Christopher Burot

Rémi Amiraux

Patricia Bonin

Sophie Guasco

Marcel Babin

Fabien Joux

Dominique Marie

Laure Vilgrain

Hermann Heipieper

Jean-Francois Rontani
email: jean-francois.rontani@mio.osupytheas.fr

Jean-François Rontani

Jean-François Rontani

Viability and stress state of bacteria associated with primary production or zooplankton-derived suspended particulate matter in summer along a transect in Baffin Bay (Arctic Ocean)

Keywords: Sea ice algae, Bacterial viability, Salinity stress, Cis-trans isomerase, EPS, Micro-and macro-zooplankton

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

Arctic sea ice shelters a huge diversity of organisms particularly well-adapted to the harsh living conditions in this ecosystem [START_REF] Boetius | Microbial ecology of the cryosphere: sea ice and glacial habitats[END_REF]. They include bacteria, viruses, archaea and diatoms [START_REF] Von Quillfeldt | High number of diatom species in first-year ice from the Chukchi Sea[END_REF][START_REF] Junge | Bacterial activity at -2 to -20°C in Arctic wintertime sea ice[END_REF]Sazhin et al., 2018). One of the numerous ecosystem services that this particular biota fulfil is the production of organic matter [START_REF] Boras | Effect of ice melting on bacterial carbon fluxes channeled by viruses and protists in the Arctic Ocean[END_REF][START_REF] Boetius | Microbial ecology of the cryosphere: sea ice and glacial habitats[END_REF]. Sympagic diatoms (diatoms inhabiting the ice matrix) are responsible for much of this production. Their contribution to annual primary production (PP) varies widely depending on the season and the region (<1-60%, e.g. [START_REF] Dupont | Impact of sea-ice biology on overall primary production in a biophysical model of the pan-Arctic Ocean[END_REF][START_REF] Fernández-Méndez | Photosynthetic production in the central Arctic Ocean during the record sea-ice minimum in 2012[END_REF], but it represents a crucial food source for the marine food web, especially in winter [START_REF] Søreide | Timing of blooms, algal food quality and Calanus glacialis reproduction and growth in a changing Arctic[END_REF].

It has been shown that Arctic sea ice contains large amounts of extracellular polymeric substances (EPSs) [START_REF] Krembs | High concentrations of exopolymeric substances in Arctic winter sea ice: implications for the polar ocean carbon cycle and cryoprotection of diatoms[END_REF], one order of magnitude higher than in the surface water [START_REF] Krembs | Abundance and variability of microorganisms and transparent exopolymer particles across the ice/water interface of melting first-year sea ice in the Laptev Sea (Arctic)[END_REF][START_REF] Meiners | Vertical distribution of exopolymer particles in sea ice of the Fram Strait (Arctic) during autumn[END_REF]. These substances are produced by both bacteria and algae, with sympagic diatoms being their primary source in sea ice [START_REF] Meiners | Vertical distribution of exopolymer particles in sea ice of the Fram Strait (Arctic) during autumn[END_REF][START_REF] Nichols | Production of exopolysaccharides by Antarctic marine bacterial isolates[END_REF]. EPSs can help protect cells against harsh environmental conditions (e.g. salinity fluctuations) and assist cell adhesion [START_REF] Cooksey | Adhesion of bacteria and diatoms to surfaces in the sea: a review[END_REF]. The release of exopolymers by the sympagic biota further influences carbon cycling by (i) providing a carbon-rich substrate that will support bacterial production and metabolic activity [START_REF] Simon | Microbial ecology of organic aggregates in aquatic ecosystems[END_REF], (ii) bypassing microbially mediated POC production by the abiotic formation of large EPS-containing particles or aggregates [START_REF] Passow | Transparent exopolymer particles (TEP) in aquatic environments[END_REF][START_REF] Thornton | Diatom aggregation in the sea: mechanisms and ecological implications[END_REF], (iii) directly contributing to the organic carbon pool, with concentrations potentially equivalent to those of particulate organic carbon (POC) in pelagic environments [START_REF] Mari | Carbon content and C:N ratio of transparent exopolymeric particles (TEP) produced by bubbling exudates of diatoms[END_REF][START_REF] Engel | Carbon and nitrogen content of transparent exopolymer particles (TEP) in relation to their Alcian Blue adsorption[END_REF], and (iv) increasing the sympagic biota sedimentation rates through aggregation [START_REF] Riebesell | Aggregation of algae released from melting sea ice: implications for seeding and sedimentation[END_REF][START_REF] Azetsu-Scott | Ascending marine particles: Significance of transparent exopolymer particles (TEP) in the upper ocean[END_REF]. From their high sedimentation rates and their good preservation during their travel J o u r n a l P r e -p r o o f toward the seafloor [START_REF] Boetius | Export of Algal Biomass from the Melting Arctic Sea Ice[END_REF][START_REF] Rontani | Monitoring abiotic degradation in sinking versus suspended Arctic sea ice algae during a spring ice melt using specific lipid oxidation tracers[END_REF][START_REF] Amiraux | Monitoring photo-oxidative and salinity-induced bacterial stress in the Canadian Arctic using specific lipid tracers[END_REF], it has been suggested that sympagic PP contributes significantly to the total PP source in sea waters, especially deep ones [START_REF] Glud | Carbon Cycling in Arctic Marine Ecosystems: Case Study Young Sound[END_REF][START_REF] Krause-Jensen | Benthic primary production in Young Sound, Northeast Greenland[END_REF]. The fate of sympagic biota in sea ice and as they sink in the water column depends mostly on grazing by zooplankton and mineralization by their attached bacteria. In the Arctic, it is estimated that the zooplankton graze about 66-79% of the new PP (including sympagic algae; [START_REF] J O U R N A L P R E -P R O O F Forest | Biogenic carbon flows through the planktonic food web of the Amundsen Gulf (Arctic Ocean): A synthesis of field measurements and inverse modeling analyses[END_REF], and since their fecal pellets generally increase the sinking rates of their food, it is estimated that zooplankton probably form most of the PP export to the aphotic zone and seafloor [START_REF] J O U R N A L P R E -P R O O F Forest | Biogenic carbon flows through the planktonic food web of the Amundsen Gulf (Arctic Ocean): A synthesis of field measurements and inverse modeling analyses[END_REF]. By contrast, bacterial activity, which is higher for attached than for free bacteria [START_REF] Hoppe | Microbial extracellular enzyme activity: a new key parameter in aquatic ecology[END_REF][START_REF] Karner | Extracellular enzymatic activity and secondary production in freeliving and marine-snow-associated bacteria[END_REF], allows cleavage of the POM into smaller pieces by extracellular enzymatic hydrolysis [START_REF] Cho | Major role of bacteria in biogeochemical fluxes in the ocean's interior[END_REF]. Such processes enhance the further enzymatic digestion of the matter and ultimately reduce its potential to reach the seafloor. However, while heterotrophic bacteria and the rest of the microbial loop process about half of the PP in low-latitude oceans [START_REF] Ducklow | Bacterial Production and Biomass in the Oceans[END_REF], their contribution at higher latitudes is assumed to be smaller. Based on bacterial activity measurements, Howard-Jones et al. (2002) suggest that a significant fraction (25-80%) of Arctic bacterioplankton is dormant or inactive in the marginal ice zone of the Barents Sea.

Recently, [START_REF] Amiraux | Monitoring photo-oxidative and salinity-induced bacterial stress in the Canadian Arctic using specific lipid tracers[END_REF] suggested that the weaker activity of bacteria in the Arctic could result from the involvement of some stress factors in ice, such as salinity. During the early stage of ice melting in spring, brine inclusions (where salinity may reach up to 150 in some ice sections collected in early spring 2015 at the GreenEdge Ice Camp, Galindo, unpublished data) become interconnected in channels and are expelled from the sea ice into the underlying seawater [START_REF] Wadhams | Ice in the Ocean[END_REF]. The ice algal bacterial community is therefore exposed to a salinity stress, which occurs over relatively short timescales (e.g. hours). In this ecosystem, prokaryotic cells subjected to high osmotic pressure have developed J o u r n a l P r e -p r o o f mechanisms to live in these extreme conditions. Various strategies are used: (i) implementation of active Na + and K + ion transport systems [START_REF] Thompson | Functions of Na + and K + in the active transport of aminoisobutyric acid in a marine Pseudomonad[END_REF], (ii) accumulation of osmocompatible compounds such as glycine betaine or proline [START_REF] Piuri | Adaptation to high salt in Lactobacillus: role of peptides and proteolytic enzymes[END_REF] or (iii) production of EPSs, which can act as a diffusion barrier [START_REF] Kim | Physiological responses of salinity-stressed Vibrio sp. and the effect on the biofilm formation on a nanofiltration membrane[END_REF]. Another major adaptive response of many microorganisms, including bacteria, is to maintain membrane fluidity through 'homeoviscous adaptation' [START_REF] Sinensky | Homeoviscous adaptation-a homeostatic process that regulates the viscosity of membrane lipids in Escherichia coli[END_REF]. The shifts in fatty acid composition of membrane lipids, and most notably by enzymatic conversion of cis-to trans-unsaturated fatty acids [START_REF] Loffeld | isomerization of unsaturated fatty acids as possible control mechanism of membrane fluidity in Pseudomonas putida P8[END_REF][START_REF] Heipieper | The cis-trans isomerase of unsaturated fatty acids in Pseudomonas and Vibrio: biochemistry, molecular biology and physiological function of a unique stress adaptive mechanism[END_REF] through the activity of cis-trans isomerases (CTIs) can be an important bacterial mechanism for modifying membrane fluidity. It has been previously suggested that trans/cis ratios > 0.1 in environmental samples may be indicative of bacterial stress [START_REF] Guckert | Phospholipid ester-linked fatty acid profile changes during nutrient deprivation of Vibrio cholerae: Increases in the trans/cis Ratio and Proportions of Cyclopropyl Fatty Acids[END_REF]. Previous analyses of sea ice and sinking particles collected in the water column during the vernal melting period showed a relatively strong CTI activity, suggesting the occurrence of salinity stress during the early stages of ice melt [START_REF] Amiraux | Monitoring photo-oxidative and salinity-induced bacterial stress in the Canadian Arctic using specific lipid tracers[END_REF]. The high trans/cis ratios observed in sinking particles was attributed to the flush of bacteria associated with ice algae from internal hypersaline ice brines (Amiraux et al., 2021b). The relative stability of these ratios with depth also suggested that bacterial communities associated with sinking sympagic algae were non-growing.

In a previous study, Amiraux et al. (2021a) studied the stress state of bacteria attached to sinking sympagic algae during a vernal melting season at a landfast ice station in Davis Strait. Their results emphasized the impact of salinity, limiting the growth state of attached bacteria at the beginning of sea ice melting, subsequently giving way to an intense free fatty acid (FFAs) stress. Indeed, the production of bactericidal FFAs by sympagic algae is enhanced by the increase in light transmittance through the ice (due to the advance melting of sea ice) resulting in a high bacterial mortality. If this study gave us valuable information on J o u r n a l P r e -p r o o f the interactions between sympagic algae and their associated bacteria in sinking samples, data on those interactions after ice melting and on suspended particles are still lacking.

In the present work, we thus monitored the salinity stress and mortality of bacteria associated with sea ice and suspended particulate matter (SPM) samples collected in 2016 along a transect from sea ice to open water in Baffin Bay (Arctic Ocean). We intend to determine if the bacteria associated with these suspended particles are also weakly active or in a poor physiological state, thus impacting the preservation of this material.

