetic bacteria LPS of bacterial cell walls (Gram-negative) Bacterial strain Mayer et al., 1974 3-OMe Man. 3-OMe L-Rha. 3-OMe Rib. 2-OMe Gal. 2,3-OMe 2 Gal. Photosynthetic bacteria, cyanobacteria LPS of bacterial cell walls (Gram-negative) Bacterial strain Tharanathan et al., 1978 6-OMe D,L Man. 2,3-OMe 2 L-Rha. 3-OMe Xyl. Emiliania huxleyi Acidic polysaccharide Coccoliths culture Fichtinger-Schepman et al., 1979 3,6 dideoxy hexose; 3-OMe Man.; 4-OMe Man.; ManN; 2,3,4-OMe 3 Ara. Cyanobacterium Synechococcus LPS of bacterial cell walls (Gram-negative) Bacterial strain Schmidt et al., 1980 Mono-, di-, and tri-methylated 6-deoxysugars; di-, tri-, tetra-methylated hexoses and pentoses Humic and fulvic acids Soil Ogner, 1980 3,4-OMe 2 Rha. 3-OMe Glc. 2-OMe Rha. 2,3-OMe 2 Fuc. Mycobacterium Glycopeptidolipid antigens Serovar Brennan et al., 1981 Mono, di, and tri-methylated 6-deoxysugars; 3,6 -dideoxy hexose; monomethylated pentoses; mono, di-methylated hexoses; heptose, 2-OMe heptose[END_REF]4,5,6-OMe 5 Gal. 15.17 59,87,89,101,117,131,161,175 a Typical fragments (unit m/z) used for structural identification of PMAAs. Although the mechanisms responsible for these changes with depth are not clear, microbial demethylation may be active [START_REF] Carpenter | Uptake of methanol to the North Atlantic Ocean surface[END_REF][START_REF] Neufeld | Substrate-specific clades of active marine methylotrophs associated with a phytoplankton bloom in a temperate coastal environment[END_REF][START_REF] Mccarren | Microbial community transcriptomes reveal microbes and metabolic pathways associated with dissolved organic matter turnover in the sea[END_REF].

On the other hand, monomethylated 6-deoxy hexoses increased with depth from 23% to 43% of total APS-F 3 sugars in the surface and deep samples, respectively (Fig. 7). This increase in relative abundance may suggest either the selective preservation of monomethyl 6-deoxysugars during APS degradation, or the addition of these sugars by mesopelagic bacteria. Monomethylated 6-deoxysugars and 6-deoxysugars (i.e. fucose, rhamnose, quinovose) have been reported in bacterial lipopolysaccharides (Table 3) and/or highly degraded material in soils as part of humic and fulvic substances [START_REF] Ogner | Analysis of the carbohydrates of fulvic and humic acids as their partially methylated alditol acetates[END_REF].

Non-methylated sugar compounds

Non-methylated sugar compounds included heptoses, tetroses, anhydrosugars, 3,6-dideoxy-hexoses, 2-deoxy-pentoses, 2-amino 6-deoxy hexoses, a branched monosaccharide (yersiniose), and 6-deoxy hexose (quinovose). With the exception of quinovose, our results showed that these compounds were mainly found in the mesopelagic sample (900 m) where they accounted 6% of the total APS-F 3 sugars (Table 2). Galactosan was the most abundant nonmethylated sugar compound accounting 1.2% of the total identified sugars followed by levoglucosan, quinovose, and 2-deoxy pentose which contributed about equally (~0.9%).

