Patricia Bonin
email: patricia.bonin@mio.osupytheas.fr

Anne-Hélène Prime

Mairie-Aimée Galeron

Sophie Guasco

Jean-Francois Rontani

Jean-François Rontani

Enhancement of biotic degradation of terrestrial particulate organic matter in an estuarine salinity gradient: Interactive effects of

Keywords: Estuaries, Terrestrial organic matter, Vascular plants, Biodegradation, Salinity, Potamoplankton

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

INTRODUCTION

The riverine export of organic matter, whether dissolved or particulate is the main land-to-ocean contributor in coastal carbon fluxes. Early estimations ranged that contribution between 0.2 and 1 PgC yr -1 [START_REF] Kempe | Carbon in the freshwater cycle[END_REF][START_REF] Meybeck | Carbon, nitrogen, and phosphorus transport by world rivers[END_REF], while more recent estimates limited that number to 0.45 PgC yr -1 [START_REF] Cole | Plumbing the global carbon cycle: integrating inland waters into the terrestrial carbon budget[END_REF] or 0.15 PgC yr -1 [START_REF] Hedges | What happens to terrestrial organic matter in the ocean?[END_REF]. Given the current importance of exhaustive and precise carbon budgets and fluxes, it is crucial to look at the behavior of organic matter upon its arrival in coastal ecosystems.

The fate of terrestrial organic matter in the ocean is not fully understood [START_REF] Handa | Land sources of marine organic matter[END_REF][START_REF] Hedges | What happens to terrestrial organic matter in the ocean?[END_REF]) and the importance of this knowledge for global carbon-cycle understanding was evidenced long ago thanks to carbon isotope mass balance models (Berner, 1989). It has long been thought that the fraction of terrestrial vascular plant derived organic matter reaching estuarine areas was refractory to further degradation, mostly due to its intense degradation on land and in the rivers and its high lignin content [START_REF] Bader | The lignin fraction of marine sediments[END_REF][START_REF] De Leeuw | The behaviour of esters in the presence of tetramethylammonium salts at elevated temperatures; flash pyrolysis or flash chemolysis?[END_REF][START_REF] Wakeham | The reactivity of plant-derived organic matter and the potential importance of priming effects along the lower Amazon River[END_REF]. However, it appears that only a small fraction of the organic matter found in coastal sediments is of terrestrial origin [START_REF] Hedges | What happens to terrestrial organic matter in the ocean?[END_REF], implying that either the estimations of current carbon reservoirs are incorrect, or, more likely, that organic matter undergoes an intense degradation upon reaching seawater [START_REF] Hedges | What happens to terrestrial organic matter in the ocean?[END_REF]. Recent estimates of CO2 efflux from streams and rivers confirmed that terrestrial organic carbon is not as recalcitrant as previously thought [START_REF] Mayorga | Young organic matter as a source of carbon dioxide outgassing from Amazonian rivers[END_REF]. It was recently estimated that over half of the Terrestrial Particulate Organic Matter (TPOM) is remineralized prior to burial [START_REF] Kandasamy | Perspectives on the terrestrial organic matter transport and burial along the land-deep sea continuum: Caveats in our understanding of biogeochemical processes and future needs[END_REF].

Over the past decade, a number of studies evidenced that TPOM discharged by rivers was prone to remineralization in coastal areas in polar, temperate and tropical climate zones [START_REF] Mayer | Input of nutritionally rich organic matter from the Mississippi River to the Louisiana coastal zone[END_REF][START_REF] Van Dongen | Differential transport and degradation of bulk organic carbon and specific terrestrial biomarkers in the surface waters of a sub-arctic brackish bay mixing zone[END_REF], Vonk et al. 2010[START_REF] Bourgeois | Distribution and lability of land-derived organic matter in the surface sediments of the Rhône prodelta and the adjacent shelf (Mediterranean Sea, France): a multi proxy study[END_REF][START_REF] Rontani | Degradation of sterols and terrigenous organic matter in waters of the Mackenzie Shelf, Canadian Arctic[END_REF]. This efficient mineralization in the marine environment could be attributed to a more efficient use of TPOM by marine bacterial assemblages than by their soil and river counterparts [START_REF] Garneau | Importance of particle-associated bacterial heterotrophy in a coastal Arctic ecosystem[END_REF]. The involvement of 'priming effects' (enhanced remineralization of TPOM in the presence of fresh substrates from algal sources; [START_REF] Steen | Novel barite chimneys at the Loki's castle vent field shed light on key factors shaping microbial communities and functions in hydrothermal systems[END_REF])) constitutes another possibility [START_REF] Bianchi | The role of terrestrially derived organic carbon in the coastal ocean: A changing paradigm and the priming effect[END_REF][START_REF] Bianchi | Positive priming of terrestrially derived dissolved organic matter in a freshwater microcosm system[END_REF][START_REF] Wakeham | The reactivity of plant-derived organic matter and the potential importance of priming effects along the lower Amazon River[END_REF]. Indeed, coastal deltaic regions, which are characterized by a high input of TPOM, a high primary production and the presence of microbial communities highly adaptable to physicochemical changes, are generally considered to have a high priming potential [START_REF] Bianchi | The role of terrestrially derived organic carbon in the coastal ocean: A changing paradigm and the priming effect[END_REF]. Unfortunately, the priming effect phenomenon has been very poorly studied by the scientific communities working on marine and continental aquatic ecosystems [START_REF] Guenet | Priming effect: bridging the gap between terrestrial and aquatic ecology[END_REF]) and studies showed contrasting results [START_REF] Bengtsson | Interactive effects on organic matter processing from soils to the ocean: are priming effects relevant in aquatic ecosystems?[END_REF]. The meta-analysis of results from the literature by [START_REF] Bengtsson | Interactive effects on organic matter processing from soils to the ocean: are priming effects relevant in aquatic ecosystems?[END_REF]) revealed a weak and non-significant priming effects across fresh water and marine ecosystems. The high photooxidation state of TPOM in some regions could also be at the origin of the intense biodegradation of this material observed in seawater [START_REF] Rontani | Degradation of sterols and terrigenous organic matter in waters of the Mackenzie Shelf, Canadian Arctic[END_REF]. Indeed, solar light-induced fragmentation can reduce structural barriers in higher plant debris and expose new microniches to bacterial colonization [START_REF] Vähätalo | Impact of solar radiation on the decomposition of detrital leaves of eelgrass Zostera marina[END_REF].