Materials and methods

Sampling

Samples were taken at three ice stations (St 403,St 409 and St 413) and one open water station (St 418) (Fig. 1) from the Canadian icebreaker CCGS Amundsen along a longitudinal transect from 68° 4' 25.32"N and 61° 36' 30.54" W to 68° 6' 52.14" N and 57° 46' 7.14" W between 25 and 28 June 2016 as part of the GreenEdge project. At the time of sampling, this transect was under the influence of the Arctic current from the North acting practically perpendicular to the transect (A. Randelhoff, Personal communication).

Consequently, advection along the transect (East-West) should be relatively limited.

The sea ice sampling was carried out using a Kovacs Mark V 14 cm diameter corer, focusing on the bottom-most 10 cm of sea ice (sub-sectioned into two further intervals: 0-3 and 3-10 cm) where most ice biota are found [START_REF] Smith | Polynyas and leads: An overview of physical processes and environment[END_REF]. To compensate for biomass heterogeneity, common in sea ice [START_REF] Gosselin | Physical control of the horizontal patchiness of sea-ice microalgae[END_REF], three or four equivalent core sections were pooled for each sampling day in isothermal containers. Pooled sea ice sections were then melted in the dark with 0.2 μm filtered seawater (FSW; 3:1 v:v) to minimize J o u r n a l P r e -p r o o f Journal Pre-proof osmotic stress on the microbial community during melting [START_REF] Bates | Fluorescence Induction and Photosynthetic Responses of Arctic Ice Algae to Sample Treatment and Salinity[END_REF][START_REF] Garrison | Organism losses during ice melting: A serious bias in sea ice community studies[END_REF].

Suspended particulate matter (SPM) samples were collected at seven depths in the first 100 m of the water column using large (20 L) Niskin bottles to accommodate any withinsample heterogeneity.

For both sea ice and SPM, samples were collected in pentaplicate (a sample for lipid analyses and four for PMA analyses) as follows. Lipid, chlorophyll a, and total particulate carbon samples were obtained by filtration through pre-weighed Whatman glass fiber filters (Buckinghamshire, UK; GF/F, porosity 0.7 μm, combusted 4 h at 450°C) and kept frozen (< -80°C). Bacterial viability samples were obtained by filtration on 0.8 μm Whatman nucleopore filters (24 mm, autoclaved 1 h at 110°C) and kept frozen (< -80°C) prior to analysis. Owing to the porosity of the filters, the analyses concerned mainly algae, particles and their attached bacteria. Bacterial abundance and productivity were measured directly onboard the CCGS Amundsen by cytometry (see Bacterial abundance) and 3 H-leucine incorporation (see Bacterial productivity).

Treatment

Chlorophyll a

Concentration of chlorophyll a and phaeopigments retained on the GF/F filters were measured before and after acidification (5% HCl) using a TD-700 Turner Design fluorometer, after 18-24 h extraction in 90% acetone at 4°C in the dark [START_REF] Parsons | A manual of chemical and biological methods for seawater analysis. Pergamon, Oxford sized algae and natural seston size fractions[END_REF]. The fluorometer was calibrated with a commercially available chlorophyll a standard (Anacystis nidulans, Sigma).

J o u r n a l P r e -p r o o f

Total particulate carbon

At Université Laval, filters were dried for 24 h at 60°C, weighed again for dry weight determination and then analyzed using a Perkin Elmer carbon-hydrogen-nitrogen-sulfur (CHNS) 2400 Series II instrument to measure TPC. Calibration was done using accurately weighed samples of acetanilide (C 8 H 9 NO).

Bacterial abundance

Samples were analyzed directly on board the CCGS Amundsen using an Accuri C6 flow cytometer equipped with 488 nm and 633 nm lasers and the standard filter setup. For enumeration of phototrophs, cells were detected on the base of their red fluorescence (FL3) and unfixed samples were analyzed 3 min at a flow rate around 65 µL min -1 . Samples were then fixed with 0.25% glutaraldehyde (final concentration) and stained for a minimum of 15 min with SYBR Green I at 1/10 000 of the commercial solution for enumeration of heterotrophic cells [START_REF] Marie | Enumeration of phytoplankton, bacteria, and viruses in marine samples[END_REF]. Trigger was set on the green fluorescence of the SYBR and samples were analyzed 2 min at a flow rate of about 35 µL min -1 .

Bacterial productivity

Bacterial production (BP) was measured for 8 to 10 depths per station, distributed in the first 350 m, by [ 3 H]-leucine incorporation [START_REF] Kirchman | Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems[END_REF] modified for microcentrifugation [START_REF] Smith | A simple, economical method for measuring bacterial protein synthesis rates in seawater using 3 H-leucine[END_REF]. Triplicate 1.7 mL aliquots were incubated with a mixture of 50/50 (v/v) [ 3 H]-leucine (Perkin Elmer) and nonradioactive leucine for 4 h at a temperature (1.5°C) close to that in situ. Samples with 5% trichloroacetic acid added prior to the isotope served as blank. Saturation and time course were performed beforehand to determine the concentration of leucine and minimum incubation time. Leucine incorporation J o u r n a l P r e -p r o o f was converted to carbon production using a conservative conversion factor of 1.5 kg C mol -1 leucine [START_REF] Simon | Protein content and protein synthesis rates of planktonic marine bacteria[END_REF].

Bacterial viability analysis

The bacterial viability analysis was conducted using a method based on propidium monoazide (PMA). PMA is a photoreactive dye that binds to DNA, inhibiting its replication by PCR. Live cells have intact membranes and are impermeable to PMA, which only influxes into cells with disrupted membranes. The combination of PMA use and PCR provides a rapid and reliable method for discriminating live and dead bacteria. The viability analysis requires two sets of the same sample, one treated with PMA (that gives the quantity of living organisms in the sample) and an untreated one (that gives the total amount of organisms in the sample).

The first step of this method consists of a treatment of the concentrated and filtrated samples with PMA, the filters are then exposed to light, allowing PMA to bind with DNA.

For a detailed protocol see Amiraux et al. (2021a).

Nucleic acids were extracted using a chloroform-based method. Filters were placed in 2 mL Eppendorf ® tubes and heat-shocked (+80°C then -80°C alternately, twice) to improve cell lysis. 100 µL of lysis solution (Tris 20 mM, EDTA 25 mM, lysozyme 1 µg.µL -1 ) was added and the samples were incubated at 37°C for 15 min. 900 µL of sterile ice-cold water and 900 µl of chloroform were then added and the samples were vortexed five times for 5 s and then centrifuged for 5 min at 10,000 × g. 700 µL of the aqueous phase was collected, transferred to a new tube and any traces of remaining chloroform removed in speed vacuum concentrator (Savant DNA 120, Thermo Scientific ™) for 15 min. Finally, 10 µL of RNase (10 mg. mL -1 ) was added to the samples, and they were incubated for 30 min at 37°C or overnight at 4°C. The DNA obtained was kept frozen at -20°C for further use.

J o u r n a l P r e -p r o o f

Absolute quantification of bacterial SSU ribosomal RNA (rRNA) gene was carried out by qPCR with SsoAdvanced™ Sybr Green Supermix on a CFX96 Real-Time System (C1000 Thermal Cycler, Bio-Rad Laboratories, CA, USA) according to the procedure described in [START_REF] Fernandes | Coupling of bacterial nitrification with denitrification and anammox supports N removal in intertidal sediments (Arcachon Bay, France)[END_REF]. For more details about the qPCR program, see Amiraux et al. (2021a).

Lipid extraction

Samples (GF/F filters) were reduced with excess NaBH 4 after adding MeOH (25 mL, 30 min) to reduce labile hydroperoxides (resulting from photo-or autoxidation) to alcohols, which are more amenable to analysis using gas chromatography-mass spectrometry (GC-MS).

Water (25 mL) and KOH (2.8 g) were then added and the resulting mixture saponified by refluxing (2 h). After cooling, the mixture was acidified (HCl, 2 N) to pH 1 and extracted with dichloromethane (DCM; 3 × 20 mL). The combined DCM extracts were dried over anhydrous Na 2 SO 4 , filtered and concentrated by rotary evaporation at 40°C to give total lipid extracts (TLEs). Aliquots of TLEs were either silylated and analyzed by gas chromatography-electron impact quadrupole time-of-flight mass spectrometry (GC-QTOF) for sterol quantification, or methylated, and then treated with dimethyldisulfide (DMDS) and analyzed by GC-MS/MS for the determination of monounsaturated fatty acid double-bond stereochemistry as previously described by [START_REF] Amiraux | Monitoring photo-oxidative and salinity-induced bacterial stress in the Canadian Arctic using specific lipid tracers[END_REF]. Cis and trans isomers of monounsaturated fatty acid (MUFA) methyl esters react with DMDS, stereospecifically, to form threo and erythro adducts, which exhibit similar mass spectra but are well-separated by gas chromatography, allowing unambiguous double-bond stereochemistry determination [START_REF] Hrudolf | Determination of double bond position in mono-unsaturated acetates by mass spectrometry of dimethyl disulfide adducts[END_REF].

Gas chromatography-tandem mass spectrometry

J o u r n a l P r e -p r o o f GC-MS and GC-MS/MS analyses were performed using an Agilent 7890A/7010A tandem quadrupole gas chromatograph 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 m film thickness) capillary column was used.

Analyses were performed with an injector operating in pulsed splitless mode set at 270°C.

Oven temperature was ramped from 70°C to 130°C at 20°C min -1 , then to 250°C at 5°C min -1 and then to 300°C at 3°C min -1 . The pressure of the carrier gas (He) was maintained at 0.69  10 5 Pa until the end of the temperature program and then ramped from 0.69  10 5 Pa to 1.49  10 5 Pa at 0.04  10 5 Pa min -1 . The following mass spectrometer conditions were used: electron energy 70 eV, source temperature 230°C, quadrupole 1 temperature 150°C, quadrupole 2 temperature 150°C, collision gas (N 2 ) flow 1.5 mL min -1 , quench gas (He) flow 2.25 mL min -1 , mass range 50-700 Dalton, cycle time 313 ms. DMDS derivatives were quantified in multiple reaction monitoring (MRM) mode. Precursor ions were selected from the most intense ions (and specific fragmentations) observed in electron ionization (EI) mass spectra. Trans/cis ratios were obtained directly from peak area measurement of threo and erythro DMDS adducts after analyses, which were carried out three times.

Gas chromatography-EI quadrupole time-of-flight mass spectrometry

Accurate mass measurements were made in full scan mode using an Agilent 7890B/7200 GC/QTOF system (Agilent Technologies, Parc Technopolis -ZA Courtaboeuf, Les Ulis, France). A cross-linked 5% phenyl-methylpolysiloxane (Macherey-Nagel; OPTIMA-5MS Accent, 30 m  0.25 mm, 0.25 m film thickness) capillary column was used.

Analyses were performed with an injector operating in pulsed splitless mode set at 270°C.

Oven temperature was ramped from 70°C to 130°C at 20°C min -1 and then to 300°C at 5°C min -1 . The pressure of the carrier gas (He) was maintained at 0.69  10 5 Pa until the end J o u r n a l P r e -p r o o f of the temperature program. Instrument temperatures were 300°C for transfer line and 230°C for the ion source. Nitrogen (1.5 mL min -1 ) was used as collision gas. Accurate mass spectra were recorded across the range m/z 50-700 at 4 GHz with the collision gas opened. The QTOF-MS instrument provided a typical resolution ranging from 8009 to 12252 from m/z 68.9955 to 501.9706. Perfluorotributylamine (PFTBA) was used for daily MS calibration.

