Vona Méléder

Martin Laviale

Bruno Jesus

Jean Luc Mouget

Johann Lavaud

Farzaneh Kazemipour

Patrick Launeau

Laurent Barillé

In vivo estimation of pigment composition and optical absorption cross-section by spectroradiometry in four aquatic photosynthetic micro-organisms

Keywords: microalgae, cyanobacteria, in vivo absorption bands, reflectance, pigments, absorption cross-section

The objective of the present study was to estimate in vivo pigment composition and to retrieve absorption cross-section values, a*, of photosynthetic micro-organisms using a non-invasive technique of reflectance spectrometry. To test the methodology, organisms from different taxonomical groups and different pigment composition were used (Spirulina platensis a Cyanophyta, Porphyridium cruentum a Rhodophyta, Dunaliella tertiolecta a Chlorophyta and Entomoneis paludosa a Bacillariophyta) and photoacclimated to two different irradiance levels: 25 µmol photon.m -2 .s -1 (Low Light, LL) and 500 µmol photon.m -2 .s -1 (High Light, HL). Second derivative spectra from reflectance were used to identify pigment in vivo absorption bands that were linked to specific pigments detected by High Performance Liquid Chromatography. Whereas some absorption bands such as those induced by Chlorophyll (Chl) a (416, 440, 625 and around 675 nm) were ubiquous, others were taxonomically specific (e.g. 636 nm for Chl c in E. paludosa) and/or photo-physiological dependent (e.g. 489 nm for zeaxanthin in the HL-acclimated S. platensis). The optical absorption cross-section, a*, was retrieved from reflectance data using a radiative transfer model previously developed for microphytobenthos. Despite the cellular Chl a decrease observed from LL to HL (up to 88 % for S. platensis), the a* increased, except for P. cruentum. This was attributed to a 'package effect' and to a greater absorption by photoprotective carotenoids that did not contribute to the energy transfer to the core Chl a.

Introduction

Marine coastal areas, particularly bays and estuaries, are amongst the most productive ecosystems on Earth (e.g. [START_REF] Kromkamp | Development in microphytobenthos primary productivity studies[END_REF]). Polyphyletic photosynthetic microbial communities (micro-algae, phytoplankton and microphytobenthos, and cyanobacteria) support this high coastal productivity. Their ecological success and adaptability to light variations in such ecosystems are partially explained by the wide plasticity of their photosynthetic apparatus constituted by a variety of light-harvesting proteins and pigments. This variability reflects the large range of photoecological responses of these micro-organisms due to different photoadaptation characteristics and photoacclimation processes [START_REF] Falkowski | Aquatic Photosynthesis -Second Edition[END_REF]. An important consequence is a variability in primary production rates, depending of species and their respective ecophysiology.

Estimation of in vivo cell absorption properties in the photosynthetic active radiation (PAR) domain (400 to 700 nm) provides important taxonomic and ecophysiological information. Namely, pigment composition allows to assess the community structure and physiology, whereas the optical absorption cross-section, i.e. a*,provides information about the potential primary production [START_REF] Johnsen | In vivo bio-optical properties of phytoplankton pigments[END_REF]. Frequently, this information is retrieved from absorption signatures measured either in vitro (i.e in solvent-extracted pigments) or in vivo (i.e. in intact cells) [START_REF] Johnsen | In vivo bio-optical properties of phytoplankton pigments[END_REF]. Pigment absorption properties are dependent on their molecular environment thus the in vitro estimations are subject to important constraints [START_REF] Bidigare | In vivo absorption properties of algal pigments[END_REF][START_REF] Hoepffner | Effect of Pigment Composition on Absorption Properties of Phytoplankton[END_REF]. Firstly, pigment absorption maxima usually shifts from in vitro measurements in comparison to in vivo measurements. This shift can vary from a few to hundreds of nanometers, depending on the pigment, the association with protein complexes (i.e. Light Harvesting Complexes, LHC) and the pigment location inside the cell [START_REF] Bidigare | In vivo absorption properties of algal pigments[END_REF][START_REF] Hoepffner | Effect of Pigment Composition on Absorption Properties of Phytoplankton[END_REF][START_REF] Barrett | The P700-chlorophyll a-protein complex and two major lightharvesting complexes of Acrocarpia paniculata and other brown seaweeds[END_REF][START_REF] Méléder | Spectrometric constraint in analysis of benthic diatom biomass using monospecific cultures[END_REF][START_REF] Porra | Metabolism and function of photosynthetic pigments[END_REF]. Consequently, the assignment of each absorption band to its respective pigment is very difficult to achieve [START_REF] Bidigare | In vivo absorption properties of algal pigments[END_REF]. Secondly, in vivo absorption is always lower than in vitro leading to a misestimation of a* [START_REF] Falkowski | Aquatic Photosynthesis -Second Edition[END_REF]. To overcome these constraints, in vivo measurements of absorbance signatures can be performed by spectrophotometry, either on cell suspensions or on thylakoids and isolated LHCs [START_REF] Ruban | Xanthophylls of the major photosynthetic light-harvesting complex of plants: identification, conformation and dynamics[END_REF][START_REF] Ruban | The superexcess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF]. Spectrophotometry assumes that the sample is homogenous, and that cell concentration (or thylakoids or LHCs) is high enough to detect absorption, but low enough to avoid self-shading. Furthermore, the absorption measurements can be affected by light scattering caused by cells in suspension [START_REF] Johnsen | In vivo bio-optical properties of phytoplankton pigments[END_REF]. Some improvements have been proposed, including the use of an integrative sphere or placing the sample (cells on wet glass-fiber filters) close to the detector [START_REF] Hoepffner | Effect of Pigment Composition on Absorption Properties of Phytoplankton[END_REF][START_REF] Bricaud | Optical efficiency factors of some phytoplankters[END_REF][START_REF] Mitchell | Chlorophyll a specific absorption and fluorescence excitation spectra for light-limited phytoplankton[END_REF][START_REF] Johnsen | Biooptical characteristics of PSII and PSI in 33 species (13 pigment groups) of marine phytoplankton, and the relevance for pulse-amplitude-modulated and fast-repetition-rate fluorometry1[END_REF]. However, these methods are constrained by the need for β-correction algorithms to remove the path length amplification effect due to the filter [START_REF] Mitchell | Chlorophyll a specific absorption and fluorescence excitation spectra for light-limited phytoplankton[END_REF]. Therefore, there is a growing interest in using non-destructive techniques to investigate micro-algal absorption properties while simultaneously limiting the scattering effect. Recently, reflectance spectroradiometric techniques have been used to assess micro-algal pigment composition [START_REF] Méléder | Spectrometric constraint in analysis of benthic diatom biomass using monospecific cultures[END_REF][START_REF] Kazemipour | Optical properties of microphytobenthic biofilms (MPBOM): Biomass retrieval implication[END_REF][START_REF] Johnsen | Optical monitoring of phytoplankton bloom pigment signautres[END_REF][START_REF] Jesus | Detection of diatom xanthophyll cycle using spectral reflectance[END_REF][START_REF] Richardson | The detection of algal photosynthetic accessory pigments using Airborne Visible-Infrared Imaging Spectrometer (AVIRIS) Spectral Data[END_REF]. Reflectance spectra can be studied using derivative analysis, a technique which is particularly useful in removing the scattering effect from absorption spectra and in resolving overlapping spectral features [START_REF] Gulyayev | First and second derivatives of the absorption spectrum of chlorophyll and associated pigments in cells of higher plants and algae at 20°C[END_REF]. Derivative analysis have been used to detect absorption bands due to electronic transitions [START_REF] Ruban | Xanthophylls of the major photosynthetic light-harvesting complex of plants: identification, conformation and dynamics[END_REF] and to assess the light-dependent (de-)epoxidation of xanthophylls [START_REF] Ruban | The superexcess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF][START_REF] Jesus | Detection of diatom xanthophyll cycle using spectral reflectance[END_REF]. Despite the great potential of this technique, few studies have used it to assess the pigment content of micro-algae and to investigate all pigment specific absorption bands [START_REF] Jesus | Detection of diatom xanthophyll cycle using spectral reflectance[END_REF][START_REF] Jesus | Spectral-radiometric analysis of taxonomically mixed microphytobenthic biofilms[END_REF][START_REF] Murphy | Estimation of surface chl a on an emersed mudflat using field spectrometry: accuracy of ratios and derivtive-based approaches[END_REF][START_REF] Murphy | Field spectrometry: New methods to investigate epilithic micro-algae on rocky shores[END_REF]. Recently, Kazemipour et al. [START_REF] Kazemipour | Optical properties of microphytobenthic biofilms (MPBOM): Biomass retrieval implication[END_REF] developed a radiative transfer model (MPBOM, MicroPhytoBenthos Optical Model) which allows the determination of microphytobenthos biofilm absorption coefficients using reflectance measurements without destroying the biofilm. This dimensionless coefficient corresponds to the optical cross-section, a*, when it is divided by the chlorophyll (Chl) a concentration (m 2 .mg Chla -1 ). Initially developed for benthic microalgae, this model can be applied to any other photosynthetic microorganisms.