The present work is focused on the Rhône Estuary, where only 34% of the POM discharged by the Rhône is deposited [START_REF] Dufois | Impact of winter storms on sediment erosion in the Rhone River prodelta and fate of sediment in the Gulf of Lions (North Western Mediterranean Sea)[END_REF]. We intend to determine if the presence of potamoplankton can enhance bacterial degradation of TPOM. Different hypothesis are tested: i) an interactive effects of OM pools mixing and/or ii) a change in the microbial community to a more adapted one with the increase of salinity. For this purpose, the degradation of suspended particulate matter (SPM) collected in the Rhône River during and after the potamoplankton bloom was studied in vitro at different salinities. Due to the overlapping between  13 C signatures of potamoplankton and terrestrial higher plants [START_REF] Tolosa | Carbon sources in suspended particles and surface sediments from the Beaufort Sea revealed by molecular lipid biomarkers and compound-specific isotope analysis[END_REF], it was problematic to discern isotopically the sources of organic matter in the SPM samples, the degradation of TPOM was thus monitored with several lipid tracers specific to terrestrial vascular plants (Table 1). At the same time, changes in the structure of bacterial communities were monitored both in the in vitro experiments and in SPM samples collected in the salinity gradient of the Rhône River mouth.

MATERIALS AND METHODS

Sampling

For the biodegradation experiments, fresh SPM samples were collected at the Arles station (43°40′44′′N, 4°37′16′′E, Fig. 1) on January 24 and March 30, 2017. At these sampling times, particulate organic matter contents were in the same range (31.6 and 36.7 g. Kg -1 , respectively). About 1000 l of Rhône River water were sampled at 1 m depth using a teflon-coated high-speed centrifuge (CEPA Z61), 42 and 56 g (wet weight) of SPM were obtained in January and March, respectively which were stored in an ice box until the return to the lab (1 h). After removal of SPM, the remaining freshwater was stored at 4°C. Collected SPM were used as bacterial inoculates and sources of TPOM upon arrival at the laboratory.

Sea water was sampled the 7 February 2017 at the station MESURHO (43° 19' 7"N,4°51'58"E) near the Rhône River mouth (Fig. 1) in order to be used to adjust salinity for incubation and to gain the inoculum of marine bacteria. At sampling time, the salinity was 20 g kg -1 (Refractometer ATAGO ATC smile). SPM samples were also collected in February 2012 along a transect following the salinity gradient in the Rhône flow in the framework of the national program MOOSE (Mediterranean Ocean Observing System for the Environment) (Fig. 1). The flow river was 980 m 3 s -1 . Five subsurface stations were sampled (between 0-3 m depth) with a Niskin bottle according to the salinity gradient: StA, StB, StC, StD and StE with 0, 15, 33, 39 and 40 g kg -1 , respectively (Fig. 1). The volume filtered (between 1 and 3 l) depended on SPM concentration at each station.

The water was filtered on pre-combusted GF/F filters (Whatman). Filters were immediately frozen at -20 °C.

Biodegradation experiments

Two sets of biodegradation experiments were performed. The first one to mimic the effect of the variations of salinity on biodegradation of TPOM with SPM collected during the potamoplancton bloom in January. The second one to compare biodegradation of TPOM in bloom and post-bloom conditions. The experiments were performed in parallel; SPM sample collected on January 24 was stored less than 15 days after its collection. We cannot totally exclude a weak degradation of TPOM during the storage of this sample.

Experimental system were composed by 30 mg (equivalent dry weight) of fresh SPM collected at the Arles station in January or March 2017 and 150 ml of water adjusted at the chosen salinity (0, 10 or 20 g kg -1 ) in 500 ml Erlenmeyer flasks. Fresh water (obtained from Rhône water after separation of SPM in January) and MESURHO sea water were used for incubations at 0 and 20 g kg -1 , respectively. Ten g kg -1 salinity was obtained by mixing sea water collected at the MESURHO station and Rhône fresh water. The dissolved organic carbon was in the same range in water from Rhône and MESURHO, it never exceeded 200 µM. The lipid content of the dissolved phase was considered as negligible relative to this of SPM.

Aerobic systems were incubated at a temperature close to the in situ temperature (15°C). To maintain aerobic conditions, flasks coated with cotton plugs were incubated in the dark on a reciprocal shaker (96 rpm, 5 cm amplitude). At each incubation time (0, 12, 24 and 38 days) three flasks (triplicates) were sampled, one for monitoring changes of bacterial biodiversity and two replicates for lipid analysis. All samples were filtered on pre-combusted GF/F filters (Whatman) and immediately frozen at -20 °C until analysis.