Compounds were identified by comparing their TOF mass spectra, accurate masses and retention times with those of standards. Quantification of each compound involved extraction of specific accurate fragment ions, peak integration and determination of individual response factors using external standards.

Statistical analysis

The collected data were analyzed using the XLStat version 22.05 software (Adinsoft TM ).

Kruskal-Wallis tests associated with a pairwise multiple comparison (using the Conover-Iman procedure) were performed on trans/cis ratios and on bacterial mortality data, at significance level α = 5%.

Results

Chlorophyll a concentration in the bottommost layer of ice (0-3 cm) was 0.44, 3.93 and 11.76 µg L -1 at St 403, St 409 and St 413, respectively. Chlorophyll a, phaeopigments and particulate organic carbon (POC) concentrations, bacterial abundance (BA) and bacterial production (BP) were measured in the upper 100 m of the water column at each station.

Though relatively weak along the whole transect, pelagic chlorophyll a concentrations were found to be highest (up to 0.58 µg L -1 ) in the upper 30 m of St 409, St 413 and St 418 (Fig. 2A). The highest phaeopigment concentrations (up to 0.50 µg L -1 ) were observed between 20 m and 40 m at St 413 (Fig. 2B). The highest POC concentrations were measured in the

J o u r n a l P r e -p r o o f

To estimate the stress state of bacteria induced by salinity in brine channels of ice, trans/cis ratios of C 16:111 (hexadec-11-enoic), oleic and C 18:111 (vaccenic) acids were measured in all the samples (Fig. 6). The results obtained showed a very strong isomerization of hexadec-11-enoic (trans/cis ratios reaching 2.1), and vaccenic (trans/cis ratios reaching 0.95) acids, but not of oleic acid in the deeper SPM samples of St 403 and St 409 (Fig. 7). A strong isomerization of hexadec-11-enoic (trans/cis ratio 0.8 and 0.78) and vaccenic acids (trans/cis ratios 0.31 and 0.18) was also observed in the 0-3 cm layer of ice at St 409 and St 413, respectively. At St 418, the three fatty acids were strongly isomerized in the upper SPM samples (trans/cis ratio reaching 0.6, 1.0 and 0.45 for hexadec-11-enoic, oleic and vaccenic acids, respectively) (Fig. 7).

The viability of bacteria was estimated with PMA in sea ice and SPM samples collected at St 409 and in SPM samples collected at St 418. At St 409, a high bacterial mortality was observed in sea ice (88.7 and 84.3% in the 3-10 and 0-3 cm layers, respectively) and in the 10 m SPM sample (62.3%), but not in the 20 m sample (Table 1). In the deeper (≥ 30 m) SPM samples collected at St 418, bacterial mortality was found to be very low, but it was relatively high in the surface and 20 m samples (23.3 and 67.5%, respectively) (Table 1).

Discussion

Composition of sea ice and SPM samples

The low chlorophyll a concentrations measured in the bottommost ice samples corresponded to only one-tenth of the values previously observed at the time of the sympagic algal bloom at the GreenEdge ice camp located near Broughton Island in Baffin Bay (Fig. 1) [START_REF] Amiraux | Temporal evolution of IP 25 and other highly branched isoprenoid lipids in sea ice and the underlying water column during an Arctic melting season[END_REF]. It thus seems that the bloom of sympagic algae took place before the start of sampling. The increase in chlorophyll a concentration observed in this layer from J o u r n a l P r e -p r o o f

St 403 to St 413 was thus attributed to the growth of the epiphytic diatom Melosira arctica, whose presence was noted during the sampling (Amiraux, unpublished data). The fatty acid profiles of all the sea ice samples were dominated by C 16:19 (palmitoleic) acid (Fig. 4) wellknown to be the main fatty acid component of sea ice-associated (sympagic or epiphytic)

diatoms [START_REF] Fahl | Lipid Content and fatty acid composition of algal communities in sea-ice and water from the Weddell Sea (Antarctica)[END_REF][START_REF] Falk-Petersen | Lipids and fatty acids in ice algae and phytoplankton from the Marginal Ice Zone in the Barents Sea[END_REF][START_REF] Leu | Increased irradiance reduces food quality of sea ice algae[END_REF]. The algal community present in the 0-3 cm sample at St 403 seemed to be mainly composed of pennate and centric (M. arctica?) diatoms and Thalassiosirales as suggested by the dominance of brassicasterol and 24-methylenecholesterol (Rampen et al., 2010) (Fig. 5). The increasing proportions of 24-methylenecholesterol observed at St 409 and St 413 were attributed to the presence of increasing amounts of M. arctica (containing a significant proportion of this sterol, Smik, unpublished data).

It is well known that sea ice retreat controls the timing of summer plankton blooms in the Arctic Ocean [START_REF] Janout | Sea-ice retreat controls timing of summer plankton blooms in the Eastern Arctic Ocean[END_REF]. From visual observation and knowledge of bloom dynamics, a chlorophyll a threshold of 0.5 µg L -1 was defined by [START_REF] Perrette | Near-ubiquity of ice-edge blooms in the Arctic[END_REF] to identify the blooms in the Arctic. The chlorophyll a concentrations measured in the water column along the transect investigated (up to 0.58 µg L -1 ) only exceeded this threshold between 0 and 20 m at St 409 and between 15 m and 35 m at St 413 (Fig. 2A), suggesting the presence of a weak ice-edge bloom at these stations. In the ice-covered water column, chlorophyll may result from: (i) the growth of pelagic phytoplankton or (ii) the release of nonaggregated sympagic or epiphytic algae during melting. The former hypothesis is well supported by the dominance of brassicasterol observed in surface SPM samples (brassicasterol/24-methylenecholesterol ratio 1.0-1.8) (Fig. 5), which contrasts with the dominance of 24-methylenecholesterol in the bottommost layer of sea ice at these stations (brassicasterol/24-methylenecholesterol ratio 0.07-0.7) (Fig. 5). This dominance of brassicasterol probably results from the presence of the prymnesiophyte Phaeocystis J o u r n a l P r e -p r o o f pouchetii, whose sterol fraction is known to consist of almost 100% brassicasterol [START_REF] Nichols | Lipids of cultured Phaeocystis pouchetii: Signatures for food-web, biogeochemical and environmental studies in Antarctica and the Southern ocean[END_REF] and which is a main component of under-ice and spring blooms across the Arctic [START_REF] Riisgaard | Impact of elevated pH on succession in the Arctic spring bloom[END_REF]. Microscopic examination of some SPM samples supported this explanation (e.g. percentage of Phaeocystis to protists at 30 m at St 413 ≈ 50%) (Babin, unpublished data). However, during sea ice melting the species composition of dispersed and aggregated algae may differ significantly [START_REF] Riebesell | Aggregation of algae released from melting sea ice: implications for seeding and sedimentation[END_REF]. Proportionately more cells of weakly aggregated pennate diatoms containing high proportions of brassicasterol (Rampen et al., 2010) may thus stay suspended, while other more aggregated ice algae (e.g.

Thalassiosirales or M. arctica) incorporated into sinking particles should rapidly sink out of the euphotic zone [START_REF] Riebesell | Aggregation of algae released from melting sea ice: implications for seeding and sedimentation[END_REF].

A high zooplanktonic grazing activity, well supported by the high abundance of C 20:111

and C 22:111 alkan-1-ols, was observed in the upper 30 m at St 413 and at the surface and between 25 m and 30 m at St 418 (Fig. 4). Wax esters are generally the main storage lipids of marine zooplankton in high-latitude species [START_REF] Lee | Lipid storage in marine zooplankton[END_REF] and the most common alkan-1ols of the wax esters found in herbivorous zooplankton are C 20:111 and C 22:111 alkan-1-ols [START_REF] Lee | Wax esters in the marine environment: origin and composition of the wax from Bute Inlet, British Columbia[END_REF][START_REF] Albers | The compositions of wax esters, triacylglycerols and phospholipids in Arctic and Antarctic copepods: evidence of energetic adaptations[END_REF]. These alcohols are only known to occur in copepods that undergo diapause [START_REF] Graeve | Diet-induced changes in the fatty acid composition of Arctic herbivorous copepods: Experimental evidence of trophic markers[END_REF], which are widely distributed in the Arctic.

In this area, dominant herbivorous zooplankton are the three large Calanus hyperboreus, C. glacialis and C. finmarchicus in addition to the smallest Pseudocalanus spp. [START_REF] Sameoto | Vertical distribution of zooplankton biomass and species in northeastern Baffin Bay related to temperature and salinity[END_REF][START_REF] Forest | Size distribution of particles and zooplankton across the shelf-basin system in southeast Beaufort Sea: combined results from an Underwater Vision Profiler and vertical net tows[END_REF]. At this period of the year, naupliis and copepodits depend on the bloom to develop into adults, and diapausing adults metabolize primary production into highly rich esters stocks [START_REF] Conover | Copepods in ice-covered seas-Distribution, adaptations to seasonally limited food, metabolism, growth patterns and life cycle strategies in polar seas[END_REF][START_REF] Falk-Petersen | Lipids and life strategy of Arctic Calanus[END_REF]. A study on the surface copepod community of Baffin Bay from Underwater Vision Profiler (UVP) data revealed that a lot a small copepods were actively feeding in the eastern J o u r n a l P r e -p r o o f and ice-free waters (including St 418) during the GreenEdge cruise (Vilgrain et al., submitted). Complementary data from net sampling showed that ice-free stations are dominated by young stages of C. finmarchicus and C. glacialis (naupliis, and stages CI to CVI) in addition with CII to CIV stages of C. hyperboreus (Fig. S1). All stages of Pseudocalanus spp. were also more abundant in St 413 and 418 with naupliis in particular (Fig. S1). The large proportion of young herbivorous stages was expected in ice-free stations according to their life cycle strategies [START_REF] Hirche | Reproduction of the Arctic copepod Calanus hyperboreus in the Greenland Sea-field and laboratory observations[END_REF]Soreide et al., 2010). Adults of all these species were generally distributed all over the Bay, but diapausing species such as Calanus spp., are probably metabolizing esters from phytoplanktonic precursors, which could explain the presence of C 20:111 and C 22:111 alkan-1-ols at St 413 and St 418.

Although the degradation of chlorophyll a to phaeopigments occurs in the guts of both large and small macro-zooplankton [START_REF] Nelson | Phytoplankton pigments in macrozooplankton feces: variability in carotenoid alterations[END_REF], these compounds could be detected in significant proportions only at 30 m at St 413 (Fig. 2B). The low concentrations of phaeopigments observed in the upper SPM samples of St 413 may be attributed to photooxidation processes, well known to degrade such pigments quickly [START_REF] Welschmeyer | Role of herbivory in controlling phytoplankton abundance: annual pigment budget for a temperate marine fjord[END_REF] and strongly favored at St 413 due to the lack of snow cover and the relatively thin ice (limited to 40 cm). Despite the very high copepod activity present at 30 m at St 418 (Fig. 5), phaeopigment concentration was found to be very weak (Fig. 2B), probably owing to a particularly intense photooxidation at this open water station. As expected, in the samples where the presence of high proportions of copepods was indicated by C 20:111 and C 22:111 alkan-1-ols, large proportions of their two main sterols [START_REF] Harvey | Biotransformation and assimilation of dietary lipids by Calanus feeding on a dinoflagellate[END_REF] cholesterol (not shown) and desmosterol (Fig. 5) could be observed.