The objective of the present study was to use reflectance data to estimate in vivo pigment composition and determination of a* values from different photosynthetic microbial species. Pigment composition was estimated from spectral reflectance using second derivative analysis and a* was calculated using the MPBOM radiative transfer model. To validate the methods, a variety of microbial species were investigated: one Cyanophyta, one Rhodophyta, one Chlorophyta and one Bacillariophyta (diatom). Intraspecific variability was also explored by cultivating all species at two different irradiance levels expected to induce photoacclimation pigment changes at the cell level.

Materials and methods

Biological material

Three species of eukaryotic micro-algae and one of cyanobacteria were chosen for their different pigment signatures: one 'green' species, Dunaliella tertiolecta E. paludosa, P. cruentum and D.tertiolecta species were grown in f/2 medium [START_REF] Guillard | Culture of phytoplankton for feeding marine invertebrates[END_REF]. S.

platensis was grown in Zarrouk medium [START_REF] Zarrouk | Contribution à l´étude d´une cyanophycée: influence de divers facteurs physiques et chimiques sur la croissance et la photosynthèse de Spirulina maxima (Setch. et Gardner) Geitler[END_REF]. Three replicates of each species were grown in a temperature-controlled culture room (14 ± 1°C, 14/10 h light/dark cycle) in 500-mL glass flasks containing 250 mL of medium at two PAR levels: 25 µmol photon.m -2 .s -1 (Low Light, LL) and 500 µmol photon.m -2 .s -1 (High Light, HL). Each culture was acclimated during 10 days, and regular dilution of the batch culture with fresh medium allowed to keep cells into exponential stage and minimize self-shading.

A thin compact layer of micro-algal cells, hereafter named 'experimental biofilm', was deposited on appropriate filters according to Kazemipour et al. [START_REF] Kazemipour | Optical properties of microphytobenthic biofilms (MPBOM): Biomass retrieval implication[END_REF]. For each culture, 5 mL were gently filtered (<1 bar) through glass microfiber filters (GF/C, 25 mm Ø, 1.2 µm porosity). This volume and the corresponding cell concentrations were sufficient to form a thin and homogenous compact layer of cells covering the filter surface (~ 1.8 cm 2 ) while reducing the distance between cells and limiting light scattering [START_REF] Kazemipour | Optical properties of microphytobenthic biofilms (MPBOM): Biomass retrieval implication[END_REF]. To estimate the amount of cells on filters, each culture was sampled.

Before any reflectance measurement was carried out, the experimental biofilms were placed 10 min under the corresponding PAR levels (i.e. HL or LL) to minimize rapid changes in pigment composition due to stress. For each PAR level, two filters were used for each culture run in triplicate (n=6). Biofilm desiccation during measurements was minimized by maintaining the filters on absorbent paper soaked with culture medium. Reflectance spectra were standardized with reference filters obtained by filtering 5 mL of the corresponding medium alone.

Radiometric measurements and interpretations

Radiometric measurements were performed using an ASD Fieldspec3 FR ® portable spectroradiometer that measures radiance (mW.cm -2 .nm -2 .sr -1 ) in the visible-infrared region of the light spectrum (between 400 and 2500 nm), with a 3 nm spectral resolution and a 1.4 nm sampling interval in the 400-1000 nm wavelength range. The light source was provided by an internal halogen lamp and the distance between the biofilm and the ASD optical fiber was kept constant by using the ASD High Intensity Contact Probe. This procedure minimizes errors associated with stray light.

Reflectance (ρ, dimentionless) was calculated as the ratio between the radiance of the cells on filters and the incident radiance measured on a perfect diffuser (Spectralon® 99%). Ten spectra were recorded and averaged for each filter.

Reflectance was standardized (ρ std ) to facilitate comparisons between the spectral signatures of cells acclimated to different light conditions following Eq. ( 1):

ρ ρ ρ ρ 925 1 × = F std (1)
whereρ std is the standardized reflectance, ρ is the reflectance of the experimental biofilm, ρ F , is the reflectance of a wet filter with no biofilm, and ρ 925 is the reflectance of the experimental biofilm at 925 nm which is insensitive to the pigment content of cells [START_REF] Méléder | Spectrometric constraint in analysis of benthic diatom biomass using monospecific cultures[END_REF][START_REF] Kazemipour | Optical properties of microphytobenthic biofilms (MPBOM): Biomass retrieval implication[END_REF].

Second derivative spectra (δδ) were calculated following Jesus et al. [START_REF] Jesus | Detection of diatom xanthophyll cycle using spectral reflectance[END_REF][START_REF] Jesus | Spectral-radiometric analysis of taxonomically mixed microphytobenthic biofilms[END_REF] and second derivative peaks were used to assign pigments to absorption wavelengths.

To facilitate the comparison between species and light-acclimation conditions, the second derivative values were standardized (δδ std ), following Eq. ( 2), to the maximum value between 650 and 690 (δδ red ), which is characteristic of Chl a red absorption:

red std δδ δδ δδ = (2)

Pigment analysis by High Performance Liquid Chromatography (HPLC)

Following radiometric measurements, the filters were immediately frozen in liquid nitrogen, and stored at -80°C until pigment extraction and analysis. Pigment analysis was performed on 3 filters for each culture and each PAR level (n=3) with a Waters® HPLC device as described in Méléder et al. [START_REF] Méléder | Spectrometric constraint in analysis of benthic diatom biomass using monospecific cultures[END_REF]. Pigments were extracted after thawing in 90% acetone, ground and left for 24 h in the dark at +4°Cas recommended by Mantoura and Llewellyn [START_REF] Mantoura | The rapid determination of algal chlorophyll and carotenoid pigments and their breakdown products in natural waters by reverse-phase highperformance liquid chromatography[END_REF]. Acetonic extracts were filtered (porosity: 0.45 µm) and diluted volume-to-volume in 1 M ammonium acetate. The HPLC protocol followed Mantoura and Llewellyn [START_REF] Mantoura | The rapid determination of algal chlorophyll and carotenoid pigments and their breakdown products in natural waters by reverse-phase highperformance liquid chromatography[END_REF] adapted by Méléder et al. [START_REF] Méléder | Spectrometric constraint in analysis of benthic diatom biomass using monospecific cultures[END_REF].

Sample volumes of 100 µL were injected and pigments were detected by photodiode array and characterized by their absorption spectra between 400 and 800 nm and their retention time [START_REF] Brotas | The use of HPLC pigment analysis to study microphytobenthos communities[END_REF][START_REF] Jeffrey | Application of pigment method to oceanography[END_REF]. Quantification was carried out at 440 nm using previously established pigment calibration with purified standards following Brotas and Plante-Cuny [START_REF] Brotas | Identification et quantification des pigments chlorophylliens et caroténoïdes des sédiments marins : un protocole d'analyse par HPLC[END_REF]. For some pigments, the contents of their derivatives were summed: Chl a and its allomers = Chl a; chlorophyll c 1 and c 1 + c 2 = Chl c; fucoxanthin and fucoxanthin by-products = Fuco. Data were presented as cellular pigment contents (µg.cell -1 ), weight to weight pigment ratios (g.g -1 ) and pigment content per unit area (mg.m -2 ).

