Philippe Juneau
email: juneau.philippe@uqam.ca

Alexandre Barnett

Vona Méléder

Christine Dupuy

Johann Lavaud

Combined effect of high light and high salinity on the regulation of photosynthesis in three diatom species belonging to the main growth forms of intertidal flat inhabiting microphytobenthos

Keywords: diatom / intertidal flat / microphytobenthos / photoprotection / photosynthesis / salinity Chl a, chlorophyll a, DD, diadinoxathin, DES, de-epoxidation state MPB, microphytobenthos, NPQ, non-photochemical quenching of chlorophyll fluorescence, PSII, photosystem II, RLC, rapid light curve, XC, xanthophyll cycle

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

Introduction

Estuarine intertidal flats belong to the most productive ecosystems on Earth [START_REF] Macintyre | Microphytobenthos: The ecological role of the 'secret garden' of unvegetated, shallow-water marine habitats. I. Distribution, abundance and primary production[END_REF][START_REF] Underwood | Primary production by phytoplankton and microphytobenthos in estuaries[END_REF] and they have a central role in structuring the foodweb of coastal areas [START_REF] Kromkamp | Developments in microphytobenthos primary productivity studies[END_REF]. A large part of the strong productivity of intertidal flats is due to the microphytobenthos (MPB) [START_REF] Admiraal | The ecology of estuarine sediment inhabiting diatoms[END_REF][START_REF] Macintyre | Microphytobenthos: The ecological role of the 'secret garden' of unvegetated, shallow-water marine habitats. I. Distribution, abundance and primary production[END_REF][START_REF] Underwood | Primary production by phytoplankton and microphytobenthos in estuaries[END_REF] which in temperate seas is mainly dominated by benthic diatoms [START_REF] Méléder | Spatiotemporal changes in microphytobenthos assemblages in a macrotidal flat (Bourgneuf Bay, France)[END_REF][START_REF] Ribeiro | Structure and diversity of intertidal benthic diatom assemblages in contrasting shores: A case study from the Tagus estuary[END_REF]. Benthic and planktonic diatoms are essential primary producers which contribute to about 40% of the marine primary production; they also play a major role in the silica and nitrogen biogeochemical cycles [START_REF] Armbrust | The life of diatoms in the world's oceans[END_REF]. The MPB diatoms constitute the bulk of the diatom diversity [START_REF] Kooistra | The origin and the evolution of the diatoms: Their adaptation to a planktonic existence[END_REF]. They can be divided in three main growth forms which mainly differ in their life in the sediment [START_REF] Kooistra | The origin and the evolution of the diatoms: Their adaptation to a planktonic existence[END_REF][START_REF] Ribeiro | Structure and diversity of intertidal benthic diatom assemblages in contrasting shores: A case study from the Tagus estuary[END_REF]: i) the epipelon comprises motile species free-living in between sediment particles [START_REF] Herlory | Microstructure of microphytobenthic biofilm and its spatio-temporal dynamics in an intertidal mudflat (Aiguillon Bay, France)[END_REF], ii) the epipsammon which lives attached to sediment particles, and iii) the tychoplankton which presumably have an amphibious life style (i.e. both sediment and water column) (e.g. [START_REF] Sabbe | A new genus, Pierrecomperia gen. nov., a new species and two new combinations in the marine diatom family Cymatosiraceae[END_REF].

Epipelon and epipsammon growth forms show distinct distribution among intertidal habitats characterised by different types of sediment [START_REF] Sabbe | Short-term fluctuations in benthic diatom numbers on an intertidal sandflat in the Westerschelde estuary (Zeeland, The Netherlands)[END_REF][START_REF] Méléder | Spatiotemporal changes in microphytobenthos assemblages in a macrotidal flat (Bourgneuf Bay, France)[END_REF][START_REF] Ribeiro | Structure and diversity of intertidal benthic diatom assemblages in contrasting shores: A case study from the Tagus estuary[END_REF]. Epipelon dominates cohesive muddy sediments (> 90% of MPB; [START_REF] Haubois | Spatiotemporal structure of the epipelic diatom assemblage from an intertidal mudflat in Marennes-Oleron Bay, France[END_REF], while epipsammon dominates less cohesive sandy sediments (> 95% of MPB; [START_REF] Méléder | Spatiotemporal changes in microphytobenthos assemblages in a macrotidal flat (Bourgneuf Bay, France)[END_REF]. Because of different habitats, epipelon and epipsammon have evolved different ways of coping with their intertidal environment. Epipelon displays vertical 'migration' following endogenous tidal/dial rhythms and environmental stimuli [START_REF] Saburova | Diatom activity within soft sediments: behavioural and physiological processes[END_REF][START_REF] Consalvey | The ups and downs of life in a benthic biofilm: migration of benthic diatoms[END_REF][START_REF] Coelho | Endogenous versus environmental control of vertical migration by intertidal benthic microalgae[END_REF]: typically, during daylight emersion, epipelic diatoms move to the sediment surface and form a dense biofilm, while before immersion they migrate downward. Epipsammon lives more or less firmly attached (stalked or adnate forms) to individual sand grain including some species able to exert micro-movements within the sphere of grains. Tychoplankton (which is sometimes considered as resuspended epipelon and/or epipsammon during immersion; [START_REF] Macintyre | Microphytobenthos: The ecological role of the 'secret garden' of unvegetated, shallow-water marine habitats. I. Distribution, abundance and primary production[END_REF] can live either as part of MPB or of phytoplankton, depending on the hydrodynamics [START_REF] Koh | Tidal resuspension of microphytobenthic chlorophyll a in a Nanaura mudflat, Saga, Arieke Sea, Japan: flood-ebb and spring-neap variations[END_REF]; it can contribute to up to one third of phytoplankton [START_REF] Guarini | Benthic contribution to pelagic microalgal communities in two semi-enclosed, European-type littoral ecosystems (Marennes-Oléron Bay and Aiguillon Bay, France)[END_REF][START_REF] Brito | Does microphytobenthos resuspension influence phytoplankton in shallow systems ? A comparison through a Fourier series analysis[END_REF].

Environmental cues can rapidly vary to an extreme in intertidal flats [START_REF] Admiraal | The ecology of estuarine sediment inhabiting diatoms[END_REF][START_REF] Paterson | Microphytobenthos in contrasting coastal ecosystems: Biology and dynamics[END_REF] and impair the photosynthetic productivity of MPB diatoms (i.e. photoinhibition) [START_REF] Blanchard | Characterizing and quantifying photoinhibition in intertidal microphytobenthos[END_REF][START_REF] Serôdio | Photosynthetic activity, photoprotection and photoinhibition in intertidal microphytobenthos as studied in situ using variable chlorophyll fluorescence[END_REF]. In order to prevent such situation, benthic diatoms have evolved diverse responses that can be distinguished in two main types: behaviour and physiology. Only epipelon can escape from a combination of sometimes harsh environmental conditions at the sediment surface by 'migrating' downward to the most optimal conditions (i.e. the so-called 'behavioural photoprotection'; [START_REF] Admiraal | The ecology of estuarine sediment inhabiting diatoms[END_REF][START_REF] Kromkamp | Determination of microphytobenthos PSII quantum efficiency and photosynthetic activity by means of variable chlorophyll fluorescence[END_REF][START_REF] Consalvey | The ups and downs of life in a benthic biofilm: migration of benthic diatoms[END_REF][START_REF] Serôdio | Microphytobenthos vertical migratory photoresponse as characterised by light-response curves of surface biomass[END_REF], especially as regards to salinity [START_REF] Sauer | Effect of salinity, light and time on the vertical migration of diatom assemblages[END_REF]. In contrast, all growth forms use physiological processes for the fast regulation of photochemistry (i.e. 'physiological photoprotection'; [START_REF] Lavaud | Fast regulation of photosynthesis in diatoms: Mechanisms, evolution and ecophysiology[END_REF][START_REF] Goss | Regulation and function of xanthophyll cycle-dependent photoprotection in algae[END_REF][START_REF] Depauw | Exploring the molecular basis of responses to light in marine diatoms[END_REF][START_REF] Lepetit | Molecular dynamics of the diatom thylakoid membrane under different light conditions[END_REF]. In diatoms, two physiological processes are important in field situation [START_REF] Brunet | Can the xanthophyll cycle help extract the essence of the microalgal functional response to a variable light environment ?[END_REF][START_REF] Lavaud | The peculiar features of non-photochemical fluorescence quenching in diatoms and brown algae[END_REF]: i) the non-photochemical quenching of chlorophyll (Chl) fluorescence (NPQ) [START_REF] Depauw | Exploring the molecular basis of responses to light in marine diatoms[END_REF][START_REF] Lepetit | Molecular dynamics of the diatom thylakoid membrane under different light conditions[END_REF][START_REF] Lavaud | The peculiar features of non-photochemical fluorescence quenching in diatoms and brown algae[END_REF], and ii) the partly related light-dependent conversion of diadinoxanthin (DD) to diatoxanthin (DT) by the DD de-epoxidase (i.e. the 'xanthophyll cycle', XC) [START_REF] Brunet | Can the xanthophyll cycle help extract the essence of the microalgal functional response to a variable light environment ?[END_REF][START_REF] Goss | Regulation and function of xanthophyll cycle-dependent photoprotection in algae[END_REF]. In benthic diatoms, NPQ and XC have been scarcely studied in situ: it varies with the diurnal and tidal cycles, season, latitude [START_REF] Serôdio | Non-photochemical quenching of chlorophyll fluorescence and operation of the xanthophyll cycle in estuarine microphytobenthos[END_REF][START_REF] Van Leeuwe | Photacclimation in microphytobenthos and the role of the xanthophylls pigments[END_REF][START_REF] Chevalier | In situ photosynthetic activity and xanthophylls cycle development of undisturbed microphytobenthos in an intertidal mudflat[END_REF][START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: Role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF], and with the position of diatom cells within the sediment and along the intertidal elevation gradient [START_REF] Jesus | Adaptations of microphytobenthos assemblages to sediment type and tidal position[END_REF][START_REF] Cartaxana | Physiological versus behavioral photoprotection in intertidal epipelic and epipsammic benthic diatom communities[END_REF]. The respective importance of behavioural and physiological responses in epipelon has received a major interest [START_REF] Mouget | Migration or photoacclimation to prevent high irradiance and UV-B damage in marine microphytobenthic communities[END_REF][START_REF] Van Leeuwe | Photacclimation in microphytobenthos and the role of the xanthophylls pigments[END_REF]Perkins et al., 2010b;[START_REF] Cartaxana | Physiological versus behavioral photoprotection in intertidal epipelic and epipsammic benthic diatom communities[END_REF][START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: Role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF]. These studies have shown that although motility is essential for an optimal response to the changes in environmental conditions, NPQ and XC remain important features, and even compensate for migration under conditions where motility is limited, to finely tune photosynthetic efficiency. Also, a recent analysis of NPQ and XC abilities among the growth forms of MPB diatoms has revealed a clear relationship between growth form and capacity for physiological photoprotection (Barnett et al., 2014), i.e. while epipsammon shows the highest NPQ and XC capacity, epipelon and tychoplankton shows the lowest ones, reflecting their respective motility and adaptation to a low light (LL) environment (i.e. tychoplankton is either buried in sediment or resuspended in a turbid water column; [START_REF] Roncarati | Photosynthetic performance, oxidative damage and antioxidants in Cylindrotheca closterium in response to high irradiance, UVB irradiance and salinity[END_REF].