The deepest SPM samples collected at St 403 were characterized by (i) very low chlorophyll a concentrations (Fig. 2A), (ii) high proportions of oleic acid (Fig. 4) and desmosterol (Fig. 5) and (iii) lack of C 20:111 and C 22:111 alkan-1-ols. Oleic acid is often J o u r n a l P r e -p r o o f enriched in secondary producers [START_REF] Falk-Petersen | Functional biodiversity of lipids in Antarctic zooplankton: Calanoides acutus, Calanus propinquus, Thysanoessa macrura and Euphausia crystallorophias[END_REF] and thus commonly interpreted as a marker of heterotrophic feeding [START_REF] Graeve | Lipids in Arctic benthos: does the fatty acid and alcohol composition reflect feeding and trophic interactions?[END_REF][START_REF] J O U R N A L P R E -P R O O F Tolosa | Distribution of pigments and fatty acid biomarkers in particulate matter from the frontal structure of the Alboran Sea (SW Mediterranean Sea)[END_REF]. Moreover, desmosterol is produced by zooplankton during the conversion of dietary phytosterols to cholesterol [START_REF] Harvey | Biotransformation and assimilation of dietary lipids by Calanus feeding on a dinoflagellate[END_REF]. Given the absence of alkanols, the presence of copepods in these samples was excluded and that of micro-zooplankton suspected. In Baffin Bay, it is well known that the ice microfauna is dominated by dinoflagellates and ciliates [START_REF] Michel | Significance of sedimentation and grazing by ice microand meiofauna for carbon cycling in annual sea ice (northern Baffin Bay)[END_REF], rarely observed in the water column, probably because prey are too scarce. These SPM samples thus seem to contain herbivorous micro-zooplankton feeding in ice and then released in the water column during melting. The presence of trans MUFAs, typical of stressed sympagic bacteria (see 4.2.), suggests the simultaneous presence of sympagic herbivorous and bacterivorous micro-zooplankton in these samples. In the deepest samples of St 409 characterized by very low amounts of sterols (Fig. 5), only bacterivorous microzooplankton, generally incapable of synthesizing or incorporating sterols [START_REF] Breteler | Role of essential lipids in copepod nutrition: no evidence for trophic upgrading of food quality by a marine ciliate[END_REF], seem present.

SPM material collected between 10 m and 20 m of the open water station St 418

exhibited relatively high proportions of hexadec-11-enoic and vaccenic acids (Fig. 4), well known to be specific to bacteria [START_REF] Lambert | Comparison of the effects of acid and base hydrolyses on hydroxy and cyclopropane fatty acids in bacteria[END_REF][START_REF] Sicre | Characterization of seawater samples using chemometric methods applied to biomarker fatty acids[END_REF]. Most of the oleic acid present in these samples also arises from bacteria (see Section 4.2.). These samples thus contained a large proportion of bacteria (Fig. 4). These observations are consistent with the highest BA measured in these samples (Fig. 3A), which is similar to those previously measured in spring in sea ice of the Chukchi Sea (0.7-2.5 10 6 cells mL -1 ; [START_REF] Meiners | Exopolymer particles: microbial hotspots of enhanced bacterial activity in Arctic fast ice (Chukchi Sea)[END_REF]. Arctic sea ice harbors large amounts of extracellular polymeric substances (EPS) in both the dissolved and particulate fractions [START_REF] Krembs | Abundance and variability of microorganisms and transparent exopolymer particles across the ice/water interface of melting first-year sea ice in the Laptev Sea (Arctic)[END_REF][START_REF] Krembs | High concentrations of exopolymeric substances in Arctic winter sea ice: implications for the polar ocean carbon cycle and cryoprotection of diatoms[END_REF][START_REF] Meiners | Vertical distribution of exopolymer particles in sea ice of the Fram Strait (Arctic) during autumn[END_REF]. Some authors [START_REF] Riedel | Seasonal study of sea-ice exopolymeric substances on the Mackenzie shelf: implications for transport of sea-ice bacteria and algae[END_REF][START_REF] J O U R N A L P R E -P R O O F Juhl | Seasonal development and differential retention of ice algae and other organic fractions in first-year Arctic sea ice[END_REF] previously demonstrated that EPS retained within the melting sea ice in the Arctic could supply a pulse J o u r n a l P r e -p r o o f of organic carbon to surface waters after most of the sea-ice algal biomass has been released into the water column. In the pelagic realm, EPS-rich particles, which have been observed to be positively or neutrally buoyant (Azetsu-Scott and [START_REF] Azetsu-Scott | Ascending marine particles: Significance of transparent exopolymer particles (TEP) in the upper ocean[END_REF][START_REF] Meiners | Exopolymer particles: microbial hotspots of enhanced bacterial activity in Arctic fast ice (Chukchi Sea)[END_REF], are densely colonized by attached bacteria and are ideal vectors for their horizontal distribution [START_REF] Meiners | Exopolymer particles: microbial hotspots of enhanced bacterial activity in Arctic fast ice (Chukchi Sea)[END_REF]. Bacteria use these particles as sites of attachment, possibly to protect them from grazers [START_REF] Salcher | Succession of bacterial grazing defense mechanisms against protistan predators in an experimental microbial community[END_REF], or as a carbon-rich substrate, which could enhance bacterial production [START_REF] Riedel | Seasonal study of sea-ice exopolymeric substances on the Mackenzie shelf: implications for transport of sea-ice bacteria and algae[END_REF]. These exopolymers could thus ascend in association with attached bacteria (Azetsu-Scott and [START_REF] Azetsu-Scott | Ascending marine particles: Significance of transparent exopolymer particles (TEP) in the upper ocean[END_REF]). The highest abundances of bacteria observed at 10 m and 20 m at St 418 (Fig. 3A) were thus attributed to the release of EPS particles heavily colonized by bacteria during sea ice melting after previous loss of the sympagic algal biomass.

Stress state of bacteria in sea ice and SPM particles

Cis-trans isomerization of MUFAs has been shown to serve as an adaptive response to chemical or osmotic stress in strains of the widespread genera Pseudomonas and Vibrio [START_REF] Okuyama | The cis-trans isomerization of the double bond of a fatty acid as a strategy for adaptation to changes in ambient temperature in the psychrophilic bacterium, Vibrio sp. strain ABE-1[END_REF][START_REF] J O U R N A L P R E -P R O O F Heipieper | Conversion of cis unsaturated fatty acids to trans, a possible mechanism for the protection of phenol-degrading Pseudomonas putida P8 from substrate toxicity[END_REF][START_REF] Molina-Santiago | Global transcriptional response of solvent-sensitive and solvent-tolerant Pseudomonas putida strains exposed to toluene[END_REF]. High trans/cis vaccenic acid ratios were previously observed in sinking particles collected during the 2015 and 2016 GreenEdge ice camps [START_REF] Amiraux | Monitoring photo-oxidative and salinity-induced bacterial stress in the Canadian Arctic using specific lipid tracers[END_REF]Amiraux et al., 2021a). These high values were attributed to release of non-growing bacteria attached to sympagic algae stressed by salinity in internal brine channels during the early stages of sea ice melting. To determine whether bacteria attached to suspended particles were also stressed by salinity, trans/cis ratios of the main monounsaturated fatty acids present in Pseudomonas sp. and Vibrio sp. (hexadec-11-enoic, oleic and vaccenic acids) (Lambert et al., 1983;Holsmtröm et al., 1998;[START_REF] Jia | Visualized analysis of cellular fatty acid profiles of Vibrio parahaemolyticus strains under cold stress[END_REF] were measured in sea ice and in SPM samples along the transect investigated. Although present in some Vibrio sp. (Lambert et al., 1983;[START_REF] Jia | Visualized analysis of cellular fatty acid profiles of Vibrio parahaemolyticus strains under cold stress[END_REF], palmitoleic acid was J o u r n a l P r e -p r o o f excluded from these measurements owing to its lack of specificity (strong dominance in sympagic and pelagic diatoms) [START_REF] Fahl | Lipid Content and fatty acid composition of algal communities in sea-ice and water from the Weddell Sea (Antarctica)[END_REF][START_REF] Leu | Increased irradiance reduces food quality of sea ice algae[END_REF].

Very high trans/cis ratios of hexadec-11-enoic and vaccenic acids were observed in the deepest SPM samples of St 403 and St 409 (Fig. 7), which seemed to be dominated by microzooplankton (see Section 4.1.). It was previously observed that the lipid composition (fatty acids and neutral lipids) of bacterivorous ciliates resembled that of their prey [START_REF] Harvey | Biotransformation and assimilation of dietary lipids by Calanus feeding on a dinoflagellate[END_REF][START_REF] Boëchat | Biochemical composition of algivorous freshwater ciliates: You are not what you eat[END_REF]. The high trans/cis values observed in these samples were thus attributed to (i) the ingestion of bacteria stressed by salinity in internal brines of sea ice by sympagic ciliates, (ii) the direct incorporation of highly isomerized dietary fatty acids and

(iii) the release of these bacterivorous ciliates in the water column during ice melting. The well-known biosynthesis of cis-oleic acid during the metabolism of ciliates [START_REF] Erwin | Lipid metabolism of ciliated protozoa[END_REF] is consistent with the relatively weak trans/cis ratio of this acid observed in these SPM samples (Fig. 7). Brine salinity, which could not be measured during the cruise, are expected to be low at the time of sampling (summer). However, high brine salinity values (ranging from 50 to 70) were measured in May 2015 and 2016 in the upper part of the ice at Qikiqtarjuaq (GreenEdge fixed station relatively close to the transect investigated, Fig. 1) [START_REF] Amiraux | Temporal evolution of IP 25 and other highly branched isoprenoid lipids in sea ice and the underlying water column during an Arctic melting season[END_REF]2021a). Non-halophilic bacteria strongly affected by these hypersaline conditions in spring could thus have been ingested by sympagic ciliates and trapped in the ice before to be released in the water column during the summer melting period.

It is well known that the uppermost section of the ice experiences the most drastic changes in brine salinity [START_REF] Ewert | Sea ice microorganisms: environmental constraints and extracellular responses[END_REF]. As a consequence, bacteria attached to sympagic algae in the bottommost ice are generally not highly affected by osmotic stress [START_REF] Rontani | Use of palmitoleic acid and its oxidation products for monitoring the degradation of ice algae in Arctic waters and bottom sediments[END_REF]Amiraux et al., 2021a). The high trans/cis ratios of hexadec-11-enoic and vaccenic acids measured in the bottommost 3 cm of ice of St 409 and St 413 (Figs. 6 and 7) were thus surprising. Given the relative similarity of these ratios with those observed in the J o u r n a l P r e -p r o o f deepest samples of St 403 and St 409 (Fig. 7), this isomerization was attributed to the presence of ciliates fed on salinity-stressed bacteria in internal brine channels and trapped during their discharge at the bottom of ice.

It is generally considered that suspended particles, which constitute most of the standing stock of particulate matter in the ocean [START_REF] Wakeham | Organic geochemistry of particulate matter in the ocean: The role of particles in oceanic sedimentary cycles[END_REF], sink very slowly through the water column. However, aggregation processes, the extent of which remains to be estimated [START_REF] Wakeham | Organic geochemistry of particulate matter in the ocean: The role of particles in oceanic sedimentary cycles[END_REF][START_REF] Hill | Controls on floc size in the sea[END_REF], can strongly increase the settling velocity of these particles and thus their contribution to the seafloor. Sympagic microzooplankton can thus contribute to the transfer of the signature of bacteria stressed by hypersaline conditions in brine channels of sea ice to the sediments.