Sources and biogeochemical importance of methylated sugars

Methylated sugars have been found in sediment trap samples [START_REF] Bergamaschi | Distributions of uronic acids and O-methyl sugars in sinking and sedimentary particles in two coastal marine environments[END_REF], diatom frustules [START_REF] Chiovitti | Variations in the substituted 3-linked mannans closely associated with the silicified walls of diatoms[END_REF], coccoliths (e.g. Emiliania huxleyi; [START_REF] Fichtinger-Schepman | Composition of a methylated, acidic polysaccharide associated with coccoliths of Emiliania huxleyi (Lohmann) Kamptner[END_REF], and mucilage material [START_REF] Evans | Studies on the synthesis and composition of extracellular mucilage in the unicellular red alga Rhodella[END_REF]. [START_REF] Klok | Carbohydrates in recent marine sediments-I. Origin and significance of deoxy-and Omethyl-monosaccharides[END_REF] reported a plethora of 52 methylated sugars in sediments from the Black Sea and Namibian continental shelf (Table 3). However, the full structure of some of these compounds was not completely resolved (tentative identification). Other studies showed that methylated sugars are common components of plant cell walls [START_REF] Popper | 3-O-methylrhamnose in lower land plant primary cell walls[END_REF] and bacterial lipopolysaccharides, where they account for 1-4% of the cell dry weight [START_REF] Weckesser | The identification of 3-O-methyl-L-rhamnose (L-acofriose) as constituent of the lipopolysaccharide of Rhodopseudomonas capsulata[END_REF][START_REF] Choma | Chemical analysis of Azospirillum lipopolysaccharides[END_REF][START_REF] Fox | Carbohydrates profiling of bacteria by gas chromatography-mass spectrometry and their trace detection in complex matrices by gas chromatographytandem mass spectrometry[END_REF].

Currently very little is known about the origins and the biogeochemical role of methylated sugars within HMWDOM. Previous studies using DT-MS showed the presence of methylated, deoxy and N-acetyl amino sugars within HMWDOM which were attributed to marine prokaryote sources [START_REF] Boon | Molecular characterization of microgram amounts of oceanic colloid organic matter by direct temperature-resolved ammonia chemical ionization mass spectrometry[END_REF]. Other studies using 1 H and 13 C NMR showed similar functional group distributions between seawater HMWDOM and HMWDOM produced by E. huxleyi, Phaeocystis sp., Thalassiosira weissflogii, and Chaetoceros neogracile [START_REF] Aluwihare | A major biopolymeric component to dissolved organic carbon in surface water[END_REF][START_REF] Aluwihare | A comparison of the chemical characteristics of oceanic DOM and extracellular DOM produced by marine algae[END_REF][START_REF] Repeta | Chemical characterization of high molecular weight dissolved organic matter in fresh and marine waters[END_REF][START_REF] Gogou | Particulate-dissolved transformations as a sink for semilabile dissolved organic matter: chemical characterization of high molecular weight dissolved and surface-active organic matter in seawater and in diatom cultures[END_REF]. In agreement with these observations, other investigations showed that E. huxleyi and Chlamydomonas eugametos (biflagellate unicellular green alga) cell walls are composed 2.

of methylated sugars pointing to a phytoplanktonic source [START_REF] Fichtinger-Schepman | Composition of a methylated, acidic polysaccharide associated with coccoliths of Emiliania huxleyi (Lohmann) Kamptner[END_REF][START_REF] Schuring | Occurrence of O-methylated sugars in surface glycoconjugates in Chlamydomonas eugametos[END_REF].

