Justyna Wojcieszek

Javier Jiménez-Lamana
email: j.jimenez-lamana@univ-pau.fr

Katarzyna Bierła

Lena Ruzik

Monika Asztemborska

Maciej Jarosz

Joanna Szpunar

Uptake, translocation, size characterization and localization of cerium oxide nanoparticles in radish (Raphanus sativus L.)

Keywords: Single particle ICP-MS, edible plants, engineered nanoparticles, laser ablation ICP-MS, ionization efficiency

Due to their unique physical and chemical properties, the production and use of cerium oxide nanoparticles (CeO2 NPs) in different areas, especially in automotive industry, is rapidly increasing, causing their presence in the environment. Released CeO2 NPs can undergo different transformations and interact with the soil and hence with plants, providing a potential pathway for human exposure and leading to serious concerns about their impact on the ecosystem and human organism. This study investigates the uptake, bioaccumulation, possible translocation and localization of CeO2 NPs in a model plant (Raphanus sativus L.), whose edible part is in direct contact with the soil where contamination is more likely to happen. The stability of CeO2 NPs in plant growth medium as well as after applying a standard enzymatic digestion procedure was tested by single particle ICP-MS (SP-ICP-MS) showing that CeO2 NPs can remain intact after enzymatic digestion; however, an agglomeration process was observed in the growth medium already after one day of cultivation. An enzymatic digestion method was next used in order to extract intact nanoparticles from the tissues of plants cultivated from the stage of seeds, followed by size characterization by SP-ICP-MS. The results

Introduction

In recent years there has been a rapid increase in the development, production and application of engineered nanoparticles (ENPs), which are currently playing a growing role in a wide range of industrial and commercial applications [START_REF] Stark | Industrial applications of nanoparticles[END_REF][START_REF] Vance | Nanotechnology in the real world: Redeveloping the nanomaterial consumer products inventory[END_REF]. Due to their unique properties and novel features, the global production of nanomaterials is significantly increasing, leading to the release of ENPs into the environment, where they can interact with higher plants.

Among the different ENPs, cerium oxide nanoparticles (CeO2 NPs) are one of the most important and promising due to their capability for undergoing a redox-cycle between the two natural oxidation states (Ce 3+ and Ce 4+ ) [START_REF] Cassee | Exposure , health and ecological effects review of engineered nanoscale cerium and cerium oxide associated with its use as a fuel additive[END_REF], with an estimated global production of up to 10 000 tons per year [START_REF] Keller | Global life cycle releases of engineered nanomaterials[END_REF][START_REF] Piccinno | Industrial production quantities and uses of ten engineered nanomaterials in Europe and the world[END_REF]. Cerium is the most abundant rare earth element in the earth's crust and belongs to the group of elements referred to as technology-critical elements that were considered just as laboratory curiosities, being now key components for the development of new technologies [START_REF] Wojcieszek | Speciation of technologically critical elements in the environment using chromatography with element and molecule specific detection[END_REF]. CeO2 NPs have special electrical, optical and thermal properties which make them widely used as polishing agents, UV-blockers, glass additives, in agricultural products and especially in automotive industry [START_REF] Stanek | Atomistic simulation of CeO 2 surface hydroxylation : implications for glass polishing[END_REF]The Project on EmergingNanotechnologies, 2019;[START_REF] Yang | Particle-specific toxicity and bioavailability of cerium oxide ( CeO 2 ) nanoparticles to Arabidopsis thaliana[END_REF]. Moreover, the high reactive oxygen species (ROS) scavenging properties gives CeO2 NPs the potential to be used as an antioxidant and radioprotective agent [START_REF] Li | Acquired Superoxide-Scavenging Ability of Ceria Nanoparticles *[END_REF]. For instance, CeO2 NPs are widely used in the production of catalysts or as a diesel fuel additive to increase fuel combustion efficiency, decrease diesel soot emissions and to reduce NOx emissions [START_REF] Cassee | Exposure , health and ecological effects review of engineered nanoscale cerium and cerium oxide associated with its use as a fuel additive[END_REF][START_REF] Johnson | Predicting contamination by the fuel additive cerium oxide engineered nanoparticles within the United Kingdom and the associated risks[END_REF]. Therefore, their release into the ecosystem is unavoidable for example through deposition along roadways that leads to their translocation to the soil and thus to plants, which has raised serious concerns about their fate and impact in the ecosystem [START_REF] Colvin | The potential environmental impact of engineered nanomaterials[END_REF][START_REF] Gardea-Torresdey | Trophic Transfer, Transformation, and Impact of Engineered Nanomaterials in Terrestrial Environments[END_REF][START_REF] Johnson | Predicting contamination by the fuel additive cerium oxide engineered nanoparticles within the United Kingdom and the associated risks[END_REF]. CeO2 NPs can interact with the soil and hence with plants, which in turn increase the risk of their bioaccumulation in the animal and human food chain [START_REF] Colvin | The potential environmental impact of engineered nanomaterials[END_REF][START_REF] Deng | Interactions between engineered nanomaterials and agricultural crops : implications for food safety *[END_REF]. As a results, and due to the importance of the physicochemical states of Ce to its food safety implications, a more extensive studies on the interaction of CeO2 NPs and higher plants is essential for a fully understanding of their environmental impact (W. Zhang et al., 2017a).

Both positive and negative effects of CeO2 NPs on plants have been demonstrated, depending on the plant species, size and concentration of NPs, exposure time and plant growth conditions [START_REF] Ma | Interactions between engineered nanoparticles ( ENPs ) and plants : Phytotoxicity , uptake and accumulation[END_REF][START_REF] Rossi | The impact of cerium oxide nanoparticles on the salt stress responses of Brassica napus L . *[END_REF][START_REF] Wang | The impact of cerium oxide nanoparticles on tomato ( Solanum lycopersicum L . ) and its implications for food safety[END_REF][START_REF] Zhao | CeO 2 and ZnO Nanoparticles Change the Nutritional Qualities of Cucumber ( Cucumis sativus )[END_REF]. Species-dependent responses of plants for CeO2 NPs treatment have been also observed by other researchers (Lopez-Moreno et al., 2010b). Additionally, it was already presented that toxicity of CeO2 NPs towards plants is concentration dependent (Lopez-Moreno et al., 2010a). On the other hand, CeO2 NPs were shown to enhance plant growth under certain exposure conditions [START_REF] Ma | Multigenerational exposure to cerium oxide nanoparticles : Physiological and biochemical analysis reveals transmissible changes in rapid cycling Brassica rapa[END_REF][START_REF] Wang | The impact of cerium oxide nanoparticles on tomato ( Solanum lycopersicum L . ) and its implications for food safety[END_REF]. CeO2

NPs have generally been considered as insoluble under environmental conditions [START_REF] Walser | Persistence of engineered nanoparticles in a municipal solid-waste incineration plant[END_REF]) [START_REF] Yokel | Biodistribution and oxidative stress effects of a systemically-introduced commercial ceria engineered nanomaterial[END_REF]. However, several studies suggest that after CeO2 NPs treatment, cerium in plants could exist in the form of Ce salts or dissolved Ce; reducing agents present in plant growth media could reduce the surface Ce 4+ of CeO2 NPs into Ce 3+ [START_REF] Dan | Single particle ICP-MS method development for the determination of plant uptake and accumulation of CeO 2 nanoparticles[END_REF][START_REF] Schwabe | Dissolution and transformation of cerium oxide nanoparticles in plant growth media[END_REF]Zhang et al., 2012;W. Zhang et al., 2017). Additionally, solubility of CeO2 NPs is also pH dependent [START_REF] Hernandez-Viezcas | In Situ Synchrotron Xray Fluorescence Mapping and Speciation of CeO 2 and ZnO Nanoparticles in Soil Cultivated Soybean (Glycine max)[END_REF]. Dissolved form of CeO2 NPs was found for example in cucumber or soybean plants [START_REF] Hernandez-Viezcas | Interactions between CeO 2 Nanoparticles and the Desert Plant Mesquite: A Spectroscopy Approach[END_REF][START_REF] Zhang | Biotransformation of Ceria Nanoparticles in Cucumber Plants[END_REF]. In contrast, other studies showed that CeO2 NPs did not undergo any transformation in cucumber, alfalfa, tomato and corn seedling (Lopez-Moreno et al., 2010b). Most of the studies performed showed that CeO2 NPs can be taken up by plants, but the majority of NPs appeared to remain in root tissues, raising concerns on their accumulation by root vegetables. However, even though the edible tissues of below-ground vegetables have direct contact with NPs contaminated soils, only little attention has been paid to this group of plants [START_REF] Zhang | Uptake and Accumulation of Bulk and Nanosized Cerium Oxide Particles and Ionic Cerium by Radish ( Raphanus sativus L.)[END_REF].

In this context, radish (Raphanus sativus L.), a popular vegetable with high global consumption, was chosen to the study since its edible part is in contact with the soil where contamination, through atmospheric dust deposition, is more likely to happen. Taking into account different, often contradictory, results presented in previous works, the main purpose of this study was to investigate the uptake, translocation, characterization and spatial localization of a commercial suspension of CeO2 NPs in a model edible plant throughout the whole process by using different mass spectrometry based techniques: the stability of the CeO2 NP in the hydroponic solution was assessed by single particle ICP-MS (SP-ICP-MS); the bioaccumulation of cerium in plants tissues and the translocation factor from roots to leaves was determined by ICP-MS; the presence of CeO2 NPs in radish tissues was investigated by SP-ICP-MS and their size distributions obtained; finally, the spatial distribution of cerium in roots was studied by laser ablation ICP-MS (LA-ICP-MS).

Material and methods

Samples and reagents

Seeds of radish (Raphanus sativus L.) were purchased from Vilmorin Garden (Komorniki, Poland). Analytical or biological reagent grade chemicals and LC-MS grade solvents were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France) unless stated otherwise. Ultra-pure water (18 MΩ cm) obtained with a MiliQ system (Millipore, Guyancourt, France) was used throughout. Hydrogen peroxide from Fisher Scientific (Hampton, NH) and nitric acid (INSTRA-Analysed) from Baker (Deventer, Netherlands) were used for samples digestion. A standard ionic solution of 1000 mg L -1 cerium was purchased from Plasma CAL standards (Teddington, UK).

Macerozyme R-10 enzyme (pectinase from Rhizopus sp., Sigma Aldrich) was used to digest plant tissues for CeO2 NPs extraction. Macerozyme R-10 is a multi-component enzyme mixture containing cellulase (0.1 unit per mg), hemicellulase (0.25 unit per mg) and pectinase (0.5 unit per mg).