Changes in light intensity and temperature are often considered as the two major forcings of the photosynthetic productivity of MPB diatoms [START_REF] Guarini | Modelling the dynamics of the microphytobenthic biomass and primary production in European intertidal mudflats[END_REF]. Surprisingly, changes in salinity have been less studied in benthic diatoms, while in planktonic diatoms it is known to induce modification of community species diversity [START_REF] Thessen | Effect of salinity on Pseudo-Nitzschia species (Bacillariophyceae) growth and distribution[END_REF][START_REF] Dijkman | Photosynthetic characteristics of the phytoplankton in the Scheldt estuary: community and single-cell fluorescence measurements[END_REF][START_REF] Muylaert | Changes in phytoplankton diversity and community composition along the salinity gradient of the Schelde estuary (Belgium/The Netherlands)[END_REF][START_REF] Petrou | Heterogeneity in the photoprotective capacity of three Antarctic diatoms during short-term changes in salinity and temperature[END_REF], and of growth and photosynthesis [START_REF] Thessen | Effect of salinity on Pseudo-Nitzschia species (Bacillariophyceae) growth and distribution[END_REF][START_REF] Dijkman | Photosynthetic characteristics of the phytoplankton in the Scheldt estuary: community and single-cell fluorescence measurements[END_REF][START_REF] Petrou | Heterogeneity in the photoprotective capacity of three Antarctic diatoms during short-term changes in salinity and temperature[END_REF]. Salinity often covaries with other environmental gradients like light and temperature in the case of high salinities (due to pore-water evaporation in the upper-layer of the sediment) and with nutrient concentrations in the case of low salinities (due to the discharge of estuarine rivers) [START_REF] Admiraal | Distribution of diatom species on an estuarine mud flat and experimental analysis of the selective effect of stress[END_REF][START_REF] Underwood | Determining the environmental preferences of four estuarine epipelic diatom taxa: growth across a range of salinity, nitrate and ammonium conditions[END_REF][START_REF] Thornton | Factors affecting microphytobenthic biomass, species composition and production in the Colne Estuary (UK)[END_REF]. Although early works stated that MPB diatoms are highly tolerant to a wide range of salinity changes [START_REF] Williams | Division rates of salt marsh diatoms in relation to salinity and cell size[END_REF][START_REF] Admiraal | Salinity tolerance of benthic estuarine diatoms as tested with a rapid polarographic measurement of photosynthesis[END_REF][START_REF] Admiraal | Distribution of diatom species on an estuarine mud flat and experimental analysis of the selective effect of stress[END_REF], further studies have shown that salinity changes, often combined with high light (HL), impairs the growth from a salinity of 40 and above [START_REF] Natana Murugaraj | Effect of salinity stress on the marine diatom Amphora coffeaeformis (Ag.) Kuetz. (Bacillariophyceae) in relation to proline accumulation[END_REF][START_REF] Scholz | Growth responses of 25 benthic marine Wadden Sea diatoms isolated from the Solthörn tidal flat (southern North Sea) in relation to varying culture conditions[END_REF], it reduces the photosynthetic performance [START_REF] Roncarati | Photosynthetic performance, oxidative damage and antioxidants in Cylindrotheca closterium in response to high irradiance, UVB irradiance and salinity[END_REF][START_REF] Le Rouzic | Changes in photosynthetic yield (Fv/Fm) responses of salt-marsh microalgal communities along an osmotic gradient (Mont-Saint-Michel Bay, France)[END_REF] via (photo-)oxidative stress [START_REF] Rijstenbil | Effects of UVB radiation and salt stress on growth, pigments and antioxidative defence of the marine diatom Cylindrotheca closterium[END_REF][START_REF] Rijstenbil | UV-and salinity-induced oxidative effects in the marine diatom Cylindrotheca closterium during simulated emersion[END_REF][START_REF] Roncarati | Photosynthetic performance, oxidative damage and antioxidants in Cylindrotheca closterium in response to high irradiance, UVB irradiance and salinity[END_REF], and it can modify the motility of epipelic diatoms in the sediment [START_REF] Sauer | Effect of salinity, light and time on the vertical migration of diatom assemblages[END_REF] via changes in the excretion of exopolysaccaharides [START_REF] Apoya-Horton | Movement modalities and responses to environmental changes of the mudflat diatom Cylindrotheca closterium (Bacillariophyceae)[END_REF]. Furthermore, although the different growth forms of MPB diatoms pertain to habitats in which the salinity can differentially vary due to the degree of cohesion of sediment [START_REF] Paterson | Microphytobenthos in contrasting coastal ecosystems: Biology and dynamics[END_REF], to our knowledge, no study explored if they show different photophysiological response to a combine high salinity-HL stress and if it correlates to their habitat-associated growth form. The objectives of the present study were therefore to determine i) if a higher salinity can increase the negative effect of HL on the photosynthetic efficiency, ii) if three representatives belonging to each of the growth forms of MPB diatoms react differently to a combined high salinity-HL stress.

Materials and methods

Sediment grain size, pore-water salinity, temperature and MPB biomass of sediment

Parameters were measured at different seasons and for two sites of the Atlantic French coast: the bay of Brouage and the bay of Bourgneuf; see [START_REF] Haubois | Spatiotemporal structure of the epipelic diatom assemblage from an intertidal mudflat in Marennes-Oleron Bay, France[END_REF] and [START_REF] Méléder | Spatiotemporal changes in microphytobenthos assemblages in a macrotidal flat (Bourgneuf Bay, France)[END_REF] for a respective characterization of the two sampling sites (see Table 1 and Figure 1 for all details). Sediment grain size was determined with a laser granulometer (Mastersizer 2000, Malvern Instruments, UK) as previously described [START_REF] Méléder | Spatiotemporal changes in microphytobenthos assemblages in a macrotidal flat (Bourgneuf Bay, France)[END_REF]. The mud fraction (grain size < 63 µm) of each sample was determined using the software Gradistat [START_REF] Blott | GRADISTAT: a grain size distribution and statistics package for the analysis of unconsolidated sediments[END_REF]. Sediment samples were centrifuged for 10 min at 3500 g and salinity was measured on the supernatant with a sensor TetraCon325 (WTW, Weilhem, Germany). The temperature at the sediment surface was measured every 30 s with a universal data logger (ULM-500, Walz Effeltrich Germany) equipped with a plane temperature sensor (accessory of the ULM-500). The sediment content of chlorophyll a (µg Chl a. g dry sediment -1 ) was used as a proxy for MPB biomass. Chl a was extracted and measured as previously described [START_REF] Herlory | Microstructure of microphytobenthic biofilm and its spatio-temporal dynamics in an intertidal mudflat (Aiguillon Bay, France)[END_REF]: spectrofluorimetric measurement (Turner TD-700 fluorometer) was performed on supernatant of sediment samples after lyophilisation, extraction (90% acetone, 12 h, 4°C, in the dark, continuous shaking) and centrifugation 10 min at 4000 g.

Diatom culture conditions

Three species belonging to the three main growth forms of MPB diatoms were used: 1) Epipelon, Navicula phyllepta (Culture Collection Yerseke-The Netherlands CCY9804, isolated in the Westerschelde estuary, North sea, The Netherlands); 2) Epipsammon, Biremis lucens (Nantes Culture Collection-France NCC360, isolated in the bay of Bourgneuf, Atlantic,France; 3) Tychoplankton, Plagiogrammopsis vanheurckii (NCC186-2, isolated in the bay of Bourgneuf). Cultures were grown in batch sterile artificial seawater F/2 medium completed with Tropic Marin artificial sea salt (Dr. Biener GmbH, Germany) at a salinity of 33, and enriched with NaHCO 3 (80 mg L -1 final concentration). Temperature was 20°C and light was 60 µmol photons m -2 s -1 (white fluorescent tubes L58W/840, OSRAM, Germany) with a 16 h:8 h light:dark photoperiod. When cultures reached exponential phase, cells were harvested by gentle centrifugation (5 min, 4000 g), resuspended to a concentration of 6  1 mg Chl a mL -1 . For this purpose, Chl a concentration was determined according to the [START_REF] Jeffrey | New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton[END_REF] spectrophotometric method. Diatom suspensions were continuously stirred under the growth conditions for at least 1 h before the high light (HL) and salinity treatments.

High light (HL) and salinity treatments

Diatom cells were exposed for 1 h to a range of increasing salinities (33, 37, 41 and 45) under the growth light intensity and under HL intensity (10x the growth light intensity, 600 µmol photons m -2 s -1 ) at 20°C. During each treatment, cells were stirred to prevent settling. Each condition was measured in triplicate. The temperature, light and salinity values/ranges were chosen according to the in situ measurements (see Table 1 and Figure 1) in order to reproduce the environment experienced by MPB diatoms in Spring (see the Results section, paragraph 3.1). Increased salinity was obtained by implementing the sterile artificial seawater F/2 medium with increasing amounts of Tropic Marin artificial sea salt (Dr. Biener GmbH, Germany). HL was provided by white fluorescent tubes (FQ 54W/865 LO, OSRAM, Germany).

Pigment analyses

At the end of each salinity and light treatments, 1 mL of diatom suspension was filtered on a membrane filter (Membrane Isopore Polycarbonate 1.2-µm RTTP filter, 25 mm diameter, Merck Millipore, Ireland), quickly frozen in liquid nitrogen and stored at -80°c until further analysis. Pigment extraction and determination of pigment content were performed as previously described (Barnett et al., 2014). Chl a.cell -1 was calculated by counting the number of cells microscopically with a Malassez's counting chamber. The de-epoxidation state (DES in %) was calculated as DES = [(DT / DD + DT) x 100], where DD is the diadinoxanthin, the epoxidized form, and DT is the diatoxanthin, the de-epoxidized form.