Our results together with literature data suggest that the surface HMWDOM sample has algal and bacterial components, based on the polydisperse diversity of the methylated sugar compounds, while the deep HMWDOM sample could have a more pronounced bacterial contribution due to the high abundance of monomethylated 6-deoxysugars which are often found in bacterial lipopolysaccharides 85,115,145,157,187,259,289,361,433 2 GalN. 37.29 900 84,102,139,144,156,193,259,318 3 2-OMe heptose a 36.92 15-1800 73, 99, 117, 145, 159, 169, 201, 261, 303, 405 4 2-OMe heptose a 36.64 15-1800 73, 99, 117, 145, 159, 169, 201, 261, 303, 405 5 GlcN 36.31 900 84, 102, 139, 144, 156, 193, 259, 318 6 2-OMe mannoheptose 36.18 15-900 73, 99, 117, 145, 159, 169, 201, 261, 303, 405 7 Gal. 30.64 15-900-180085, 115, 145, 170, 187, 217, 259, 289, 361 8 Glc. 30.36 15-900-180085, 115, 145, 170, 187, 217, 259, 289, 361 9 Man. 29.90 15-900-180085, 115, 145, 170, 187, 217, 259, 289, 361 10 3-OMe Gal. 29.20 15-900-180087, 99, 129, 145, 189, 217, 261, 333 11 4-OMe Glc. 28.89 15-900-180087, 99, 127, 129, 159, 189, 201, 217, 261, 333 12 3-OMe Man. 28.57 15-900-1800 87, 99, 127, 129, 159, 189, 201, 217, 261, 333 13 3-OMe Glc. 28.43 15-900 87, 99, 127, 129, 159, 189, 201, 217, 261 85, 103, 115, 127, 145, 158, 170, 187, 217, 289 33 Qui. 20.96 15-900-180099, 103, 115, 128, 145, 157, 170, 217, 289, 303 34 Ara. 20.80 15-900-180085, 103, 115, 127, 145, 158, 187, 217, 289 35 Fuc. 20.60 15-900-180099, 103, 115, 128, 145, 157, 170, 217, 289, 303 36 Rib. 20.48 15-900 85, 103, 115, 127, 145, 158, 187, 217, 289 37 Rha. 20.28 15-900-180099, 103, 115, 128, 145, 157, 170, 217, 289, 303 38 4-OMe Fuc. 19.23 15-900 85, 87, 99, 129, 131, 143, 159, 189, 261 39 3-OMe Fuc. 19.16 15-900 87, 101, 117, 129, 143, 159, 189, 203, 261 40 3-OMe Xyl. 18.97 15-900-1800 87, 99, 117, 129, 159, 189, 203, 261 polysaccharides of several species of gram-negative bacteria (Table 3). However, it is worth noting that the presence of these compounds in other organisms, such as planktons, algae, photosynthetic bacteria etc. has not been studied. Yersiniose has been reported in bacterial lipopolysaccharides of diazotrophs isolated from soils, roots, and plant leaves [START_REF] Mattos | Nitrogen-fixing Burkholderia brasiliensis produces a novel yersiniose Acontaining O-polysaccharide[END_REF], and further investigations are warranted to determine if it is also present in marine diazotrophs. Anhydrosugars (levoglucosan, galactosan, and mannosan) were detected in the 900 m sample. These compounds are generally found in the airborne particulate matter as the thermal degradation products of cellulose (levoglucosan) and hemicelluloses (mannosan and galactosan) [START_REF] Simoneit | Levoglucosan, a tracer for cellulose in biomass burning and atmospheric particles[END_REF] and have never been detected in seawater. Reported levoglucosan concentrations in atmospheric samples over the ocean vary between 0.0008 and 0-15 ng m -3 [START_REF] Simoneit | Organic tracers from biomass burning in atmospheric particulate matter over the ocean[END_REF], while >1000 ng m -3 values were recorded in atmospheric samples of urban air sheds. Although we have detected anhydrosugars in the APS-F 3 fraction, our preliminary results suggest that anhydrosugars were concentrated in the APS-F 5 fraction (Fig. 2). Therefore, anhydrosugars were not quantified in this study. Nevertheless, these results indicate that the HMWDOM-900 m sample might include carbon from terrestrial biomass burning. Additional measurements of δ 13 C of pure levoglucosan in the sample and rigorous tests of the analytical protocol are necessary to establish the source of anhydrosugars in HMWDOM.