Absorption cross-section estimation

The MPBOM quantitative radiative transfer model was used to estimate a* using the programming language IDL (Interactive Data Language) on ENVI 4.3 software. This model requires the input of biofilm reflectance spectra and the background reflectance spectra (i.e. wet filters without cells). One of the model outputs model is the absorption coefficient (α). This is related to the weighted concentration (C i ) and to the specific extinction coefficient (ε i ) at any wavelength of the m pigments present in the cells (Eq. 3):

Ci m i i ∑ = ε α (3)
α is dimensionless but when divided by the amount of Chl a (in mg.m -2 ) it corresponds to the absorption cross-section, a* (m 2 .mg Chla -1 ). * a corresponds to the averaged optical cross-section for the PAR domain, whereas red * a corresponds to the average for the domain of the red absorption of Chl a between 670 and 685 nm.

For comparison between light treatments, * a ∆ was calculated for each species. This measures the variation in absorption properties between LL and HL conditions and is associated with differences in Chl a (∆Chl a) cellular content.

All statistical analyses and graphical results were carried out in R statistical computing environment (2.6.1 [START_REF] Ihaka | R: a language for data analysis and graphics[END_REF]). Data normality was tested with the Shapiro-Wilk test while the effects of light acclimation (HL vs. LL) were tested using Student's ttest.

Results

Pigment analysis by HPLC

Pigment composition

In total, 14 liposoluble pigments (3 chlorophylls and 11 carotenoids) were detected and identified in this study (Table 1). D. tertiolecta showed the most diverse pigment composition (8 pigments), followed by E. paludosa and S. platensis (6 pigments) while only 3 pigments were detected in P. cruentum. Chl a and β-carotene (β-car)

were detected in all organisms, while other pigments were species-specific: The cellular Chl a content for each species significantly decreased (t-test, p < 0.05) from LL to HL conditions, illustrating the photoacclimation effect (Table 2).

Considering the other detected pigments, most of their cellular content followed the same trend with asignificant decrease (t-test, p < 0.05) from LL to HL condition, with the exception of DT in E. paludosa, VX in S. platensis and ZX in P. cruentum which significantly increased (p < 0.05), AX in D. tertiolecta and DD in E. paludosa which remained stable (p > 0,05) (results not shown).The resulting pigment/Chl a ratios for each species at each irradiance are presented in Table 3. Notably, only Fuco:Chl a and Chl c:Chl a ratios (in E. paludosa) were not affected by the growth irradiance.

Pigment content on filters

The amount of Chl a collected on the different filters varied from 0.56 mg.m -2 ± 0.09 on S. platensis HL-filters to 18.98 mg.m -2 ± 1.26 on E. paludosa LL-filters (Figure 1).

The amount of cells obtained by filtration on LL-acclimated filters was lower or similar to HL filters (Table 2), thus the significant increase of pigment contents on LL-filters could only be the result of the increase in pigment cellular contents as a consequence of the light treatments. Only for D. tertiolecta and P. cruentum few pigments remained stable on filters (t-test, p = 0.5, figure 1).

For D. tertiolecta filters, Chl b, VX, and β-car followed the same trend as Chl a, and the opposite of AX (Figure 1a). Moreover, Lut, ZX and NX contents were comparable in HL and LL filters (p = 0.48, 0.23 or 0.25, respectively). In E. paludosa, the content of Fuco, Chlc and β-car was higher in LL-filters, while it was similar for DD (p = 0.6) and significantly higher in HL-filters for DT (Figure 1b). S. platensis showed the largest Chl a decrease (up to 5.5 times) from LL to HL-filters and all the other pigments followed the same trend (p ≤ 0.05) (Figure 1c). Finally, pigment contents of HL and LL-filters of P. cruentum were not significantly different (p > 0.1).

Radiometric analysis

Standardized reflectance spectra showed a similar shape whatever the species and the growth light conditions (Figure 2). A large absorption band in the blue domain, from 400 to 550/600 nm, was clearly visible followed by a transmittance peak and a second absorption band in the red domain around 675 nm. The transmittance peak varied in shape and was notched by smaller absorption bands: three in D.tertiolecta 2a), two in E. paludosa (Figure 2b), and one in S. platensis and P. cruentum 2c andd). Overall, reflectance values were lower in LL-acclimated cells indicating a higher absorbance. Almost no marked differences between species or light acclimation conditions could be inferred from the reflectance spectra, with the exception of the more pronounced 480 nm absorption band in LL-acclimated D.tertiolecta cells, and the specific 636 nm absorption band in E. paludosa (Figure 2).

(Figure
(Figures
The analysis of second derivative spectra improved the comparison between samples because each absorption peak could be assigned to an absorption band (Figure 3). Some of the second derivative peaks were detected in all species, whatever the growth irradiance, while others were species-and growth irradiance-specific.

Ubiquitous peaks were (Figure 3 detected in the red domain (~ 675 nm) in all species but E. paludosa (Figure 3). This double peak appeared in both photoacclimation conditions and was separated by a dip centered at 675 nm. The first peak (671 nm) was always smaller than the second one (683 nm) (Figure 3). Specific peaks (Figure 3, upward arrows) were: 1) ~ 489 nm in D.tertiolecta cells, which was smaller in LL-acclimated cells (Figure 3a) and was also present in HL-acclimated S. platensis cells (Figure 3c) and P. cruentum cells whatever the photoacclimation conditions (Figure 3d); 2) ~ 650 nm in D.tertiolecta cells (Figure 3a) and in HL-acclimated S. platensis cells (Figure 3c); 3) ~ 497 nm in LL-acclimated cells of E. paludosa (Figure 3b); 4) ~ 636 nm in E. paludosa cells (LL and HL) (Figure 3b); 5) ~ 524 nm in S. platensis (Figure 3c) and 6) ~570 nm in P. cruentum (LL and HL), which was smaller in LL-acclimated cells (Figure 3d).

Light absorption properties

Discussion

In many ecophysiological studies dealing with primary production and photosynthetic organisms with different pigment contents and photoacclimation strategies, the determination of the quantity of light absorbed and used to drive photosynthesis is a real challenge to estimate the photosynthetic efficiency, especially for field studies.

The method herein described offers a simple way to determine a key factor in photobiology and photosynthetic studies, the optical absorption cross-section, a* [START_REF] Falkowski | Aquatic Photosynthesis -Second Edition[END_REF], by combining HPLC and reflectance data.

Advantages of using reflectance and second derivative spectra to assess the absorption properties of photosynthetic micro-organisms

In vivo measurement of the absorption properties of photosynthetic micro-organisms using spectroradiometry on cells settled as a biofilm is an efficient alternative to more classic approaches using spectrophotometry which suffer from light scattering and/or the need forβ-correction [START_REF] Johnsen | In vivo bio-optical properties of phytoplankton pigments[END_REF][START_REF] Hoepffner | Effect of Pigment Composition on Absorption Properties of Phytoplankton[END_REF][START_REF] Bricaud | Optical efficiency factors of some phytoplankters[END_REF][START_REF] Mitchell | Chlorophyll a specific absorption and fluorescence excitation spectra for light-limited phytoplankton[END_REF][START_REF] Johnsen | Biooptical characteristics of PSII and PSI in 33 species (13 pigment groups) of marine phytoplankton, and the relevance for pulse-amplitude-modulated and fast-repetition-rate fluorometry1[END_REF]. Kazemipour et al. [START_REF] Kazemipour | Optical properties of microphytobenthic biofilms (MPBOM): Biomass retrieval implication[END_REF] showed that pigment reflectance mirrors well its absorbance when measurements are performed on a thin homogenous compact layer of micro-algal cells limiting distance between cells deposited on filters [START_REF] Kazemipour | Optical properties of microphytobenthic biofilms (MPBOM): Biomass retrieval implication[END_REF], but not thick enough to avoid saturation of reflectance measurements [START_REF] Méléder | Spectrometric constraint in analysis of benthic diatom biomass using monospecific cultures[END_REF]. The biofilms used in the current study fitted these requirements because if there had been any scattering between cells, the model MPBOM would have not fitted the data. Additionally, the biomass was low enough to limit selfshading because in our experimental biofilms this is only observed at biomasses higher than 100 mg.Chl a.m -2 [START_REF] Méléder | Spectrometric constraint in analysis of benthic diatom biomass using monospecific cultures[END_REF].

Since light scattering and inherent reflectance are minimized, the reflectance measured this way corresponds to a double transmittance through the layer of cells.

The incident light (i.e. PAR) is depleted by the absorbing fraction during the first transmission, reflected by the background, then transmitted again with a second depletion (see [START_REF] Kazemipour | Optical properties of microphytobenthic biofilms (MPBOM): Biomass retrieval implication[END_REF] for details). When the background reflectance is removed by standardization, the remaining reflectance signal contains only the cell absorption information.