SPM material collected in the topmost waters of St 418 seems to be composed of EPS particles retained in sea ice and discharged in seawater after the initial release of algal biomass [START_REF] Riedel | Seasonal study of sea-ice exopolymeric substances on the Mackenzie shelf: implications for transport of sea-ice bacteria and algae[END_REF][START_REF] J O U R N A L P R E -P R O O F Juhl | Seasonal development and differential retention of ice algae and other organic fractions in first-year Arctic sea ice[END_REF]. Such EPS particles contain high amounts of bacteria [START_REF] Meiners | Exopolymer particles: microbial hotspots of enhanced bacterial activity in Arctic fast ice (Chukchi Sea)[END_REF] that may be of sympagic or pelagic origin. The very high trans/cis ratios of hexadec-11-enoic, vaccenic and oleic acids observed in these SPM samples (Fig. 7) demonstrate that the bacteria attached to EPS particles are strongly stressed by salinity and thus arise from sea ice. The strong isomerization of oleic acid observed also attests to the bacterial origin of this acid. In the presence of osmotic stress, CTI activity is used by bacteria as an urgent response to guarantee survival, before other adaptive mechanisms [START_REF] Heipieper | Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems[END_REF]. Consequently, with no osmotic stress (as is the case in the water column) the trans/cis ratio of bacteria stressed by salinity in brine channels of sea ice should decrease to the basic level [START_REF] Fischer | The trans/cis ratio of unsaturated fatty acids is not applicable as biomarker for environmental stress in case of long-term contaminated habitats[END_REF]. Since conversion of trans to cis fatty acids is not catalyzed [START_REF] Eberlein | Immediate response mechanisms of Gram-negative solvent-tolerant bacteria to cope with environmental stress: cis-trans isomerization of unsaturated fatty acids and outer membrane vesicle secretion[END_REF], recovery of the regularly low trans/cis ratio needs de novo synthesis of cis fatty acids and thus depends on bacterial growth rates. The very high values of the trans/cis ratio observed in the topmost waters of St 418 (Fig. 7) are thus J o u r n a l P r e -p r o o f indicative of the non-growing state of bacteria attached to EPS particles. This assumption is well supported by the relatively weak BP measured in these samples (Fig. 3B) exhibiting the highest BA (Fig. 3A).

Viability of attached bacteria in sea ice and SPM particles

PMA treatment showed that most of the bacteria associated with sympagic algae at St 409 had disrupted membranes and so were dead (Table 1). These results are consistent with the high mortality of attached bacteria measured in sea ice at the end of the 2016 GreenEdge ice camp (Amiraux et al., 2021a) and attributed to the bactericidal properties of free fatty acids (FFAs) released by sympagic algae under the effect of light stress. The toxicity of FFAs results from their insertion into the bacterial inner membrane, increasing its permeability and letting internal contents leak from the cell, which can result in death [START_REF] J O U R N A L P R E -P R O O F Boyaval | Effects of free fatty acids on propionic acid bacteria[END_REF][START_REF] Shin | Antibacterial activity of bioconverted eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) against foodborne pathogenic bacteria[END_REF]. A high mortality was also observed in the 10 m SPM sample (Table 1).

This suggests that the algal material present in this sample (Fig. 5) results from the release of non-aggregated and FFA-producing sympagic diatoms during ice melting rather than from the growth of pelagic algae. By contrast, the viability of bacteria attached to suspended particles collected at 20 m and dominated by micro-zooplankton was found to be very good (Table 1).

Concerning SPM samples collected at St 418, PMA revealed a very low mortality of attached bacteria in the deeper (≥30 m) samples (Table 1). This good viability is probably due to the presence of unstressed bacteria associated with copepod or micro-zooplankton material, which dominated these samples (see Section 4.1.). By contrast, a lower viability was observed in the samples collected at the surface and at 20 m (23.3 and 67.5% of mortality, respectively)

(Table 1). The 20 m sample mainly composed of EPS particles thus contained a mixture of dead bacteria (in which the integrity of cell membranes could not be maintained) and nongrowing bacteria (where cis-trans isomerization of monounsaturated fatty acids ensured J o u r n a l P r e -p r o o f membrane stiffness but not growth). The presence of a significant proportion of zooplanktonic material (potential supports of unstressed bacteria) in the surface sample (Fig. 4) could explain the lower mortality (relative to the 20 m sample) observed (Table 1).

Considerations about the preservation and transfer of sympagic material to the seafloor

The preservation of sympagic algae during their transfer in the water column depends mostly on grazing by zooplankton and mineralization by their attached bacteria. We previously demonstrated that during the early stages of ice melting, bacteria associated to sinking sympagic material have been strongly stressed in hypersaline brine channels and are thus mainly non-growing in these particles [START_REF] Amiraux | Monitoring photo-oxidative and salinity-induced bacterial stress in the Canadian Arctic using specific lipid tracers[END_REF]. In contrast, during the advances stages of melting most bacteria associated to sinking ice algae appeared to be stressed by free fatty acids and dead (Amiraux et al., 2021a).

Whereas only a small part of the sympagic material is released during the early stages of ice melting, i.e. when bacteria are stressed by hypersaline conditions (Amiraux et al., 2021a(Amiraux et al., , 2021b)), a strong cis-trans isomerization of MUFAs was previously observed in Arctic sediments [START_REF] Rontani | Degradation state of organic matter in surface sediments from the Southern Beaufort Sea: a lipid approach[END_REF][START_REF] Amiraux | Monitoring photo-oxidative and salinity-induced bacterial stress in the Canadian Arctic using specific lipid tracers[END_REF]. The results obtained during the present study allow to propose an explanation to this paradox. Indeed, during the early stages of ice melting feeding of sympagic microzooplankton on stressed bacteria results to the incorporation and transfer of that stress signal (after aggregation) to the deeper waters, whereas at the advanced stages of melting copepods intensively assimilate the sympagic material (EPS-rich particles and sea ice algae) released in the water column. Due to the good healthy state of bacteria associated to the resulting copepod fecal pellets, this material should be degraded intensively within the water column contributing only weakly to the sediments. It thus appears that trophic relationships between sea-ice algae, their associated bacteria and zooplanktonic grazers are strongly intricate and need to be more investigated.

J o u r n a l P r e -p r o o f

Conclusions

Lipid analyses and propidium monoazide (PMA) method allowed the monitoring of the stress and viability of attached bacteria in sea ice and SPM samples collected during the GreenEdge 2016 cruise in Baffin Bay, along a transect from sea ice to open water. Our results are summarized in a conceptual trophic network scheme (Fig. 8).

At the western stations ice covered St 403 and St 409 lipid analyses showed a strong cis-trans isomerization of MUFAs attributed to the presence of sympagic bacterivorous microzooplankton (ciliates) incorporating trans fatty acids after ingestion of bacteria osmotically stressed in hypersaline brine channels of ice (Fig. 8). At the St 409, the high bacterial mortality measured in sea ice is consistent with that previously observed during the GreenEdge 2016 ice camp (Amiraux et al., 2021a) and is likely due to the release of bactericidal FFAs by sympagic algae under the effect of light stress (Fig. 8).

At the eastern ice-covered station St 413 and open water station St 418 the lipid analysis showed a high proportion of macro-zooplankton (copepods) (Fig. 8). SPM material collected in shallower waters at the open water St 418 seems to be mainly composed of EPSrich particles retained in sea ice and discharged in seawater after the initial release of algal biomass. In those waters, most of the bacteria associated to this material appeared to be either dead or in a non-growing state, while these attached to deeper SPM of St 418 (dominated by zooplanktonic material) were in good healthy state (Fig. 8).

2021a2021b

  J o u r n a l P r e -p r o o f topmost waters of St 409, St 413 and St 418 (values reaching 200 mg L -1 at the surface of St 418 and at 15 m in the case of St 413) (Fig.2C). BA was found to be relatively low at St 403 and St 409, but increased from St 413 to St 418, reaching 1.9 10 6 cells mL -1 in the upper 30 m of water of St 418 (Fig.3A). The highest values of BP (up to 0.33 µgC L -1 d -1 ) were observed at the surface of St 409 and at 10 m for St 413 (Fig.3B).To learn more about the nature of the organisms present in the different ice and SPM samples, the relative proportions of the main monounsaturated fatty acids (MUFAs) (C 16:19 , C 16:111 , C 18:19 , C 18:111 , C 20:111 and C 22:111 ), and alcohols (C 20:111 and C 22:111 ) were measured (Fig. 4). All the ice samples collected at St 403, St 409 and St 413 were dominated by C 16:19 (palmitoleic) acid. By contrast, C 18:19 (oleic) acid appeared to be the dominant MUFA of most of the SPM samples, except for those collected in the upper 30 m at St. 413 and at the surface and between 25 m and 30 m at St 418 containing a high abundance of C 20:111 and C 22:111 acids and alcohols (Fig. 4).The main algal sterolscholest-5,24-dien-3-ol (desmosterol), 24-methylcholesta-5,22Edien-3-ol (brassicasterol), 24-methylcholesta-5,24(28)-dien-3-ol (24methylenecholesterol), and 24-ethylcholest-5-en-3-ol (sitosterol)were quantified in ice and water samples at the different stations to confirm the nature of the material present in SPM. Cholesterol, a contaminant often introduced in the samples during their withdrawal and treatment, was excluded from this comparison. Sea ice was found to be dominated by brassicasterol and 24-methylenecholesterol at St 403 and by 24-methylenecholesterol at St 409 and St 413 (Fig. 5). The deeper SPM samples collected at St 403 showed a dominance of desmosterol, while most of those collected at St 409 and at St 413 were dominated by brassicasterol. Relatively high proportions of desmosterol were also observed in the surface and 20 m samples of St 413 and in the surface and 30 m samples of St 418 (Fig. 5).

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. Chlorophyll a (A), phaeopigments (B) and total particulate carbon (C) in seawater

Figure 3 .

 3 Figure 3. Bacterial abundance (BA) (A) and bacterial production (BP) (B) in seawater at the

Figure 4 .

 4 Figure 4. Relative proportion of the main monounsaturated fatty acids (FA) and alcohols (ol)

Figure 5 .

 5 Figure 5. Relative proportions of brassicasterol, desmosterol, sitosterol and 24-

Figure 6 .

 6 Figure 6. MRM chromatograms (m/z 217 → 185 and m/z 245 → 213) of DMDS derivatives

Figure 7 .

 7 Figure 7. Mean Trans/cis ratio of vaccenic (C 18:111 ), oleic (C 18:19 ), and hexadec-11-enoic

  Figure1

Figure 8

 8 Figure8

Table 1

 1 Mean percentage of dead attached bacteria in sea ice and SPM samples collected at St 409 and St 418 (n = 3). For each station, significantly different values are annotated with different letters (P < 0.05).

	Station	Sample	Dead bacteria (%)
	St 409	Ice (3-10 cm)	88.7 ± 4.7 (b)
	St 409	Ice (0-3 cm)	84.3 ± 9.7 (b)
	St 409	SPM 10 m	62.3 ± 22.06 (ab)
	St 409	SPM 20 m	0 ± 11.9 (a)
	St 418	SPM surface	23.3 ± 11.3 (b)
	St 418	SPM 20 m	67.5 ± 4.1 (c)
	St 418	SPM 30 m	3.8 ± 39.0 (ab)
	St 418	SPM 40 m	0.6 ± 15.5 (ab)
	St 418	SPM 100 m	0 ± 4.6 (a)

Acknowledgments

This work was supported by the Bacstress (INSU-EC2CO-Microbien) and GreenEdge projects. The GreenEdge project is funded by the following French and Canadian programs and agencies: ANR (Contract #111112), CNES (project #131425), IPEV (project #1164), J o u r n a l P r e -p r o o f

CSA, Fondation Total, ArcticNet, LEFE and the French Arctic Initiative (GreenEdge project).