Aqueous suspension of CeO2 NPs (40 wt %) with a nominal size of 30-50 nm was purchased from US Research Nanomaterials, Inc. (Houston, TX). A diluted suspension of CeO2 NPs was prepared daily in ultrapure water by accurately weighing aliquots of the stock suspension after one minute sonication. After dilution and before each analysis, the suspensions were sonicated for approximately 1 min.

Instrumentation

Homogenization of the plant tissues was performed by a Vibracell 75115 ultrasonic probe (Bioblock Scientific, Illkirch, France) offering a nominal power of 500 W. Incubation of samples was performed in a Grant OLS-200 water bath (Keison Products, Essex, UK). A Branson B2510 ultrasonic bath (Branson, Danbury, CT) was used for sonication of nanoparticles suspensions and enzymatic extracts before SP-ICP-MS analysis. A Digi-Prep system from SCP Science (Quebec, Canada) was used for acid digestion of plant tissues and extracts for the total cerium determination. A centrifuge 5415R (Eppendorf, Hamburg, Germany) was used for the extraction procedure. pH was adjusted by using a FiveEasy pH meter from Mettler Toledo (Columbus, OH).

The determination of the total concentration of cerium in the plant samples was carried out using an Agilent 7500ce ICP-MS (Agilent, Tokyo, Japan) instrument fitted with Ni cones and a 2.5 mm i.d. injector torch. The position of torch and nebulizer gas flow was adjusted each day of work.

The working conditions of ICP MS were optimized daily using a 1 µg L -1 solution of 7 Li + , 89 Y + and 205 Tl + in 2% (v/v) HNO3.

Single particle ICP-MS method

An Agilent 7900 ICP MS equipped with Single Nanoparticle Application Module was used for the characterization of CeO2 NPs. The default instrumental and data acquisition parameters are listed in Table 1. During analysis 140 Ce with a natural abundance of about 88.5 % was monitored. 140 Ce is interference-free in ICP-MS analysis which makes this isotope the best choice for analysis of Ce based nanoparticles. SP-ICP-MS analyses were performed in TRA mode using a dwell time of 100 µs, with a total time of analysis of 60 s. The dwell time is a critical parameter when working in single particle mode and the use of microsecond dwell time can improve particle sizing accuracy and counting [START_REF] Abad-Álvaro | Evaluation of number concentration quantification by single-particle inductively coupled plasma mass spectrometry: microsecond vs. millisecond dwell times[END_REF]. Gold nanoparticle standard reference material with a nominal diameter of 56 nm (RM 8013) was obtained from NIST (Gaithersburg, USA) and was used for determination of transport efficiency, which was calculated by the particle frequency method described by [START_REF] Pace | Determining Transport Efficiency for the Purpose of Counting and Sizing Nanoparticles via Single Particle Inductively Coupled Plasma Mass Spectrometry[END_REF]. The sample flow rate was calculated daily by measuring the mass of water taken up by the peristaltic pump for 2 min (this operation was repeated 3 times). After each sample analysis, the software automatically processed the raw data and generated the particle size, particle concentration, size distribution and information about concentration of dissolved metal. The size distributions were prepared in Origin 8.5 software (Northampton, MA) and adjusted to lognormal distributions in order to obtain the median diameter. Mass fraction 0.8139

Laser ablation ICP-MS method

A NewWave UP-213 laser ablation (LA) system (NewWave Research, Freemont, USA) was coupled to an Agilent model 7700x (Agilent, Tokyo, Japan) using a 60 cm Tygon tube (5.0 mm i.d.).

The laser ablation system was operated in a focused spot mode at the repetition rate of 20 Hz with a spot size of 250 μm and a scan speed of 100 μm s -1 . The ablated matter was transported into the ICP with He gas (800 mL min -1 ) and mixed in a T-connector with carrier gas of ICP MS delivered at 1.1 L min -1 aerosol (a Micromist nebulizer and a double pass Scott spray chamber were removed from the configuration). No collision/reaction gas was used during analyses. A 1.5 mm i.d. injector torch and Pt cones were used. Pulse energy was of 30% and fluence was 1.45 J cm -2 . The ICP-MS instrument was optimized in wet plasma condition before coupling with laser in order to obtain the maximum Ce signal-to-noise ratio.

Procedures

Plant cultivation

Portions of radish seeds (ca. 1 g) were germinated in distilled water in darkness for 3 days and then the seedlings were transferred to 350 mL opaque containers with a Knop nutrient solution (pH 6.8) and placed in a growth chamber. After 4 days, the CeO2 NPs suspension was added to the medium at a cerium concentration of 5 mg L -1 . As a control variant, plants were left in the nutrient solution without the addition of cerium. Cultivation was carried out for the next 7 days in a growth chamber. The plants were grown at with a temperature varying from 21-25 °C, light intensity 200 microeinsteins m -2 s -1 , 12 h photoperiod and 50-60% relative humidity. All the time the solutions in the containers were aerated. Each variant of the cultivation was performed in three replicates. After cultivation, the plants were harvested and the roots were gently rinsed with deionized water. The plants were divided into roots and above ground organs (leaves plus stems) and lyophilized. Dried plant material was ground in a mortar before further analysis.

For LA-ICP-MS analysis, radish plants were initially cultivated in typical garden soil in the greenhouse for 4 weeks to obtain well-formed tubers. Next, adult plants of radish, with the taproot diameter of ca. 1.5 cm were conditioned in deionized water for 24 hours and then transferred to opaque containers with suspensions of CeO2 NPs (50 mL) at concentrations of 5 mg L -1 or 50 mg L -1 for 24 and 48 h. After finishing the cultivation, taproots were gently rinsed with deionized water and prepared for further analysis. Each variant of the cultivation was performed in three replicates.

Determination of total cerium content

Samples of leaves and roots (0.025 g) were digested by adding 2.5 mL HNO3 (c) in a DigiPrep tube and the following temperature program: 30 min of heating up to 65°C and then keeping temperature at 65°C for 4 h. Afterwards, 1 mL of H2O2 (c) was added to the samples and the digestion was continued for the next 4 h using the same program. After digestion, the mixtures were cooling down and diluted with ultrapure water to a final volume of 25 mL. Further dilutions were prepared with 2% HNO3 (v/v), directly before ICP-MS analysis. Two isotopes of cerium ( 140 Ce, 142 Ce) were monitored during analysis. The analytical blanks were analyzed in parallel. Quantification was performed by external calibration; a 5 points calibration curve was prepared for cerium concentration in the investigated range from 0.0 to 5.0 ng mL -1 . The content of cerium in each sample was calculated as the mean of results obtained for the monitored isotopes, from three replicates of each sample. In order to verify the accuracy of the method, a recovery study was performed. A known amount of CeO2 NPs suspension (at the same concentration as used for plant cultivation) was spiked into the controls of roots and leaves plus stems and the acid digestion method described above was applied.

Enzymatic digestion method

After cultivation performed on radish seeds, plant tissues were digested enzymatically as reported in previous works [START_REF] Jiménez-Lamana | Single particle ICP-MS characterization of platinum nanoparticles uptake and bioaccumulation by Lepidium sativum and Sinapis alba plants[END_REF][START_REF] Kińska | Study of the uptake and bioaccumulation of palladium nanoparticles by Sinapis alba using single particle ICP-MS[END_REF]. Briefly, grounded samples of leaves and roots (0.025 g) were homogenized with 8 mL of 2 mM citrate buffer (pH 4.5;

adjusted with citric acid) by using an ultrasonic probe. After the end of homogenization, 2 mL of Macerozyme R-10 solution (0.01 g of enzyme powder for roots and 0.05 g of enzyme powder for leaves plus stems, dissolved in 2 mL of ultrapure water) was added to samples and they were next shaken at 37 °C for 24 hours in a water bath with continuous shaking. After digestion, the samples were settle down for approximately 15 min and the obtained suspensions were filtered with a 0.45 µm syringe filter (Sigma Aldrich). The filtered samples were next analyzed by SP-ICP-MS.

Study of the spatial distribution of CeO2 NPs in radish roots by LA-ICP-MS

After the end of cultivation performed on adult plants, radish roots were washed with ultrapure water in order to remove nanoparticles adsorbed on the surface. Afterwards, fresh roots samples were cut into 150 μm thick slices using the Leica VT1000 S vibrating blade microtome (Leica Biosystems, Wetzlar, Germany). The radish slices were directly mounted onto glass slides coated with double-sided tape to enhance adhesion and kept in a fridge before further treatment. Four repetitions of each root sample were prepared. The prepared slices were next analyzed by means of LA-ICP-MS. The tissue section was systematically scanned (line by line) by a focused laser beam.

Results and discussion

Cultivation of radish in the presence of nanoparticles and determination of total content of cerium

During cultivation, radish plants appeared tolerant to the applied concentration of CeO2 NPs since no visible phytotoxic effects were observed. Biomass production and tissues hydration did not differ substantially between the control and the CeO2 NPs treated plants. Some additional studies on the content of photosynthetic pigments showed that plants were not affected by nanoparticles.

The determination of the total cerium content in radish tissues was the first step done after the plant cultivation with the CeO2 NPs suspension, in order to investigate its distribution among the different tissues. Control and CeO2 NPs treated samples of leaves (together with stems) and roots of radish were analyzed by standalone ICP-MS after samples mineralization. The quantification limit, calculated as 10 times the standard deviation of the blank (n = 3) divided by the sensitivity, was 0.43 ng g -1 . The results of cerium content were obtained from three independent experiments and are presented in Table 2.

Table 2.

Total cerium content in radish tissues from control and CeO2 NPs treated plants (mean ± standard deviation).

Control plant samples /µg g -1 CeO2 NPs treated samples /µg g -1

Leaves + stems < LOQ 32.9 ± 0.4

Roots < LOQ 1948 ± 9

The obtained results showed that cerium is accumulated in analyzed radish tissues. However, the majority of cerium was found in radish roots and only minor part of cerium was transported to above ground organs, which is in good agreement with many studies as discussed below. The translocation factor (TF), defined as the ratio of cerium content in leaves with respect to roots, was 0.017. It has been reported that the translocation from roots to shoots is independent of the cerium content in the roots and of the supplied form of cerium [START_REF] Yang | Particle-specific toxicity and bioavailability of cerium oxide ( CeO 2 ) nanoparticles to Arabidopsis thaliana[END_REF]. Taking into account that roots as edible part of radish are in direct contact with the soil where contamination is more likely to happen, the results of the total content are especially important from the point of view of food safety.

To determine the accuracy of the proposed acid digestion method, a recovery study was made.

Control plant samples (leaves plus stems, and roots) were spiked with a known amount of CeO2 NPs and the acid digestion method was applied. Good recovery values were obtained for both leaves plus stems (103.2%) and roots (102.7%). Regardless of the total content of cerium determined in plant tissues in previous works, its translocation from roots to the aerial part was always in very low level.