Chl fluorescence yield and rapid light curves (RLCs)

For a complete overview of the definition, measurement and calculation of the fluorescence levels and of the photophysiological parameters, see Table 2. Chl fluorescence yield was monitored with a Diving-PAM fluorometer (Walz, Germany) on a 2.5 mL stirred and 20°C controlled diatom suspension (see [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[END_REF]. Before measurement, the cells were dark-adapted for 15 min, and a saturating pulse (3600 µmol photons m -2 s -1 , duration 0.4 ms) was fired to measure F 0 , F m and F v /F m . For RLCs (Perkins et al., 2010a), the diatom suspension was exposed to 8 successive increasing intensities (29-1038 µmol photons.m -2 .s -1 ) of 60 s each. At the end of each RLC-light step exposure, F m ' was measured. RLCs allow constructing rETR vs. E curves; from the fitted rETR-E curves [START_REF] Eilers | A model for the relationship between light intensity and the rate of photosynthesis in phytoplankton[END_REF], rETR m , α, E k can be extracted.

Results

Pore-water salinity and MPB biomass in different sediments of the French Atlantic coast

The changes in pore-water salinity in the upper layer (first 1 cm) was measured over different seasons at two sites of the French Atlantic coast characterised by two sediment types: 1) a site with 95% of cohesive muddy sediment which is known to be dominated by a community of epipelic diatoms, and especially Navicula phyllepta, throughout the year [START_REF] Haubois | Spatiotemporal structure of the epipelic diatom assemblage from an intertidal mudflat in Marennes-Oleron Bay, France[END_REF]; 2) a site with a mix of muddy and sandy (thus less cohesive) sediment which is known to be dominated by a community of epipsammic diatoms where Biremis lucens and Plagiogrammopsis vanheurckii are typical [START_REF] Méléder | Spatiotemporal changes in microphytobenthos assemblages in a macrotidal flat (Bourgneuf Bay, France)[END_REF]. Overall, pore-water salinity varied between 29 and 48 during the 3 h emersion period (Table 2). As expected, variations over an emersion were higher in summer than in winter, and over seasons in cohesive than in less cohesive sediment with an overall minimum and maximum variation during an emersion of 2.3 and 8.3, respectively.

The changes in pore-water salinity were further deciphered at the two sites during the course of an emersion in Spring at two depths in the upper layer of sediments (Fig. 1). Large changes in pore-water salinity occurred within only 1.5 h: mean Δ 5.1 and Δ 3.5 for the muddy and the muddy-sandy sediment, respectively. Nevertheless, these changes were mainly (muddy sediment, Fig. 1A) and even exclusively (muddy-sandy sediment, Fig. 1B) observed in the first 0.5 cm where most of the MPB biomass was present (Fig. 1C). In the deeper sediment layer (-0.5-1 cm), a high equivalent MPB biomass (40.5  3.5 and 48.5  5.3 µg Chl a g sediment -1 in muddy and muddy-sandy sediment, respectively) was observed and the pore-water salinity was close to 33: 34.0  0.7 in mud and 33.0  1.3 in muddy sand.

Photophysiological response of Navicula phyllepta (epipelon), Biremis lucens (epipsammon) and Plagiogrammopsis vanheurckii (tychoplankton) to a combined high salinity-HL stress

The PSII activity of the three species was assessed by measuring F v /F m and ΦPSII, as well as the RLCs photophysiological parameters (, rETR m and E k ) (see Table 2). While in LL-acclimated cells F v /F m and ΦPSII did not change significantly with salinity (Tables 3 and5), HL treatment induced a significant mean decrease in F v /F m (N. phyllepta, -8.3  1.0 % < B. lucens, -11.3  1.7 % < P. vanheurckii, -37.3  1.7 %) and in ΦPSII (N. phyllepta, -6.3  1.5 % < B. lucens, -10.0  3.3 %) independent of salinity (Tables 3 and5). Only in P. vanheurckii ΦPSII changes were salinity-dependent, i.e. -11.5  3.5 % from 33-37 and -28.5  1.5 % for 41-45 (Table 3). The RLCs photophysiological parameters (, rETR m and E k , see Table 2) changed differently depending on the species and salinity/light treatments. N. phyllepta cells were not significantly affected either by changes in salinity nor by the HL treatment (Fig. 2 and Table 5). In B. lucens LL acclimated cells, E k and rETR m were significantly affected by salinity (Fig. 2 and Table 5) and, as expected, α and E k were significantly lower and higher, respectively, after HL treatment (Table 5) although only for a limited range of salinities: up to 41 for α and up to 37 for E k . In HL cells, further salinity raising induced an increase in α (+25.5  3.5 %) and a subsequent decrease in E k (-39  3 %) (Fig. 2). It was in P. vanheurckii , rETR m and E k showed the most pronounced changes with salinity and HL treatment (Fig. 2 and Table 5). α decreased in both LL acclimated cells (-23  14 %) and after HL treatment (-39  5 %, especially from a salinity of 33 to 37). rETR m followed an opposite trend, and significantly increased with salinity by a mean factor of up to 1.60  0.13, and independent of the light treatment (Table 5). As a consequence, E k significantly increased together with salinity for both light treatments (x 1.96  0.3 and x 1.53  0.3 for HL and LL cells, respectively).

The photoprotective response of the species was assessed by measuring NPQ and the XC components and operation (Tables 3 and4). NPQ m of LL acclimated cells (Table 3) was on average 0.224  0.135 (N. phyllepta), 0.332  0.018 (B. lucens) and 0.645  0.105 (P. vanheurckii) and, as expected, it significantly increased during the HL treatment (Tables 3 and5) as follows: P. vanheurckii (x 1.6  0.3 for all salinities) < N. phyllepta (x 1.8  0.3 for all salinities except 45, x 2.5) < B. lucens (x 2.2  0.4 for all salinities). Only in P. vanheurckii LL acclimated cells, NPQ m significantly decreased by -30% from a salinity of 33 to 45 (Tables 3 and5). DES was much higher in LL acclimated cells of P. vanheurckii (40  4 %) than in N. phyllepta (12  3 %) and B. lucens (7  1 %) (Fig. 3). During the HL treatment, and independent of salinity, DES significantly increased to a different extent (P. vanheurckii, 58  2 % > B. lucens, 39  3 % > N. phyllepta, 31  2 %) (Fig. 3 and Table 5). Interestingly, in N. phyllepta, while diatoxanthin (DT) significantly increased during HL (i.e. due to diadinoxanthin, DD, de-epoxidation), DD did not similarly decreased (Tables 4 and5), as it would have been expected, arguing for a de novo synthesis of DD during HL. Out from DD and DT, there was no additional significant pigment changes in the three species whatever the treatment except a significant HL-salinity independent decrease in Chl a.cell -1 (-20.3  4.5 %) in N. phyllepta.

The higher sensitivity of P. vanheurckii to salinity alone was further illustrated by the fact that the combination with the HL treatment did not significantly change some of the photophysiological parameters (, rETR m , E k , NPQ m ) in contrast to N. phyllepta and B. lucens (Table 5).

Discussion

Pore-water salinity changes in Atlantic French coast intertidal flats and their potential effects on intertidal MPB diatoms

In the field, the upper layer sediment pore-water salinity can highly (Δ 5) and rapidly (within 1.5 h) increase, and reach values as high as 48 in Summer (values up to 55-60 were even reported elsewhere, [START_REF] Roncarati | Photosynthetic performance, oxidative damage and antioxidants in Cylindrotheca closterium in response to high irradiance, UVB irradiance and salinity[END_REF][START_REF] Serôdio | Photosynthetic activity, photoprotection and photoinhibition in intertidal microphytobenthos as studied in situ using variable chlorophyll fluorescence[END_REF]. Nevertheless, high salinity events are not restricted to hot sunny days and also occur at moderate temperature (16-20°C), as shown here in Spring, due to wind-driven desiccation [START_REF] Williams | Division rates of salt marsh diatoms in relation to salinity and cell size[END_REF][START_REF] Sauer | Effect of salinity, light and time on the vertical migration of diatom assemblages[END_REF] in the first 0.5 cm of the sediment (where the bulk of the MPB biomass inhabits). Changes in the sediment pore-water salinity depend on the sediment cohesion with higher values and amplitude in cohesive sediment probably due to the trapping (and subsequent higher evaporation) of pore-water at the surface [START_REF] Paterson | Microphytobenthos in contrasting coastal ecosystems: Biology and dynamics[END_REF][START_REF] Sauer | Effect of salinity, light and time on the vertical migration of diatom assemblages[END_REF].

Therefore, although temperature may be optimal (20-25 °C, [START_REF] Blanchard | Seasonal effect on the photosynthesic capacity of intertidal microphytobenthos and temperature[END_REF][START_REF] Scholz | Growth responses of 25 benthic marine Wadden Sea diatoms isolated from the Solthörn tidal flat (southern North Sea) in relation to varying culture conditions[END_REF], HL and high salinity conditions may occur in the sediment upper layer during Spring-early Summer emersion by the Atlantic French coast. These conditions may differentially impair the photosynthetic efficiency of the main growth forms of MPB as regards to the sediment cohesion of their respective habitat or their amphibious life. The HL-high salinity combination has been rarely studied before [START_REF] Rijstenbil | Effects of UVB radiation and salt stress on growth, pigments and antioxidative defence of the marine diatom Cylindrotheca closterium[END_REF][START_REF] Rijstenbil | UV-and salinity-induced oxidative effects in the marine diatom Cylindrotheca closterium during simulated emersion[END_REF][START_REF] Roncarati | Photosynthetic performance, oxidative damage and antioxidants in Cylindrotheca closterium in response to high irradiance, UVB irradiance and salinity[END_REF]. Most previous works focused on low salinity stress combined to nutrient gradient due to estuarine rivers discharge [START_REF] Admiraal | Distribution of diatom species on an estuarine mud flat and experimental analysis of the selective effect of stress[END_REF][START_REF] Underwood | Determining the environmental preferences of four estuarine epipelic diatom taxa: growth across a range of salinity, nitrate and ammonium conditions[END_REF][START_REF] Thornton | Factors affecting microphytobenthic biomass, species composition and production in the Colne Estuary (UK)[END_REF] and on the long-term effect of salinity changes (most often measured by specific growth, [START_REF] Williams | Division rates of salt marsh diatoms in relation to salinity and cell size[END_REF][START_REF] Jackson | The effect of salinity on growth and amino acid composition in the marine diatiom Nitzschia pungens[END_REF][START_REF] Underwood | Determining the environmental preferences of four estuarine epipelic diatom taxa: growth across a range of salinity, nitrate and ammonium conditions[END_REF][START_REF] Natana Murugaraj | Effect of salinity stress on the marine diatom Amphora coffeaeformis (Ag.) Kuetz. (Bacillariophyceae) in relation to proline accumulation[END_REF][START_REF] Scholz | Growth responses of 25 benthic marine Wadden Sea diatoms isolated from the Solthörn tidal flat (southern North Sea) in relation to varying culture conditions[END_REF], instead of effects (including short-term exposure) on the photosynthetic efficiency [START_REF] Admiraal | Salinity tolerance of benthic estuarine diatoms as tested with a rapid polarographic measurement of photosynthesis[END_REF][START_REF] Admiraal | Distribution of diatom species on an estuarine mud flat and experimental analysis of the selective effect of stress[END_REF][START_REF] Roncarati | Photosynthetic performance, oxidative damage and antioxidants in Cylindrotheca closterium in response to high irradiance, UVB irradiance and salinity[END_REF].