Chemical analysis

After defrosted, filters were placed in methanol (20 ml) with excess NaBH4 (70 mg; 30 min at 20 °C). This operation was carried out in order to reduce hydroperoxides present in the samples [START_REF] Galeron | Seasonal survey of the composition and degradation state of particulate organic matter in the Rhône River using lipid tracers[END_REF], which could induce autoxidative damages of lipids during the hot saponification step. After NaBH4 reduction, 20 ml water and 2.8 g KOH were added and the mixture directly saponified by refluxing (75°C) for 2 h. After cooling, samples were acidified with HCl (pH 1) and extracted (3×) with dichloromethane (DCM). The combined DCM extracts were dried, evaporated to dryness and the total lipid extract (TLE) was dissolved in 300 μl of pyridine/N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA; Supelco; 2:1, v:v) and silylated for 1 h at 50 °C to convert OH-containing compounds to trimethylsilyl -ether or ester derivatives. After solvent removal under a stream of N2, the derivatized residue was dissolved in 100 μl BSTFA (to avoid desilylation of FAs) and 600 µl of solvent (n-hexane).

GC-quadrupole time of flight mass spectrometry (GC-QTOF)

Accurate mass measurements were carried out in full scan mode with an Agilent 7890B/7200 GC-QTOF System (Agilent Technologies, Parc Technopolis -ZA Courtaboeuf, Les Ulis, France). A cross-linked 5% phenyl-methylpolysiloxane (Agilent; HP-5MS ultra inert) (30 m  0.25 mm, 0.25 µm film thickness) column was used. Analysis was performed with an injector operating in pulsed splitless mode set at 270 °C and the oven temperature was programmed from 70 °C to 130 °C at 20 °C min -1 and then to 300 °C at 5 °C min -1 . The carrier gas (He) was maintained at 0.69 × 10 5 Pa until the end of the temperature program. Instrument temperature was 300 °C for the transfer line and 230 °C for the ion source. Accurate mass spectra were recorded across the range m/z 50-700 at 4 GHz. The instrument provided a typical resolution ranging from 8009 to 12252 from m/z 68.9955 to 501.9706. Perfluorotributylamine (PFTBA) was utilized for daily MS calibration ( < 2 ppm).

Standards

Quantification of lipids was carried out using GC-QTOF with external standards. Sitosterol, sitostanol, -amyrin, -amyrin, lupeol, vanillic, syringic and 4-hydroxycoumaric acids were obtained from Sigma-Aldrich. Cutin components (isomeric dihydroxyhexadecanoic acids) were quantified with a standard of 9-10-dihydroxyhexadecanoic acid obtained by oxidation of palmitoleic acid with OsO4 [START_REF] Mccloskey | Mass spectra of O-isopropylidene derivatives of unsaturated fatty esters[END_REF].

Analysis of the bacterial communities associated to SPM

DNA extraction and PCR amplification

Genomic DNA was obtained from the filters using lysozyme-sodium dodecyl sulfate (SDS) followed by phenol-chloroform extraction and subsequent precipitation using ethanol. DNA concentration was determined spectrophotometrically following treatment with RNase. PCR amplification of bacterial 16SrRNA gene for DGGE analysis was performed using the GML5F -907MR primer set for Bacterial community as previously described [START_REF] Bonin | Anaerobic biodegradation of squalene: using DGGE to monitor the isolation of denitrifying bacteria taken from enrichment cultures[END_REF][START_REF] Goregues | Molecular, biochemical, and physiological approaches forunderstanding the ecology of denitrification[END_REF]. For the ribosomal diversity analysis, the V4 region of the bacterial and archaeal 16S rRNA genes were amplified using a universal primer set, 515F-Y (5′-GTGYCAGCMGCCGCGGTAA-3′, [START_REF] Parada | Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples[END_REF]) and 806RB (5′-GGACTACNVGGGTWTCTAAT-3′, [START_REF] Apprill | Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton[END_REF]. PCR amplification was performed with 50 ng of target DNA and 0,5 µM (1 µL à 10 pmol/µL) of each primer. The reaction mixture contains 10 μL of GoTaq® PFU (Promega) in a final volume of 50 μl. PCR amplification is done in a minicycle (Biorad T100 Thermal Cycler) for 35 cycles. The matrix DNA is initially denatured for 3 minutes at 94°C. Each cycle includes a step of denaturing the double-stranded DNA (45 seconds, 94°C), a step oligonucleotide hybridization (60 seconds, 50°C) and an elongation step (90 seconds, 72°C). The amplification ends with a final elongation step at 72°C for 10 minutes. Amplification products are detected by electrophoresis on agarose gel 1.5%

(Promega) followed by bromide staining ethidium (colored intercalant) (1μg.ml -1 ).The 16S rRNA gene amplicons were sequenced by the MiSeq Illumina (paired end 2* 250) platform Get of Genotoul.

Diversity analysis

The analysis of the biodiversity was performed by DGGE for the incubation samples whereas prokaryotic diversity and community structure analysis was based on 16S rDNA-sequencing for the salinity profile samples.

Denaturing Gradient Gel Electrophoresis (DGGE) was performed using a D-code Universal Mutation Detection System (Bio-Rad Laboratories Inc.). Banding patterns across the gels were normalized using a previously constructed ladder composed of seven different bands that was loaded several times on each gel. Fingerprints observed on the gels were analyzed using

GelCompar II (Applied Maths, St-Martens-Latem, Be). After normalization, automatic band detection was performed using a minimum profiling level of 5% relative to maximum. Band comparison between different profiles was settled with a 2% position tolerance and a 1% optimization to allow for tolerance of bands shifts within and among lanes. For DGGE patterns, calculation of the pair-wise similarities of densitometric profiles was based on Pearson's correlation coefficient [START_REF] Michener | A quantitative approach to a problem in classification[END_REF] to construct dendrograms using the Unweighted Pair-Group Method Analysis (GelCompar II software, Applied Maths, St-Martens-Latem, Be).