To date, reflectance or absorbance spectra have been used to identify only a few specific pigment absorption features (e.g. the 650 nm band characteristic of the Chlorophyta or the 636 nm band of the Bacillariophyta). One of the major difficulties of assessing the in vivo pigment absorption properties is the possibility of existing multiple absorption bands for each pigment. Several factors may influence the pigment absorption properties in cells, e.g. the light scattering by the algal cell (or any particle), the optical properties of each pigment, the spatial arrangement of pigments in LHCs and the spatial arrangement of cells within a sample [START_REF] Johnsen | In vivo bio-optical properties of phytoplankton pigments[END_REF]. All these factors can affect each other. Derivation of reflectance spectra helps to remove unwanted signals and improve the separation of overlapping pigment absorption features [START_REF] Demetriades-Shah | High resolution derivative spectra in remote sensing[END_REF]. It can thus facilitate the identification of micro-organism absorption bands and their corresponding pigments.

In this study, the HPLC analysis of the 4 species revealed pigment signatures which are in agreement with current literature, for example regarding the clear effect of light observed on pigment ratios ( [START_REF] Laviale | Relationships between pigment ratios and growth irradiance in 11 marine phytoplankton species[END_REF] and references therein). Overall, most of the pigments detected by HPLC could be linked to specific derivative peaks. However, when a pigment/Chl a ratio was lower than 0.1, the pigment absorption was not strong enough to generate any derivative peak. This is why, for instance, VX absorption showed no second derivative peaks, although it is well-known to absorb at 510 nm in vivo [START_REF] Van Amerongen | Light-Harvesting in Photosystem II[END_REF].

Identification of chlorophyll absorption features

Second derivative reflectance peaks detected in each species and light condition were assigned to specific pigment absorption features on the basis of in vitro absorption bands and by the pigment changes induced by light acclimation (Table 1).

The peaks most easily identified were those due to the ubiquitous pigment Chl a. In organic solvents, Chl a absorption bands are commonly detectable at 417, 431, 618 and 665 nm [START_REF] Roy | Phytoplankton Pigments -Characterization, Chemotaxonomy and Applications in Oceanography[END_REF] and could be easily linked to the in vivo bands detected in all species at 416, 440, and 625 nm and to the often-observed second derivative double peak in the red domain (671 and 683 nm). This observation is similar to what was observed by Hoepffner and Sathyendranath [START_REF] Hoepffner | Effect of Pigment Composition on Absorption Properties of Phytoplankton[END_REF] using in vivo phytoplankton absorption spectra decomposed by a Gaussian function. These bands correspond to the major absorption bands according to the four-orbital model of Gouterman [START_REF] Gouterman | Spectra of porphyrins[END_REF], i.e. the blue absorption bands (B-bands or Soret bands) and the red absorption bands (Q-bands) [START_REF] Falkowski | Aquatic Photosynthesis -Second Edition[END_REF][START_REF] Parson | Optical Spectroscopy in Photosynthetic Antennas[END_REF]. The first two bands (416 and 440 nm) correspond to the Soret bands (B y and B x , respectively) whereas the last two bands (625 and around 675 nm) correspond to the Q-bands (Q x and Q y, respectively). The Q y Chl a is responsible for all photochemical reactions due to its de-excitation to the ground state [START_REF] Falkowski | Aquatic Photosynthesis -Second Edition[END_REF]. It is also in the Q y transition band that a double peak was always observed at 671 and 683 nm, except in the diatom E. paludosa. However, this double absorption band was described by Jesus et al [START_REF] Jesus | Spectral-radiometric analysis of taxonomically mixed microphytobenthic biofilms[END_REF] in the same species, and by Serôdio et al [START_REF] Serôdio | Effects of chlorophyll fluorescence on the estimation of microphytobenthos biomass using spectral reflectance indices[END_REF] in natural biofilms. The double Q y absorption peak is rarely mentioned in the literature and its origin remains unclear. Gulyayev and Litvin [START_REF] Gulyayev | First and second derivatives of the absorption spectrum of chlorophyll and associated pigments in cells of higher plants and algae at 20°C[END_REF] were the first to report it at the same wavelengths as in the present study, using a variety of photosynthetic organisms, but they did not explain its origin. The same is true for more recent reports (e.g [START_REF] Falkowski | Aquatic Photosynthesis -Second Edition[END_REF]) that also provided no explanation. To our knowledge, only Serôdio et al. [START_REF] Serôdio | Effects of chlorophyll fluorescence on the estimation of microphytobenthos biomass using spectral reflectance indices[END_REF] have suggested that the two peaks are the result of Chl a fluorescence emission centered at 683 nm. The chlorophyll fluorescence emitted at this wavelength would affect the radiometric data by increasing the reflectance at this wavelength and decreasing the absorption peak in the second derivative spectra [START_REF] Serôdio | Effects of chlorophyll fluorescence on the estimation of microphytobenthos biomass using spectral reflectance indices[END_REF]. However, it is possible that fluorescence emission is not the single factor affecting the dip between the two peaks. Another possible explanation for the double peak are absorption variations caused by photosystem (PS) stoichiometry. This is supported by previous observations that variations in PS stoichiometry (PSI and PSII) may cause a shift in the absorption from 674 to 678nm [START_REF] Hoepffner | Effect of Pigment Composition on Absorption Properties of Phytoplankton[END_REF][START_REF] Johnsen | Biooptical characteristics of PSII and PSI in 33 species (13 pigment groups) of marine phytoplankton, and the relevance for pulse-amplitude-modulated and fast-repetition-rate fluorometry1[END_REF]. In fact, because the PSI Chl a core absorbs at 683 nm and the core of PSII Chl a at 670 nm, a change in the relative amount of the PSI against the PSII is likely to induce this shift [START_REF] Hoepffner | Effect of Pigment Composition on Absorption Properties of Phytoplankton[END_REF]. The consequence in second derivative spectra should be the double peak, attributed to the two photosystem cores. The PSI:PSII ratio is often different from 1:1 and varies with species and with environmental light conditions [START_REF] Hoepffner | Effect of Pigment Composition on Absorption Properties of Phytoplankton[END_REF][START_REF] Fujita | Chromatic Variation of the Abundance of PSII Complexes Observed with the Red Alga Prophyridium cruentum 10.1093/pcp/pce164[END_REF][START_REF] Smith | Response of the Photosynthetic Apparatus in Dunaliella salina (Green Algae) to Irradiance Stress[END_REF][START_REF] Walters | Towards an understanding of photosynthetic acclimation[END_REF]. This mechanism is a longterm photoacclimation process that requires hours to days to occur. It allows the excitation energy within the photosynthetic machinery to be regulated by changing the relative amount of the two photosystems [START_REF] Dietzel | Photosynthetic acclimation: State transitions and adjustment of photosystem stoichiometry as functional relationships between short-term and long-term light quality acclimation in plants[END_REF]. For example, in this study, the Chlorophyta species showed the largest difference between light conditions with the second derivative peak at 671 nm increasing more than the 683 nm in the HL treatment. This supports the hypothesis that HL-acclimated D. tertiolecta cells decreased their PSI:PSII ratio as proposed by Walters et al. [START_REF] Walters | Towards an understanding of photosynthetic acclimation[END_REF]. The Rhodophyta and Cyanophyta species showed similar height for the two peaks in both light conditions, which could be explained by a stable PSI:PSII ratio. This might be cause by a parallel decrease in both photosystems when the cells are HL-acclimated as reported previously for Rhodophyta [START_REF] Gantt | Antenna Systems of Red Algae: Phycobilisomes with Photosystem II and Chlorophyll Complexes with Photosystem I[END_REF]. Although the assessment of PS stoichiometry by radiometry looks very promising further studies are still required to test our hypothesis, using for example, reflectance analyses of cells with controlled amount of PSI and PSII Three ubiquitous peaks (466, 546 and 586 nm) may also be assigned to Chl a as they are minor absorption bands observed in vitro in organic solvent, with a shift [START_REF] Jeffrey | Data for the identification of 47 key phytoplankton pigments[END_REF].