Differential photosynthetic and photoprotective response to a combined high-salinity-HL stress in diatoms representative of the main growth forms of intertidal MPB

The photosynthesis of the three examined species responded differently to the combination of HL and salinity stress. In our conditions, N. phyllepta, the photosynthetic efficiency was not largely impaired neither by high salinity nor HL alone or both in combination (i.e. changes in PSII and RLCs photophysiological parameters were not > 10 % on average). Noticeably, HL induced a decrease in Chl a. cell -1 that was not observed in the two other species. It shows the ability to lower the overexcitation of the whole photosynthetic machinery under HL stress [START_REF] Brunet | Pigments and photoacclimation processes[END_REF]. In contrast, in B. lucens, the photosynthetic electron transport rate (rETR m ) was slightly but significantly affected by high salinity alone. Additionally, the photoacclimatory-coupled decrease in  and increase in E k , that illustrates the decrease of the excitation pressure on PSII [START_REF] Perkins | Light response curve methodology and possible implications in the application of chlorophyll fluorescence to benthic diatoms[END_REF][START_REF] Cruz | Relationship of rapid light curves of variable fluorescence to photoacclimation and non-photochemical quenching in a benthic diatom[END_REF][START_REF] Lefebvre | Duration of rapid light curves for determining in situ the photosynthetis of microphytobenthos biofilms[END_REF], were abolished by higher salinities (from 37 on). Nevertheless, in our moderately stressful conditions, it did not largely impaired PSII activity (decrease in F v /F m and ΦPSII at maximum ~11%). The high salinity-dependent inhibition of photoacclimation was not observed in P. vanheurckii for which the decrease of the excitation pressure on PSII was obviously a key response in all conditions. While under HL alone,  and E k modulation was enough, under high salinity alone and combined with HL, the additional increase in rETR m , possibly through a stronger activation of the Calvin cycle enzymes [START_REF] Nymark | An integrated analysis of molecular acclimation to high light in the marine diatom Phaeodactylum tricornutum[END_REF], was necessary to cope with the stress. These changes in the photosynthetic efficiency were sufficient for P. vanheurckii to cope with salinity stress alone but not when it was it was combined with HL (strong ΦPSII decrease for salinities > 41 on), supporting its HL sensitivity (strong F v /F m decrease) due to its adaptation to a LL environment (low  and E k ; see also Barnett et al., 2014).

In parallel to modification in PSII-related photophysiological parameters, all species exerted a photoprotective response but to a different extent. In contrast to prolonged high salinity exposure [START_REF] Rijstenbil | UV-and salinity-induced oxidative effects in the marine diatom Cylindrotheca closterium during simulated emersion[END_REF], HL DES increase was independent of salinity in all species.

Hence, the de-epoxidase enzyme, responsible for the light-dependent conversion of DD to DT, does not seem to be influenced by a short (1 h) salinity stress. DT is well-known to i) scavenge reactive oxygen species (ROS) and to help preventing the peroxidation of lipids of the thylakoid membrane [START_REF] Lepetit | Evidence for the existence of one antenna-associated, lipid-dissolved and two protein-bound pools of diadinoxanthin cycle pigments in diatoms[END_REF], ii) participate to NPQ [START_REF] Goss | Regulation and function of xanthophyll cycle-dependent photoprotection in algae[END_REF][START_REF] Lavaud | Silencing of the violaxanthin de-epoxidase gene in the diatom Phaeodactylum tricornutum reduces diatoxanthin synthesis and non-photochemical quenching[END_REF][START_REF] Lavaud | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF]. In contrast to the two other species, in N. phyllepta DD de-epoxidation was accompanied by DD de novo synthesis, a way to enhance the capacity to further synthesize DT in case of prolonged stress [START_REF] Lepetit | High light acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF]. Probably due to the shortness of high salinity exposure, there was no significant increase in fucoxanthin and β-carotene, a well-known pigment response to oxidative stress additional to the XC [START_REF] Dambeck | Antioxidative activities of algal keto carotenoids acting as antioxidative protectants in the chloroplast[END_REF]Tefler, 2014). As expected, NPQ m was higher in B lucens than in N. phyllepta, while it was higher than previously observed in P. vanheurckii probably due to i) different growth light conditions which generated a higher DES, and ii) the different way of measuring NPQ (RLCs vs. Non-Sequential Light Curves-NSLCs) (Barnett et al., 2014).

HL NPQ increased was impacted by salinity depending on the species: while NPQ increase was similar (about 1.6x) for all salinities in P. vanheurckii, it was higher (about 2.2x) in B lucens, and it reached an even higher level (2.5x) for a salinity of 45 in N. phyllepta. These observations clearly illustrate how i) NPQ helped to dissipate the excess of light excitation energy in PSII when the photosynthetic machinery was slowed-down by salinity, ii) N. phyllepta appeared to be insensitive to all high salinities lower than 45. Strikingly, in P. vanheurckii, NPQ decreased linearly (-0.018  0.001 NPQ unit. salinity unit -1 ) illustrating the high salinity-dependent NPQ inhibition disregard of the high amount of DT synthesised in this species. Most probably here, NPQ decrease and discrepancy between DES and NPQ might be due to a stronger involvement of DT in the prevention of lipid peroxidation by ROS [START_REF] Lepetit | Evidence for the existence of one antenna-associated, lipid-dissolved and two protein-bound pools of diadinoxanthin cycle pigments in diatoms[END_REF][START_REF] Lepetit | High light acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF].

Relationship between the response of intertidal MPB diatoms to a combined high salinity-HL stress and their habitat-related growth form

The photophysiological response of N. phyllepta, B. lucens and P. vanheurckii to the combined high salinity-HL conditions fitted well with their respective growth form and original habitat.

The relationship between photophysiology and the different growth forms of MPB diatoms to light alone was already documented before (Barnett et al., 2014).

Epipelon

N. phyllepta photochemistry was not affected neither by the high salinity stress alone, nor by the combination of HL and high salinity, illustrating an adaptation to potentially extreme conditions of light and salinity at the surface of cohesive (muddy) sediment. This is in agreement with previous reports on the high tolerance to salinity changes of Navicula sp.

representatives [START_REF] Underwood | Determining the environmental preferences of four estuarine epipelic diatom taxa: growth across a range of salinity, nitrate and ammonium conditions[END_REF][START_REF] Scholz | Growth responses of 25 benthic marine Wadden Sea diatoms isolated from the Solthörn tidal flat (southern North Sea) in relation to varying culture conditions[END_REF], and to a larger extent of epipelon representatives [START_REF] Williams | Division rates of salt marsh diatoms in relation to salinity and cell size[END_REF][START_REF] Admiraal | Salinity tolerance of benthic estuarine diatoms as tested with a rapid polarographic measurement of photosynthesis[END_REF][START_REF] Admiraal | Distribution of diatom species on an estuarine mud flat and experimental analysis of the selective effect of stress[END_REF][START_REF] Clavero | Salinity tolerance of diatoms ftom Thalassic hypersaline environments[END_REF]. In response to light stress, epipelic diatoms use both vertical motility in the sediment and physiology [START_REF] Mouget | Migration or photoacclimation to prevent high irradiance and UV-B damage in marine microphytobenthic communities[END_REF][START_REF] Van Leeuwe | Photacclimation in microphytobenthos and the role of the xanthophylls pigments[END_REF]Perkins et al., 2010b;[START_REF] Cartaxana | Physiological versus behavioral photoprotection in intertidal epipelic and epipsammic benthic diatom communities[END_REF][START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: Role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF]Barnett et al., 2014). Although in our experiments motility was abolished, the photophysiological response of N. phyllepta confirms the likeliness of an equivalent balance between motility and physiology to respond to salinity stress. Surprisingly, N. phyllepta did not deploy a strong photophysiological response pointing out to other intra-cellular means that explain its relative insensitivity to high salinity (at least up to 45). For instance, they use proline to adjust their osmotic balance [START_REF] Natana Murugaraj | Effect of salinity stress on the marine diatom Amphora coffeaeformis (Ag.) Kuetz. (Bacillariophyceae) in relation to proline accumulation[END_REF]. Most importantly, cells surround themselves with exopolysaccharides (EPS) to minimize the negative impact of desiccation and high salinity [START_REF] Sauer | Effect of salinity, light and time on the vertical migration of diatom assemblages[END_REF] on motility: i) it was shown on a natural assemblage that a shift in salinity from 35 to 45 generated a -30 % migration of the cells at the surface of sediment [START_REF] Sauer | Effect of salinity, light and time on the vertical migration of diatom assemblages[END_REF]; ii) in controlled laboratory conditions, motility can be even abolished at a salinity of 50 [START_REF] Apoya-Horton | Movement modalities and responses to environmental changes of the mudflat diatom Cylindrotheca closterium (Bacillariophyceae)[END_REF]. This phenomenon is based on the decrease of the gliding speed of the cells [START_REF] Apoya-Horton | Movement modalities and responses to environmental changes of the mudflat diatom Cylindrotheca closterium (Bacillariophyceae)[END_REF] and its rapidity (5 s; [START_REF] Apoya-Horton | Movement modalities and responses to environmental changes of the mudflat diatom Cylindrotheca closterium (Bacillariophyceae)[END_REF]) might be related to intracellular calcium responses [START_REF] Falciatore | Perception of environmental signals by a marine diatom[END_REF][START_REF] Apoya-Horton | Movement modalities and responses to environmental changes of the mudflat diatom Cylindrotheca closterium (Bacillariophyceae)[END_REF].

Excretion of EPS during high salinity events allows cell attachment, a prerequisite for cell gliding [START_REF] Apoya-Horton | Movement modalities and responses to environmental changes of the mudflat diatom Cylindrotheca closterium (Bacillariophyceae)[END_REF] that, in field conditions, indeed supports the vertical cell migration to apparently escape extreme salinities. Therefore, the motility response of epipelic diatoms was speculated to be part of an adaptive strategy to respond the sometimes highly changing environment, including light and salinity, at the surface of cohesive sediment, [START_REF] Admiraal | The ecology of estuarine sediment inhabiting diatoms[END_REF].