This project was made possible by the support of the Hamlet of Qikiqtarjuaq and the members of the community and of the Inuksuit School and its Principal Jacqueline Arsenault. The

Supplementary material


INTRODUCTION: 1 2

The use of large biomedical implants has demonstrated a decisive turn over the last 30 3 years with the popularization of hip and knee joint replacement [START_REF] Tsaras | Incidence, 660 secular trends, and outcomes of prosthetic joint infection: a population-based study, olmsted 661 county, Minnesota, 1969-2007[END_REF]. Fast aging in industrial 4 societies, increased life expectancy and quality along with functional demands from the elder 5 population account for the rise of both invasive and non-invasive procedures comprising 6 biomaterial implantation [START_REF] Kurtz | Projections of primary and revision hip 663 and knee arthroplasty in the United States from 2005 to 2030[END_REF]. Substantial progress was made in terms of mechanical properties, 7 biocompatibility, and biointegration resulting in excellent functional outcomes in multiple 8 surgical specialties. On the other hand, there is to this date no consensus for an anti-infectious 9 strategy in order to prevent biofilm formation on implanted surfaces. The consequences of 10 nosocomial infections arising from surgical implants still represent a heavy burden on both 11 patients and healthcare systems worldwide [START_REF] Kurtz | Economic burden of 666 periprosthetic joint infection in the United States[END_REF]. In spite of the advancements of antisepsis 12 procedures, a significant and fixed proportion of nosocomial infections are still reported (from 13 1 to 2% respectively for hip and knee joint replacement). Biofilm is an exopolysaccharidic 14 matrix comprising bacteria with reduced antibiotic sensitivity and poor mechanical accessibility 15 (4)(5) [START_REF] Donlan | Biofilms: microbial life on surfaces[END_REF]. Biofilm formation plays a major role in the failure of conservative treatments 16 (antibiotic use, wound debridement and lavage) for implant-related infections [START_REF] Urish | Antibiotic-675 tolerant Staphylococcus aureus Biofilm Persists on Arthroplasty Materials[END_REF]. A common 17 rule is to perform implant removal to treat biofilm-related infections if a proper conservative 18 treatment has previously failed, which explains the high morbidity caused by the management 19 of infections on implanted devices [START_REF] Moyad | Evaluation and management of the infected total hip 678 and knee[END_REF]. This represents the rationale behind the development of 20 coating strategies for the prevention of implant-related infections [START_REF] Tiller | Designing surfaces that kill bacteria on 680 contact[END_REF]. Indeed, since the first 21 stage of biofilm formation is believed to be bacterial adherence, there has been a tremendous 22 enthusiasm about the development of new coatings and surface modifications aimed at repelling 23 bacteria or killing bacteria on contact. Numerous in vitro studies showed the interest of biocidal 24 polymers grafted on plastics or metals in the medical field [START_REF] Chouirfa | Review of titanium 682 surface modification techniques and coatings for antibacterial applications[END_REF]. They target bacterial 25 colonization, bacterial encapsulation or both. Polymer-based coatings may either reduce 26 bacterial adherence or directly kill bacteria without altering the mechanical and thermic 27

properties of implanted devices. The agents preventing bacterial colonization are antiadhesive 28 polymers inhibiting bacterial adhesion (polyvinyl sodium sulfonate, polyethylene glycol, PEG) [START_REF] Tiller | Polymer surfaces derivatized with 728 poly(vinyl-N-hexylpyridinium) kill airborne and waterborne bacteria[END_REF] and biocidal substances such as antibiotic coatings and silver-impregnated surfaces. Currently, 30 the most concerning drawbacks of antibacterial polymers which relies on antiadhesive effects 31 are their transitory local efficacy, potential toxicity through progressive leaching and the low 32 or absent bacteriostatic or bactericidal effect. Therefore, bactericidal polymeric agents have 33 been introduced. The most used are high-density quaternary ammonium salts (chitosans [START_REF] Muxika | Chitosan as a bioactive 685 polymer: Processing, properties and applications[END_REF]), phosphonium salts [START_REF] Pugachev | Bis-phosphonium salts of pyridoxine: the relationship between structure and 689 antibacterial activity[END_REF], sulfonium salts [START_REF] Tashiro | Antibacterial and Bacterium Adsorbing Macromolecules[END_REF], pyridinium salts [START_REF] Madaan | Quaternary Pyridinium Salts: A Review[END_REF] and mono or biguanidium 35 salts [START_REF] Budhathoki-Uprety | Synthesis of Guanidinium 695 Functionalized Polycarbodiimides and Their Antibacterial Activities[END_REF]. These high-density cationic polymers feature a non-selective bacteria contact-killing 36 effect [START_REF] Kügler | Evidence of a charge-density threshold for 698 optimum efficiency of biocidal cationic surfaces[END_REF]. While they are extensively described in the literature, the biocidal mechanisms 37 remain poorly detailed. In fact, most studies focused on killing mechanisms occurring in 38 solutions of varying densities [START_REF] Crismaru | Survival of Adhering Staphylococci during Exposure to a Quaternary Ammonium 704 Compound Evaluated by Using Atomic Force Microscopy Imaging▿[END_REF] [START_REF] Stratton | In vitro biocompatibility studies of 707 antibacterial quaternary polymers[END_REF] and not on grafted high-density QAP. A quaternized polyvinylpyridine (Q-PVP) statistical polymer was synthesized following the 56 introduction of specific chains on the polymeric chain in a single-step reaction in boiling 57 methanol. Five specific changes were carried out in the synthesis process. Polymers synthesized 58 with no bromobutane chain were labelled Q-PVP-iodo. Reaction time also varied in order to 59 modulate the ammonium quaternization rate. Polymers synthesized in 36h, 2 days and 4 days 60 were respectively labelled Q-PVP-36h, Q-PVP-2days, Q-PVP-4days. When polyethylene 61 glycol (PEG) replaced 1-bromobutane in the synthesis (24h), the product was labelled Q-PVP-62 PEG. The iodopropyltrimethoxysilane chain was used as a "hook" in order to immobilize the 63 polymeric chain on the activated surface through a covalent linkage between the surface and 64 the polymeric moiety. The butyl chain was used to quaternize the rest of the Q-PVP chain. A 65 fixed 5% proportion of iodopropyltrimethoxysilane was critical to reach storability of the 66 polymer since higher proportions cause reticulation in volume and therefore prevent long-term 67 storage. 68 69

2) Polymer grafting 70 71 1cm 2 titanium plates (99,6% purity, Goodfellow, Cambridge Ltd., Huntington, United 72 Kingdom) were successively polished on 3 grains (P800, P2000 and P4000 grit paper using a 73 Saphir 320, ATA GmbH, Mammelzen, Germany) for respectively 1 minute, 2 minutes and 3 74 minutes. Following abundant lavage and a 5-minute sonication process with distilled water in 75 order to remove residual particles, the plates were placed in a piranha solution (96% sulfuric 76 acid/30% hydrogen peroxide (2/1, v/v) for two hours so as to obtain hydroxylation and surface 77 chemical abrasion (hence forming activated surfaces). All plates were subsequently rinsed, 78 sonicated in distilled water and dried under nitrogen flux. Polymer grafting was achieved either 79 by one or six spin-coating rounds of a diluted 125μL polymer drop (100μL polymer, 25μL 3) Surface characterization 94 95 X-ray Photoelectron Spectroscopy (XPS): 96 XPS analyses were performed using an Omicron Argus X-ray photoelectron spectrometer. The 97 monochromated AlKα radiation source (hν = 1486.6 eV) had a 300 W electron beam power. 98

The emission of photoelectrons from the sample was analyzed at a takeoff angle of 90° under 99 ultra-high vacuum conditions (≤ 10 -10 Torr). Spectra were carried out with a 100eV pass energy 100 for the survey scan and 20 eV pass energy for the C1s, O1s, N1s, S2p regions. Binding energies 101 were calibrated against the aliphatic C1s contribution binding energy at 284. [START_REF] Moyad | Evaluation and management of the infected total hip 678 and knee[END_REF] was described elsewhere [START_REF] Vallée | BSA adsorption on aliphatic 709 and aromatic acid SAMs: investigating the effect of residual surface charge and sublayer 710 nature[END_REF]. All reported spectra were recorded at 8 cm -1 resolution by co-112 addition of 128 scans; using a modulation of polarization enabled us to perform rapid analyses 113 of the samples after immersion without purging the atmosphere or requiring a reference 114 The second conclusion that can be drawn from these data is the equivalent thickness of the 262 functionalized surface. In fact, one can estimate that thickness by looking at the decrease of the 263 Ti signal after functionalization, taking into account that the mean free path () of TiO2 264 electrons onto organic films is around =2.85 nm. In the case of the dip coated method, the 265 XPS Ti2p signal is completely extinguished suggesting an equivalent thickness of the Q-PVP-266 4 days film superior to 3, i.e. superior to 8.5 nm, while for the spin coating method, the TiO2 267 signal is still faintly visible, suggesting an equivalent thickness of 8 nm for the Q-PVP-4 days 268

films. 269

Finally, chemical composition details can be obtained when looking at the different atomic 270 percentages of the different films as compared to non-functionalized control, Polyvinylpyridine quaternization was modulated depending on reaction time (Q-PVP-282 iodo in 12 hours, Q-PVP-36h, Q-PVP-2days, Q-PVP-4days) leading to grafted polymers with 283 increasing 𝑁 + 𝑁 ratios along with increased reaction time. The 𝑁 + 𝑁 ratio was determined through 284 PM-IRRAS spectra using the area ratio between the stretching mode of the C-N + band (1640 285 cm -1 ) and C-N band (1600 cm -1 ) by looking at the very specific IR marker present at 1640 cm - 286 1 assigned to the protonated amino groups. One can notice that this contribution is increasing 287 as a function of reaction time (Figure 2 Imperfections in polishing were more easily visible on control plates and grafted plates after 318 BHI sedimentation and fixation. Despite this, all surfaces were relatively homogeneous. Neither 319 the BHI sedimentation nor the fixation protocol seemed to cause further chemical abrasion of 320 titanium surfaces (Figure 3).