The 16S amplicons were sequenced by the MiSeq Illumina (paired end 2 * 250) platform Get of Genotoul. The paired-end raw reads were firstly overlapped and merged by platform GeT of Genotoul. The analysis of the 16S-assembled data was relying on the use of QIIME 1.91 [START_REF] Kuczynski | Using QIIME to analyze 16S rRNA gene sequences from microbial communities[END_REF] Core Team, 2008).

Statistical analyses

The results were analyzed using the R® (GNU) software. Multivariate analyses (main components (PCAs) and multiple factorials (MFAs)) were carried out in order to combine the results of chemical and microbiological extractions. The influence of the different conditions and incubation times were verified by mean (ANOVA) comparisons and Pearson or Spearman correlations (normal data or not).

RESULTS AND DISCUSSION

Lipid composition of SPM samples collected at the Arles station

In SPM samples from the Rhône River (Fig. 1, StA), fatty acid (FA) profiles are generally dominated by saturated FA and these of sterols by cholest-5-en-3-ol (cholesterol) and 24ethylcholest-5-en-3-ol (sitosterol) [START_REF] Galeron | Seasonal survey of the composition and degradation state of particulate organic matter in the Rhône River using lipid tracers[END_REF]) (See appendix). In contrast, the sample collected in January appeared to contain high proportions of polyunsaturated FA (C16:3, C16:4, C18:4, C20:3 and C20:5), cholesta-5,24(25)-dien-3-ol (desmosterol), 24-methylcholesta-5,22E-dien-3-ol (brassicasterol) and 24-methylcholesta-5,24(28)-dien-3-ol (24methylenecholesterol) (Fig. S1A) (See appendix). This typical algal lipid composition confirmed the presence of the yearly potamoplanktonic bloom event, which was previously observed in the Rhône River [START_REF] Galeron | Seasonal survey of the composition and degradation state of particulate organic matter in the Rhône River using lipid tracers[END_REF]. The timing (ranging from January to March)

and the intensity of this event seem to be strongly dependent on meteorological conditions (temperature, rainfall intensity and cloud cover thickness). On the basis of the high proportions of desmosterol and 24-methylenecholesterol detected, this bloom was attributed to freshwater diatoms. Indeed, 24-methylenecholesterol is the most common sterol in diatoms [START_REF] Volkman | Sterols in microorganisms[END_REF][START_REF] Rampen | A comprehensive study of sterols in marine diatoms (Bacillariophyta): implications for their use as tracers for diatom productivity[END_REF]) and desmosterol was proposed as a taxonomic marker for some species of diatoms, particularly for Rhizosolenia, where it is the dominant sterol [START_REF] Barrett | Sterols of 14 species of marine diatoms (Bacillariophyta)[END_REF]). This assumption is well supported by the drop of silica concentration observed at the same period (MOOSE data 2017, unpublished data).

In contrast, the SPM sample collected on March 7, 2017 exhibited a more classical lipid profile with very small proportions of PUFA, desmosterol and 24-methylenecholesterol attesting to the disappearance of the bloom (Fig. S1B).

The two samples (January and March) also contained: (i) isomeric dihydroxyhexadecanoic acids arising from the hydrolysis of cuticular waxes (von Wettstein-Knowles 1993, Otto et al.

2005) , (ii) a variety of pentacyclic triterpenoids with structures based on the ursene (-amyrin and ursolic acid), oleanene (-amyrin and oleanolic acid) or lupene (lupeol) hydrocarbon skeletons common in higher plant materials such as leaves, bark, roots and wood [START_REF] Pancost | The palaeoclimatic utility of terrestrial biomarkers in marine sediments[END_REF][START_REF] Otto | A comparison of plant and microbial biomarkers in grassland soils from the Prairie Ecozone of Canada[END_REF]) and (iii) phenolic acids (vanillic, syringic, ferulic, 4hydroxycoumaric acids) (Fig. S1). These last acids are generally considered as characteristic of lignin from non-woody vascular plant tissues (e.g., grass leaves, conifer needles) [START_REF] Hedges | The characterization of plant tissues by their lignin oxidation products[END_REF]. They may also constitute ester-bound aromatic constituents of suberin [START_REF] Kolattukudy | Hydrophobic layers attached to cell walls. Cutin, suberin and associated waxes[END_REF][START_REF] Riederer | Occurrence, distribution and fate of the lipid plant biopolymers cutin and suberin in temperate forest soils[END_REF][START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF]. It may be noted that ferulic acid and 4-hydroxycoumaric acid were also identified as components of ligno-cellulose complexes of grasses linking carbohydrates and lignin via ester bonds [START_REF] Lam | Bonding of hydroxycinnamic acids to lignin: ferulic and p-coumaric acids are predominantly linked at the benzyl position of lignin, not the βposition, in grass cell walls[END_REF]). Since the applied alkaline hydrolysis treatment cleaves esters, but not ethers, the phenolic acids detected are likely derived from ester-bound moieties of suberin and ligno-cellulose rather than from the ether-linked lignin macromolecule [START_REF] Otto | A comparison of plant and microbial biomarkers in grassland soils from the Prairie Ecozone of Canada[END_REF]. 4-Hydroxycoumaric and ferulic acids (C) are only present in non-woody tissues of vascular plants, while syringic acid (S) is unique to angiosperms and vanillic acid (V) exists in all vascular plants (Table 1) (Sun et al., 2016). On the basis of the ratio C/V and S/V [START_REF] Hedges | The characterization of plant tissues by their lignin oxidation products[END_REF] observed, TPOM carried by the Rhône River seems dominated by debris of angiosperm and grass leaves.