For instance, [START_REF] Yang | Particle-specific toxicity and bioavailability of cerium oxide ( CeO 2 ) nanoparticles to Arabidopsis thaliana[END_REF] reported that the TF of cerium from roots to above ground organs in Arabidopsis thaliana plant is 0.042, while [START_REF] Rossi | The impact of cerium oxide nanoparticles on the salt stress responses of Brassica napus L . *[END_REF] found a TF of around 0.002 after analysis of Brassica napus L. In the case of desert plant mesquite, the TF of cerium was 0.2 and 0.25 for plants treated with 500 and 4000 mg L -1 , respectively (Hernandez-Viezcas et al., 2016). In the case of plants grown in soils, a TF of 0.02-0.03 was found in kidney bean plants [START_REF] Majumdar | Science of the Total Environment Soil organic matter in fl uences cerium translocation and physiological processes in kidney bean plants exposed to cerium oxide nanoparticles[END_REF].

It should be also mentioned that no translocation of cerium to above ground organs was observed in the case of tomato or wheat plants [START_REF] Antisari | Uptake and translocation of metals and nutrients in tomato grown in soil polluted with metal oxide ( CeO 2 , Fe 3 O 4 , SnO 2 , TiO 2 ) or metallic ( Ag , Co , Ni ) engineered nanoparticles[END_REF][START_REF] Schwabe | Dissolved cerium contributes to uptake of Ce in the three crop plants †[END_REF]. The TF is dependent also on the size of CeO2 NPs. It was showed that after analysis of pumpkin leaves and roots, the TF for CeO2 NPs of 9 nm was 0.0004 whereas no cerium translocation was observed in the case of NPs of 64 nm [START_REF] Schwabe | Dissolved cerium contributes to uptake of Ce in the three crop plants †[END_REF].

Study of ionization efficiency of CeO2 NPs in the plasma

The calculations behind SP-ICP-MS theory lie on some assumptions, being a plasma ionization efficiency for nanoparticles comparable to the corresponding dissolved species one of them [START_REF] Pace | Determining Transport Efficiency for the Purpose of Counting and Sizing Nanoparticles via Single Particle Inductively Coupled Plasma Mass Spectrometry[END_REF]. However, while this has proven to be true for silver or gold nanoparticles [START_REF] Hu | A New Strategy for Highly Sensitive Immunoassay Based on Single-Particle Mode Detection by Inductively Coupled Plasma[END_REF][START_REF] Laborda | Selective identification, characterization and determination of dissolved silver(i) and silver nanoparticles based on single particle detection by inductively coupled plasma mass spectrometry[END_REF], it might not be the case for nanoparticles of a different nature, like it has been reported for selenium nanoparticles [START_REF] Jiménez-Lamana | Detection and characterization of biogenic selenium nanoparticles in selenium-rich yeast by single particle ICPMS[END_REF], and needs to be first investigated. A ionization efficiency lower than 100% was reported for CeO2 NPs with sizes from 1 to 10 nm [START_REF] Sánchez-García | Size determination and quantification of engineered cerium oxide nanoparticles by flow field-flow fractionation coupled to inductively coupled plasma mass spectrometry[END_REF]. However, this value might be different in the case of the CeO2

NPs used in this study, since the ablation and ionization may strongly vary while increasing the size of the nanoparticle. In order to calculate the ionization efficiency, the response of the ICP-MS towards the nanoparticulated form of cerium was studied, by determining and comparing the total cerium concentration of digested nanoparticles and a suspension of undigested nanoparticles. The calibration was achieved with aqueous standards of cerium in 2% HNO3 for the digested nanoparticles and in ultrapure water and in 2% HNO3 for the direct analysis of the suspension, in order to investigate the influence of the medium on the cerium sensitivity. The concentrations determined in ultrapure water and in 2% HNO3 for the undigested CeO2 NPs suspension were 9.7 ± 0.9% and 10.2 ± 1.2% with regard to the concentration determined after acid digestion, respectively (Table 3).

Table 3. Determination of cerium concentration in digested and undigested suspension of CeO2 NPs (mean ± standard deviation).

Sample Total Cerium content / mg L -1

Acid digestion 237.9 ± 3.0 Suspension in ultrapure water 23.1 ± 2.1

Suspension in 2 % HNO3 (v/v) 24.4 ± 2.9

These results show that ICP-MS sensitivity depends on the physicochemical form of cerium and thus a particle ionization efficiency, defined as the ratio of the ionization efficiency of the particle to the ionization efficiency of the corresponding dissolved metal solution [START_REF] Pace | Determining Transport Efficiency for the Purpose of Counting and Sizing Nanoparticles via Single Particle Inductively Coupled Plasma Mass Spectrometry[END_REF], needs to be taken into account. According to the results obtained in this study, a particle ionization efficiency of 10% was applied for sizing CeO2 NPs. In addition, it was shown that the medium has no significant influence on the ionization of CeO2 NPs.

The suspension of CeO2 NPs used in this study was then analysed by means of SP-ICP-MS at a concentration of around 1x10 8 NP L -1 taking into account the ionization efficiency calculated. The size distribution obtained (Fig. 1) showed a polydisperse distribution with its central part within the size range provided by the manufacturer: 30-50 nm.

Investigation of CeO2 NPs stability

Stability in growth medium

Before the analysis of plant samples, the stability of the suspension of CeO2 NPs in the growth medium was investigated. A suspension of CeO2 NPs of 5 mg L -1 was spiked into the nutrient solution and the size distribution of the nanoparticles was determined by SP-ICP-MS immediately upon preparation and after 1, 2, 4 and 7 days. Each suspension analysed was diluted accordingly in order to measure the same number of events. The median diameters obtained from the corresponding size distributions of nanoparticles at the different times analysed are shown in Table 4. From the results obtained, it can be concluded that CeO2 NPs undergo agglomeration after 1 day of cultivation in growth medium, with an increase of the median diameter from 56.0 nm to 128.3 nm, whereas the size distribution does not vary significantly from 1 to 7 days of cultivation (Fig. S1). The agglomeration of CeO2 NPs has already been observed in hydroponic solution, without the addition of a stabilizing agent [START_REF] Schwabe | Influence of two types of organic matter on interaction of CeO 2 nanoparticles with plants in hydroponic culture[END_REF][START_REF] Schwabe | Dissolution and transformation of cerium oxide nanoparticles in plant growth media[END_REF]. Remarkably, no background signal corresponding to dissolved cerium was observed on the SP-ICP-MS time scans at any of the incubation times tested in the present study. This finding can be explained by two facts: on one hand, the occurrence of CeO2 NPs agglomerates prevent from cerium dissolution, since agglomeration reduces the effective specific surface area of nanoparticles, which results in less cerium dissolution;

on the other hand, the release of cerium ions is size dependent: in a study performed with CeO2 NPs in Hoagland medium, [START_REF] Schwabe | Dissolution and transformation of cerium oxide nanoparticles in plant growth media[END_REF] showed that the smallest particles (9 nm) released the highest amount of cerium, whereas the release of cerium by the largest particles (64 nm) was almost negligible.

Influence of enzymatic digestion and plant matrix

The influence of the enzymatic procedure on the size distribution of the CeO2 NPs suspension was also studied. For this purpose, a suspension of CeO2 NPs (5 mg L -1 ) with no plant tissue was treated with the same conditions of enzymatic digestion. As it can be shown in Fig. 2, the size distribution obtained for enzyme-treated CeO2 NPs was in good agreement with the size distribution obtained for CeO2 NPs stock suspension freshly prepared and hence the integrity of CeO2 NPs is not affected by the enzyme used in the digestion procedure.

In addition, the influence of the plant matrix was investigated. Control leaves plus stems and control roots of radish were spiked with 5 mg L -1 of the CeO2 NPs suspension, submitted to the enzymatic digestion procedure and the corresponding nanoparticle size distributions obtained by SP-ICP-MS. The size distributions obtained for both spiked tissues were in good agreement with those obtained for freshly enzyme-treated CeO2 NPs (Fig. 2). These results showed that the procedure proposed can be used to extract CeO2 NPs from plant tissues without causing their transformation.

Single particle ICP-MS analysis of leaves plus stems and roots of radish

In order to identify the form of cerium inside the radish tissues cultivated from the stage of seeds, samples of leaves plus stems and samples of roots were subjected to the enzymatic procedure followed by SP-ICP-MS analysis. In a first step, roots and leaves plus stems of control plants were analyzed and the corresponding time scans obtained (Fig. 3a and3b, respectively). As expected, only a few pulses above the background were observed for control plants. The presence of these pulses may be explained by a slight contamination of the sonication probe during the enzymatic digestion procedure, but their occurrence is not significant (less than 10 pulses out of 600,000 readings) and hence does not hamper the further analyses. Afterwards, the SP-ICP-MS analyses of CeO2 NPs treated samples were performed. Samples of roots were diluted 3000 times before analysis whereas samples of leaves plus stems were diluted 200 times. Each tissue was analyzed by triplicate. Time scans obtained showed a significant number of pulses in both plants tissues (Fig. 3c and3d), proving that radish is able to uptake CeO2 NPs and transports them into the aerial part of the plant. The presence of CeO2 NPs has been already reported in tomato (S. lycopersicum L.), cucumber (C. sativus), pumpkin (Cucurbita pepo), and soybean (Glycine max) shoots [START_REF] Dan | Single particle ICP-MS method development for the determination of plant uptake and accumulation of CeO 2 nanoparticles[END_REF][START_REF] Zhang | Uptake and distribution of ceria nanoparticles in cucumber plants[END_REF]Zhang et al., 2017b), in Arabidopsis thaliana shoots [START_REF] Yang | Particle-specific toxicity and bioavailability of cerium oxide ( CeO 2 ) nanoparticles to Arabidopsis thaliana[END_REF] and in romain lettuce shoots (P. Zhang et al., 2017a). According to plus stems (Fig. 4b), showed a significant number of nanoparticles at higher diameters than in the case of roots (median diameter: 60.3 nm). Since it was found that plants are not able to take up big nanoparticles this finding may be explained by an agglomeration process that takes place at the endpoint of transport of CeO2 NPs from roots to above ground organs. Once CeO2 NPs have entered the plant, they are translocated up to above-ground organs through the xylem system, driven by the transpiration stream [START_REF] Zhao | Influence of CeO2 and ZnO nanoparticles on cucumber physiological markers and bioaccumulation of Ce and Zn: A Life Cycle Study[END_REF]. The endpoint of this transport pathway is the leaves [START_REF] Zhao | Influence of CeO2 and ZnO nanoparticles on cucumber physiological markers and bioaccumulation of Ce and Zn: A Life Cycle Study[END_REF], where nutrients and water arrive through the veins. At this point, nanoparticles will be locally more concentrated and hence agglomeration by contact is more likely to occur. Similarly, particle aggregates have been found in leaves of Arabidopsis thaliana treated with CeO2 NPs [START_REF] Yang | Particle-specific toxicity and bioavailability of cerium oxide ( CeO 2 ) nanoparticles to Arabidopsis thaliana[END_REF]. The biotransformation of CeO2 NPs inside plants remains a controversial issue. Some authors have suggested the uptake of Ce(III) followed by re-precipitation as pathway in CeO2 NPs uptake by plants [START_REF] Schwabe | Dissolved cerium contributes to uptake of Ce in the three crop plants †[END_REF]. However, our study showed that the dissolution of cerium is not significant and thus this mechanism can be discarded. On the other hand, cerium has been shown to be present in the shoots of cucumber and lettuce plants as CeO2 and cerium carboxylates [START_REF] Zhang | Biotransformation of Ceria Nanoparticles in Cucumber Plants[END_REF]Zhang et al., 2017a), although the majority of studies have shown that cerium mostly remained as CeO2 NP in plants tissues [START_REF] Majumdar | Exposure of cerium oxide nanoparticles to kidney bean shows disturbance in the plant defense mechanisms[END_REF]Zhang et al., 2017b). For instance, in a study conducted with romaine lettuce, the amount of cerium carboxylates in leaves was reported at 3.5% (P. Zhang et al., 2017a). These previous findings make us state that the cerium signal observed in radish leaves plus stems is due to CeO2 NPs and/or aggregates.