2) Surface charge determination by fluorescein test: 345 346 SLSC Q-PVP-36h showed systematically higher absorbance than plates that were spin-coated 347 in a single round with Q-PVP-4days (0.296 ± 0.034 or 2.31 x 10 17 charges/cm 2 , n=6 vs 0.133 ± 348 0.026 or 1.04 x 10 16 charges/cm 2 , n=6). SLSC Q-PVP-2days plates averaged 0.130 ± 0.040 349 (n=3) with an estimated surface charge density of 1.02 x 10 17 charges/cm 2

4) Biocompatibility assessment (MTT-assay) 365 366

Mean MC3T3 absorbance on control titanium plates was 0.42 ± 0.02, arbitrarily considered 367 100% viability. MC3T3 cell viability decreased on plates displaying higher surface cationic 368 densities compared with controls (from Q-PVP-iodo (118% ± 5.4 % survival) to Q-PVP-4 days 369 (73% ± 5.1 %). If the initial reaction did not include a bromobutane chain and lasted only 12 370 hours (Q-PVP-iodo), cells deposited on grafted titanium surfaces persistently showed higher 371 cell survival at 72h than cells on control titanium plates (118% ± 5.4 %). Of all tested samples, 372 Q-PVP-PEG surfaces displayed the lowest viability rate (60.1% ± 2. Mean L929 absorbance on control titanium plates was 0.27 ± 0.002, arbitrarily considered 385 100% viability. L929 cell viability gradually decreased on plates displaying higher cationic 386 surface densities, from Q-PVP-iodo (96.3 ± 2.2 %) survival to Q-PVP-4 days (87% ± 1.6 %). 387 Q-PVP-iodo did not increase L929 survival at 72h. 388

For each given surface cationic density, L929 cells demonstrated better survival compared with 389 plates showed statistically significant differences in absorbance values compared with control 395 (P<0.05) except for Q-PVP-iodo. The red-dotted line represents the minimum acceptable 396 viability (70%). 397 398

5) Biocompatibility assessment (SEM-FEG): 399 400

Qualitative morphological assessment showed that L929 cell behavior differed in terms of 401 adhesion, morphology and shape on depending on surface charge density. Surfaces of increased 402 charge density decreased cell adhesion and shape. Indeed, a gradual transition in the shape of 403 cells was observed between Q-PVP-4 days, the highest density polymer (cells mostly round, 404 few spindle cells, few pseudopods) and Q-PVP-iodo, the lowest density polymer (mostly 405 spindle cells, numerous pseudopods) (Figure 8). Surfaces were ranked in terms of cell-adhesion 406 In vitro assays on MRSA demonstrated a fast (within one hour) and highly efficient killing 441 activity on surfaces grafted with Q-PVP-4 days, especially on dip-coated titanium (complete 442 sterilization, Figure 10). Under proliferating conditions (37°C in a rich medium, BHI), the 443 observed bacterial growth inhibition was more modest, though significant (Figure 11). hours) on dip-coated titanium plates (Q-PVP-4 days). The effect was prolonged despite 466 systematic medium replacement and enriching with BHI every 24 hours. At seven days, the 467 observed biofilm appeared multi-layered and richer on control plates compared with grafted 468 plates. On grafted plates, biofilm was scattered across the surface and made the underlying 469 titanium support still visible on numerous areas (Figure 12). Quaternary ammonium compounds have been known as an antibacterial agent in solution for 500 almost a century [START_REF] Domagk | A new class of disinfectant[END_REF]. Their primary and main use to this date is surface disinfection [START_REF] Sykes | Disinfection and Sterilization[END_REF]. Their 501 mechanism of action in solution has been already extensively described (24)(25)(26) [START_REF] Russell | The mechanism of action of some antibacterial agents[END_REF]. 502

promotion: Q-PVP-iodo > Control > Q-PVP-36h > Q-PVP-2 days > Q-PVP-
Because of their efficacy in solution, toxicity thresholds were defined, especially regarding 503 hemolysis [START_REF] Stratton | In vitro biocompatibility studies of 707 antibacterial quaternary polymers[END_REF]. Also, there have been efforts to mitigate the risk of cytotoxicity by associating 504 quaternary ammonium compounds with various molecules in solution [START_REF] Stratton | Effect of steric hindrance on the 725 properties of antibacterial and biocompatible copolymers[END_REF]. However, 505 quaternary ammonium polymers (QAP) in solution are of limited use for implantable devices 506 as they do not permit any lasting antibacterial effect on surfaces due to rapid dispersion of the 507 polymers. The immobilization of antibacterial polymers on implantable surfaces led to the 508 discovery of various techniques that allowed the use of smaller polymer quantities with local 509

TREATED CONTROL

In our opinion, two main reasons account for this: 1) it had the highest achievable 𝑁 + 𝑁 ratio in 543 our setting 2) the coating thickness was increased. Yet these findings disregard the fact that Q-544 PVP-4 days decreased both tested cell line viabilities close to the critical viability threshold 545 (70%). Following L929 and MC3T3 MTT survival assessment, Q-PVP-2 days demonstrated 546 the best compromise in terms of preliminary biocompatibility testing (L929 and MC3T3 547 survival) and surface cationic density (systematically above 10 15 cations/cm 2 ). Therefore, we 548 postulate that the best compromise of viability and antibacterial activity would be using Q-549 PVP-2 days as a ready-to-use and covalently graftable coating. Regarding coating thickness, it 550 seemed to be an important factor to modulate as it impacted leaching severity. The leaching 551 mechanism in this study depended on coating thickness and probably butyl group lengths. A 552 longer carbon-chain may have decreased hydrolysis by distilled water of the silane bond from 553 the surface. In this study, Only SLSC plates in distilled water demonstrated leaching that was 554 significant enough to drop below the biocidal effect threshold (10 15 charges/cm 2 ). Thicker 555 coatings may also prevent water from interacting with the titanium-silane bond. Therefore, 556 optimizing coating thickness and cationic density through reaction time modulation is expected 557 to lead to durable grafting and persistence in vivo, which should be confirmed in animal studies. 558 559 This is the first AFM study visually demonstrating the 3D killing effect of surfaces covalently 560 grafted with high-density QAP. The displayed effects were perforation and shrinking 561 dramatically impacting the external shape of bacteria (MRSA). In contrast, AFM studies on 562 QAP in solution raise the issue of the external stress (AFM tip) that may enhance QAP action 563 and eventually destroy bacterial cell membranes [START_REF] Crismaru | Survival of Adhering Staphylococci during Exposure to a Quaternary Ammonium 704 Compound Evaluated by Using Atomic Force Microscopy Imaging▿[END_REF]. In this study, high cationic density was 564 sufficient to cause bacterial cell perforation and death. 565 566

Limitations of the study: 567 568

We have not explained why PEGylated polymers performed poorly in terms of cell viability. 569

There was a seemingly contradictory result concerning fluorescein test measurements regarding 570 SLSC with Q-PVP-4 days compared with Q-PVP-B36h. It was expected that Q-PVP-4 days 571 would display a higher absorbance, which appeared to be systematically the opposite. This 572 warrants further investigation too. Also, biocompatibility studies were performed on spin-573 coated surfaces while biofilm studies relied on dip-coated samples, which may introduce a bias 574 artificially maintaining a high viability due to thinner surfaces. 575

39 AFigure 1 .

 391 novel ready-to-use and scalable QAP, Q-PVP (quaternized polyvinylpyridine) was 40 synthesized in a methanolic solvent in a single step. The objective of the study was to 41 characterize grafted titanium surfaces, measure their in vitro antibacterial activity quantitatively 42 and qualitatively, assess biofilm inhibition and in vitro biocompatibility. Quaternized polyvinylpyridine polymer synthesis 50 n: 1Eq 51 A: iodopropyltrimethoxysilane, 20 to 50mEq 52 B: 1-bromobutane, 1Eq 53 x depends on reaction time 54 55

5 ) 6 )

 56 by fluorescein test: 117 118 Surface cationic density (NH4/cm 2 ) was calculated by a fluorescein test. It is estimated 119 that each fluorescein molecule (negatively charged) strongly binds quaternary ammonium 120 molecules (positively charged) belonging to the polymeric chain. Treated plates were placed in 121 a 2% fluorescein aqueous solution for five minutes. They were then rinsed and sonicated for 122 five minutes in order to remove physisorbed fluorescein. Plates were rinsed in distilled water a 123 second time, immersed in a test tube containing CTAB 0,1% (aqueous solution of cetyl 124 trimethylammonium bromide) diluted in PBS (Phosphate-Buffered Saline) (90% CTAB/10% 125 PBS) and vortexed for 10 seconds. Fluorescein was then dissolved, allowing an optical density 126 measurement using a spectrophotometer at 501 nm wavelength (CamSpec M550, Spectronic 127 Camspec Ltd, Leeds, UK). Measurements were repeated three times. 128 129 Surface cationic density was estimated according to the following formula: 130 131 132 A: fluorescein optical density at 501 nm 133 V: volume 134 NA: Avogadro number (6.022140857×10 23 mol -1 ) 135 ε: fluorescein molar absorptivity or molar extinction coefficient (L×mol -1 x cm -1 ). 136 137 The impact of coating thickness on fluorescein tests was also assessed. Spin-coating was either 138 performed in a single round (125μL in 40 seconds) or six rounds (six 125μL drops, 40 seconds 139 each). Plates were either labelled SLSC (single-layer spin-coating) or MLSC (multi-layer spin-140 coating). 141 142 Polymer leaching: 143 Eight plates spin-coated with Q-PVP-4 days, either SLSC or MLSC, underwent a 1-week 144 leaching test at 37°C and were immersed in two different mediums: SLSC plates in distilled 145 water (n=2), SLSC in rabbit serum (Sigma-Aldrich, Saint-Louis, MO, USA) (n=2), MLSC 146 plates in distilled water (n=2), MLSC plates in rabbit serum (n=2). A fluorescein test was 147 performed to determine the remaining surface charge density percentage (RSCD) after one 148 week. Measurements were repeated three times. 149 150 Atomic force microscopy (AFM) surface analysis: 151 Grafted and control titanium plate imaging was performed using a commercial atomic 152 force microscope (Bruker Nano Inc.-Nano Surfaces Division, Santa Barbara, CA, USA) 153 equipped with a J scanner (150 x 150 x 5 μm). Images were analyzed in "QNM ®" mode 154 (Quantitative Nanomechanical Property Mapping) in air or "intermittent-contact" mode in a 155 quartz measure cell. Sharps were made of silicon nitride (Si3N4) and had a theoretical stiffness 156 constant of 0.05 N.m -1 and a curve ray of 20 nm. Observations were done at a constant speed 157 of 1 Hz, 512 lines of 512 pixels each, being recorded for each image. Data were analyzed using 158 Nanoscope Analysis software (Bruker, Santa Barbara, CA, USA). The displayed images are 3D 159 reconstructions of height and DMT modulus images. 160 161 Scanning Electron Microscopy with Field Emission Gun (SEM-FEG) surface analysis: 162 163 SEM images were recorded with a Hitachi SU-70 field emission gun scanning electron 164 microscope. The samples were fixed on an alumina SEM support with a carbon adhesive tape 165 and were observed without metallization. An in-lens secondary electron detector (SEUpper) was 166 used to characterize our samples. The accelerating voltage was 1 kV, and the working distance 167 was around 5 mm. At least five different locations were analysed on each surface, arising to the 168 observation of a minimum of 100 single cells. 169 170 Irradiation test: 171 172 Dip-coated titanium plates were sterilized by gamma irradiation from a ⁶⁰Co source 173 with a total delivered dose of 27kGy (minimum threshold 25kGy, ISO 11137-2: 2013) (21). 174 The irradiation was performed at 0°C for 4 hours (BBF Sterilisationsservice GmbH, Kernen, 175 Germany) to prevent denaturation of the polymer during the sterilization process. Infra-red 176 spectra were compared before and after irradiation to verify the integrity of the polymer at the 177 precursor cell line (MC3T3-E1) was cultured in MEM-α (Gibco 184 Invitrogen, France) enriched with 10% FBS (fetal bovine serum), 2mM glutamine, 100 U/mL 185 penicillin and 100 μg/mL streptomycin. Murine fibroblasts (L929) were also cultured 186 separately in DMEM (Dulbecco's Mod Eagle Medium) supplemented with 10% FBS, 2mM 187 glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. 188 189 5x10 5 cells of each cell line were seeded on each titanium plate in their respective medium 190 (1mL) in 24-well plates. Five triplicate conditions were tested (control, Q-PVP-iodo, Q-PVP-191 36h, Q-PVP-2days, Q-PVP-4days, Q-PVP-PEG) in order to further elucidate the consequences 192 of the introduction of specific chains in the polymer on cell viability. All tested surfaces were 193 spin-coated. In both cases, following incubation at 37°C in humid atmosphere and 5% CO2, a 194 trypsin EDTA treatment was applied in order to retrieve adherent cells. The protocol was 195 repeated three times for each cell line. Finally, an MTT assay comparing optical densities at 196 550nm was then performed in order to measure cell viability at 72h (Wallac 1420 Victor2 ™ 197 microplate reader, Perkin Elmer, Waltham, MA, USA). 70% cell viability was considered to be 198 the critical threshold. Measurements were averaged on each triplicate and presented with mean 199 ± SEM (standard error of the mean). 200 201 SEM-FEG cell adhesion and morphological analysis: 202 203 L929 and MC3T3 cells were cultured on titanium plates spin-coated and grafted with Q-PVP-204 iodo, Q-PVP-36h, Q-PVP-2days, Q-PVP-4days until 72h under the conditions described 205 previously. All titanium plates underwent the following fixation protocol: plates were rinsed 206 three times with PBS 5% and fixed in 2,5% glutaraldehyde (2 hours). The fixative was then 207 drained off and plates were immersed in ethanol at room temperature as follows: 25% EtOH (5 208 minutes), 50% EtOH (5 minutes), 75% EtOH (5 minutes), 100% EtOH (5 min) and finally dried 209 under a fume hood. Cell adhesion behavior was assessed by SEM-FEG. 210 211 Antibacterial activity: 212 213 An ST 2012-238 MRSA strain (Lyon, France) isolated from a patient with a PJI 214 (prosthetic joint infection) was cultured in Brain Heart Infusion (BHI) at 37°C overnight. 215 According to a modification of the 22196:2011 ISO norm, a 10 7 CFU/mL bacterial suspension 216 of 20 μL in rich medium (BHI) was simultaneously deposited and applied with cover slips on 217 titanium plates (control versus grafted with Q-PVP-iodo, Q-PVP-36h, Q-PVP-2 days, Q-PVP-218 4 days, Q-PVP-PEG using spin coating or dip coating) inside 24-well culture plates for 219 triplicate. Culture plates were then transferred to a stove at 37°C. Cultures were sequentially 220 stopped after 1h (bacterial "killing" at 37°C) and 24h (growth inhibition at 37°C), diluted in 221 0.9% saline and vortexed for detachment of live bacteria and bacterial counting. 222 223 Antibiofilm activity: 224 225 Q-PVP-4days dip-coated titanium plates were placed into 24-well culture plates. A 10 6 226CFU/mL bacterial suspension in 1mL of BHI (prepared as described above) was used to fill 227 wells containing titanium plates. Culture plates were then transferred to a stove for culture at 228 37°C. Cultures were then stopped sequentially each time for two control and two grafted 229 titanium plates after respectively 6h, 12h, 24h, 72h, and 7 days (medium was removed and 230 replaced with sterile BHI every 24h). An in vitro biofilm of gradual maturity was therefore 231 created on dip-coated titanium plates and controls. In order to determine the influence of BHI 232 on plate surface morphology, two other control titanium plates were prepared: one was 233 immersed in sterile BHI throughout the experiment and underwent fixation while the other was 234 activated by piranha treatment and underwent fixation afterwards. All titanium plates 235 underwent the fixation protocol described above. SEM-FEG imaging on treated and control 236 titanium plates covered with 6 h to 7 day biofilm were obtained. was performed on Q-PVP-4days plates (controls versus treated) at 20°C following 241 a three-hour contact with a 10 7 CFU/mL MRSA inoculum. For the latter, AFM imaging was 242 two means, a Student t-test was used. All statistical analyses were 247 performed with Prism 5 (Graphpad software). P-values below 0.05 were considered significant 248 and p-values were represented as follows: *=P<0.05, **=P<0.01. performed on both dip and spin-coated quaternized surfaces (Q-PVP-257 4days) compared to bare surfaces before any functionalization. First, one can notice on the 258 control surface that most of layer of the sample is composed of TiO2 oxidized titanium, with 259 the main contribution of the Ti2p3/2 signal being centered at 459.0 eV and only a very small 260 contribution (less than 3 %) of metallic titanium visible at 454.1 eV. 261