Isomeric dihydroxyhexadecanoic acids, -and -amyrins, lupeol, vanillic, syringic and 4hydroxycoumaric acids were thus selected as tracers (Table 1) to monitor the degradation of TPOM during the different incubations. Sitosterol is also commonly associated with terrestrial higher plant inputs [START_REF] Lütjohann | Sterol autoxidation: from phytosterols to oxyphytosterols[END_REF]), but some microalgae (and notably diatoms) are also known to produce this sterol [START_REF] Volkman | Sterols in microorganisms[END_REF][START_REF] Rampen | A comprehensive study of sterols in marine diatoms (Bacillariophyta): implications for their use as tracers for diatom productivity[END_REF]. Due to the expected presence of high proportions of diatoms during the bloom event, a significant part of this sterol could not be traced back to higher plant material in SPM samples collected the 24 th January, so we thus discarded the use of this widespread and ambiguous tracer during our in vitro studies of TPOM degradation.

Ex situ experiments to mimic variation of salinity and fresh algae input on bacterial degradation of TPOM

The bacterial degradation of TPOM was monitored with selected specific lipid tracers (Table 1) during the incubation of SPM collected in the Rhône River in January (during the bloom event) at different salinities (0, 10 and 20 g kg -1 ) and at 15 °C. The decreasing concentrations of 3-hydroxy-urs-12-en-11-one and 3-hydroxy-olean-12-en-11-one (specific tracers of and -amyrin autoxidation, respectively) ( [START_REF] Galeron | Seasonal survey of the composition and degradation state of particulate organic matter in the Rhône River using lipid tracers[END_REF][START_REF] Galeron | Oxidation products of α-and β-amyrins: potential tracers of abiotic degradation of vascular-plant organic matter in aquatic environments[END_REF]) observed during these incubations allowed us to exclude the involvement of autoxidative artifacts. This demonstrates that changes in the composition of these lipids observed during our incubations were microbially mediated.

Cuticular wax components (isomeric dihydroxyhexadecanoic acids) and specific triterpenes of vascular plants (-and -amyrins and lupeol) appeared to be degraded more efficiently at the highest salinity (Table S1, Fig. 2A-C). It is interesting to note that this degradation took place without accumulation of intermediate catabolites likely due to rapid turnover. In the case of phenolic acids (vanillic, syringic and 4-hydroxycoumaric acids), a decrease of their concentrations was observed at all salinities towards the end of the incubation (Fig. 2D-F). However, in the case of the highest salinity an increase in the concentration could be observed at the end of the incubation (Fig. 2D-F). This increase can be attributed to the bacterial degradation of lignin, suberin or ligno-cellulose, well known to produce these acids [START_REF] Otto | A comparison of plant and microbial biomarkers in grassland soils from the Prairie Ecozone of Canada[END_REF]. Indeed, in the literature there are a number of reports of bacteria (Streptomyces, Nocardia, Sphingomonas, Pseudomonas, Rhodococcus …) that can break down lignin structures and produce phenolic acids (for a review see [START_REF] Bugg | Pathways for degradation of lignin in bacteria and fungi[END_REF].

During the incubations, bacterial degradation also affected lipids of the algal components of the bloom event. As in the case of TPOM, the extent of degradation of these algal lipids increased with salinity (C20:5 FA, desmosterol and phytol degradation percentages ranging from 90, 70 and 40% at 0 g kg -1 to 95, 90 and 70% at 20 g kg -1 , respectively (data not shown)).

A global analysis of the evolution of the structure of the bacterial community associated to Rhône SPM by DGGE clearly shows the impact of salinity on bacterial communities.

Changes in salinity allow for the establishment of specific communities for each salinity, dominated by different phylotypes (Fig. 3). The communities observed after incubation of Rhône SPM at salinity 20 g kg -1 (T12, T24, T38) are close to those observed in situ at the same salinity (greater than 50% similarity) (Fig. S2). This community seems to harbor bacteria possessing higher capacities to degrade algal and vascular plant components than these initially present within the river.

Interactive effects of the presence of freshwater algae on the bacterial degradation of TPOM

Comparison of the bacterial TPOM biodegradation after incubation with the two kinds of SPM (collected in January during the potamoplankton bloom or in March after this event) clearly showed a faster degradation of cuticular wax components and triterpenes in the presence of potamoplankton after incubation at the same salinity (20 g kg -1 ) (Table S1, Fig. 4A-C).

Interestingly, the increase in phenolic acid concentration (attributed to lignin or suberin degradation) observed at the end of the incubations with January SPM was not observed in the case of incubations with March SPM (Table S2, Fig. 4D-F). The presence of freshwater phytoplankton (dominated by diatoms) thus enhances TPOM remineralization. TPOM was more intensively degraded with January SPM in the presence of high level of potamoplankton, which could have a beneficial interactive effect on these processes or induce a change in microbial communities.