Tychoplankton

Similar to epipelon, tychoplankton movement modalities, when it is buried in sediment at low tide [START_REF] Roncarati | Photosynthetic performance, oxidative damage and antioxidants in Cylindrotheca closterium in response to high irradiance, UVB irradiance and salinity[END_REF], are strongly influenced by salinity changes [START_REF] Apoya-Horton | Movement modalities and responses to environmental changes of the mudflat diatom Cylindrotheca closterium (Bacillariophyceae)[END_REF]. Additionally, high salinity drives the detachment of cells from their substratum, which could be a strategy to avoid longer exposure for this amphibious group [START_REF] Apoya-Horton | Movement modalities and responses to environmental changes of the mudflat diatom Cylindrotheca closterium (Bacillariophyceae)[END_REF]. Nevertheless, as reported before [START_REF] Roncarati | Photosynthetic performance, oxidative damage and antioxidants in Cylindrotheca closterium in response to high irradiance, UVB irradiance and salinity[END_REF] and here, the physiological response to salinity (combined or not with HL) of tychoplankton seems to be more complex than the one of epipelon. They appear highly sensitive to salinities > 35 [START_REF] Underwood | Determining the environmental preferences of four estuarine epipelic diatom taxa: growth across a range of salinity, nitrate and ammonium conditions[END_REF] including drastic growth limitation at salinities > 40 [START_REF] Rijstenbil | Effects of UVB radiation and salt stress on growth, pigments and antioxidative defence of the marine diatom Cylindrotheca closterium[END_REF][START_REF] Scholz | Growth responses of 25 benthic marine Wadden Sea diatoms isolated from the Solthörn tidal flat (southern North Sea) in relation to varying culture conditions[END_REF]. In our conditions, salinities from 35 on generated a strong photophysiological response in P. vanheurckii: its photochemical machinery acclimated just like high salinities would render it more light sensitive (see paragraph 4.2.). This general response was likely related to the linear lowering of NPQ with high salinities together with the anti-oxidative stress response (i.e. strong DT synthesis). It supports the obvious salinity (and light) sensitivity of P. vanheurckii. This is confirmed by previous studies on another tychoplankton representative (Cylindrotheca closterium) [START_REF] Rijstenbil | Effects of UVB radiation and salt stress on growth, pigments and antioxidative defence of the marine diatom Cylindrotheca closterium[END_REF][START_REF] Rijstenbil | UV-and salinity-induced oxidative effects in the marine diatom Cylindrotheca closterium during simulated emersion[END_REF][START_REF] Roncarati | Photosynthetic performance, oxidative damage and antioxidants in Cylindrotheca closterium in response to high irradiance, UVB irradiance and salinity[END_REF]. In response to the high salinity-and/or HL-dependent ROS generation, the intracellular pools and activity of important players of the oxidative stress response (i.e. the reduced glutathione-GSH, the superoxide dismutase-SOD enzyme) increased. Albeit such protective response, cells could not avoid significant lipid peroxidation [START_REF] Roncarati | Photosynthetic performance, oxidative damage and antioxidants in Cylindrotheca closterium in response to high irradiance, UVB irradiance and salinity[END_REF]. Peroxidation of lipids of the thylakoid membrane disturbs osmoregulation [START_REF] Rijstenbil | Effects of UVB radiation and salt stress on growth, pigments and antioxidative defence of the marine diatom Cylindrotheca closterium[END_REF][START_REF] Rijstenbil | UV-and salinity-induced oxidative effects in the marine diatom Cylindrotheca closterium during simulated emersion[END_REF] which might explain the synthesis of intracellular osmoregulators like free sugars (mannose, [START_REF] Paul | Osmoregulation in the marine diatom Cylindrotheca fusiformis[END_REF], amino acids (taurine, [START_REF] Jackson | The effect of salinity on growth and amino acid composition in the marine diatiom Nitzschia pungens[END_REF]and proline, Natana Murugaraj and Jeyachandran, 2007). Moreover, leakage of the thylakoid membrane can impair the build-up of the transthylakoid ΔpH (i.e. loss of membrane potential, [START_REF] Rijstenbil | UV-and salinity-induced oxidative effects in the marine diatom Cylindrotheca closterium during simulated emersion[END_REF], which would well explain the NPQ decrease with increasing salinities [START_REF] Lavaud | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF][START_REF] Lavaud | The peculiar features of non-photochemical fluorescence quenching in diatoms and brown algae[END_REF]. All together, these observations fit well with an adaptation of tychoplankton to salinities ~33 as it is mostly the case in the water column (when cells are resuspended at high tide) or buried in sediment (when cells settle down at low tide) as observed here from -0.5 cm down (see paragraph 4.1.).

Epipsammon

The response of epipsammon to salinity changes is much less documented. To our knowledge, only [START_REF] Scholz | Growth responses of 25 benthic marine Wadden Sea diatoms isolated from the Solthörn tidal flat (southern North Sea) in relation to varying culture conditions[END_REF] investigated the negative impact of salinity on the growth of episammic species like Achnantes spp. and Amphora spp.. Because it lives attached to sediment particles and the light penetration is deeper in (less cohesive) sandy sediment, the epipsammon photophysiological response to HL is efficient (Barnett et al., 2014). Here, under LL, B. lucens was relatively insensitive to high salinity. Nevertheless, and although DES and NPQ were high, the ability to decrease the excitation pressure on PSII during HL exposure was partially abolished by salinities > 37-41. As a consequence, rETR m decreased, thus potentially impairing the photosynthetic productivity. While B. lucens is well adapted to cope with HL and with high salinity, it appears less well adapted to the combination of the two. This fits well with the fact that in its natural habitat, even if the light climate can be extreme, changes in salinity remain moderate i) even in the first 0.5 cm of sediment, and ii) especially deeper where a significant part of the epipsammon biomass inhabits, as shown here (see paragraph 4.1.).

Conclusions

The photophysiology of representatives of the three main groups of intertidal MPB diatoms (i.e. epipelon, epipsammon and tychoplankton) differentially responded to a high salinity stress alone or combined with moderate HL exposure. While the representative of epipelon was relatively insensitive to these conditions, the tychoplankton representative was highly sensitive to both, and the epipsammon representative was sensitive mainly to the stress combination.

These specific responses fitted well with i) their natural habitat (i.e. more or less cohesive sediment) for which light climate and changes in salinity differ, ii) their growth form (i.e. motile, immotile or amphibious) which determines their probability to be confronted to a combined high salinity-HL stress, and their capacity to eventually escape from it (i.e. epipelon). Although light and temperature are regarded as major drivers of the photosynthetic productivity of MPB in Western Europe intertidal mudflats [START_REF] Kromkamp | Developments in microphytobenthos primary productivity studies[END_REF], salinity increase during emersion obviously can non-negligibly modulate the MPB photosynthesis when it is combined with HL (and temperature) according to the weather conditions and sediment type. It nevertheless appears mostly restricted to epipsammon and tychoplankton, and in field conditions, although likely stronger than in the present study, its effect probably remains negligible compared to HL stress.

P . v a n h e u r c k ii

S a lin ity

  Figure legends

Figure 2

 2 Figure 2

Figure 3

 3 Figure 3

F

  photochemistry vs. E

Figure

  Figure 1_Juneau et al.

Table 1_Juneau et al. Pore-water salinity measured during emersion in the upper sediment layer (first 1 cm) in two sites of the French Atlantic coast with two different sediment types and at different seasons.

 1_Juneau Values are averages  standard deviation (n = 9 to 12).

	Table 2_Juneau et al.
	Photophysiological parameters used in this study, their meaning and how they were measured. Abbreviations: Chl, chlorophyll;
	DD, diadinoxanthin; DT, diatoxanthin; E, light intensity; PSII, photosystem II; RLCs, Rapid Light Curves. See the Materials and
	Methods section for further details.
	Parameter Unit		Definition		Photophysiological meaning	Measurement conditions
		Sediment type	Season	Min	Max	Emersion Δ	Emersion Δ
	F 0		No units		Minimum PSII Chl	Used to calculate F v /F m	Measured with RLCs after 15
				period			max	mean
		Mud		Winter	29.0  1.2 fluorescence yield 34.1  1.1 (see below) 2.3	1.3  0.6	min of dark acclimation
	F m	(95.1  0.1 % mud/ No units	02/19-02/24	Maximum PSII Chl	Used to calculate F v /F m and NPQ	Measured with RLCs during a
		4.9  0.1% sand)	Spring	32.5  1.1 fluorescence yield 38.8  1.1 (see below) 5.1	4.4  1.0	saturating pulse after 15 min of
				04/19-04/22			dark acclimation
				Summer	35.8  0.2	48.2  0.7	8.3	4.6  2.7
	F v /F m	No units		Maximum	PSII
				07/13-07/26
					quantum yield;
		Muddy sand	Spring	30.8  1.0	35.4  2.8	3.2	1.9  1.2
		(57.9  7.9% sand/	04/05-07/05
		42.1  7.9% mud)	Fall	32.8  0.4	37.3  2.7	3.8	3.7  0.2
				09/30-10/02

Table 3_Juneau et al. Photochemical potential and non-photochemical fluorescence quenching in Navicula phyllepta (N.p.), Biremis lucens (B.l.) and Plagiogrammopsis vanheurckii (P.v.) exposed to different salinities.

 3_Juneau Abbreviations: LL, growth low light (60 µmol m -2 s -1 photons); HL, high light (600 µmol m -2 s -1 photons). Definitions and conditions of measurement of F v /F m , ΦPSII and NPQ m are listed in Table2. Values are averages  standard deviation (n = 3).