Summary and concluding remarks

This study showed the presence of a much broader suite of sugars (in total 50 compounds; Table 2; Fig. 6) within HMWDOM carbohydrate than previously recognized. Our results suggest that methyl sugars (APS-F 3 ) account for approximately 2-3% of APS, and that the relative distribution of sugars changes significantly with depth. Mono-and di-methylated hexoses and monomethylated 6-deoxysugars represent ~90% of the methylated sugars identified in our samples. Mono-and di-methylated hexoses dominated the surface sample (64% of the total methylated compounds), while at 1800 m depth monomethylated 6-deoxysugars were the most abundant sugars (42% of the total methylated compounds) indicating a net loss of diversity and mass of methylated sugars with depth. Other sugar compounds identified in this study comprised the heptoses, deoxy hexoses (quinovose), amino deoxy hexoses, deoxy pentoses, dideoxy hexoses, anhydrosugars, and branched sugars (yersiniose) and accounted 1-6% of the total identified compounds.

Methylated-, deoxy-, amino deoxy-, dideoxy-sugars, and heptoses are broadly distributed within plants and bacteria and currently their presence in HMWDOM cannot provide information to distinguish specific sources of DOM (algal and bacterial contributions; Table 3). Nevertheless, very few studies have identified and quantified these compounds in marine samples (e.g. phytoplanktonic organisms, sediment trap material, prokaryotic bacteria) and therefore, their potential as biomarkers requires a more thorough examination of potential sources.

Much more work is left to be done on the polysaccharide component (APS) of HMWDOM. For example, the F 1 fraction (Fig. 1) is not hydrolyzed by strong acid, and has not yet structurally characterized. Nevertheless, 1 H NMR suggests that it is composed by 6-deoxysugars [START_REF] Panagiotopoulos | Characterization of methyl sugars, 3-deoxysugars and methyl deoxysugars in marine high molecular weight dissolved organic matter[END_REF] including perhaps quinovose, identified in this study. Anhydrosugars, also appear to be a part of the HMWDOM carbohydrate fraction. Do they have a terrestrial or a marine origin?

Fig. 1 .

 1 Fig.1. Silver ion chromatography of the APS component of the HMWDOM from the 1800 m sample. Sugars were separated on two Ag + cation exchange columns connected in series at 80 °C and eluted with high purity water at 0.5 ml min -1 . The detection was made by refractive index (RI) and the fractions F 1 -F 5 were collected manually for further chemical characterization.

Fig. 3 .

 3 Fig.3. Chromatogram of a standard of PMAAs of galactose. The number on each peak corresponds to the compound name given in Table1. Peaks were identified as a function of their mass spectra and comparison with literature data[START_REF] Sassaki | Rapid synthesis of partially O-methylated alditol acetate standards for GC-MS: some relative activities of hydroxyl groups of methyl glycopyranosides on Purdie methylation[END_REF][START_REF] Wang | An alternative method for the rapid synthesis of partially O-methylated alditol acetate standards for GC-MS analysis of carbohydrates[END_REF].

bFig. 4 .

 4 Fig. 4. Mass spectra and fragmentation patterns (primary & secondary fragmentation) of 4,6-OMe 2 mannose.

Fig. 5 .

 5 Fig.5. GC-MS chromatograms for the APS-F 3 sample at 15, 900, and 1800 m depths. The numbers on the peaks correspond to the compounds in Table2.

APS-F 3 Sample preparation Standard preparation of alditol acetates

	Standard preparation of
	partially methylated alditol
	acetates (PMAA)
	Monosaccharide standards (i.e.	Monosaccharide standards (i.e.
	glucose, mannose, arabinose,	glucose, mannose, arabinose,
	heptose …..)	heptose …..)
	Methylation
	MeI
	Acid hydrolysis
	HCl
	Reduction
	(NaBH 4 or NaBD 4 )
	Acetylation
	Ac 2 O
	GC-MS analysis

Fig.

2

. Summary of the experimental procedures used in this study.

Table 1

 1 Partially methylated alditol acetates (PMAAs) of galactose identified by GC-MS. Abbreviations: Gal.: galactose, RT: retention time.