In situ behavior of TPOM in the salinity gradient of the Rhône River

The stable carbon isotopic  13 C) signatures of the main lipids (fatty acids and sterols) were measured previously in the SPM samples collected along the salinity gradient of the Rhône River [START_REF] Galeron | Lipoxygenase-induced autoxidative degradation of terrestrial particulate organic matter in estuaries: A widespread process enhanced at high and low latitude[END_REF]. Sitosterol (-27.1‰) was depleted in 13 C relative to 24methylenecholesterol (-21.6‰) indicating a mainly terrestrial origin. We therefore used the percentage of sitostanol relative to the parent sitosterol for in situ TPOM bacterial degradation estimates. Indeed, stanols serve as useful indicators of bacterial degradation of  5 -sterols [START_REF] Gagosian | Vertical transport of steroid alcohols and ketones measured in a sediment trap experiment in the equatorial Atlantic Ocean[END_REF][START_REF] Wakeham | Reduction of stenols to stanols in particulate matter at oxic-anoxic boundaries in sea water[END_REF][START_REF] De Leeuw | The behaviour of esters in the presence of tetramethylammonium salts at elevated temperatures; flash pyrolysis or flash chemolysis?[END_REF]. In the SPM samples collected in the Rhône Estuary, the percentage of sitostanol appeared to increase logarithmically from 0.4 to 15.4 % (Table S3) with the proportion of algal sterols (brassicasterol and 24methylenecholesterol) relative to sitosterol (Fig. 5). The plateau observed at high proportions of algal material may be attributed to the well-known inhibition effect observed when cosubstrates (algal sterols) and substrate (sitosterol) compete for the same enzymes (Dalton, 1982). Indeed, reduction of brassicasterol, 24-methylenecholesterol and sitosterol to the corresponding stanols involves the same enzymatic sequence. This enhancement of the biodegradation of sitosterol (arising mainly from terrestrial vascular plants in the Rhône River) [START_REF] Galeron | Seasonal survey of the composition and degradation state of particulate organic matter in the Rhône River using lipid tracers[END_REF] with an increasing proportion of algal material (co-substrate) suggests that the presence of algal material favors TPOM degradation in the Rhône Estuary. Concerning the origin of the algal material present in these SPM samples, the relatively enriched  13 C signature of 24-methylenecholesterol (a well-known diatom marker) [START_REF] Volkman | Sterols in microorganisms[END_REF][START_REF] Rampen | A comprehensive study of sterols in marine diatoms (Bacillariophyta): implications for their use as tracers for diatom productivity[END_REF]) allowed to exclude the presence of freshwater phytoplankton, which typically exhibits more depleted  13 C values (-34‰ to -36‰) [START_REF] Tolosa | Carbon sources in suspended particles and surface sediments from the Beaufort Sea revealed by molecular lipid biomarkers and compound-specific isotope analysis[END_REF]. The enhancement of the biotic degradation of TPOM in the salinity gradient of the Rhône River seems thus to result from the presence of marine diatoms.

The bacterial degradation of vascular plant material in the Rhône Estuary is well supported by: (i) the depleted carbon isotopic  13 C) signatures (ranging from -30.9‰ to -27.8‰) of palmitoleic acid mainly arising from bacteria in the SPM samples investigated and

(ii) the  13 C signatures of the specifically bacterial vaccenic (-30‰) and branched pentadecanoic (-29‰) acids previously measured by [START_REF] Bourgeois | Distribution and lability of land-derived organic matter in the surface sediments of the Rhône prodelta and the adjacent shelf (Mediterranean Sea, France): a multi proxy study[END_REF] in sediments from the same deltaic region of the Rhône River. These different isotopic data confirm that bacteria present in the Rhône Estuary grew on 13 C depleted C3 terrestrial plant material and not 13 C enriched marine algae.

In situ biodiversity along the salinity gradient

The prokaryotic biodiversity was analyzed at different sampling sites along the salinity gradient in the Rhône River mouth. Five subsurface stations were sampled StA, StB, StC, StD and StE corresponding to the following salinities: 0, 15, 33, 38 and 39 g kg -1 . From the five libraries, 342123 sequences of 16S rRNA gene were analyzed (Table 2). Good's index showed that the sequencing depths were sufficient to cover 72.86 to 85.70 % of the microbial diversity (Table 2). The subdivision into operational taxonomic units (OTUs) sharing >97 % and after discarding low-quality read identity allowed the detection of the different OTUs (Table 2). A drastic decrease of species diversity was observed at the boundary between the two water masses (StA in the Rhône River and sea water, StB salinity 15g kg -1 ) since both Shannon's diversity (H') and Simpson's diversity (D1) indexes (H' = 5.87, D1 = 0.93) were lower than in Rhône water (H' = 7.75 , D1 = 0.98) (Table 2). For higher salinities (StC, StD, StE) the diversity increases again, corresponding to a change in communities.

Rhône River communities was dominated by Proteobacteria (Alphaproteobacteria, Gammaproteobacteria and Betaproteobacteria). At intermediate salinity (15 g kg -1 ),

Betaproteobacteria strongly increased and new taxonomic classes were observed (Cytophagia, Flavobacteria). This community is also characterized by the presence of Actinobacteria. At salinities close to that of seawater Flavobacteria, Alphaproteobacteria, Gammaproteobacteria.

and Thaumarchaeota dominated, while Betaproteobacteria were not detected (Figs 6 and7).

Within the Proteobacteria classes, different families were observed within the salinity gradient.