This assumption is based on the fact that Chl a was the only pigment found in the four species. However, some uncertainty remains in the diatom species since Fuco is known to exhibit a broad absorption band around 550 nm in vivo [START_REF] Goodwin | Carotenoids in higher plants[END_REF]. Thus, a spectral data. For instance, Jesus et al. [START_REF] Jesus | Detection of diatom xanthophyll cycle using spectral reflectance[END_REF] observed a shift from 487 to 508 nm associated with the operation of the xanthophyll cycle which corresponds to the HL enzymatic conversion of DD to DT, while Ruban et al. [START_REF] Ruban | The superexcess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF] and Lavaud and Lepetit [START_REF] Lavaud | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF] have shown that a thin spectral band around 520 nm is related to the DT molecules which effectively involved in the photoprotective dissipation of excess energy (the so-called NPQ, i.e. the non photochemical fluorescence quenching).

In the Cyanophyta species, the absorption features between 450-550 nm showed the largest differences between the two light acclimation treatments. The increase in the 489 nm absorption band from LL to HL was probably caused by the increase in ZX.

The specific absorption band at 524 nm increased from LL to HL, which could be caused by increases in either Myx or Osc. However, Myxis the most likely source of this peak because the amount of Osc was 5 times lower than Myx and was probably too low to be retrieved from the spectral data in a reliable way.

Identification ofphycobiliproteins (PBPs) absorption features

Although PBPs were not quantified in the current study, some absorption bands couldbe linked to this pigment group. The Cyanophytaspecies showed a series of absorption bands from ~585 to ~630 nm,whichcould correspond to the absorption domains of the blue PBPsphycocyanins (610-620 nm) and allophycocyanins (650-655 nm) [START_REF] Bermejo Roman | Recovery of pure B-phycoerythrin from the microalga Porphyridium cruentum[END_REF]. Allophycocyanin peaks appeared to show an unexpected increase in HL-acclimated cells of the Cyanophytaspecies, which is not consistent with their known light-harvesting function [START_REF] Zhao | Phycobiliproteins[END_REF]. However, under high light condition a phosphorylation cascade is induced in cells activating specific polypeptides that degrade PBPs [START_REF] Grossman | Regulation of Light Harvesting in Cyanobacteria[END_REF], which is accompanied by a Chl a decrease. A greater allophycocyanin/Chl a ratio illustrated by a higher absorption peak at 650 nm suggests that the decrease of the PSI and PSII (containing Chl a) was faster than the decrease of PBPs, whereas the expected trend is a decrease of the ratio with light [START_REF] Gantt | Antenna Systems of Red Algae: Phycobilisomes with Photosystem II and Chlorophyll Complexes with Photosystem I[END_REF]. The same unexpected trend was observed with phycocyaninin the Rhodophyta species, where HL-acclimated cells showed higher absorption peaks around 570 nm corresponding to the absorption by phycoerythrin [START_REF] Bermejo Roman | Recovery of pure B-phycoerythrin from the microalga Porphyridium cruentum[END_REF].

Estimation of optical absorption cross-section (a*)

One goal of this study was to demonstrate the possibility of assessing a* from reflectance data using the MPBOM [START_REF] Kazemipour | Optical properties of microphytobenthic biofilms (MPBOM): Biomass retrieval implication[END_REF] regardless of the taxonomic algal group considered. This parameter is required for the calculation of the absolute photosynthesis quantum yield which is essential to estimate primary productivity from incident PAR. Also, it must be estimated in vivo because a* is highly dependent on cell size and pigment composition as well as on plastid size and number, which drive the pigment-package effects, i.e. self-shading, light scattering, etc. [START_REF] Falkowski | Aquatic Photosynthesis -Second Edition[END_REF]. By settling the micro-algal cells on a filter, the light scattering effects observed in a cell suspension are suppressed and thus allowing to use reflectance to estimate a* in vivo. This approach avoids the use of complex apparatus, as an integrating sphere. Values of * a and red * a , estimated by the MPBOM were in accordance with published values, as well as changes observed with cells grown under LL or HL increase has also been reported before [START_REF] Johnsen | Biooptical characteristics of PSII and PSI in 33 species (13 pigment groups) of marine phytoplankton, and the relevance for pulse-amplitude-modulated and fast-repetition-rate fluorometry1[END_REF]. The efficiency of light absorption in HL-acclimated cells is regulated on a long-term scale (i.e. several days, depending on the growth rate) by a decrease in the number of thylakoids per plastid generating a decrease in the plastid volume and/or by a decrease in the number of plastids per cell (in species with many small plastids), and also by changes in the cellular pigment content. Both processes lead to a decrease in the "package effect" [START_REF] Falkowski | Aquatic Photosynthesis -Second Edition[END_REF]. Often, these phenomena occur together with a reduction in LHCs size, depending on the light conditions [START_REF] Falkowski | Aquatic Photosynthesis -Second Edition[END_REF]. The diatom species showed the highest * a increase with light, suggesting that this group might be the most efficient to acclimate to changing light conditions, possibly due to its strong potential for the regulation of photosynthesis in fluctuating light conditions [START_REF] Brunet | Pigments and photoacclimation processes[END_REF][START_REF] Lavaud | Fast regulation of photosynthesis in diatoms : Evolution, regulation and ecophysiology[END_REF]. In contrast, the Rhodophyta species showed a stability in a* values instead of the expected increase due to package effect (rearrangement of plastids, thylakoids and LHCs). In spite of the photoacclimation status (i.e. decrease in Chl a cellular content from LL to HL), the cells showed the same absorption cross-section values and these results suggest that there was no rearrangement of plastids, thylakoids and/or LHCs for this taxonomical group. This can be explained by a specific photoacclimation strategy described for P. cruentum [START_REF] Gantt | Antenna Systems of Red Algae: Phycobilisomes with Photosystem II and Chlorophyll Complexes with Photosystem I[END_REF] whereby Chl a and phycobilisome content per thylakoid area remain relatively constant even though the overall pigment content decreases in HL-acclimated cells.

Another relevant result observed in the Chlorophyta and the Cyanophyta species was the a* increase from LL to HL, which was highest when the average was calculated over the PAR domain in comparison to what was measured in Chl a absorption red domain. Between 670 and 680 nm, it is known that only Chl a absorbs the light energy, whereas in the rest of the PAR spectrum, carotenoids also absorb, including the photoprotective ones [START_REF] Johnsen | Biooptical characteristics of PSII and PSI in 33 species (13 pigment groups) of marine phytoplankton, and the relevance for pulse-amplitude-modulated and fast-repetition-rate fluorometry1[END_REF] as for instance violaxanthin. Thus, the coupled increase in photoprotective pigments in HL photoacclimated cells and the Chl a decrease, could explain the bigger * a values in comparison to the red * a values.

Consequentially, the a* increase seems to be not only due to the rearrangement of the package effect, but also to the increase in photoprotective pigments, although they could not participate in the energy transfer to the cores Chl a [START_REF] Johnsen | Biooptical characteristics of PSII and PSI in 33 species (13 pigment groups) of marine phytoplankton, and the relevance for pulse-amplitude-modulated and fast-repetition-rate fluorometry1[END_REF]. Indeed, some of these pigments are only involved in the xanthophyll cycle, as AX, ZX and DT.

It should also be noted that red * a did not shown significant difference between LL and HL conditions in the Chlorophyta species in contrast to * a . This implies that the * a increase was only caused by the carotenoid increase and in this case the package effect affected only red * a , reducing its decrease in spite of the cellular Chl a content. This result was in accordance with those found by Johnsen and Sakshaug [START_REF] Johnsen | Biooptical characteristics of PSII and PSI in 33 species (13 pigment groups) of marine phytoplankton, and the relevance for pulse-amplitude-modulated and fast-repetition-rate fluorometry1[END_REF] that observed that Chlorophyta are also the species with the lowest absorption-cross section coefficients.

