Marion Schiavone

Nathalie Sieczkowski

Mathieu Castex

Etienne Dague

Jean Marie François

Effects of the strain background and autolysis process on the composition and biophysical properties of the cell wall from two different industrial yeasts

Keywords: Cell wall, autolysis, atomic force microscopy, β-glucans, chitin, yeast

The Saccharomyces cerevisiae cell surface is endowed with several technological properties, among which its effective biosorption capacity to reduce undesirable molecules, such as volatiles phenols in wine or mycotoxins in food. Since cell surface properties are intimately linked to cell wall structure, the aim of this study was to investigate effects of autolyzing/drying process on the biochemical composition of cell wall and on the nanomechanical properties of cell surface. To achieve these goals, we used a recently developed method to analyze the biochemical composition, and we took advantage of Atomic Force Microscopy to access the nanomechanical properties. We choose to work on two industrial strains because of their difference in winemaking applications and sorption properties. We found that the autolysis/drying process of the two strains did not significantly modify the biochemical composition of their cell wall. It, however, caused severe changes in cell surface topography characterized by a 4-fold increase of the roughness and by a global increase in adhesion characteristics of the autolyzed/dried samples. Though this process had some effects on biomechanical properties, the two strains natively harbored differences in biophysical properties that could be accounted by difference in cell wall composition, as the strain with the highest mannoproteins content was also characterized by the presence of highly adhesive patches forming nanodomains,. Comparative transcriptome analysis uncovered a strong upregulation of flocculin encoding FLO11 gene in this industrial strain that corroborated with higher interaction to concanavalin A-functionalized AFM tip, leading to the idea that this protein is responsible for the patches formation.

Introduction

The yeast Saccharomyces cerevisiae is used since millennia in traditional biotechnological purposes, such as the production of wine, beer and bread. In addition to this role in fermented foods and beverages, this yeast species is attracting increased attention because of other relevant biological properties, such as antimicrobial and biosorption activities, that make them promising candidates for a wide range of applications not limited to the food sector. Indeed, the antimicrobial activities against undesirable bacteria and mold have been recognized since a long time to provide probiotic properties of yeasts, although the mechanisms underlying these antagonistic activities remain unclear {Hatoum, 2012 5788 /id}. Biosorption is a cell surface property that is defined as the physicochemical process wherein undesirable molecules which can be a toxin, heavy metals or volatile compounds interact and accumulate at the surface of microbial cells {Petruzzi, 2014 5810 /id;Fomina, 2014 5809 /id}. Since this property is displayed by inactive or dead cells and does not require intracellular energy, the physicochemical properties of the microorganism's surface are determining factor in the nature of the interaction and in the sorption capacity. The surface of yeast cell corresponds to a thick wall of about 120-180 nm, exhibiting an ultrastructure of two distinct layers as visualized by electron microscopy {Osumi, 1998 235 /id}: an outer layer mainly constituted of mannoproteins (30-50% of cell wall dry mass) and an inner layer made of chitin (2 -5 % of cell wall dry mass) and -glucans (40 -60 % of cell wall dry mass). Extensive biochemical analysis allowed to propose a supramolecular structure of the mature cell wall in which the chains of -(1,3) glucose residues are branched to -(1,6)-glucans, forming a fibrillar network, that serves as backbone to which are linked chitin, -(1,6)-glucan and mannoproteins. In addition, the outer layer of mannoproteins can be linked to the inner layer through -(1,6)-glucan via a remnant of a GPI anchor {Lesage, 2006 3212 /id}. It is therefore suggested that the sorption capacity for a given molecule can be linked to a specific component of the yeast cell wall. This suggestion has been verified in very few cases. For example, a major role of mannoproteins in the retention of volatile aroma by yeast cell walls has been reported {Lubbers, 1994 551 /id;Pradelles, 2008 5792 /id}, whereas -glucan was showed as a valuable microbiological binder of the mycotoxin zearalenone {Yiannikouris, 2004 3122 /id}. However, these studies were mostly carried out with pure fractions of polysaccharides and did take into account neither the complexity and variability of the cell wall composition, that can depend on culture conditions {Aguilar-Uscanga, 2003 1148 /id} and strains background {Nguyen, 1998 432 /id}, nor the process by which yeast cells are prepared and used as biosorbant. In this context, Pradelles et al {Pradelles, 2008 5792 /id} investigated effects of cell wall composition on the sorption of the undesirable 4-ethylphenol. While they confirmed a predominant role of mannoproteins in the sorption capacity of this volatile aromatic compound, they also highlighted the fact that drying the yeast biomass greatly increased the sorption capacity of this molecule, which was in part correlated with increased surface hydrophobicity of the dried biomass rehydrated subsequently in water {Pradelles, 2009 5793 /id}. Taken together, these results indicate that the drying process and more generally the method to prepare yeast cell wall fractions for biosorption applications may have a strong impact on physico-chemical properties of the cell surface. This question is particularly pertinent when we consider that yeast cells employed for biosorption applications are obtained from cultures that are autolysed and air-dried at 55°C, and commercially supplied as 'yeast cell wall' fraction or YCW. Autolysis is a term that describes the breakdown of cell constituents by action of endogenous enzymes. It can occur naturally when yeast have completed their growth cycle and entered the death phase, or it can be induced at high temperature and low pH {White, 2002 4963 /id} {Martinez-Rodriguez, 2009 455 /id}. Proteases, -glucanases and chitinases are among autolytic hydrolases that are implicated in this process, and therefore, it can be expected that this process may cause important change in cell wall composition. Accordingly, Martinez-Rodriguez et al {Martinez-Rodriguez, 2001 4964 /id} reported important ultrastructural changes during the autolysis of yeast cells using Low Temperature Scanning Electron Microscopy (LTSEM), whereas Aerosol Flow Tube -Fourier Transform Infrared Spectroscopy (AFT-FTIR) that was used to monitor global biochemical changes during autolysis revealed hydrolysis of mannans and -glucans. In spite of these apparent hydrolyses, microscopic observation of autolyzed cells showed that the cell shape was fully retained suggesting that the cell wall structure was not destroyed {Hernawan, 1995 4965 /id}. Altogether, these studies raised fundamental questions about the effects of industrial processes on structure and properties of yeast cell surface. These questions can be addressed using Atomic Force Microscope (AFM) which is a very powerful force microscope technology allowing tridimensional images of cell surface and quantitative measurement of nanomechanical properties such as roughness and elasticity (Young modulus) {Dague, 2007 443 /id;Dufrene, 2010 5053 /id}. We used recently this technology to image and quantify surface properties of yeast cell wall mutants {Dague, 2010 5052 /id}, to investigate effects of antifungal drug caspofungin and heat shock on yeast cells (Formosa et al., 2013;Pillet et al., 2014). These studies led to the finding that the nanomechanical properties of the cell surface are merely dependent on the molecular architecture of the cell wall and not on a specific polysaccharide component of the wall. To gain deeper knowledge on the molecular organization of cell wall, the single molecule force spectroscopy (SMFS) can be employed. This method consists in probing the cell with an AFM tip that is functionalized with a specific ligand that could interact with some proteins present at the cell surface {Hinterdorfer, 2012 463 /id}. Accordingly, probing the yeast cell surface with AFM tip functionalized with concanavalin A (conA tip), which is a lectin protein that interacts with D-mannose and glucose residues (ref), has provided quantitative information about distribution, frequency of adhesion events, flexibility and extension of mannans on the surface of different yeast strains {Dufrene, 2013 459 /id}. With this approach, Alsteens et al. {Alsteens, 2008 457 /id} investigated the surface properties of a bottom-fermenting and a top-fermenting yeast. They showed that the latter species has higher adhesion frequency and more extended polysaccharides than the former one, consistent with the fact that the surface of top-fermenting yeast cells is richer in proteins and more hydrophobic.