Alphaproteobacteria are dominated by Rhodobacteraceae in freshwater and then Pelagibacteraceae (at StB, 15 g kg -1 ) and finally Rhodospirillaceae was detected as the salinity increased (at StC, 33 g kg -1 ). Among Gammaproteobacteria, Sinobacteraceae dominated in freshwater, while Halomonadaceae and Alteromonadaceae were detected at salinities over 15 g kg -1 . Within Betaproteobacteria at StB, Rhodocyclaceae present in freshwater are substituted by Comamonadaceae and Moraxellaceae, which then was progressively not detected with increasing salinities (Fig. 6). Results indicated a significant change in the composition of the microbial population all along the salinity gradient. In freshwater, the communities are dominated by Betaproteobacteria; at StB, the communities are characterized by the presence of Actinobacteria, whereas at the highest salinities Flavobacteria, Alphaproteobacteria and Gammaproteobacteria were to be the most abundant. The dominance of Gamma and Alphaproteobacteria in seawater and that of Betaproteobacteria in freshwater is in concordance with the observations of [START_REF] Newton | A guide to the natural history of freshwater lake bacteria[END_REF] previously reported.

To unravel the relationships, the composition of the microbial community at the class level taxonomy and the percentage of TPOM biodegradation, estimated from the ratio sitostanol/sitosterol and the ratio (brassicasterol and 24-methylene cholesterol)/sitosterol reflecting the amount of algal in the salinity gradient between, we performed a bi-plot MFA (Fig. 7). The proportion of algal material strongly influences the biodegradation of sitosterol (Fig. 7), which derives mainly from terrestrial plants in this area [START_REF] Galeron | Seasonal survey of the composition and degradation state of particulate organic matter in the Rhône River using lipid tracers[END_REF]. This phenomenon seems to be of more relevance in seawater (at salinities of 39-40 g kg -1 ) (Fig. 7).

The assimilative capacity of this recalcitrant organic material is therefore different according to the communities present in the environment [START_REF] Bianchi | The role of terrestrially derived organic carbon in the coastal ocean: A changing paradigm and the priming effect[END_REF]. Interestingly, Crenarchaeota, more abundant at intermediate salinities (33 g kg -1 ) and well-known to degrade proteins and products released by diatoms (co-substrate) more efficiently than bacteria [START_REF] Kirchman | Standing stocks and activity of Archaea and Bacteria in the western Arctic Ocean[END_REF]) could be also responsible for the enhancement of the degradation of TPOM. According to these nutritional strategies, bacteria more efficient at degrading recalcitrant organic matter would be located in the estuarine zone. Indeed, on the basis of the competitive exclusion principle, generalist species (degrading recalcitrant compounds through priming and tolerant to changes) should dominate diversified habitats (sea), while specialized species (in the degradation of the TPOM) should live in a simplified environment (river) [START_REF] Pimm | Food web patterns and their consequences[END_REF][START_REF] Kassen | The experimental evolution of specialists, generalists, and the maintenance of diversity[END_REF].

CONCLUSIONS

In order to determine what phenomena are at the origin of the increase TPOM biodegradation in seawater, SPM samples collected in the Rhône River during and after a bloom of potamoplankton were incubated at different salinities (0, 15 and 20 g kg -1 ). The degradation of TPOM was monitored with specific lipid tracers (triterpenes, phenolic acids, cuticular wax components). It appeared that TPOM was degraded more efficiently at the highest salinity and in the presence of potamoplankton. The communities observed after incubation at the highest salinity (20 g kg -1 ), which are close to those observed in situ at the same salinity, degraded algal and vascular plant components more efficiently than that present within the river. The enhanced degradation of TPOM observed in the presence of potamoplankton (dominated by diatoms) is likely due to the presence of an algal co-substrate or/and a more efficient bacterial community during the bloom period. The bacterial consumption of TPOM observed in SPM samples collected in 2012 in the salinity gradient of the Rhône River was attributed to: (i) the involvement of priming effect, this process being likely induced by the presence of important proportions of marine diatoms, or (ii) a more efficient use of TPOM by marine bacterial assemblages than by their river counterparts. Indeed, the increase of salinity in the river mouth results in an enrichment in Gammaproteobacteria, which seem to be more efficient degraders of TPOM than Betaproteobacteria dominating the communities in the river. Samples were identified as their incubation time (T0, T12, T24 or T38 incubation days) followed by their salinity (0 g kg -1 (S0), 10 g kg -1 (S10) or 20 g kg -1 (S20)). Rhône SPM (Post-bloom samples) T0S20 2.9 0.8 2.9 T0S20 4.0 2.3 7.1 T12S20 1.5 0.9 3.4 T12S20

1.1 0.9 2.1 T24S20 1.4 1.2 3.2 T24S20 1.3 0.9 2.7 T38S20 1.2 1.0 3.8 T38S20

1.1 0.9 2.5 a T = incubation time (0, 12, 24 or 38 day), S = Salinity (0 , 10 or 20 g kg -1 )

Figure captions Figure 1 :

 captions1 Figure captions

Figure 2 :

 2 Figure 2: Concentrations (ng mg -1 ) of isomeric dihydroxyhexadecanoic acids (cuticular wax

Figure 3 :

 3 Figure 3: Multiple Factor Analysis (MFA), correlation biplot showing 16 samples (individual

Figure 4 :

 4 Figure 4: Concentrations (ng mg -1 ) of isomeric dihydroxyhexadecanoic acids (cuticular wax

Figure 5 :

 5 Figure 5: Biodegradation percentage of TPOM (estimated with the ratio sitostanol / sitosterol)

Figure 6 :

 6 Figure 6: Percentages of abundance of OTUs, classed at family level and ranked according to

Figure 7 :

 7 Figure 7: Multiple Factor Analysis (MFA), correlation biplot showing 5 samples (collected in

Figure S1 :

 S1 Figure S1: TOF mass chromatograms of the silylated total lipid extract of SPM collected in the

Figure S2 :

 S2 Figure S2: Dendrogram from DGGE analyses of the evolution of the bacterial community