Study of spatial distribution of CeO2 NPs in radish roots by LA-ICP-MS

The results obtained by SP-ICP-MS analysis showed that the great majority of CeO2 NPs remain in the form of nanoparticles both in roots and leaves. Therefore, in order to check if CeO2 NPs have the ability to enter and be accumulated inside the radish tissues, the localization of CeO2 NPs in the radish roots, as edible part of the plant, was determined by LA-ICP-MS. Analyses were performed after cultivation of radish carried out on adult plants. It is worth to mention that radish belongs to the group of plants with root system in type of taproot, with one central and dominant root. Additionally, taproot of radish is in the shape of fusiform root -The primary root of the system is the widest in the middle with secondary root tapers towards the bottom. Optimisation of a LA-ICP-MS analysis in terms of sensitivity and S/N ratio allowed to choose optimum scan speed of 100 µm s -1 and spot size of 250 µm; increase in the laser beam spot size resulted in a considerable gain in sensitivity and homogeneity of the signal as more material is introduced into the plasma per time unit. After analysis of control plants cultivated for 2 days, no signal corresponding to cerium was observed (Fig. 5). The results obtained for radish samples treated with 5 mg L -1 of CeO2 NPs (Fig. 5A) showed peaks from cerium only at the external part of the analysed slices. Nanoparticles were found at a depth of about 1.2 mm leading to the conclusion that CeO2 NPs have the ability to enter and be accumulated into the radish tissues, which is in good agreement with another study performed with radish in soil (W. Zhang et al., 2017b). Similar situation was observed in the case of radish samples treated with 50 mg L -1 CeO2 NPs (Fig. 5B). Signals from cerium were registered just below the skin surface, with higher intensity than those observed for samples treated with 5 mg L -1 CeO2 NPs. However, one additional signal was observed in the central part of samples after 1 day and 2 days of treatment, which suggest that cerium is accumulated and transported by secondary roots from bottom towards the central part of radish roots, not only through the surface. Accumulation of the metals in the taproot of plants was already observed after a LA-ICP-MS analysis of another root vegetables such as carrot [START_REF] Yudasari | The 1064 nm laser-induced breakdown spectroscopy (LIBS) inspection to detect the nutrient elements in freshly cut carrot samples[END_REF]. In addition, two additional signals were observed for samples treated with CeO2 NPs for two days, suggesting that after accumulation, nanoparticles can be translocated within analysed radish tissues. Moreover, it was observed that the intensity of cerium signals increased with time for both treatments, showing that a higher content of cerium can be accumulated in radish roots with a longer exposure time; a relevant information in terms of food safety taking into account the persistence of CeO2 NPs in the environment. A good reproducibility of the cerium spatial distribution was achieved after comparing results of the different slices of the same radish root or from different radish roots with the same exposure concentration and time.

Conclusions

The uptake, bioaccumulation, translocation, physico-chemical characterization and localization of CeO2 NPs in radish was investigated by using different mass spectrometry based techniques. Our results showed that after cultivation, the majority of cerium remain in roots, with a low transportation up to leaves and stems (TF 0.017) as it has been observed in other studies. In order to get a correct particle size distribution, the behavior of CeO2 NPs in plasma was investigated,

showing that ICP-MS sensitivity depends on the physicochemical form of cerium and thus the ionization efficiency must be taken into account. Although CeO2 NPs underwent agglomeration in hydroponic medium after 1 day, the SP-ICP-MS analysis indicated that radish only takes up small nanoparticles, since the presence of bigger nanoparticles and/or aggregates was not observed. The studies performed discarded interaction with plant matrix or effect of the enzymatic digestion procedure that affected the size of the nanoparticles. However, the analysis of leaves plus stems showed the presence of nanoparticles with bigger sizes, which suggests that nanoparticles undergo agglomeration at the endpoint of their transportation. Finally, the analysis by LA-ICP-MS in radish roots showed that the accumulation of CeO2 NPs occurs mainly below the skin surface but after accumulation they have the ability to enter and be translocated within the tissue. The results obtained in this work can be consider meaningful from the point of view of food safety.

Fig. 1 .

 1 Fig. 1. Size distribution of the commercial suspension of CeO2 NPs used in the cultivation studies.

Fig. 2 .

 2 Fig. 2. Size distributions of: CeO2 NPs stock suspension freshly prepared (blue); CeO2 NPs after enzymatic digestion procedure (orange); control leaves plus stems (green) and roots (yellow) spiked with 5 mg L -1 CeO2 NPs suspension. Size distributions were adjusted to lognormal distributions (grey line) and the median diameter ± the standard deviation obtained.

Fig. 3 .

 3 Fig. 3. Time scans obtained for samples of: a) roots, and b) leaves of control plants and for c) roots and d) leaves of plants treated with 5 mg L -1 of CeO2 NPs.

Fig. 4 .

 4 Fig. 4. Size distributions obtained for a) roots, and b) leaves plus stems of radish

Fig. 5 .

 5 Fig. 5. LA-ICP-MS analysis of radish roots treated with A) 5 mg L -1 CeO2 NPs B) 50 mg L -1 CeO2 NPs. Light blue line: control plant; dark blue line: plants treated for 1 day; green line: plants treated for 2 days

Table 1

 1 Default instrumental and data acquisition parameters for SP-ICP-MS.

	Instrumental parameters
	RF Power	1550 W
	Argon gas flow rate
	Plasma	15 L min -1
	Auxiliary	0.9 L min -1
	Nebulizer	1.10 L min -1
	Sample uptake rate	0.35 mL min -1
	Data acquisition parameters
	Dwell time	100 µs
	Readings per replicate	600000
	Total acquisition time	60 s
	Analyte	Ce
	Mass (amu)	140
	Density	7.13 g cm -3

Table 4 .

 4 Median diameters of CeO2 NPs suspended in nutrient solution after different contact times.

	Incubation time Median diameter / nm
	0	56.0 ± 1.9
	1 day	128.3 ± 19.1
	2 days	119.1 ± 4.6
	4 days	112.5 ± 3.9
	7 days	134.6 ± 22.7

Acknowledgements

This work was financially supported by the National Science Centre, Poland (grant n° 2015/18/M/ST4/00257) as well as by a STSM Mobility Grant (JW) COST Action TD1407 (grant n° 42007) and the project AQUITRACES Region Aquitaine 20131206001-13010973. Authors would like to thank Wojciech Kurek (Department of Botany, Warsaw University of Life Sciences (SGGW)) for preparation of samples for LA-ICP-MS analysis.

the number of pulses detected, the nanoparticle number concentration in roots and in leaves plus stems was calculated as 3.3 x 10 11 and 2.4 x 10 10 NP L -1 , respectively. The largest number of number concentration of nanoparticles observed in roots could be explained by a direct contact of roots with CeO2 NPs suspension. Remarkably, the intensity of the pulses observed in the time scan obtained for leaves is significantly higher than for roots, which suggested the presence of bigger nanoparticles. In addition, no signal corresponding to dissolved cerium was observed for leaves, suggesting that CeO2 NPs did not underwent dissolution during their transport, although small signal coming from dissolved form of cerium was observed for roots. This is in good agreement with other studies that have shown that the dissolution of CeO2 NPs occur at the root surface rather than inside plants (P. Zhang et al., 2017b). For instance, [START_REF] Ma | Where Does the Transformation of Precipitated Ceria Nanoparticles in Hydroponic Plants Take Place?[END_REF] shown that dissolution of CeO2 NPs only occurred at root surfaces, whereas Ce (IV) was not reduced in the tissues in hydroponic cucumber plants. Other authors have reported that plants roots activity have an impact on the dissolution of CeO2 in hydroponically grown wheat, pumpkin, and sunflower plants [START_REF] Schwabe | Dissolved cerium contributes to uptake of Ce in the three crop plants †[END_REF].

Due to the small signal coming from dissolved form of cerium observed after analysis of roots by SP-ICP-MS, a speciation study was performed by means of size exclusion chromatography (SEC) coupled to ICP-MS. The chromatogram obtained (Fig. S1) showed just one signal coming from high molecular weight cerium compounds which could be explained by the ability of metal ions to create agglomerates even with low molecular weight ligands. However, the extraction efficiency of cerium from radish roots (procedure explained in Supplementary Information), was determined at only 0.19% and therefore, due to negligible presence of cerium compounds, the speciation study of cerium was not continued.

From time scans and by applying the ionization efficiency factor calculated before, the size distributions of CeO2 NPs in radish tissues were obtained. As it can be observed, the particle size distribution obtained for radish roots was narrower (Fig. 4a), with a median diameter of 42.7 nm, suggesting a preferential uptake of NPs at smaller sizes and that the agglomerates present in growth medium already after one day are not taken up by the plant. The finding that plant uptake of CeO2 NPs depends on the particle size and smaller particles are more readily taken up by plants has been previously suggested [START_REF] Zhang | Uptake and distribution of ceria nanoparticles in cucumber plants[END_REF]. However, the particle size distribution obtained for leaves Appendix A. Supplementary data Electronic supplementary information (ESI) available: procedure of extraction and fractionation of Ce species from radish samples by SEC-ICP-MS; size distributions of CeO2 NPs in grow medium at different incubation times; SEC-ICP-MS chromatogram of ammonium acetate extract of radish roots.