			 0.020  0.008  0.024  0.041  0.039  0.332
			0.703	0.560	0.357	0.627	0.530	0.782
			 0.010	 0.019	 0.103	 0.019	 0.038	 0.271
	P.v.		0.588	0.312	0.791	0.370	0.265	1.003
			 0.034	 0.024	 0.141	 0.032	 0.031	 0.048
			0.539	0.292	0.622	0.352	0.270	1.178
			 0.009	 0.095	 0.129	 0.028	 0.021	 0.103
			LL 0.555	0.291	0.625	HL 0.336	0.213	0.908
	Species Salinity F v /F m  0.021	ΦPSII NPQ m F v /F m  0.017  0.011  0.051	ΦPSII NPQ m  0.032  0.163
	N.p.	33	0.724 0.577	0.599 0.316	0.306 0.542	0.667 0.359	0.551 0.221	0.547 0.870
			 0.011  0.055	 0.030  0.041	 0.060  0.019	 0.028  0.018	 0.031  0.066	 0.053  0.143
		37	0.727	0.597	0.315	0.659	0.568	0.485
			 0.019	 0.031	 0.044	 0.007	 0.041	 0.070
		41	0.730	0.600	0.250	0.662	0.559	0.542
			 0.010	 0.068	 0.022	 0.006	 0.038	 0.017
		45	0.724	0.600	0.259	0.674	0.571	0.653
			 0.015	 0.051	 0.038	 0.009	 0.019	 0.016
	B.l.	33	0.694	0.563	0.323	0.629	0.504	0.588
			 0.010	 0.009	 0.097	 0.009	 0.003	 0.142
		37	0.694	0.554	0.315	0.602	0.498	0.864
			 0.012	 0.016	 0.071	 0.010	0.040	 0.186
		41	0.689	0.569	0.332	0.607	0.496	0.736

Table 4_Juneau et al. Pigments in Navicula phyllepta (N.p.), Biremis lucens (B.l.) and Plagiogrammopsis vanheurckii (P.v.) exposed to different salinities.

  Abbreviations: LL, growth low light (60 µmol m -2 s -1 photons); HL, high light (600 µmol m -2 s -1 photons). Chl a, chlorophyll a; Chl c, chlorophyll c; Fx, fucoxanthin; DD, diadinoxanthin; DT, diatoxanthin; ß-car, ß-carotene. Chl a is in pg. cell -1 ; other pigments are in mol. 100 mol Chl a -1 . Values are averages  standard deviation (n = 3).

	B.l.		1.830	27.1	76.8	7.1	19.5	1.7	1.642	25.7	72.5	2.2	14.5	8.6
			 0.280	 2.7	 4.5	 6.2	 0.2	 0.8	 0.201	 1.2	 4.4	 1.9	 2.1	 0.2
			1.661	24.3	70.0	2.8	17.7	1.2	1.626	24.4	69.2	3.5	13.8	9.7
			 0.105	 3.2	 6.6	 1.4	 0.9	 1.0	 0.186	 2.9	 8.9	 0.5	 2.1	 0.8
			1.883	29.0	87.3	3.6	23.4	1.1	1.554	22.8	63.9	3.5	12.8	8.9
			 0.210	 4.6	 7.3	 0.5	 0.8	 0.9	 0.088	 0.9	 3.5	 0.1	 2.2	 1.1
			LL 1.749	26.6	76.7	4.1	18.9	1.1	HL 1.731	25.5	72.7	3.7	15.7	8.8
	Species Salinity Chl a  0.220	Chl c  2.8	Fx  7.9	ß-car  1.2	DD  0.3	DT  1.3	Chl a  0.157	Chl c  0.9	Fx  2.7	ß-car  0.6	DD  1.7	DT  0.1
	N.p. P.v.	33	0.830 2.010	33.2 29.6	151.0 97.8	8.6 1.9	33.7 13.5	3.5 7.3	0.716 2.070	37.1 30.8	134.2 97.9	13.0 1.9	30.8 9.9	9.1 13.4
			 0.020  0.410	 0.9  4.4	 0.9  12.2	 8.3  0.0	 2.3  2.2	 0.2  2.3	 0.019  0.419	 3.1  3.2	 3.5  3.9	 5.2  0.3	 1.0  0.9	 1.9  1.4
		37	0.821 2.366	42.2 30.9	157.0 103.5	8.3 2.1	34.1 14.5	3.1 7.8	0.621 1.679	35.9 27.1	131.8 90.9	13.9 2.2	29.1 8.8	9.9 11.4
			 0.065  0.441	 8.9  0.9	 11.4  6.4	 8.5  0.3	 1.1  0.0	 0.1  2.1	 0.043  0.503	 2.8  2.7	 1.7  9.5	 3.7  0.1	 2.9  1.6	 1.8  3.0
		41	0.812 2.034	42.7 25.6	152.1 86.2	14.0 1.4	33.8 11.8	3.0 6.2	0.652 1.586	34.8 26.8	127.8 89.7	12.2 1.9	29.6 8.5	9.4 11.9
			 0.016  0.488	 0.8  2.4	 5.8  4.7	 6.4  0.1	 5.6  0.4	 0.4  1.7	 0.053  0.508	 2.4  1.7	 1.7  2.3	 6.7  0.3	 1.6  0.4	 2.6  0.1
		45	0.793 2.095	40.9 26.8	150.1 88.3	14.1 3.4	33.2 11.0	3.0 8.6	0.607 1.456	33.2 23.9	127.5 78.4	11.3 2.2	28.8 7.4	11.8 10.7
			 0.040	 1.9	 6.1	 5.8	 5.0	 0.2	 0.048	 0.6	 1.4	 5.3	 0.2	 2.6

Table 5_Juneau et al. Results of the 2 factor ANOVA procedure for the comparison of the parameters measured in Navicula phyllepta (N.p.), Biremis lucens (B.l.) and Plagiogrammopsis vanheurckii (P.v.) exposed to different salinities and lights

  . Code: white + n.s. (non significant): p > 0.05; light grey + *: p < 0.05; medium grey + **: p < 0.01; dark grey + ***: p < 0.001; arrow up, increase of values; arrow down, decrease of values. The two factor ANOVA analysis was performed on data shown in Tables3 and 4, Fig.2 and 3.

	Salinity					Light					Light x Salinity
		N.p.	B.l.	P.v.			N.p.	B.l.	P.v.			N.p.	B.l.	P.v.
	F v /F m n.s.	n.s.	n.s.		F v /F m *** ↓ *** ↓ *** ↓	F v /F m n.s.	n.s.	n.s.
	FPSII n.s.	n.s.	n.s.		FPSII *	↓ *** ↓ *	↓	FPSII n.s.	n.s.	n.s.
		n.s.	n.s.	*	↓		n.s.	*** ↓ ** ↓		n.s.	n.s.	n.s.
	rETR m n.s.	*	↓ *** ↑	rETR m n.s.	n.s.	n.s.		rETR m n.s.	n.s.	n.s.
	E k	n.s.	*	↓ ** ↑	E k	n.s.	** ↑ *	↑	E k	n.s.	*	n.s.
	NPQ m n.s.	n.s.	*	↓	NPQ m *** ↑ *** ↑ *** ↑	NPQ m **	n.s.	n.s.
	DES	n.s.	n.s.	n.s.		DES	*** ↑ *** ↑ *** ↑	DES	n.s.	n.s.	n.s.
	DD	n.s.	n.s.	n.s.		DD	n.s.	*** ↓ *** ↓	DD	n.s.	n.s.	n.s.
	DT	n.s.	n.s.	n.s.		DT	*** ↑ ** ↑ *** ↑	DT	n.s.	n.s.	n.s.
	Chl a n.s.	n.s.	n.s.		Chl a *** ↓ n.s.	n.s.		Chl a n.s.	n.s.	n.s.
	Chl c n.s.	n.s.	n.s.		Chl c n.s.	n.s.	n.s.		Chl c n.s.	n.s.	n.s.
	Fx	n.s.	n.s.	n.s.		Fx	n.s.	n.s.	n.s.		Fx	n.s.	n.s.	n.s.
	ß-car n.s.	n.s.	n.s.		ß-car n.s.	n.s.	n.s.		ß-car n.s.	n.s.	n.s.

Acknowledgements

The authors acknowledge the Centre National de la Recherche Scientifique-CNRS (program 'chercheurs invités' 2011), the University of La Rochelle-ULR (ACI 'chercheurs invités' 2011), the Region Poitou-Charentes (program 'chercheurs invités' 2013), the Contrat Plant Etat Région-CPER Littoral (2007-13), and the Natural Science and Engineering Research Council of Canada-NSERC (grant #262210-2011) for their financial support.  0.388  5.1  11.1  2.1  4.7  0.5  0.371  5.1  12.8  0.5  1.0  1.3


Asma Mabrouk
email: asmamabrouk@gmail.com

Nathalie Lorrain

Mohamed Lazhar

Maherzi L Haji

Oueslati

M Oueslati

Correlation between optical properties surface morphology of porous silicon electrodeposited by Fe3+ ion

Keywords: Silicon, Nanocristallites, Radiative recombination, Potential fluctuations

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

In recent years, Porous silicon (PS) layer has been intensively investigated due to its strong photoluminescence (PL) properties in the visible range [1] at room temperature.

For its large surface area and high chemical reactivity, PS is also considered as a promising material for different applications such as photovoltaic conversion [2,[START_REF] Vitanov | Solar Energy Mater. Solar Cell Solar Energy[END_REF], silicon-insulator (SOI) [4,5] and sensor technologies [6,7]. However, the inefficiency and instability [8,9,10] of the optical properties of the PS create many problems in the application area such as the decrease of PL intensity [9,10] and the shift of the energy of maximum of PL band [9,10] due to the surface oxidation of PS crystallites. To solve this problem, new nano-composites lines have been developed by the insertion of atomic or molecular species such as hydrocarbons, polymers, semiconductors and transition metals [11][12][13][14][15]. Herino et al [START_REF] Herino | Impregnation of porous silicon in: Properties of Porous Silicon[END_REF] investigated the deposition of metals and other conducting materials in relation to their influence on the luminescent properties of PS and to their modification of its conductivity. The metal incorporation has been carried out by electrochemical or electroless methods and also by evaporation and revealed an increase of the electroluminescence after the deposition of In and Al into porous silicon. Galvanostatic and electroless deposition of Au, Ni and Cu has been used and it is accompanied by an oxidation of the porous silicon surface whereas cathodic deposition does not generate oxide formation at the pore surface [START_REF] Ronkel | [END_REF]. Silicon dots have been also obtained using liquid and vapor phase preparation methods, which the formation has been evidenced using SEM (Scanning Electron Microscopy) and AFM (Atomic Force Microscopy) [18,19].

Electro-deposition of Au, Pt and Pd into macro-pores has been studied [20,21]. It has shown that macro-porous silicon filling with Pd and Pt starts growing from the pore bottom.

On the other hand, for the deposition of Au, no condition could be found to achieve this behavior. The deposition is performed under cathodic conditions by choosing an appropriate supporting electrolyte which induces a shift of the open-circuit potential to negative values and inhibits oxidation of the silicon. Since the equilibrium potential of Au is more positive compared with Pt and Pd, the rate of hole injection is faster and this is the result of the greater difference between the equilibrium potential and the silicon valence band.