Conclusion and future prospects

The non-invasive radiometric methodology proposed in this study was shown to be a good alternative to the already existing techniques for the assessment of the pigment absorption properties of photosynthetic micro-organisms. Namely, in vivo pigment absorption bands could be identified from second derivative reflectance spectra in a reliable way. More importantly, by coupling this approach to the MPBOM model, we demonstrated the feasibility of measuring the absorption cross-section (a*) of photosynthetic microorganisms in vivo, which is a key parameter for the estimation of the absolute photosynthetic efficiency and the primary productivity. In agreement with previous works, the pattern of variation of a* observed in this study suggests that these photosynthetic microorganisms display a high plasticity in response to light changes, and that the differences in responses between taxonomic groups are large [START_REF] Perkins | The application of variable chlorophyll fluorescence to microphytobenthic biofilms[END_REF]. Last but not least, this non-invasive methodology will facilitate the investigation of pigment absorption properties of microphytobenthos biofilms in vivo and in situ. An example of an original application could be the mapping of microphytobenthos light absorption efficiency using remote sensing imagery. This would strongly help assessing coastal primary production at the habitat/ecosystem scale, such as intertidal mudflats.

  diadinoxanthin (DD), diatoxanthin (DT), fucoxanthin (Fuco) and Chl c were present in E. paludosa only; neoxanthin (NX), antheraxanthin (AX), lutein (Lut) and Chl b only in D. tertiolecta; oscillaxanthin (Osc) and myxoxanthophyll (Myx) only in S. platensis. In contrast, P. cruentum did not exhibit any specific liposoluble pigments showing only Chl a, β-car and zeaxanthin (ZX). 3.1.2 Pigment changes in response to light Light did not seem to have a large effect on the qualitative pigment composition. Only few pigments were detected exclusively in one of the two light conditions: violaxanthin (VX) in HL-acclimated S. platensis cells, Chl b in LL-acclimated D.tertiolecta cells and β-car in LL-acclimated cells for all the species except P. cruentum. It was assumed that the apparent lack of Chl b and β-car detection in HLacclimated species was due to concentrations below the PDA detection threshold, rather than a true absence of pigments in the cells.

  , downward arrows): ~ 416 nm; ~ 440 nm; ~ 462 (except LL-acclimated D.tertiolecta and S. platensis cells); ~ 546 nm and ~ 586 nm; ~ 625 nm (with a high variability from 615 to 631 nm), with two shoulders detected at 615 and 631 nm for LL-acclimated P. cruentum cells. An absorption double peak was

Figure 1 .

 1 Figure1. Pigment concentration expressed in mg.m -2 in the four species acclimated at the two light (PAR) intensities, LL (black) and HL (gray). For each pigment (mean ± 95 % CI; n = 3), horizontal bar indicates no significant differences between HL and LL conditions (t test p ≥ 0.05).

Figure 2 .

 2 Figure 2. Spectra of standardized reflectance (ρ 925 ) measured for the four species acclimated at the two light (PAR) intensities, LL (black) and HL (gray). Arrows indicate absorption bands notching the transmittance peak of the 'green gap'.

Figure 3 . 2 )

 32 Figure 3. Spectra of second derivative (δδ) calculated for the four species acclimated at the two light (PAR) intensities, LL (black) and HL (gray). Downward arrows indicate ubiquitous absorption peaks while upward arrows indicate specific peaks.

Table 4 )

 4 Chla -1 in HL-acclimated cells (Table4). In the Chl a red absorption domain, i.e. from 0.008 (E. paludosa) to 0.063 (S. platensis) m 2 .mg Chl a -1 in LL-acclimated cells and from 0.027 (E. paludosa) to 0.106 (S. platensis) m 2 .mg Chl a -1 in HL-acclimated cells. Globally, values were greater in HLwhile in P. cruentum there was no significant changes in

	between 670 and 685 nm,
	acclimated cells, and	a	*	red	values were greater than * a values, expect S. platensis
	(Table 4). E. paludosa and P. cruentum showed a similar decrease in cellular Chl a
	content (-67 %, Table 4) but contrasting results regarding * a and	a	*	red	. In E.
	paludosa this decrease was accompanied by a 3-fold increase in * a and	a	*	red
	(reaching 226 %, * a and red * a regardless of the photoacclimation state.

Average absorption cross-section values through the PAR domain ( * a ) estimated using the MPBOM varied from 0.007 (E. paludosa) to 0.047 (P. cruentum) m 2 .mg Chl a -1 in LL-acclimated cells and from 0.024 (E. paludosa) to 0.117 (S. platensis) m 2 .mg red * a vary

Table 1 .

 1 Absorbance maxima for all pigments detected in the four strains analyzed by HPLC (in vitro) and by radiometry (in vivo). Pigments are ordered following their HPLC elution time from the earliest to the latest. n.d.: in vivo absorbances not detected by radiometry. References for in vitro absorbances: Jeffrey et al.[START_REF] Jeffrey | Application of pigment method to oceanography[END_REF], except (*) in Brotas and Plante-Cuny,[START_REF] Brotas | The use of HPLC pigment analysis to study microphytobenthos communities[END_REF]. In vivo absorbance are from this study.

	Sources	E. paludosa	E. paludosa	S. platensis	D. tertiolecta	D. tertiolecta, S. platensis	S. platensis	E. paludosa	D. tertiolecta	E. paludosa	D. tertiolecta	D. tertiolecta, S. platensis (HL), P. cruentum	D. tertiolecta	All 4 strains	All 4 strains
		636											650	671/ 683
	vivo													625
	Maxima in		550	n.d.	n.d.	n.d.	524	DD+DT: 496	n.d.	496 DD+DT:	500	489		440 416	499 466
		630											646	665
	vitro Maxima in	573 442	(465) 451	529 498 (469)	466 437 (414)	469 440 (417)	508 475 (451)	477 447	471 444	481 453	474 446	478 452	596 458	618 (581)	481 453
								(420)	(421)		(421)

Table ( s)

 (

Table 2 .

 2 Cellular content of Chl a in the four studied species photoacclimated at the two PAR intensities, HL and LL. For each value (mean ± 95 % CI; n = 3), differences between LL and HL are significant (t test p ≤ 0.05).

	Strain	Light acclimation	Cellular content of Chl a (µg.10 -6 cell)	Amount of cells on filters
		LL	4.37 ± 1.08	2.4 10 6 ± 2.0 10 5
	D. tertiolecta	HL	1.02 ± 0.15	5.5 10 6 ± 2.7 10 5
		LL	11.45 ± 0.76	2.9 10 6 ± 1.5 10 5
	E. paludosa	HL	3.78 ± 0.62	2.3 10 6 ± 2.2 10 5
		LL	0.69 ± 0.26	1.0 10 7 ± 3.1 10 5
	S. platensis	HL	0.09 ± 0.01	1.2 10 7 ± 3.5 10 5
		LL	0.65 ± 0.13	4.0 10 6 ± 3.2 10 5
	P. cruentum	HL	0.21 ± 0.01	1.3 10 7 ± 1.1 10 6

Table 3 .

 3 Pigment content expressed in ratio against Chl a in the four studied species photoacclimated at the two PAR intensities, HL and LL. For each ratio (mean ± 95 % CI; n = 3), differences between LL and HL are significant (t test p ≤ 0.05), except (n.s.).

	Ratio ± std	D. tertiolecta LL	D. tertiolecta HL
	Lut / Chl a	0.27 ± 0.01	0.57 ± 0.05
	Chl b / Chl a	0.13 ± 0.03	n.d.
	ZX / Chl a	0.11 ± 0.005	0.17 ± 0.005
	VX / Chl a	0.07 ± 0.003	0.02 ± 0.003
	NX / Chl a	0.03 ± 0.001	0.05 ± 0.003
	car / Chl a	0.08	n.d.
	AX / Chl a	0.02 ± 0.003	0.06 ± 0.003
		E. paludosa LL	E. paludosa HL
	Fuco / Chl a (n.s.)	0.61 ± 0.03	0.69 ± 0.07
	Chl c / Chl a (n.s.)	0.11 ± 0.007	0.11 ± 0.01
	DD / Chl a	0.03 ± 0.002	0.12 ± 0.01
	DT / Chl a	0.01 ± 0.002	0.14 ± 0.02
	car / Chl a	0.02 ± 0.008	n.d.
		S. platensis LL	S. platensis HL
	ZX / Chl a	0.57 ± 0.02	1.46 ± 0.15
	Myx / Chl a	0.34 ± 0.02	0.88 ± 0.08
	car / Chl a	0.16 ± 0.04	n.d.
	Osc / Chl a	0.06 ± 0.01	0.16 ± 0.04
	VX / Chl a	n.d.	0.09 ± 0.04
		P. cruentum LL	P. cruentum HL
	ZX / Chl a	0.37 ± 0.13	0.63 ± 0.1
	car / Chl a	0.03	0.07 ± 0.02

Table 4 .