Thierry Tran

Céline Lafarge

Pascale Winckler

Rémi Pradelles

Nathalie Cayot
email: nathalie.cayot@agrosupdijon.fr

Camille Loupiac

Ex situ and in situ investigation of protein / exopolysaccharide complex in Porphyridium cruentum biomass resuspension

Introduction

The extraction of B-phycoerythrin from Porphyridium cruentum has been intensively investigated [START_REF] Bermejo | Recovery of pure B-phycoerythrin from the microalga Porphyridium cruentum[END_REF][START_REF] Bermejo | Recovery of B-phycoerythrin using expanded bed adsorption chromatography: Scale-up of the process[END_REF][START_REF] Benavides | Simplified two-stage method to B-phycoerythrin recovery from Porphyridium cruentum[END_REF] because this pigment protein is used as fluorescent biomarker [START_REF] Glazer | Phycobiliproteins-a family of valuable, widely used fluorophores[END_REF].

P cruentum is a spherical unicellular red alga (Rhodophyta) without an organized cell wall [START_REF] Jones | Studies on the growth of the red alga Porphyridium cruentum[END_REF]. P. cruentum's ultrastructure was firstly determined using electron microscopy from 1959 [START_REF] Gantt | The ultrastructure of Porphyridium cruentum[END_REF][START_REF] Brody | Observations on cellular structures of Porphyridium cruentum[END_REF]. This microalga is photoautotrophic and its photosynthesis is achieved by the lamellashaped thylakoids that host the photosynthetic material [START_REF] Gantt | The ultrastructure of Porphyridium cruentum[END_REF]. But besides chlorophyll, P. cruentum hosts numerous fluorescent substances such as phycobiliproteins including Bphycoerythrin, R-phycocyanin and allophycocyanin whose absorption and fluorescent properties have been well studied [START_REF] French | Fluorescence-Spectrum Curves of Chlorophylls, Pheophytins, Phycoerythrins, Phycocyanins and Hypericin[END_REF][START_REF] Gantt | Further evidence for a phycobilisome model from selective dissociation, fluorescence emission, immunoprecipitation, and electron microscopy[END_REF]. Those molecules are all located in the thylakoids: the phycobiliproteins are organized in a hemispherical structure called phycobilisome located on the outside of this thylakoid membrane. The chlorophyll is on the inside part of the thylakoid [START_REF] Gantt | Phycobilisomes of Porphyridium cruentum[END_REF][START_REF] Glazer | Phycobilisomes: structure and dynamics[END_REF]. Quantitatively, there is roughly ten times more B-phycoerythrin than Rphycoerythrin and allophycocyanin separately in P. cruentum [START_REF] Fuentes | Outdoor continuous culture of Porphyridium cruentum in a tubular photobioreactor: quantitative analysis of the daily cyclic variation of culture parameters[END_REF]. This microalga is also encapsulated in a sheath generated by the continuous secretion of anionic sulfated exopolysaccharides (EPS) [START_REF] Ramus | The correlation of Golgi activity and polysaccharide secretion in Porphyridium[END_REF][START_REF] Geresh | Characterization of the extracellular polysaccharide of Porphyridium sp.: molecular weight determination and rheological properties[END_REF][START_REF] Geresh | Fractionation and partial characterization of the sulphated polysaccharide of Porphyridium cruentum[END_REF][START_REF] Malis) Arad | Red microalgal cell-wall polysaccharides: biotechnological aspects[END_REF]. Their composition has been determined. They host a high diversity of sugar residues including D-xylose, D-and L-galactose, D-glucose, D-glucuronic acids [START_REF] Geresh | Characterization of the extracellular polysaccharide of Porphyridium sp.: molecular weight determination and rheological properties[END_REF][START_REF] Jones | Extracellular mucilage of the red alga Porphyridium cruentum: Mucilage of Porphyridium[END_REF][START_REF] Patel | Separation and fractionation of exopolysaccharides from Porphyridium cruentum[END_REF], and are covalently linked to sulfate groups and peptides [START_REF] Heaney-Kieras | The Covalent Linkage of Protein to Carbohydrate in the Extraceliular Protein-Polysaccharide from the Red Alga Porphyridium cruentum[END_REF]. The composition of those EPS are dependent on the cultural conditions of microalgae [START_REF] Soanen | Improvement of exopolysaccharide production by Porphyridium marinum[END_REF][START_REF] De | Influence of sulphate on the composition and antibacterial and antiviral properties of the exopolysaccharide from Porphyridium cruentum[END_REF][START_REF] Adda | Effect of nitrate on growth and production of cellwall polysaccharide by the unicellular red alga Porphyridium[END_REF].

The EPS are often seen as an obstacle to the extraction and purification of B-phycoerythrin (B-PE). In fact, the interaction between proteins and EPS can lead to a loss of extraction yield. Some studies often highlighted the difficulty to isolate B-PE from EPS [START_REF] Bernaerts | Molecular and rheological characterization of different cell wall fractions of Porphyridium cruentum[END_REF] and even suggested the existence of B-PE / EPS complexes [START_REF] Jubeau | High pressure disruption: a two-step treatment for selective extraction of intracellular components from the microalga Porphyridium cruentum[END_REF]. To our knowledge, no research was published on the topic of B-PE / EPS interactions, and even less in situ on microalgae biomass. The interactions between proteins such as bovine serum albumin, ovalbumin, whey protein and sulfated anionic polysaccharides (carrageenans, dextran sulfate) have been extensively investigated in model systems by using calorimetry, spectroscopy, or rheology.

Those studies have shown that these interactions were electrostatic interactions induced by the polysaccharide charge density at low ionic strength [START_REF] Galazka | Interactions of ovalbumin with sulphated polysaccharides: effects of pH, ionic strength, heat and high pressure treatment[END_REF]. Those complexes could then be dissociated by the addition of salt. The main parameters influencing these associations are pH, ionic strength and temperature [START_REF] Galazka | Interactions of ovalbumin with sulphated polysaccharides: effects of pH, ionic strength, heat and high pressure treatment[END_REF][START_REF] Mulloy | The specificity of interactions between proteins and sulfated polysaccharides[END_REF][START_REF] Galazka | Complexes of bovine serum albumin with sulphated polysaccharides: effects of pH, ionic strength and high pressure treatment[END_REF][START_REF] Galazka | Changes in protein-protein and proteinpolysaccharide interactions induced by high pressure[END_REF][START_REF] De Kruif | Complex coacervation of proteins and anionic polysaccharides[END_REF][START_REF] De Kruif | Polysaccharide protein interactions[END_REF][START_REF] Gurov | Equilibrium and nonequilibrium complexes between bovine serum albumin and dextran sulfate-I. Complexing conditions and composition of non-equilibrium complexes[END_REF][START_REF] Weinbreck | Complexation of Whey Proteins with Carrageenan[END_REF].

In order to better understand B-PE / EPS interactions, the present study aimed to examine the behavior of B-PE towards EPS in resuspension of P. cruentum biomass submitted to several pH and ionic strengths.

Moreover, following the approach of overcoming the obstacle of the EPS interactions during the extraction of B-PE, two extraction processes were tested: successive macerations and high pressure homogenizer treatment. This choice is based firstly on the hypothesis that those methods can reduce the interaction between B-PE and EPS and secondly on their ability to be up scaled for industrial purpose. These methods were also chosen because they were mechanical (and non-chemical or biological), and were supposed to limit the denaturation of protein pigments.

As maceration is a simple and common extraction method, it was carried out as a single step in a lot of reported studies [START_REF] Bermejo | Recovery of pure B-phycoerythrin from the microalga Porphyridium cruentum[END_REF][START_REF] Benavides | Simplified two-stage method to B-phycoerythrin recovery from Porphyridium cruentum[END_REF][START_REF] Tang | One-step chromatographic procedure for purification of B-phycoerythrin from Porphyridium cruentum[END_REF][START_REF] Marcati | Extraction and fractionation of polysaccharides and B-phycoerythrin from the microalga Porphyridium cruentum by membrane technology[END_REF] and did not allow to reduce the amount of EPS in the sample. On the contrary, successive macerations are expected to solubilize and dilute the EPS after each step, thus allowing further extraction of B-PE. This is the modality chosen for this study by using three successive macerations.

High pressure homogenizer is a more aggressive extraction method since it is expected to disrupt complexes along with the cells themselves by applying shear forces on the system. High pressure homogenizer treatments (HPH) for the extraction of B-PE from P. cruentum have already been studied by Jubeau et al. [START_REF] Jubeau | High pressure disruption: a two-step treatment for selective extraction of intracellular components from the microalga Porphyridium cruentum[END_REF]. In this work, the effect of HPH with one step and two steps was studied. After the use of these two methods of extraction (maceration and HPH), the B-PE content was determined by spectrophotometry. More recently, the use of high hydrostatic pressure was applied to study the extraction of B-PE [START_REF] Tran | Effect of high hydrostatic pressure on extraction of B-phycoerythrin from Porphyridium cruentum: Use of confocal microscopy and image processing[END_REF].

In the present work, we used microscopy to follow the B-PE and bound EPS in situ. The advantage to work with P. cruentum is that this microalgae offers interesting conditions for confocal microscopy because of the size of its cells of 7 to 16 µm [START_REF] Gantt | The ultrastructure of Porphyridium cruentum[END_REF] and, the fluorescence properties of phycobiliproteins and chlorophyll with excitation maximum wavelengths ranging from 545 to 675 nm with distinct emission peaks [START_REF] French | Fluorescence-Spectrum Curves of Chlorophylls, Pheophytins, Phycoerythrins, Phycocyanins and Hypericin[END_REF]. Confocal laser scanning microscopy (CLSM) allows the simultaneous observation of transmitted light and fluorescence signal based on one excitation wavelength. A spectral detection enables differentiation of fluorescent emission into several wavelength ranges [START_REF] Wolf | Phycobiliprotein fluorescence of Nostoc punctiforme changes during the life cycle and chromatic adaptation: characterization by spectral confocal laser scanning microscopy and spectral unmixing[END_REF][START_REF] Webb | Confocal optical microscopy[END_REF][START_REF] Norton | Using confocal laser scanning microscopy, scanning electron microscopy and phase contrast light microscopy to examine marine biofilms[END_REF][START_REF] Zimmermann | Spectral imaging and its applications in live cell microscopy[END_REF]. CLSM was already reported as a powerful method to study the phycobiliproteins composition of cells in situ and follow the changes in intracellular phycobiliprotein content through the life cycle of another photosynthetic microorganism (Nostoc punctiforme) [START_REF] Wolf | Phycobiliprotein fluorescence of Nostoc punctiforme changes during the life cycle and chromatic adaptation: characterization by spectral confocal laser scanning microscopy and spectral unmixing[END_REF]. Then, to localize and study the B-PE / EPS complexes, the native autofluorescent compounds of the cells and fluorescence markers for the EPS (bound or not) were used.