Even if the electro-deposition process is well described in the literature [START_REF] Herino | Impregnation of porous silicon in: Properties of Porous Silicon[END_REF][START_REF] Ronkel | [END_REF]20,21], the physical properties of the obtained compound depend on fixed parameters of the sample preparation including the chemical nature of the semiconductor, crystal orientation, doping type and density, electrolyte composition and applied current or voltage. Systematic investigations with different electro-deposition parameters are required in order to find new and better physical properties. Iron has been successfully used to stabilize PS layers and interesting results has been obtained [22]. Direct impregnation of SP in Fe(NO 3 ) 3 solution [22] has been used to showed an improvement and stabilization of PS photoluminescence (PL) after only three weeks. Rahmani et al. [22] demonstrated by EDX analysis that atomic percentage of iron has decreased from the top to the bottom of the layer indicating that iron deposition occurs mainly at the pore bottom. Rahmani et al attributed this stability of the PL to the passivation of the Si nano-crystallites by iron. Beside, a gradient of the metal content towards the pore tips has been observed by Ronkel et al. [START_REF] Ronkel | [END_REF]. Fe-deposition into the pores carried out to achieve electrical contact to porous silicon starts with the nucleation at the pore bottom because of the cathodic standard potential of Fe that is close to the one of silicon [START_REF] Ronkel | [END_REF].

In the present work, the filling of porous silicon matrices with iron nanostructures is carried out electrochemically in a second process step after the anodization of the silicon wafer. Here we report the influence of iron electro-deposition parameters including iron concentration and current density on optical and morphological properties of PS electropassivated by iron ions (PSF). The correlation of the optical and morphological properties of samples has been investigated using photoluminescence study.

Experimental details

An ohmic contact was first formed by coating the backside of the silicon wafer with aluminum (Al) and subsequently annealed at 300°C for 30 min. PS layer was created from boron-doped p-type Si(100) substrate by electrochemical etching in 2:1:1 of 40% hydrofluoric acid, ethanol and water mixture with a current density of 10mA/cm 2 for 6 minutes. After anodization process, the samples were rinsed in pure water and dried by argon gas. The electro-deposition of iron was carried out in an aqueous electrolyte containing FeCl 3 (0.3M) for15 min at different current densities and was followed by drying with argon gas to remove residual impurities. Five samples PSF3, PSF11, PSF22, PSF28 and PSF32 have been prepared

for the following electro-deposition current densities J c =3µA/cm 2 , J c =11µA/cm 2 , J c =22µA/cm 2 , J c = 28µA/cm 2 and J c = 32µA/cm 2 .

Structural and morphological qualities of both PS and PSF samples prepared for different current densities were investigated by scanning electron microscope (SEM) (JEOL JSM -5600

LV equipped with X-ray (EDX) spectrometer used for compositional investigation on the cross-section of the samples. The minimum iron concentration which can be detected is about 0.1 at%. The estimated error for each concentration variation was at most 0.3% taking into account the doping inhomogeneity of the sample. The scanned surface is about 1µm² with a resolution of 1µm.

PL measurements were carried out in a variable temperature (10-300 K) closed-cycle cryostat with Jobin-Yvon T64000 spectrometer. We used the 488 nm wavelength of spectra Physics 2017 argon laser and an incident laser power of 5 mW at the surface of the sample.

The Raman shift was determined with an accuracy of 0.25 cm -1 (1800 gr/mm grating mode).

Results and discussions

Characterization of porous silicon/iron composites by SEM and EDX

SEM images of the surface were taken before (PS as prepared sample) and after iron electro-deposition (PSF3 and PSF22 samples) in order to study the evolution of the surface morphology (Fig. 1). After Fe ion electro-deposition, regular cracks were formed leaving isolated regions or "platelets" of nearly uniform thickness. These cracks become more pronounced for relative high current densities J c (Fig. 1C) and disappear for very high J c (Fig. 1D) due to the increase of the corrosive effect of the electro-deposition.

The SEM images of the cross section of samples etched with different J c are shown in Fig. 2.

It seems that the cracks are formed at a region of high stress that we associate to lattice defects. The presence of pores favors the formation of cracks through the structure, leaving distinctive "islands" where the porous silicon network has collapsed laterally parallel to the Si substrate.

The platelets or islands are loosely bound to the substrate (see Fig . 2 B andC) and show slight curling around their edges for samples etched for J c  22µA/cm 2 . The degree of curling increases with increasing J c and becomes even higher for J c = 32µA/cm 2 (Fig. 2D), this effect induces an increase in diameters due to the total dissolution of Si nano-crystallites. At low current densities, pores unzip normally to the surface inducing lateral collapse of the platelets as seen in Fig. 2B. The increase of J c would induce both lateral and normal stresses which would be responsible for the sponge structure of samples. For high J c , the formation of curled platelets proves the corrosive behavior of electro-deposition.

Complementary studies using Energy dispersive X-ray analysis were carried out to estimate the iron concentration through the depth of the porous layer. In Fig. 3, we show that Fe atomic percentage for PSF3 sample decreases from the top to the bottom of the layer. This proves that iron is well incorporated into the PS layer but the amount is not homogeneously distributed in the porous layer. By an electro-deposition method, Iron is mainly deposited in the first micrometer of the porous layer from the surface, and not at the pore bottom as in the impregnation method.

Photoluminescence studies 3.2.1. Electro-deposition current density dependence of PL at T=300K

Fig. 4 shows the PL spectra of PS and PSF samples for different current density J c . We observed an enhancement of the PL intensity (I PL ) with increasing J c . The I PL intensity reached a maximum for J cM = 22μA/cm 2 . For J c ≤ J cM , we note a slight blue-shift of the PL band energy which we attributed to the increasing confinement in dots and wires that we attribute to the decrease of the nano-crystallite sizes [23,24]. As we have already discussed (See Fig. 1), the pores unzip normal to the surface inducing lateral collapse of the platelet and causing a change in the pore-morphology. So, a corrosive behavior of electro-deposition occurs for high J c , leading to a complete dissolution of silicon nano-crystallites and accompanied of a decrease of the I PL (Fig. 4).

PL and shape of crystallites

It has been proven in several studies [25,26] that the porous p-type layer is formed by cylindrical and spherical crystallites shapes. Thus, PS is described as a mixture of quantum dots (QD) and quantum wires (QW) of different concentrations and sizes. The theoretical PL spectra could be calculated by using a simple quantum confinement-based model [27,28] where the visible red photoluminescence (PL) is only produced in QWs and QDs. The total PL intensity (I PL ) of PS is the sum of contributions of wire and dot emission.

I PL = + (1)
Where and represent the intensity contributions of wires and dots in the PL spectra respectively and they are expressed by [27,28]:

= Δ . Χ exp [-( ) ( . [Δ ] . -1) ] (2)
= Δ . Χ exp [-( ) ( . [Δ ] . -1) ] (3)
k W and k D are suitable normalization constants. and are the concentrations of luminescent wires and dots, respectively, and their root mean square and. We assume a Gaussian distribution of the diameters and of the wire and the dots, centered on their mean values and , respectively. We have adopted the dependence of [ ( ) ] . [29] for the energy gap versus the crystallite diameter d W(D) . E W(D) are the confinement contributions of QWs and QDs on the energy gap of the PS.

From the fitting of theoretical PL spectra to experimental ones (See Fig. 5), we have determined all the optimized fitting parameters  W ,  D , d 0W, d 0D ,  W and  D characterizing the QWs and the QDs (See Table 1).

In Fig. 5, we present the variation of QD (QW) diameter d D (d W ) as function of electrodeposition current density J c . For low J c , we observe a decrease of the diameters d D and d W , illustrating an etching effect of Si atoms and a reducing of crystallite sizes which induces a blue shift of the PL band (See Fig. 4). However, for J c which is relatively high (28µA/cm²), d D and d W do not change. This behavior is accompanied by a corrosive behavior as it has been

shown by SEM images (See Fig. 1). As for the adjusting theoretical PL spectra, an increase of the PL intensity ratio [I PL (QW)/I PL (QD)] is first observed with electrodeposition process. This proved that the etching process reduced the sizes of both wires and dots and preferentially destroys the spheres. The reduction of the sizes induced a blue shift of the total PL band for low current density (etching mechanism) and then a decrease of PL intensity occurs for high J c due to corrosive mechanism as clearly illustrated in Fig. 4.

PL temperature dependence

In Fig. 7 . This S-shape behavior has already been observed by several authors [30][31][32][33] and has been attributed to localized exciton (LE) by potential fluctuations induced by random distribution of impurities, defects and alloy disorder in samples [34]. The S-shape disappears in the case of PSF22 sample where E PL (T) presents a temperature variation according to Bose-Einstein model [35] (See Fig. 7c). In Fig. 7(a and b), the first S-shape redshift between 10 and 100 K is attributed to thermal relaxation of LE to absolute potential minimum [30]. With an additional increase in the temperature, the excitons are thermally delocalized and transferred to the higher-energy states (blueshift) above the mobility edge (ME) in the band-tail until reaching the conduction-band edge. With a larger increase in temperature, the thermal energy prevents the exciton localization, and the PL band follows the variation of the energy gap according to Varshni law (second redshift). Thus, the S-shaped behavior of the PL band (E PL (T)) is the signature of the competition processes between the recombination of localized and delocalized excitons. We have analyzed the S-shape using the theoretical band-tail model [36], which is given by the following equation:

( ) = - - (4)
E g (T) plays the role of the optic gap depending on temperature T. E g0 is E g (T=0), α and β the Varshni coefficients [37,38], and K B the Boltzmann constant (K B = 8.6173324 10 -5 eV.k -1 ).

The third term comes from the localization effect in which σ indicates the degree of localization, i.e., the large value of σ means a strong localization effect. We present in Fig. 7.a (b) the fitting of experimental values E g (T) using the equation (4) for PSF3(PSF11).

The adjusting values of E g0 , α, β and are reported in table 3. Note that for T=300K, all the excitons are delocalized due to the thermal excitation. Thus, the energy gap is more important in the PSF11 (E gap =1.88eV) sample than in the PSF3 (E gap =1.78eV) sample.

As temperature rises, the PL maximum band energy [E PL (T)] which is shown in Fig. 7.c. shift to lower energy side and each intensity decreases with increasing temperature.

This behavior is caused by the decrease in the gap with the increase of the temperature. In fact there are two factors responsible for the variation of the energy gap with temperature.

First, because of thermal expansion the band structure and energy band gap decreases with increasing temperature. Second the effects of lattice vibrations and electron phonon interaction, which induce a link of the valence and conduction band and reflect the temperature dependence of the distribution of phonons. To describe this behavior there are several empirical laws (Varshni, Bose-Einstein, etc).