Figure 4 :

 4 Grafted and control surfaces presented a near identical roughness (controls: 23.0 nm vs grafted: 329 22.6 nm). Grafted surfaces were chemically homogeneous. AFM imaging of a control titanium plate (1μm scale) 334

Figure 3 :Figure 5 :

 35 Figure 3: SEM-FEG of grafted (A&B) vs control ( C ) titanium plates A : grafted titanium plate after glutaraldehyde fixation B : grafted titanium plate after BHI sedimentation and glutaraldehyde fixation C : control titanium plate after glutaraldehyde fixation

Figure 6 .

 6 MTT-assay: M3T3 cell survival at 72h on surfaces of various cationic densities 378 Controls: n=9 ; Q-PVP-iodo: n=9 ; Q-PVP-36h: n=9 ; Q-PVP-2 days: n=8 ; Q-PVP-4 days: 7 ; 379 Q-PVP-PEG: n=3. All data are represented as mean ± standard error. All treated plates showed 380 statistically significant differences in absorbance values compared with control (P<0.05) except 381 for Q-PVP-36h. The red-dotted line represents the minimum acceptable viability (70%). 382 383 384

Figure 7 .

 7 MTT assay: L929 cell survival after 72h on surfaces of various surface charge 392 densities. Controls: n=6 ; Q-PVP-iodo: n=6 ; Q-PVP-36h: n=6 ; Q-PVP-2 days: n=6 ; Q-PVP-393 4 days: 3 ; Q-PVP-PEG: n=3. All data are represented as mean ± standard error. All treated 394

Figure 8 :Figure 9 :

 89 Figure 9: SEM-FEG imaging displaying gradual adherence decrease of MC3T3 cells on surfaces of

Figure 10 :Figure 11 :

 1011 In vitro killing (1 hour) in rich medium (BHI) at 37°C. Inoculum: n=1 represents 449 the bacterial count (CFU/mL) of the bacterial suspension initially deposited on titanium 450 substrates ; Control: n=9 ; Spin-coated: n=9 ; Dip-coated: n=9 ; All data are represented as 451 mean ± standard error. All treated plates showed statistically significant differences in 452 bacteriological count values compared with control (P<0.01). In vitro growth inhibition (24 hours) in rich medium (BHI) at 37°C. Inoculum: 458 n=1 ; Control: n=9 ; Spin-coated: n=9 ; Dip-coated: n=9 ; All data are represented as mean ± 459 standard error. All treated plates showed statistically significant differences in bacteriological 460 count values compared with control (P<0.01). imaging revealed a rapid-onset bacterial adherence reduction on a young biofilm(6 465

Figure 12 : 8 )Figure 14 :

 12814 MRSA biofilm growth on Q-PVP-4 days dip-coated surfaces vs control. 475 476 Specific effects on bacterial morphology: bacterial killing 477 478 The latter grafted surfaces exhibited signs of direct bacterial destruction by splitting or 479 shrinkage (Figure 13A, left). Loss of volume occurred in tandem with bacterial cell wall 480 perforation exhibited by sphericity loss (Figure 13B right). 481 482 Figure 13: (A) left, (B) right: SEM-FEG imaging of MRSA bacteria on control vs treated 483 surfaces (dip-coated Q-PVP-4 days) after 24 hours. These findings were also verified using 3D AFM. Bacterial perforation and volume loss was 486 observed on killed bacteria after a 3 hours contact with Q-PVP-4 days dip-coated titanium 487 compared with control. 3D AFM imaging of bacterial killing (MRSA) after a 3 hour-contact with dip-492 coated Q-PVP-4-days. 493 Yellow: spherical bacteria, appropriate height (live) 494 Red: loss of height and perforation of bacteria (dead)

  silanized polyvinylpyridine in solution, especially when synthesized 579 in 48 hours, may represent a good compromise of biocompatibility and bactericidal activity 580 against MRSA when grafted on titanium. If ultimately confirmed in vivo, these findings could 581 pave the way for easier synthesis and grafting to render surfaces permanently biocidal and 582

  MD, MS (Conceptualization, Data curation, Formal analysis, Funding 625 acquisition, investigation, methodology, Software, Writing original draft), Azzam Saleh-626 Mghir, PhD (Conceptualization, Data curation, Formal analysis, Investigation, Methodology, 627 Project administration, Resources, Supervision, Validation, Visualization), Claire Valotteau, 628 PhD (Data curation, Formal analysis, investigation, methodology, Software, Resources, 629 Visualization), Chahrazad Cherifi, PhD (Data curation, Formal analysis, investigation, 630 methodology, Software, Resources, Visualization), Narjes Hafsia, PhD (Data curation, Formal 631 analysis, investigation, methodology, Software, Resources, Visualization), Martine Cohen-632 Solal, MD, PhD (Methodology, Project Administration, Resources, Supervision), Charles 633 Court, MD, PhD (Project Administration), Anne-Claude Crémieux, MD, PhD 634 (Conceptualization, Formal analysis, Funding Acquisition, Investigation, Methodology, Project 635 Administration, Resources, Supervision, Validation, Visualization, Review-editing ), Vincent 636 Humblot, PhD (Conceptualization, Data curation, Formal analysis, Investigation, 637 Methodology, Project Administration, Resources, Software, Supervision, Validation, 638 Visualization, Writing -review & editing). 639 We would like to thank Pr. Philippe Hernigou for his logistic help. 640 641 FUNDING STATEMENT: 642 This work was supported by IMEA -Fondation Internationale Léon Mba and Société Française 643 de Chirurgie Orthopédique. The funders had no role in study design, data collection and 644 analysis, decision to publish, or preparation of the manuscript. 645 646 DISCLOSURE: 647 Houssam Bouloussa, MD, MS declares that he is co-inventor of issued patent US 648 US10238110B2 and owns stocks from DeBogy Molecular, Inc. (current assignee).

model 30, Memmert GmgH, Schwabach, Germany) for 30 minutes for 84 reticulation. The plates were then sonicated in pure ethanol for five minutes for adequate 85 removal of any remaining physisorbed particles. Control plates underwent the same protocol 86

	80
	81	distilled water) on activated titanium plates (40 seconds, 3000 rpm, Laurell WS 650 Spin
	82	Coater, Laurell Technologies Corp., North Wales, PA, USA) or by dip-coating (diluted polymer
	83	solution with 40% distilled water). Plates were immediately transferred to a pre-heated stove at
	87	110°C (Memmert, with distilled water instead (spin-coating or dip-coating, heating, sonication).
	88
	89
	90
	91
	92
	93

Table 1 ,

 1 showing 271 that similar amount of Q-PVP-4days is grafted on the surface, regardless of the deposition 272

	273	method.
	274

Table 1 :

 1 Atomic percentage obtained from XPS data for spin and dip coated films as compared 275

	276	to non functionalized surface.
		Atomic %	Ti2p	O1s	C1s	N1s
		TiO2 control surface 17.5	41.4	40.2	0.9
		Spin-coated surface 0.3	6.5	87.6	5.6
		Dip-coated surface	ND	4.3	89.8	5.9
	277	ND=non detectable; % are obtained as mean of two measurements, the standard deviation of
	278	the measure is around 0.2 %.
	279
	280	Polarization Modulation Infra-Red Reflection Absorption Spectroscopy (PM-IRRAS):
	281

Table 2 : % of quaternization of the copolymer as a function of reaction time. 313

 2

	).

𝑁

ratio are presented on Table

2

for each reaction, and one can see that a stable regime is 309 achieved after 48 hours of reaction time, with no more possible quaternization even with twice 310

Table 3 .

 3 Fluorescein test measurements with leaching and RSCD calculations are reported in Table3. 360 Surface charge determinations on titanium plates grafted with Q-PVP-4 days before 362 and after a 7-day immersion in either rabbit serum or distilled water. 363

	.

Complete Manuscript

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