	Peak	PMAA galactose	RT	Characteristic fragments a
	no	standard	(min)
	1

Table 2

 2 Identified monosaccharides within the APS-F 3 fraction. In bold all the new compounds. Abbreviations: Man.: Mannose; Gal.: Galactose; Glc.: Glucose; Xyl.: Xylose; Ara.: Arabinose; Fuc.: Fucose; Quin.: Quinovose; Rha.: Rhamnose; Rib.: Ribose; GlcN.: Glucosamine; GalN.: Galactosamine; FucN.: Fucosamine; QuiN: Quinovosamine; RT.: Retention time.

	Peaks	Monosaccharides	RT (min)	Depth (m)	Characteristic fragments (m/z)
	1	Mannoheptose	39.41	15-900

connected in series at 80 °C and eluted with high purity water at 0.5 ml min -1 . The detection was made by refractive index (RI) and the fractions F 1 -F 5 were collected manually for further chemical characterization.

C. Panagiotopoulos et al. / Marine Chemistry 154 (2013) 34-45

Acknowledgments

We thank the staff at the Natural Energy Laboratory in Kona, Hawaii for their assistance in providing facilities for sample collection. We also thank F. Vaultier and J. Girardin for analytical assistance. This manuscript benefited from the advice and comments given by B. Charrière, N. and V. Ragoussis and the two anonymous reviewers. Funding was provided by CNRS/INSU LEFE/Cyber grant (DORADE project), region Provence Alpes Côte d'Azur (MANDARINE project) and the Aix Marseille University (ORANGE project). D.J.R. received support from the National Science Foundation Center for Microbial Research and Education (CMORE) DBI 0424599 and the Gordon and Betty Moore Foundation.

 3-OMe Rha. 18.95 15-900-180087, 101, 117, 129, 143, 159, 189, 203, 261 42 2-OMe Xyl. 18.80 15-900-1800 85, 87, 99, 117, 127, 159, 201, 261 434-OMe Rha. 18.65 15-900 85, 87, 99, 129, 131, 143, 159, 189, 261 44 2-OMe Ara. 18.42 15-900 85, 87, 99, 117, 127, 159, 201, 261 45 2-OMe Fuc. 18.29 15-900-180087, 117, 129, 141, 159, 173, 201, 231, 275 46 2-OMe Rha. 18.16 15-900-1800 87, 117, 129, 141, 159, 173, 201, 231, (Table 3). Previous investigations based on amino acid measurements in HMWDOM also showed that HMWDOM contains algal and bacterial components and are in agreement with the present study [START_REF] Mccarthy | Major bacterial contribution to marine dissolved organic nitrogen[END_REF][START_REF] Dittmar | Origin and biogeochemical cycling of organic nitrogen in the eastern Arctic Ocean as evident from D-and L-amino acids[END_REF][START_REF] Jones | An assessment of the microbial contribution to aquatic dissolved organic nitrogen using amino acid enantiomeric ratios[END_REF][START_REF] Kaiser | Major bacterial contribution to the ocean reservoir of detrital organic carbon and nitrogen[END_REF].

Sources and biogeochemical importance of non-methylated sugars

In contrast to methylated sugars, non-methylated sugars (quinovose, yersiniose, dideoxy hexoses, heptoses, deoxy-pentoses, amino deoxy hexoses) have been reported almost exclusively in O-antigenic 2. Mono-and di-methylated pentose results are pooled. The two most abundant sugars at each depth are also given.

Table 3

Summary of methylated sugar markers, identified in different environmental sources as alditol acetates, on GC-MS analysis. In bold the compounds identified in this study. Same abbreviations as in Table 2. ManN.: Mannosamine; GlcA.: Glucuronic acid.; LPS.: Lipopolysaccharides. §19 out of 52 compounds were identified tentatively.

Compound

Species Location Environmental source


Optical simulations

Mono-junction photovoltaic cells have intrinsic limitations:

• Non-absorption of low energy photons (<Eg);

• Thermalization of high energy photons (>Eg);

• Heating of the cell while operating, which reduces its efficiency. We use numerical simulations to study isolated nickel nanoparticles of different shapes and sizes, in order to find the structure which is best suited for IR solar harvesting.