The most frequently used empirical relation for numerical fittings of Eg(T) was first suggested by Varshni [37,38] and given as follows :

= - (5)
Where is the transition energy at 0 K, and are the Varshni coefficients.

Vina et al [35] proposed an empirical relation, which can be expressed as:

= -/ (6)
Where the value of the energy gap at 0 K is , is an empirical constant and approximates the Debye temperatures [39].

The evolution of the peak energy of sample as a function of temperature simulated using the Bose-Einstein (Vina law) and the Varshni equation is presented in Fig. 7c. The adjustment parameters between 10K and 300K for the sample are listed in Table 3.

In our case, the Vina model is more accurate to the experiment data and reflects the behavior at low and high temperature revealing the domination of the electron-phonon interactions.

PL intensity study

In Fig. 8, we display the normalized integrated PL intensity for PSF3, PSFe11 and the PSF22 samples as a function of temperature. When the temperature increases, we observe a monotonic intensity decrease of the PSF22 sample while the PSF3 and PSF11 samples witnessed two intensity decrease regions. In the low temperature region, the increasing PL intensity indicates that more carriers were captured by the local potential fluctuations. At higher temperatures, the carrier thermal escape from the local potential fluctuations becomes dominant, leading to a decrease in the PL intensity. Therefore, the competition between these two processes will exhibit a maximum PL intensity at a certain temperature T M . It can be seen from Fig. 8 that these samples exhibit different maximum intensity temperatures. The confinement energies for PSF11 (E = 1.88eV) are typically shallower than for SPF3 samples (E=1.78eV), therefore, carriers in the PSF11 will thermally escape from the local potential fluctuations at lower temperatures, which will result in a lower T M (T M =140K) for the PSF11 sample.

At a low temperature of 10K, carriers in the PSF3 sample are randomly distributed among the potential minima (Fig. 8). As the temperature increases from 10 K up to T ≈ 120 K, weakly localized carriers are thermally activated and relax down into other strongly localized states and reach a saturated redistribution, resulting in the initial redshift of the peak energy and a decrease of PL intensity. In the 120-160 K interval, increasing temperature enable carriers to achieve the thermal equilibrium with the lattice and to occupy higher-energy levels of the localized states, resulting thus in the blueshift of the peak energy toward the free-exciton ground state up to T≈170 K and an increase of the IPL. With a higher increase in temperature, the thermal energy prevents the exciton localization and the line shape of the PL spectrum becomes a characteristic line shape of the delocalized exciton recombination resulting in a decrease of the PL intensity. In fact there are two kinds of carrier transfer processes:

i-tunneling transfer [40,41] which occurs when the carrier energy is lower than the barrier height. ii-classical activation transfer [40] due to thermal activation across the barrier which is negligible at the low temperatures. Due to the effect of the different carrier injection processes the emission intensity of samples could be changed. Increasing temperature could enhance the tunneling rate. However, the thermal dissociation of excitons increases with temperature promoting the escape of excitons from QD to QW.

The integrated PL intensity of samples as a function of reciprocal temperature is displayed in fig 9. On the basis of the excitons -tunneling and dissociation model, the temperature dependence of the PL intensity from the QD and QW can be expressed by the equation bellow [42]:

= + + . (7)
The first and second terms of equation represent the excitons tunneling and the thermal dissociation respectively. A, B, C and D are constants, E 1 is the LO-phonon energy of tunneling assistance, E 2 is an activation energy of thermal dissociation and K B is Boltzmann's constant. By fitting the experimental data for the PSF3 (PSF11) samples (fig9), we obtain A<0, E1= 15.8meV (E 1 = 12.83meV) and E 2 = 16.8meV (E 2 = 13.45meV).

It is easy to find that the contribution of the exciton tunneling to the emission from the PSF3 (PSF11) samples is negative, which implies that the exciton tunneling can reduce the emission.

By fitting the experimental data for PSF22, we obtain E 1 = 66meV and E 2 = 68meV. The value of E 1 is close to the LO-phonon energy of silicon barrier (63meV). However the value of activation energy is much larger than that of PSF3 and PSF11 and the binding energy of the exciton in the Si bulk (the exciton binding of silicon=15meV). Therefore, in a nanoparticle exciton cannot be broken up by thermal phonons, thus, the higher activation energy possibly associated to the exciton tunneling process [43].

Conclusions

In summary, we have studied PL of PSF at different temperatures and current densities. According to this study, we observed anomalous emission behavior due to the presence of strong potential fluctuations in the material, which could be at the origin of the Sshape of the PL band temperature dependence. Typical surface morphology is observed in PSF. The observed PL behavior was associated to the competition between localized exciton and free exciton recombination. The PL is explained using quantum confinement model which well described the typical observed morphology of PSF surface. The increase of electro-deposition current density promotes the transformation of spherical crystallites to cylindrical ones and induced an important fluctuating potential. Temperature dependent PL of SPF3 and SPF11 samples show the thermal quenching with increasing temperature. PL peak energy shows S-shaped behavior, i.e., a redshift-blueshift-redshift with increasing temperature. This S-shaped behavior of PL peak energy can be explained by localized exciton model. At low temperature, recombination occurs due to the localized states. At high temperature, a blueshift occurs due to carriers escape into the LE states. With further increase of temperature, the band gap evolution follows the Bose-Einstein model. The S-shaped temperature dependence was not observed for the rest of samples.

Figures and tables captions

Table 1:

Optimized fitting parameters giving the theoretical curves shown in Fig. 5.

Table 2:

Optimized fitting parameters giving the theoretical curves shown in figure 7. SEM of cross-section of PS, PSF3, PSF22 and PSF32 structures for J = 0µA/cm 2 (A), J = 3µA/cm 2 (B), J = 22µA/cm 2 (C) and J = 32µA/cm 2 (D) respectively.

Tables caption

Table 1: Optimized fitting parameters giving the theoretical curves shown in Fig. 5.

Table 2:

Optimized fitting parameters giving the theoretical curves shown in figure 7.

  , we present the evolution of the PL maximum band energy [E PL (T)] of samples PSF3, PSF11 and PSF22 as a function of temperature in the range [10 -300K] under a constant excitation power of 300 W/cm -2 . The E PL (T) of PSF3 and PSF11 show S-shape variation which corresponds to a red shift with increasing temperature in the range [10 -60 K], a blue shift in the range [60 -180 K] and then a red shift in the range [180 -300 K]

Figure 1 :

 1 Figure 1: SEM of PS as prepared (A), PSF3 [J=3µA/cm 2 ] (B), PSF22 [J= 22µA/cm 2 ] (C) and PSF32 [J= 32µA/cm 2 ] (D).

Figure 2 :

 2 Figure 2:SEM of cross-section of PS, PSF3, PSF22 and PSF32 structures for J = 0µA/cm 2 (A), J = 3µA/cm 2 (B), J = 22µA/cm 2 (C) and J = 32µA/cm 2 (D) respectively.

Figure 3 :

 3 Figure 3: Variation of Fe atomic percent as a function of the deep in the porous layer of SPF3.

Figure 4 :

 4 Figure 4:PL spectra of PS and PSF samples for different electro-deposition current.

Figure 5 :

 5 Figure 5:Theoretically calculated PL contributions of dots (QD) and wires (QW) in the experiment PL spectra (full curve) at T=300K for different electro-deposition current density J c : a-J c = 0 µA/cm 2 (PS), b-J c =22µA/cm 2 (PSF22), c-J c =32µA/cm 2 (PSF32).

Figure 6 :

 6 Figure 6: Adjusted diameters of dots (continuous line) and wires (dashed line) of samples etched at different electro-deposition current intensities.

Figure 7 :

 7 Figure 7:PL band energy (E PL ) as a function of temperature of samples PSF3, PSF11 and PSF22.The solid curve is calculated using the Gaussian band-tail model described in Ref[36] for samples of PSF3 and PSF11. Note that Bose-Einstein model is a very good agreement with experimental data for sample PSF22.

Figure 8 :

 8 Figure 8:The temperature dependence of the normalized integrated PL intensities for samples PSF3, PSF11 and PSF22.

Figure 9 :

 9 Figure 9: Integrated PL intensities as a function of the reciprocal temperature for PSF3, PSF11

Figure 1 :

 1 Figure 1

Figure 2 :

 2 Figure 2:

Figure 3 :

 3 Figure 3: Variation of Fe atomic percent as a function of the deep in the porous layer of SPF3.

Figure 4 :

 4 Figure 4:PL spectra of PS and PSF samples for different electro-deposition current.

Figure 5 :

 5 Figure 5:Theoretically calculated PL contributions of dots (QD) and wires (QW) in the experiment PL spectra (full curve) at T=300K for different electro-deposition current density J c : a-J c = 0 µA/cm 2 (PS), b-J c =22µA/cm 2 (PSF22), c-J c =32µA/cm 2 (PSF32).

Figure 6 :

 6 Figure 6: Adjusted diameters of dots (continuous line) and wires (dashed line) of samples etched at different electro-deposition current intensities.

Figure 7 :

 7 Figure 7:PL band energy (E PL ) as a function of temperature of samples PSF3, PSF11 and PSF22.The solid curve is calculated using the Gaussian band-tail model described in Ref[36] for samples of PSF3 and PSF11. Note that Bose-Einstein model is a very good agreement with experimental data for sample PSF22.

Figure 8 :

 8 Figure 8:The temperature dependence of the normalized integrated PL intensities for samples PSF3, PSF11 and PSF22.

Figure 9 :

 9 Figure 9: Integrated PL intensities as a function of the reciprocal temperature for

Table 3 :

 3 Adjustment

Parameter for Varshni relation and the Bose-Einstein statistics (Vina law).

Table 1

 1 Samples J c (µA/cm 2 )

			(%)	(%)	d (nm)	d (nm) I PL (QW)/ I PL (QD)
	PS	-	0.79	0.21	3.2	2.7	0.4
	SPF11	11	0.42	0.57	3.1	2.5		2.04
	SPF22	22	0.77	0.23	3.2	2.6	0.49
	SPF28	28	0.50	0.50	3.1	2.5	1.52
	SPF32	32	0.71	0.23	3.1	2.6	0.49

Table 3

 3

	Varshni law	Vina law
	Pic position : 1.8216 eV
	E g0 =1.8342 eV	E g0 =1.8288 eV
	=0.6 meV/K	λ=0.693 eV
	β=799 K	θ=799.8 K

Table 3 :

 3 Adjustment Parameter for Varshni relation and the Bose-Einstein statistics (Vina law).

Acknowledgements

We would like to acknowledge to Ahmed Souissi and Radhouane Chtourou from CNRTe -Borj Cedria for PL measurements at different temperatures. The authors would like to thank also Zeineb Dkhili for her correction of English language.