To sum-up, in order to better understand the B-PE / EPS interactions, the present study aimed to examine these interactions ex situ and in situ in resuspension of P. cruentum biomass submitted to several pH, ionic strengths and using two different extraction processes.

Materials and methods

Biological material and culture conditions

The marine Rhodophyta Porphyridium cruentum (1380-1C) was provided by the SAG Culture Collection (Göttingen, Germany). The microalga was grown by Microphyt (Baillargues, France) in 5,000 L photobioreactor (PBR) consisting in 1.2 km of glass tubes with co-circulation of liquid medium and CO2 enriched air [START_REF] Muller-Feuga | Appraisal of a horizontal two-phase flow photobioreactor for industrial production of delicate microalgae species[END_REF][START_REF] Muller-Feuga | Réacteur photosynthétique pour la culture de microorganismes et procédé de culture de microorganismes[END_REF]. The PBR was set under a greenhouse in order to control temperature (between 22 and 28°C) and the intensity of natural light using curtains. The pH set point of 7.5 was automatically controlled by CO2 injection and monitored by an inline Fermprobe F-235 pH probe from Broadley James (Silsoe, United Kingdom). Air was injected continuously at rate of 35 L/min. The culture medium used is a marine type medium, corresponding to a modified Hemerick's medium [START_REF] Hemerick | Culture methods and growth measurements[END_REF]. This medium was modified by addition of N and P up to 20 and 4 mmol/L respectively. Cultivation was conducted in semi-continuous conditions, thus maintaining the exponential growth phase during 85 days. Cells were harvested by bowl centrifugation at 6000 rpm during 60 min at room temperature (maximum reachable temperature of 35°C) using a KG 8006 from GEA (Oelde, Germany) and concentrated at the rate of around 10-13% dry weight. The biomass was frozen at -20°C in polyethylene bags, heat sealed, and stored at -20°C. The biomass was then dispatched in aliquots in airtight glass bottles at -18°C away from light. The total number of freezing / thawing cycles was 2 with freezing at -18°C and thawing at room temperature.

Materials

KH2PO4 (CAS Number 7778-77-0), and NaOH (CAS Number 1310-73-2) were purchased from Sigma-Aldrich (Saint Louis, USA). Isolectin GS-IB4 from Griffonia simplicifolia was purchased from Thermofischer Scientific, USA (Alexa Fluor TM 488 Conjugate). Isolectin was used as fluorescence probe for labeled EPS. Water used in all experiments was deionised and obtained from an Elga Ionic system PURELAB Option.

Effect of pH and ionic strength

P. cruentum biomass was resuspended at 0.2 g dry biomass / 100 mL in different buffers and let one hour under magnetic stirring at room temperature, away from direct light exposure. To investigate the influence of pH, the different buffers used were 0.1M KH2PO4 at pH 7, 5 and 8.5. For each of these buffers, a version with 1M NaCl was made to investigate the influence of ionic strength. The NaCl content of the raw material being unknown, the final NaCl concentration of the samples might be higher. All suspensions were done in triplicate.

Impact of three successive macerations with deionized water

(1) The impact of successive macerations was assessed by resuspending 0.02 g dry biomass in 10 mL of deionized water during one hour under magnetic stirring, away from direct light exposure. All macerations were done in triplicate. One mL of this resuspension was withdrawn and used for CLSM.

(2) The suspension was then centrifuged at 2570 rpm during 20 minutes at room temperature. The centrifuge was a 541/R with F45-30-11 rotor from Eppendorf (Hambourg, Germany). After centrifugation, the whole supernatant was harvested for further analysis whereas the pellet was resuspended in the same volume of distilled water as for the first resuspension (10 mL) and mixed using a vortex during two minutes. One mL of resuspension was withdrawn and used for microscopy.

(3) Step (2) was then repeated one more time.

Characterization of supernatant

B-phycoerythrin content determination

The absorbance spectrum between 220 and 800 nm was measured using a Monaco UVmc1 from Safas (Monaco) and semi-micro UV-cuvettes Brand (Wertheim, Germany). Extract samples were diluted by 4 with the appropriate buffer to respect the Beer-Lambert law and each modality was measured in triplicate.

The quantification of B-phycoerythrin (B-PE) in aqueous extracts was calculated using the following equations firstly used by [START_REF] Bermejo | Recovery of pure B-phycoerythrin from the microalga Porphyridium cruentum[END_REF] [START_REF] Bermejo | Recovery of pure B-phycoerythrin from the microalga Porphyridium cruentum[END_REF]:

[R-phycocyanin (R-PC)] (mg/mL) = (OD620-0.7*OD650)/7.38 (Equation 1)

[Allophycocyanin (APC)] (mg/mL) = (OD650-0.19*OD620)/5.65 (Equation 2)

[B-phycoerythrin] (mg/mL) = (OD565-2.8*[R-PC]-1.34*[APC]
)/12.7 (Equation 3) OD620, OD650, and OD565 correspond to the optical densities measured respectively at 620 nm, 650 nm and 565 nm.

A purity index can also be calculated through the OD545/OD280 ratio. The extract is considered to have a high purity level beyond a value of 4 [START_REF] Camara-Artigas | pH-dependent structural conformations of B-phycoerythrin from Porphyridium cruentum[END_REF].

The B-PE content was determined in mg/mL and was then converted in mg/g dry biomass.

pH determination

The pH was measured using a HI 2209 pH meter from Hanna instruments (Lingolsheim, France) and a DJ133 pH probe from VWR (Radnor, USA). The device was calibrated using reference buffer solution. The result corresponds to the value stable after 10 seconds during measurement.

High pressure homogenizer

P. cruentum biomass was resuspended at the rate of 1.5 g dry biomass / 100 mL of deionized water overnight at 5°C, away from direct light exposure and under magnetic stirring. This resuspension was treated by a Microfluidizer LM10 high pressure homogenizer from Microfluidics (Westwood, USA). Ice was used to cool down the system in order to avoid protein denaturation. The different treatments involved a one shot treatment at 70 MPa and a two-steps treatment at 70 MPa and 120 MPa.

Confocal fluorescence microscope

Transmission and fluorescence images of P. cruentum resuspensions were captured on a Nikon C1Si Eclipse TE 2000 U confocal microscope (Nikon, Japan). Imaging was carried out with a ×100 PlanApo objective (NA: 1.4, oil, Nikon, Japan). The 561 nm laser diode was chosen depending on: (i) the maximum excitation wavelength of B-PE at 545 nm and (ii) the maximum excitation wavelength of isolectin at 488 nm. The fluorescence probe was added to the sample at the rate of 40 µL/mL of resuspension sample. Fluorescence properties of phycobiliproteins and chlorophyll from P. cruentum have been well studied [START_REF] French | Fluorescence-Spectrum Curves of Chlorophylls, Pheophytins, Phycoerythrins, Phycocyanins and Hypericin[END_REF][START_REF] Gantt | Phycobilisomes of Porphyridium cruentum. Pigment analysis[END_REF][START_REF] Murata | Fluorescence of Chlorophyll in Photosynthetic Systems[END_REF][START_REF] Murata | Light-Induced Change of Chlorophyll a Fluorescence in Porphyridium cruentum[END_REF][START_REF] Brody | Action Spectra for Sensitization of Ligth Emission from Monomeric and Aggregated Chlorophyll at Physiological and Liquid Nitrogen Temperatures[END_REF]. Table 1 describes the wavelength range chosen for the detection of fluorescent substances of interest. The fluorescence emission signal was collected on C1Si multispectral detector with a spectral resolution of 8 nm. Wolf and Schüssler [START_REF] Wolf | Phycobiliprotein fluorescence of Nostoc punctiforme changes during the life cycle and chromatic adaptation: characterization by spectral confocal laser scanning microscopy and spectral unmixing[END_REF] reported the normalized fluorescence emission spectra from phycobiliproteins and chlorophyll. It is worth noting, that the fluorescence emission peaks of phycocyanin, allophycocyanin and chlorophyll overlap between 625 and 725 nm, meaning that the channels associated with phycocyanin and chlorophyll quantitatively over estimate phycocyanin and chlorophyll fluorescence emission respectively. This is not the case for B-PE. The choice was made to not investigate allophycocyanin, because of the strong overlapping of fluorescence emission peaks.

Five µL of resuspension were mounted between slid and slip cover. Calibration of detected fluorescence regarding B-PE concentration for a given laser power was made by acquisition of distilled water (blank) and different dilutions of P. cruentum extract (cell-free supernatants from resuspension). The fluorescence signal varied proportionally to the dilution rate (data not shown). The distilled water sample did not show any fluorescence emission higher than noise signal background (data not shown). The two used fluorescence probes were tested individually and together with all lasers to determine false positive fluorescence emission (possibly from autofluorescence of P. cruentum). It was observed that the chloroplast very slightly emitted fluorescence between 495 and 515 nm using the 488 nm laser without using isolectin (data not shown). Besides that, no interference or false positive were detected.

Statistical treatment

The statistical treatment was processed by XLStat v.14.0. by Addinsoft (Paris, France).

Analysis of variance (ANOVA) was performed to determine significant differences between the samples for a given parameter. Significance was established at p < 0.05. ANOVA showing significant differences lead to the use of Newman-Keuls pair test.

Results and discussion

Confocal fluorescence microscopy image description

The observation in transmitted light picture of resuspension of P. cruentum (Figure 1A)

showed round cells with inhomogeneous content delimited by a membrane that appeared as a white line. The whole system seemed to be kept together by an extracellular matrix, but it was hardly visible because it appeared translucent on the picture. The presence of such matrix was perceived during microscopic observation because of the coordinated motion of the cells. The blurry cells and white spots resulted from cells outside the focal plane of the objective. When looking at the merged image of transmitted light and fluorescence emission of all channels selected for this study (channels 1, 2 and 3 (Figure 1B)), fluorescence was detected in three areas: intracellular area, extracellular area, and spherical objects of about 2 µm diameter with high fluorescence emission intensity in extra and intracellular areas. In this study, these objects will be named vesicles for more convenience. They were already observed in previous work [START_REF] Tran | Effect of high hydrostatic pressure on extraction of B-phycoerythrin from Porphyridium cruentum: Use of confocal microscopy and image processing[END_REF].