• The absorption and scattering cross-sections of these particles are extracted from discrete dipole approximation (DDA) calculations, using the open-source code DDSCAT [START_REF] Draine | Discrete dipole approximation for scattering calculations[END_REF]. • The absorption spectra are compared to the AM1.5G spectrum to calculate the absorbed solar power over the range 0.875-2.5 µm.

All nanoparticles have a height of 150 nm.

Perfect structures

We developed a process to fabricate Ni nanoantennas on Si substrate using e-beam lithography, and study the impact of the fabrication defects.

Smaller nanoparticles tend to have a better absorption-to-volume ratio.

Choice of Ni diabolo as a promising structure. Several studies aimed at optimizing the efficiency of PV-TE by including a photothermal interface (PTI) in between the PV cell and the thermoelectric generator [START_REF] Park | Photothermally Activated Pyroelectric Polymer Films for Harvesting of Solar Heat with a Hybrid Energy Cell Structure[END_REF][START_REF] Kim | High-Performance Monolithic Photovoltaic-Thermoelectric Hybrid Power Generator Using an Exothermic Reactive Interlayer[END_REF].

Thermal model

Realistic structures

Objective of the PhD: to design a PTI using thermoplasmonic nanoantennas to convert solar IR radiation into heat.

Characteristics of an optimal PTI:

• High absorption and matching with the solar IR spectrum;

• High thermal conductivity;

• Electrically insulating. Poor spectral matching between absorption and solar irradiance.

➢ Identification of a nanoantenna design capable of efficient IR solar harvesting.

➢ Development of a proven process to fabricate metallic nanoantennas on various substrate (Si, glass), and study of the fabrication defects:

• Need to optimize the dimensions of the fabricated nanoantennas;

• Working on alternative fabrication processes (electrochemical deposition, nanoimprint) to reduce the surface roughness or the fabrication time.

➢ The thermal model can be improved to estimate the temperature of a photothermal interface integrated in a hybrid PV-TE device.

➢ The optical and thermal properties of the PTI need to be experimentally characterized:

• IR spectroscopy;

• Measure of localized heating using Raman spectroscopy.

1

  / Laboratoire d'analyse et d'architecture des systèmes du CNRS Harvesting of IR solar energy by thermoplasmonic nanoantennas S. Hanauer 1 , A. Mlayah 1 , F. Carcenac 1 , J.-B. Doucet 1 , E. Daran 1 , A. Dmitriev², I. Massiot 1 Laboratoire d'Analyse et d'Architecture des Systèmes (LAAS-CNRS), 7 Avenue du Colonel Roche, 31400 Toulouse, France ²Department of Physics, University of Gothenburg, 41296 Göteborg, Sweden

Figure 1 :

 1 Figure 1: Scheme of the intrisic losses in a PV cell and importance of the different phenomena for a GaAs cell with regard to the AM 1.5G solar spectrum.

Figure 2 :

 2 Figure 2: Schematic representation of a PV-TE device including a photothermal interface made of thermoplasmonic nanoantennas.

Figure 5 :

 5 Figure 5: (A) SEM image of Ni diabolos on Si and (B) Simulation target extracted from AFM measurements to represent a real Ni diabolo.

Figure 4 :

 4 Figure 4: Calculated interaction cross-sections for a perfect Ni diabolo and AM 1.5G solar spectrum, below (red) and above (blue) the energy gap of a GaAs PV cell.Figure6: Comparison between the interaction cross-section spectra of a perfect Ni diabolo and the associated realistic structure.

Figure 6 :

 6 Figure 4: Calculated interaction cross-sections for a perfect Ni diabolo and AM 1.5G solar spectrum, below (red) and above (blue) the energy gap of a GaAs PV cell.Figure6: Comparison between the interaction cross-section spectra of a perfect Ni diabolo and the associated realistic structure.

Figure 3 : 3 Figure 7 :

 337 Figure 3: Calculated solar absorption over the range 0.875-2.5 µm, normalized by volume, for isolated nickel nanoantennas of different shapes. The circled entry corresponds to the presented diabolo.