 4 Absorption cross-section estimated from MPBOM for the four studied strains photoacclimated at the two PAR intensities, HL and LL. a *: absorption cross-section in m 2 .mg Chl a -1 , averaged on the PAR domain ± 95 % CI; a *red : absorption cross-section in m 2 .mg Chl a -1 , averaged between 670 and 685 nm;,  a*red and Chl a: variation from LL to HL conditions in cells. For each strain, differences between LL and HL are significant (t test p ≤ 0.05), except n.s ( and  a*red were minimized to zero).

	Strain	Light acclimation	a * (m 2 .mg Chl a -1 )	a*red Chl a -1 ) (m 2 .mg			a*red  Chl a
	D. tertiolecta	LL HL	0.017 ± 0.003 0.027 ± 0.003	0.023 ± 0.004 0.030 ± 0.004	57 %	0 % n.s	-71 %
		LL	0.007 ± 0.000	0.008 ± 0.000
	E. paludosa				226 % 225 % -67 %
		HL	0.024 ± 0.005	0.027 ± 0.005
		LL	0.054 ± 0.002	0.063 ± 0.002
	S. platensis				117 % 68 %	-88 %
		HL	0.117 ± 0.021	0.106 ± 0.017
	P. cruentum	LL HL	0.047 ± 0.013 0.035 ± 0.002	0.040 ± 0.011 0.027 ± 0.001	0 % (n.s.)	0 % (n.s.)	-67 %

pigment mixture including Chl a could also explain the presence of these minor ubiquitous bands.

The other chlorophylls (b and c) also show a Q y band (636 nm for Chl c and 650 nm for Chl b) without any double peak feature. These Q y absorption bands are in accordance with previous findings [START_REF] Hoepffner | Effect of Pigment Composition on Absorption Properties of Phytoplankton[END_REF][START_REF] Méléder | Spectrometric constraint in analysis of benthic diatom biomass using monospecific cultures[END_REF][START_REF] Kazemipour | Microphytobenthos biomass mapping using the optical model of diatom biofilms: Application to hyperspectral images of Bourgneuf Bay[END_REF]. The other major absorption bands (Soret bands and Q x ) were not observed, possibly because of their small height and/or because of their overlap with other absorption peaks.

Identification of carotenoid absorption features

Carotenoids are structurally and functionally the most diverse pigment group. They cover part of the 'green gap' with absorption bands from 450 to 550 nm [START_REF] Scheer | The Pigments[END_REF]. Several carotenoids are involved in photoacclimation and photoregulation processes, e.g. xanthophyll cycles and structural adjustment of the LHCs [START_REF] Dietzel | Photosynthetic acclimation: State transitions and adjustment of photosystem stoichiometry as functional relationships between short-term and long-term light quality acclimation in plants[END_REF]. Xanthophylls are important in micro-algal photoprotective processes [START_REF] Brunet | Pigments and photoacclimation processes[END_REF], which explains the increase in the xanthophyll:Chl a ratio in the Chlorophyta (VX, AX and ZX pool) and the diatom (DD and DT pool) under HL. This led to a higher absorption in the 489-500 nm region. In the Chlorophyta, the absorption increase at 489 nm was caused by the presence of the xanthophylls ZX and Lut, with absorption bands at 485 nm [START_REF] Dietzel | Photosynthetic acclimation: State transitions and adjustment of photosystem stoichiometry as functional relationships between short-term and long-term light quality acclimation in plants[END_REF] and 494 nm [START_REF] Ruban | Xanthophylls of the major photosynthetic light-harvesting complex of plants: identification, conformation and dynamics[END_REF][START_REF] Laviale | Relationships between pigment ratios and growth irradiance in 11 marine phytoplankton species[END_REF], respectively. The 489 nm peak was also observed in the Cyanophyta and the Rhodophytaspecies, both containing ZX. In the diatom species, the absorbance was centered at 497 nm. This was probably due to the joint absorption of DD and DT at 487 and 508 nm, respectively [START_REF] Ruban | The superexcess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF][START_REF] Jesus | Detection of diatom xanthophyll cycle using spectral reflectance[END_REF][START_REF] Olaizola | Short-term response of the diadinoxanthin cycle and fluorescence yield to high irradance in diatoms (Bacillariophyceue)[END_REF]. The case of the xanthophyll pigments illustrates very well the difficulty in deconvoluting the individual contribution of pigments with very similar reflectance spectrum shapes. Nevertheless, some authors have demonstrated the possibility of obtaining very detailed information from Highlights :

• In vivo estimation of the pigment absorption bands and the optical absorption cross-section, a* • Four photosynthetic micro-organisms cultivated at two PAR levels were analyzed • A new methodology was proposed: spectroradiometry measuring reflectances.

• Some absorption bands were ubiquous, others were taxonomically specific and/or photo-physiological dependent. • A double peak feature at 671 and 683 nm, supposed to be caused by PSII and PSI, was detected


Pierre Tattevin
email: pierre.tattevin@chu-rennes.fr

Emanuele Durante-Mangoni

Moses Massaquoi

Pierre Tattevin Emanuele

Durante-Mangoni Moses

Massaquoi P Tattevin

WHAT'S NEW IN INTENSIVE CARE Does this patient have Ebola virus disease ?

published or not. The documents may come

Does this patient have Ebola virus disease?

Introduction

Ebola virus is one of the most virulent human pathogens. Since 1976, Ebola virus disease (EVD) has caused more than 20 outbreaks in Africa, with case fatality rates of 30%-90%, in the absence of any approved treatment or vaccination [START_REF] Feldmann | Ebola haemorrhagic fever[END_REF]. It is transmitted by direct contact through broken skin or mucous membranes with blood, urine, saliva, feces, vomit, and other body fluids of symptomatic infected patients or convalescent persons, or through contaminated needle sticks [START_REF] Feldmann | Ebola haemorrhagic fever[END_REF][START_REF] Colebunders | Ebola haemorrhagic fever--a review[END_REF]. The current outbreak in West Africa probably begun in December 2013 in Guinea [START_REF] Baize | Emergence of Zaire Ebola Virus Disease in Guinea -Preliminary Report[END_REF], and causes unprecedented concerns for the following reasons:

i) It is due to a strain with 97% homology with Zaire ebolavirus, the most virulent species, with prior fatality rates as high as 90% ; ii) As of August 22 nd , four countries have been involved, with 2,615 suspected cases, 1,528 laboratory confirmed cases, and 1,427 related deaths: this is already much more than the largest epidemic reported to date (425 cases in Uganda, 2000Uganda, -2001)), and the situation is unlikely to be resolved soon [START_REF] Fauci | Ebola -Underscoring the Global Disparities in Health Care Resources[END_REF][START_REF] Chan | Ebola Virus Disease in West Africa -No Early End to the Outbreak[END_REF]; iii) The 2014 West Africa outbreak affects rural as well as urban areas, and recently reached the most populous African country (Nigeria); iv) Experienced governmental and non-governmental organizations, including Médecins Sans Frontières (MSF), have been active on the field since March 2014 [START_REF] Baize | Emergence of Zaire Ebola Virus Disease in Guinea -Preliminary Report[END_REF], but failed to control the epidemic.

Many factors contributed to this failure, including population poverty and authorities' distrust , disease denial in the context of strong religious beliefs, porous borders, weaknesses in public health systems, inadequate salaries and lack of adequate protection for health care workers. On August 8, nine months after the first documented cases, the World Health Organization (WHO) has declared this outbreak a public health emergency of international concern and has called for a strong and coordinated international response, stating that 'all nations should be prepared to detect, investigate, and manage Ebola cases' [START_REF] Hawkes | Ebola outbreak is a public health emergency of international concern, WHO warns[END_REF].

Ebola virus disease outside Africa

The risk that this outbreak will establish a foothold in high-income countries outside Africa is very low, as human-to-human transmission of EVD does not occur when adequate infection control procedures are implemented. However, patients with EVD acquired in West Africa have already been transferred to America and Europe, and this is likely to become more common as the disease spreads. Hence, physicians working in emergency departments or intensive care units (ICU) outside Africa must be able to identify patients with possible EVD, and manage them appropriately, with two main objectives: i) prevent any human-tohuman transmission of Ebola virus in contacts, including health care workers, relatives, and other patients; ii) ensure all EVD suspected patients receive appropriate care, whether or not affected. We aimed to describe epidemiological, clinical and biological clues that may be used to classify patients with suspected EVD.