  . The removal of low quality bases and chimera sequences were performed by QIIME 1.91 script and UCHIME(Edgar et al., 2011), respectively. The highquality sequences were next clustered into Operational Taxonomic Unit (OTU) using the UCLUST algorithm and a threshold of 97% of sequence identity. The taxonomic assignment of representative sequences of OTUs was performed using the green genes database(Edgar, 2010). The OTU table was filtered for low abundance OTUs(Bokulich et al., 2013). Finally, sub-sampling normalization, alpha and beta diversity were characterized by Phyloseq, an open-

source software package project for R (www.r-project.org)

(McMurdie and Holmes, 2013; R

Table 2 :

 2 Phylum-level taxonomic distribution of bacterial and archeal assemblages from sampling stations along salinity gradient at the Rhône river mouth

		salinity	Number of trimmed sequences	Number of observed OTU (97%)	Good's coverage (%)	shannon index	Simpson index
	StA	0	74780	52161	85.35	7.75	0.98
	StB	15	69753	57801	71.53	5.87	0.93
	StC	33	73376	68842	85.70	7.02	0.97
	StD	39	55065	42430	72.86	6.39	0.96
	StE	40	69146	54486	82.75	6.83	0.97

Table S1

 S1 Evolution of the concentration (ng mg -1 ) of vascular plant components during incubations at different salinity conditions

			Sample a	α-Amyrin β-Amyrin	Lupeol	Isomeric dihydroxy
							C 16:0 acids b
			T0S0	0.7	0.8	0.8	30.1
			T0S0	0.5	0.9	1.0	44.6
			T12S0	0.3	0.5	0.9	24.5
			T12S0	0.2	0.3	0.3	48.5
			T24S0	0.4	0.3	0.8	75.3
			T24S0	0.2	0.2	0.2	11.7
			T38S0	0.2	0.2	0.3	15.8
			T38S0	0.6	0.7	0.8	4.1
	Rhône SPM (Bloom samples)	T0S10 T0S10 T12S10 T12S10 T24S10 T24S10 T38S10 T38S10 T0S20	0.5 0.2 0.5 0.5 0.2 0.5 0.4 0.4 1.1	0.6 0.3 0.5 0.6 0.2 0.6 0.6 0.5 1.4	0.5 0.3 0.6 0.6 0.3 0.7 0.8 0.7 1.3	24.6 15.5 30.3 10.5 5.2 16.1 4.0 4.9 22.0
			T0S20	1.1	1.3	1.5	63.2
			T12S20	1.0	1.1	1.0	36.8
			T12S20	3.7	1.5	1.9	17.2
			T24S20	0.4	0.4	0.4	8.3
			T24S20	0.2	0.3	0.2	12.6
			T38S20	0.3	0.4	0.4	8.9
			T38S20	0.3	0.5	0.5	11.4
	Rhône SPM (Post-bloom	samples)	T0S20 T0S20 T12S20 T12S20 T24S20 T24S20 T38S20 T38S20	0.1 0.3 0.2 0.3 0.2 0.2 0.1 0.1	0.2 0.3 0.4 0.5 0.3 0.3 0.2 0.2	0.2 0.6 0.3 0.5 0.3 0.3 0.3 0.3	7.3 7.3 8.6 1.5 6.4 5.4 4.7 6.2

a T = incubation time (0 ; 12 ; 24 or 38 days), S = Salinity (0 ; 10 or 20 g kg -1 ) b Cuticular wax components

Table S2

 S2 Evolution of the concentration (ng mg -1 ) of phenolic acids during the different incubations

		Sample a	Vanillic acid	Syringic acid	4-Hydroxycoumaric acid
		T0S0	3.5	2.4	6.6
		T0S0	4.9	3.1	8.4
		T12S0	2.5	2.5	6.6
		T12S0	1.6	1.6	5.5
		T24S0	2.5	2.0	5.3
		T24S0	1.6	1.0	2.9
		T38S0	1.5	1.0	4.8
		T38S0	1.3	0.9	1.9
	Rhône SPM (Bloom samples)	T0S10 T0S10 T12S10 T12S10 T24S10 T24S10 T38S10 T38S10 T0S20	4.4 2.7 1.2 0.5 1.5 1.8 1.1 2.1 3.9	1.9 1.7 1.0 0.7 0.9 1.7 1.3 1.2 2.1	4.8 4.0 4.2 1.6 3.3 3.3 2.2 6.6 6.6
		T0S20	3.6	2.1	6.8
		T12S20	1.9	1.7	5.1
		T12S20	1.1	1.2	2.9
		T24S20	1.1	0.9	2.3
		T24S20	1.2	0.8	4.6
		T38S20	1.9	1.2	4.5
		T38S20	2.3	1.6	5.2

Acknowledgements

This work was supported by the LEFE-CYBER (Les Enveloppes Fluides et l'Environnement) national program, as part of the MORTIMER (Matière ORganique Terrestre rejetée par les fleuves et les rIvières en MER) research program. This study is also a contribution to the Labex OT-Med (through ANR-11-LABX-0061) funded by the French Government "Investissements d'Avenir" program of the French National Research Agency (ANR) through the A*MIDEX project (ANR-11-IDEX-0001-02). Thanks are due to the FEDER OCEANOMED (N° 1166- 39417) for the funding of the apparatus employed. Additional data were provided by "MOOSE" (Mediterranean Oceanic Observing System for the Environment) with the support of the "Agence de l'Eau Rhône-Méditerranée-Corse". We thank two anonymous referees for their useful and constructive comments.

Percent relative abundance