Intracellular fluorescence was observed in channels 1, 2 and 3 (Figure 1C, 1D and 1E) and could be attributed to the phycobiliproteins (B-PE and R-phycocyanin (R-PC)) and chlorophyll, which is consistent with what is described in the literature [START_REF] Gantt | Phycobilisomes of Porphyridium cruentum. Pigment analysis[END_REF][START_REF] Redlinger | Photosynthetic Membranes of Porphyridium cruentum: An Analysis of Chlorophyll-Protein Complexes and Heme-Binding Proteins[END_REF]. On the fluorescence emission pictures (Figure 1B, 1C and 1D), the chloroplast appeared as a fluorescent rounded shaped object with bright spots which are likely attributable to phycobilisomes. The intracellular fluorescence emission area was clearly separated from the extracellular fluorescence emission by a black area corresponding to the surface of the cell by comparison with transmitted light picture (Figure 1B). This excluding space around the cells was larger than the space delimited by the membrane (by comparison with transmitted light picture, Figure 1A) and so, can be attributed to P. cruentum's well-known bound exopolysaccharide sheath [START_REF] Gantt | The ultrastructure of Porphyridium cruentum[END_REF].

The extracellular fluorescence indicates the presence of phycobiliproteins (Figure 1B, 1C and 1D) and, at a much lesser extent, of chlorophyll (Figure 1E), that were both released outside of the cells. These extracellular B-PE and R-PC fluorescence emissions are uniformly distributed. On the opposite, the vesicles showed a distinct round shape, with sometimes a "capsule-like" aspect conveyed by the weaker fluorescence emission intensity in the middle (Figure 1B, 1C and 1D). It is unclear if chlorophyll was also part of this structure (Figure 1E).

On the other hand, the vesicles might actually have included allophycocyanin since the other two phycobiliproteins B-PE and R-PC were clearly present (Figure 1C and1D). The presence of chlorophyll, which is a hydrophobic compound in the aqueous phases such as the extracellular space, could be explained by protein-chlorophyll complexes naturally occurring in P .cruentum's photosynthetic membranes [START_REF] Redlinger | Photosynthetic Membranes of Porphyridium cruentum: An Analysis of Chlorophyll-Protein Complexes and Heme-Binding Proteins[END_REF] and so opens the possibility for proteinprotein associations.

The addition of isolectin allowed the marking of the bound exopolysaccharide matrix but not that of the sheath, which stayed visible from its lack of fluorescence (Figure 2A). The probe interacted strongly with the bound EPS resulting in the change of aspect of the extracellular matrix, which was different from the native aspect of the sample without isolectin. Indeed while the extracellular matrix appeared translucent without the addition of isolectin (Figure 1A), it seemed to be solidified due to the marking and appeared clearly surrounding the cells on the transmitted light picture (Figure 2B). Therefore, both samples, with and without isolectin are important to consider. Regarding the channels attributed to phycobiliproteins and to chlorophyll, no important change was seen compared to the sample without the addition of isolectin (Figure 2C

Effect of pH and ionic strength variations on the stability of vesicles

We hypothesize that this aggregation of phycobiliproteins and chlorophyll could result from an association of proteins with the EPS. This kind of association between these two families of molecules and the spherical "capsule-like" aspect of these objects could point to identify them as coacervates [START_REF] Sanchez | Stability and structure of protein-polysaccharide coacervates in the presence of protein aggregates[END_REF][START_REF] Turgeon | Protein-polysaccharide interactions: phase-ordering kinetics, thermodynamic and structural aspects[END_REF]. Coacervation is defined as the separation between two liquid phases in colloidal systems [51] leading to a concentrated colloid phase called "coacervate" and a less concentrated phase being the equilibrium phase. In practice, the coacervate can be solid-like with high viscosity and can be seen as a precipitate [START_REF] De Kruif | Complex coacervation of proteins and anionic polysaccharides[END_REF]. The association occurring is driven by electrostatic interactions and the main parameters influencing the stability of coacervates are pH and ionic strength, acting respectively on the charges of compounds and on entropy [START_REF] De Kruif | Complex coacervation of proteins and anionic polysaccharides[END_REF][START_REF] Turgeon | Protein-polysaccharide complexes and coacervates[END_REF]. Regarding sulfated polysaccharides, including microbial exopolysaccharides, the stability of the precipitates is framed by narrow physicochemical conditions. Optimal conditions appear to be a pH value close to the isoelectric point of protein and NaCl concentration below 0.1 M [START_REF] Galazka | Interactions of ovalbumin with sulphated polysaccharides: effects of pH, ionic strength, heat and high pressure treatment[END_REF][START_REF] Galazka | Complexes of bovine serum albumin with sulphated polysaccharides: effects of pH, ionic strength and high pressure treatment[END_REF][START_REF] Weinbreck | Complexation of Whey Proteins with Carrageenan[END_REF]. For that reason, in this study, the biomass was resuspended in deionized water and in 0.1 M phosphate buffer with different pH values: 5, 7 and 8.5. The pH values of 5 and 8.5 are close to the fluorescence stability limits of B-PE [START_REF] Munier | Physicochemical factors affecting the stability of two pigments: Rphycoerythrin of Grateloupia turuturu and B-phycoerythrin of Porphyridium cruentum[END_REF]. It is noteworthy to mention that the isoelectric point of phycoerythrin ranges between 4.5 and 5.1 depending on what specie it was isolated from [START_REF] Bryant | Phycoerythrocyanin and Phycoerythrin: Properties and Occurrence in Cyanobacteria[END_REF]. Also, for each pH value, 1 M of NaCl was added to the buffer to be in high ionic strength conditions.

For better readability, CLSM pictures presented in Figures 3, 4 and 5 were generated by merging channel 1 (B-PE, yellow), channel 3 (chlorophyll, red), and channel 4 (isolectin, green) when appropriate. B-PE was rather ubiquitous in all the samples. Chlorophyll typically was found in the chloroplast of the cell, and isolectin was meant to label the EPS. By comparing channel 1 and 2 (data not shown), it could be stated that R-PC was associated with the presence of B-PE, so we hypothesized that so was also allophycocyanin. Given the poor amount of R-PC and allophycocyanin compared to B-phycoerythrin in P. cruentum, it was chosen to keep only channel 1 to mark the presence of the phycobiliproteins.

When performing ANOVA on the samples based on water and phosphate buffer with or without NaCl, significant differences of B-PE content and purity index values were found (Table 2, p = 0.003 and p < 0.0001 respectively). However, in the case of the samples based on water and the samples based on pH7 phosphate buffer without addition of NaCl, the B-PE contents and purity indexes of the supernatants were not significantly different. Likewise, the visual observations made by CLSM of those resuspensions were similar (Figure 3). The use of water or pH7 0.1 M phosphate buffer seemed to be equivalent from the perspective of the studied parameters.

In phosphate buffer with and without NaCl, B-PE content and purity index of the samples supernatants were significantly lower at pH5 without NaCl and pH8.5 with and without NaCl compared to the pH7 supernatants with and without NaCl (Table 2). This suggests that pH variations had a stronger impact on the extracellular B-PE content than the addition of NaCl.

When looking at the CLSM images, the only visible change was a more "packed" aspect of the vesicles at pH8.5 without NaCl (Figure 3). Nevertheless, this phenomenon did not occur at pH8.5 with the addition of NaCl. It can be hypothesize that the interactions induced by higher pH leading to the aggregation of the vesicles were suppressed by the higher ionic strength induced by the addition of NaCl.

The fact that vesicles were still present with addition of NaCl is surprising because, in theory, the increase of the ionic strength induced by the addition of NaCl should suppress electrostatic interactions and thus prevent complex formation between molecules [START_REF] Turgeon | Protein-polysaccharide interactions: phase-ordering kinetics, thermodynamic and structural aspects[END_REF][START_REF] Weinbreck | Complex Formation of Whey Proteins: Exocellular Polysaccharide EPS B40[END_REF].

In conclusion for this group of samples, the major change was seen at pH8.5 without NaCl addition with an agglomeration of the vesicles. This suggests that the interactions between the compounds involved in these structures (presumably phycobiliprotein and EPS) were strengthened. Also, the addition of 1 M NaCl could prevent the agglomeration of vesicles at pH8.5 but could not visibly dissociate the vesicles at any pH value. This is contradictory with the theory built around coacervates that states that interactions leading to coacervation and complex formation are strengthened at lower pH, closer to the isoelectric point of protein and at moderate ionic strength [START_REF] Galazka | Interactions of ovalbumin with sulphated polysaccharides: effects of pH, ionic strength, heat and high pressure treatment[END_REF][START_REF] Galazka | Complexes of bovine serum albumin with sulphated polysaccharides: effects of pH, ionic strength and high pressure treatment[END_REF][START_REF] Weinbreck | Complexation of Whey Proteins with Carrageenan[END_REF]. The vesicles should then likely be categorized as precipitates.

With the isolectin dyeing (Figure 4), the extracellular matrix appeared clearly although its aspect was different in phosphate buffer compared to in water, regardless of the pH or NaCl addition. The pH variations and addition of NaCl in phosphate buffer did not seem to impact the cohesion of the extracellular matrix. However, the disappearance of extracellular B-PE fluorescence in sample made of phosphate buffer compared to the sample without isolectin could suggest an adsorption of B-PE by isolectin and thus the existence of protein / protein interactions in those specific conditions (Figure 4). Since this was not the case in the sample in water, this phenomenon could be induced by the higher ionic strength or the presence of ions present in the buffer and could lead to a bias in the observation. However, in all samples including the one made in water, there was a clear juxtaposition of B-PE fluorescence emission with the isolectin marked extracellular matrix, suggesting a trapping of the protein in the matrix network.

To conclude, pH had the strongest impact on the resuspensions in terms of B-PE content in supernatants and organization of the vesicles. Ionic strength variations might play a less impactful role on the interactions between the vesicles, but higher ionic strengths could not manage to meaningfully alter the structure of the vesicles. Moreover, the resuspension in water showed the same B-PE concentration and purity index in the supernatant than the sample obtained with phosphate buffer at pH7 and showed the highest B-PE content in supernatants. Resuspension in deionized water was chosen for the next experiments.

Effect of extraction processes on the stability of vesicles

Successive macerations

The successive macerations lowered significantly the extracellular B-PE content at each step as shown in Figure 5 and Table 2 (p < 0.0001). The abundance and fluorescence emission intensity of vesicles also decreased but some of them were still present after three macerations steps. Most of the B-PE was extracted during the first step (85 mg/g dry biomass). At the second step, only a very small quantity of the previous amount (8 mg/g dry biomass) was extracted. The third step allowed the extraction of a significantly lower amount of B-PE (Table 2). Also, the purity index significantly dropped at each step along the B-PE concentration (p < 0.0001). The same observation could be made for the pH value that did not decrease further than 6.7 (p = 0.0016) (Table 2).