Epidemiological clues

As of August 2014, EVD has been essentially transmitted in three countries during the current outbreak: Guinea, Liberia, and Sierra Leone (Fig. 1). A fourth country, Nigeria, was more recently added to the list of countries where transmission of Ebola virus occurs, which documents that spread may occur in countries not bordering the original epidemic area. For case definition, the Centers for Disease Control and Prevention (CDC) consider that residence in, or travel to an area where EVD transmission is active, is a risk factor for EVD (http://www.cdc.gov/vhf/ebola/hcp/case-definition.html). Although this makes sense for operational reasons, two issues deserve consideration. Firstly, outbreaks are dynamic processes, and this is particularly true for this EVD epidemic. Hence, we have no guarantee that countries bordering with areas of active transmission (e.g. Mali, Ivory Coast), or even more remote African countries, will remain EVD-free. This remark is not an invitation to prematurely increase the list of countries to be considered at risk for EVD exposure, but a word of caution to governments and clinicians: in the context of the current outbreak, a patient returning from countries neighbouring those mentioned above, and presenting with fever, a severe illness, haemorrhagic symptoms, and no alternative diagnosis, should raise attention. Secondly, in subjects coming back from one of the four EVD countries, the level of exposure may differ from almost zero (e.g. airlines staff or passengers in transit who spent a few hours in a local airport), to very high (e.g. health care staff working in an Ebola treatment unit).

The second epidemiological clue is the incubation time or window period, conservatively defined as [3-21 days]. These limits allow to rule out EVD in any patient with fever onset later than 21 days after returning from an endemic area. More reliable data, obtained in settings where a single well-defined exposure had occurred, indicated a range of 3-13 days for the incubation period (mean, 6-8 days). However, the mean incubation period may vary according to the inoculum and the route of infection, averaging at 9.5 days for contact exposures, and 6.3 days after injection during the 1976 Zaire ebolavirus outbreak [START_REF]Ebola haemorrhagic fever in Zaire, 1976[END_REF][START_REF]Ebola haemorrhagic fever in Sudan, 1976. Report of a WHO/International Study Team[END_REF].

Clinical findings suggestive of Ebola virus disease

High-grade fever is a necessary criteria for EVD in most case definitions. Indeed, in all published series, fever was present in > 97% of cases [START_REF] Feldmann | Ebola haemorrhagic fever[END_REF][START_REF] Colebunders | Ebola haemorrhagic fever--a review[END_REF][START_REF] Pittalis | Case definition for Ebola and Marburg haemorrhagic fevers: a complex challenge for epidemiologists and clinicians[END_REF][START_REF] Hartman | Ebola and marburg hemorrhagic fever[END_REF][START_REF] Macneil | Proportion of deaths and clinical features in Bundibugyo Ebola virus infection, Uganda[END_REF][START_REF] Borchert | Ebola haemorrhagic fever outbreak in Masindi District, Uganda: outbreak description and lessons learned[END_REF][START_REF] Kortepeter | Basic clinical and laboratory features of filoviral hemorrhagic fever[END_REF][START_REF] Sureau | Firsthand clinical observations of hemorrhagic manifestations in Ebola hemorrhagic fever in Zaire[END_REF][START_REF] Bwaka | Ebola hemorrhagic fever in Kikwit, Democratic Republic of the Congo: clinical observations in 103 patients[END_REF][START_REF] Khan | The reemergence of Ebola hemorrhagic fever, Democratic Republic of the Congo, 1995. Commission de Lutte contre les Epidemies a Kikwit[END_REF][START_REF] Ndambi | Epidemiologic and clinical aspects of the Ebola virus epidemic in Mosango, Democratic Republic of the Congo[END_REF][START_REF] Richards | Unexpected Ebola virus in a tertiary setting: clinical and epidemiologic aspects[END_REF][START_REF]Outbreak of Ebola hemorrhagic fever Uganda, August 2000-January 2001[END_REF], and a literature review found that 791 of 796 cases of EVD presented with a body temperature > 101°F, or 38.3°C [START_REF] Pittalis | Case definition for Ebola and Marburg haemorrhagic fevers: a complex challenge for epidemiologists and clinicians[END_REF]. Typically, fever is of abrupt onset, associated with flu-like symptoms (chills, malaise, arthromyalgias), and soon followed by digestive manifestations [START_REF] Feldmann | Ebola haemorrhagic fever[END_REF][START_REF] Colebunders | Ebola haemorrhagic fever--a review[END_REF]. In the current West Africa outbreak, severe diarrhea and vomiting have been invariably observed during the early disease course [START_REF] Baize | Emergence of Zaire Ebola Virus Disease in Guinea -Preliminary Report[END_REF]. Subsequent signs and symptoms are variable, mostly respiratory (chest pain, shortness of breath, cough), cutaneous (in 25-50% of cases, a mostly non-itchy, diffuse maculopapular rash with secondary desquamation occurs), and neurological (confusion).

Brain involvement translates into a significant occupational risk for health care workers, as patients may become aggressive. Haemorrhagic manifestations, once considered the hallmark of EVD, arise during the peak of the illness in 50 to 70% of patients, and may vary from petechiae to multiple foci of mucosal haemorrhage, up to uncontrolled bleeding after injections or vessel punctures. In fatal cases, death usually occurs between day 6 and day 16, due to a combination of hypovolaemic and septic shock, massive bleeding (typically gastrointestinal), and multiorgan failure.

Laboratory abnormalities

Due to limited access to laboratory tests in involved countries, and the risk associated with improper handling of blood samples, less is known on laboratory features of EVD [START_REF] Feldmann | Ebola haemorrhagic fever[END_REF][START_REF] Kortepeter | Basic clinical and laboratory features of filoviral hemorrhagic fever[END_REF].

Most patients show early moderate leukopenia, with deep lymphopenia attributed to massive apoptosis. Later, neutrophilia, and/or atypical lymphocytes appear. Other common findings include moderate thrombocytopenia (50,000-100,000 cells per µL), highly raised liver enzymes (especially aspartate aminotransferase), hyperproteinemia (possibly related to capillary leak), and prolongation of prothrombin and thromboplastin times, often in the context of disseminated intravascular coagulation. No specific abnormalities have been reported at later stages, when multiorgan failure ensues.

Differential diagnosis

The definite EVD diagnosis hinges on detection of viral antigen by enzyme-linked immunosorbent assay or viral RNA by polymerase-chain-reaction. IgM and IgG antibodies may become positive later in the course of illness. Alternative diagnoses must be carefully considered and ruled out, especially when early treatment may favourably impact prognosis (e.g. malaria, any bacterial severe sepsis or septic shock, enteric fever, leptospirosis, rickettsiosis, African trypanosomiasis). This is one of the main challenges in the management of suspected EVD, as the recommended infection control measures for collecting and handling biological specimens may delay necessary laboratory testing (e.g. traditional chemistry, hematology, blood cultures, malaria tests, serology), and jeopardize patient care, if not promptly performed. Likewise, basic imaging studies (ultrasound, chest X-ray) should not be deferred, provided adequate protection is used, so that the necessary process to rule out EVD in suspected cases will not translate into a loss of opportunity for those with alternative diagnosis. Of note, common detergents and hypochlorite are effective in neutralising Ebola virus.

Conclusions

The 2014 EVD outbreak in West Africa is a public health emergency of international concern [START_REF] Hawkes | Ebola outbreak is a public health emergency of international concern, WHO warns[END_REF]. Given the extent of this outbreak, and the flow of transcontinental exchanges, every physician active in emergency departments or ICU worlwide may turn out to be involved in the care of patients suspected of EVD. Take-home messages from this paper, as of August 2014, are i) suspect EVD in any patient who presents with fever within three weeks after a stay in Guinea, Sierra Leone, Liberia, or Nigeria; ii) while implementing infection control procedures to prevent any secondary cases (in case EVD is confirmed), ensure that all plausible differential diagnoses are appropriately considered and managed.

Fig. 1 .

 1 Fig. 1. Distribution of EVD cases (confirmed, probable and suspected) by district in the affected countries from December 2013 to 16 August 2014

Declaration of interest

The authors declare that they have no conflict of interest.