Through the isolectin dyeing, a decrease of the extracellular matrix occurrence leading to the scattering of P. cruentum cells without their enveloping matrix was observed with the increase of macerations steps (Figure 5). After the third resuspension step, few vesicles were still visible between the cells and showed fluorescence emission from B-PE and isolectin (Figure 5). That means that extracellular B-PE bound to EPS was still present in the sample and this highlights the important role that bound EPS plays as an obstacle in the extraction of this protein from P. cruentum.

High pressure homogenizer treatment

Figure 6 shows the results obtained after HPH treatment. The untreated control sample is shown in Figure 6A. Both one-step (Figure 6B) and two-steps treatments (Figure 6C) had similar profiles. While some cells remained whole, a lot of fluorescence-less crescent-looking objects were visible, resulting from shear forces destructing the cells and ripping off bits of the sheath that appear as fluorescence-less crescents. Debris with higher fluorescence emission was also visible (Figures 6B and6C). The treatment by HPH dissociated the extracellular matrix through the application of shear forces, scattering extracellular matrix bits and cells (Figure 6D). With the isolectin dyeing, debris could be identified as bits of bound EPS matrix, and so did chloroplasts, identified by their intense B-PE and chlorophyll fluorescence (Figure 6D). Besides this, very rare vesicles present in the control (Figure 6A)

were seen after one step treatment (Figure 6B) and no vesicles were observed in two-steps treatments (Figure 6D). This observation confirmed the efficiency of the HPH treatment regarding the disruption of cells and extracellular matrix, both representing the biggest obstacles to the extraction of B-PE from P. cruentum. This is consistent with previous reported studies that were made at molecular scale. In fact, some studies [START_REF] Villay | Comparison of polysaccharide degradations by dynamic high-pressure homogenization[END_REF][START_REF] Ye | High pressure homogenization to improve the stability of caseinhydroxypropyl cellulose aqueous systems[END_REF][START_REF] Paquin | Technological properties of high pressure homogenizers: the effect of fat globules, milk proteins, and polysaccharides[END_REF] showed that HPH treatment reduced molecular weight of polysaccharides, thus limiting viscosity.

Surprisingly, there was no significant extraction of B-PE after one-step treatment but only after two-steps treatments where B-PE concentration significantly decreased (p = 0.0115, Table 2). The reasons could be a protein denaturation caused by the high pressure, the elevation of temperature in the system, or both. The purity index significantly dropped after both treatments, but this drop was not significantly different whether the one-step or two-step treatment was applied (p < 0.0046) (table 2). This suggests that the 70 MPa high pressure treatment could extract other intracellular compounds by disrupting the cells, but that the second step at 120 MPa did not make any further effect besides denaturing B-PE.

Conclusion

Benefits of this study are numerous: development of labeling method for the observation of EPS in microalgae, understanding of protein-EPS interactions in situ, impact of some mechanical extractions on these interactions, and, regarding the application purpose, the importance of the bound EPS for extraction yields.

The experiments combining confocal laser scanning microscopy with fluorescent isolectin probe showed that the observed fluorescent objects consisted of an association of phycobiliproteins (mainly B-PE) and EPS. Their stability towards pH and ionic strength variations arouses interests since the understanding of this system could help building more stable protein / polysaccharide complexes. The observed fluorescent vesicles do not fit in the theoretical behavior of coacervates and may rather be stable precipitates.

Still, this experiment was made in the context of a complex environment and some uncontrolled parameters such as the presence of endogenous ions or other proteins could play a role in the stability of the vesicles. Further understanding of the molecular interactions involved between B-PE and EPS of P. cruentum could be investigated by working in model conditions, using purified protein and EPS.

Also, this study highlights for the first time in detail the importance of bound EPS matrix towards the extraction of target compounds (such as B-PE) as an obstacle in the case of P. cruentum biomass: this extracellular matrix, encapsulating the cells and the smaller vesicles, acts as a shelter against disruption processes and is likely to trap target compounds both by physical entrapment and through molecular interactions. Successive macerations with water or HPH treatment are efficient methods to free the P. cruentum cells and B-PE from the bound EPS matrix. However, maceration is a simple method and provides the extract with the greatest amount of BPE and the best purity index within our experimental conditions.

Another way to improve extraction could be to frame appropriate cultural conditions and growth phase in order to harvest microalgae with limited amount of bound EPS.

Abstract:

During the extraction processes of B-phycoerythrin from P. cruentum, the possibility of the occurrence of extraction-inhibiting B-phycoerythrin / exopolysaccharide (EPS) complexes was highlighted. The use of confocal laser scanning microscopy allowed the observation of the cells including their sheath and their chloroplast, the bound EPS matrix, and extracellular vesicles involving phycobiliproteins and EPS in P. cruentum biomass resuspensions. The observations were performed using autofluorescence and fluorescence marking.

pH variations showed more impact than ionic strength increase induced by NaCl addition on the B-phycoerythrin content and the organization of vesicles. Namely, agglomeration of the vesicles could be observed at pH8.5 without NaCl addition. With addition of 1 M NaCl, this phenomenon was prevented but the vesicles were not visibly dissociated for any pH value.

These results point towards a precipitate rather than a coacervate, which are expected to have narrow stability conditions regarding pH and ionic strength.

In an attempt to dissolve or break the vesicles and the extracellular matrix, successive macerations in water and use of high pressure homogenizer process in one-step or two-step treatments were applied to the resuspensions. The successive macerations could free the cells and the vesicles from the extracellular matrix, whereas the homogenizer treatment disrupted the extracellular matrix, the vesicle and a part of the cells. In our case, only the successive macerations showed a positive extraction since the high pressure treatment caused unwanted denaturation.

It is concluded that more experiments in model conditions could help understand the nature of the interactions between phycobiliproteins and EPS leading to the formation of the vesicles.

Porphyridium cruentum, confocal fluorescence microscopy, complexation, B-phycoerythrin, exopolysaccharide.

Graphical abstract

follows: Thierry Tran, Céline Lafarge, Pascale Winckler, Rémi Pradelles, Nathalie Cayot, Camille Loupiac. Medium used for resuspensions pH B-phycoerythrin (mg/g dry biomass)

Purity Index (A545/A280) 0.2 g dry biomass / 100 mL biomass in Deionized water 6.9 ± 0.0 e 85 ± 1 a 1.5 ± 0.0 abc 0.1 M phosphate buffer pH 7

7.1 ± 0.0 c 86 ± 6 a 1.5 ± 0.0 a 0.1 M phosphate buffer pH 5 5.4 ± 0.0 f 79 ± 4 abc 1.5 ± 0.0 ab 0.1 M phosphate buffer pH 8.

  , 2D and 2E versus Figure1C, 1D and 1E). According to Figures2C, 2Dand 2E, the extracellular matrix, corresponding to the bound EPS matrix, was wrapped around cells and vesicles, keeping the whole structure together. Also, vesicles happened to be lightly marked by isolectin as well, suggesting that EPS were involved in the vesicle composition (Figure2C, 2D and 2E). It is plain to imagine that this extracellular matrix can prevent the extraction of B-PE and other compounds by protecting the cells and possibly hold back substances, by physical entrapment or by involving molecular interactions.

Figure 1 :

 1 Figure 1: Fluorescence emission images obtained by CLSM from P.cruentum resuspension at 0.2 g dry biomass / 100 mL in deionized

Figure 2 :

 2 Figure 2: Fluorescence emission images obtained by CLSM from P.cruentum resuspension at 0.2 g dry biomass / 100 mL deionized water

Figure 3 :

 3 Figure 3: Image of mixed 1 and 3 fluorescence emission channels (channel 1 attributed to fluorescence emission of B-phycoerythrin and

Figure 4 :

 4 Figure 4: Image of mixed 1, 3 and 4 fluorescence emission channels (channel 1 attributed to fluorescence emission of B-phycoerythrin,

Figure 5 :

 5 Figure 5: Image of mixed 1, 3 and 4 fluorescence (when appropriate) emission channels (channel 1 attributed to fluorescence emission of

Figure 6 :Table 2 :

 62 Figure 6: Image of mixed 1, 3 and 4 fluorescence (when appropriate) emission channels (channel 1 attributed to fluorescence emission of

Acknowledgement

This work was supported by the Regional Council of Bourgogne -Franche Comté and the "Fonds Européens de Développement Régional" (FEDER) for the "PARI Alim+" project.

Highlights

-P. cruentum chloroplast and EPS were labelled and observed by confocal microscopy.

-Phycobiliproteins/EPS vesicles were identified.

-Vesicles were found stable towards pH and ionic strength.

-EPS inhibited the extraction of B-phycoerythrin.

Declaration of authors contributions

Camille Loupiac, Nathalie Cayot, Céline Lafarge and Thierry Tran designed the experiment. Thierry Tran and Céline Lafarge carried out the experiment, acquired, processed and interpreted the data. Pascale Winckler provided access and assistance to the use of confocal microscopy. Rémi Pradelles provided the microalgal biomass. Thierry Tran took the lead in writing the manuscript but all authors provided critical feedbacks and helped shape the research, analysis and manuscript.

Conflict of interest statement

The authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Statement of Informed Consent, Human/Animal Rights

No conflicts, informed consent, human or animal rights are applicable for this work.

Declaration of authors agreement to authorship and submission of the manuscript for peer review.

The authors declare that this manuscript is original, has not been published before and is not currently being considered for publication elsewhere. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship. We further confirm that the order of authors listed in the manuscript has been approved by all of us. The Corresponding Author is the sole contact for the Editorial process. She is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. Signed by all authors as a : according to [START_REF] French | Fluorescence-Spectrum Curves of Chlorophylls, Pheophytins, Phycoerythrins, Phycocyanins and Hypericin[END_REF][START_REF] Gantt | Phycobilisomes of Porphyridium cruentum. Pigment analysis[END_REF] for phycobiliproteins and [START_REF] Murata | Fluorescence of Chlorophyll in Photosynthetic Systems[END_REF][START_REF] Murata | Light-Induced Change of Chlorophyll a Fluorescence in Porphyridium cruentum[END_REF][START_REF] Brody | Action Spectra for Sensitization of Ligth Emission from Monomeric and Aggregated Chlorophyll at Physiological and Liquid Nitrogen Temperatures[END_REF] for chlorophyll and for [START_REF] Murphy | Five alpha-D-galactopyranosyl-binding isolectins from Bandeiraea simplicifolia seeds[END_REF] isolectin.