The purpose of this study was therefore to investigate effects of industrial autolysis and drying process on cell wall composition and cell surface nanomechanical properties of two industrial yeast strains that bear differences in winemaking properties. Effects of this process on adhesive properties and on polysaccharides distribution using conA tip of the two strains were also analyzed 120 by comparing whole active-living from rehydrated dried yeast cells. 121

Material and Methods

Strains and culture conditions

Two diploid industrial winemaking Saccharomyces cerevisiae strains, L71 and L69, from Lallemand Inc. (Blagnac, France) were studied. Differences between the two strain was mainly that the L69 strain has a higher mannoproteins content, expresses different sensorial notes during wine fermentation and presents good capacity to interact with volatile undesirable phenols (Lallemand Inc., unpublished data). The strains were propagated under standard laboratory conditions, which corresponded to growth in 200 ml YPD medium (1% [w/v] yeast extract, 2% [w/v] bactopeptone and 2% [w/v] glucose) in 1 liter shake flasks at 30°C under shaking at 200 rpm. They were also propagated under industrial standard protocol, involving batch and fed-batch growth on molasses based medium, collected, and concentrated to produce packed yeast cells termed 'cream'. The latter cream was then subjected to autolysis (20 hr at 55°C), followed by separation and then by spraydrying to produce the dry "YCW" (industrial term meaning Yeast Cell Wall fraction that is used for this type of yeast packaging). This autolytic/drying process leads to > 99.9 % of cell mortality as estimated by methylene blue coloration test {Cot, 2007 5468 /id}. YCW were provided in 30 g packets sealed under vacuum and stored unopened at 4°C until required. Rehydration was performed according to the manufacturer's guidelines (Lallemand Inc., Blagnac, France): 0.1 g of YCW was sprinkled onto 10× its weight of temperature equilibrated (30°C) sterile water in a tube. The YCW was left to absorb water slowly for 20 min, mixed gently to form slurry and the temperature was maintained at 30°C throughout rehydration.

AFM measurements

Sample preparation

Cells collected from exponential growth in YPD or after rehydration of YCW were washed two times in sodium acetate (18 mM CH 3 COONa, 1 mM CaCl 2 and, 1 mM MnCl 2 , pH 5.2), and immobilized on polydimethylsiloxane (PDMS) stamps prepared as described in {Dague, 2011 442 /id}. Briefly, freshly oxygen activated microstructured PDMS stamps were covered by 100 µL of yeasts sample at OD 600 around 1.0. The cells were then deposited into the microstructures of the stamp by convective/capillary assembly.

AFM imaging

Images and force-distance curves were recorded at room temperature in acetate solution using an AFM Nanowizard III (JPK Instruments, Berlin, Germany) and MLCT AUWH cantilevers (Bruker, Santa Barbara, USA). The spring constants of the cantilevers were systematically measured by the thermal noise method according to {Hutter JL, 1993 5529 /id} and were found to be in the range of 0.01-0.02 N.m -1 . AFM height and adhesion images were recorded in Quantitative Imaging TM mode {Chopinet, 2013 454 /id}, and the maximal force applied to the cell was limited to 1.5 nN.

Surface roughness measurements

To measure cell surface roughness, images were obtained by scanning at high resolution (1µm x 1µm areas) on independent cell samples (at least 5) in the contact mode. In contact mode, the tip is in contact with the surface of the sample and scans this surface horizontally with a constant force, which is lower as possible. Height and vertical deflection images were both recorded with the contact mode. Height images with the same center of offset were processed and analyzed with the power spectral density method (JPK data processing software) consisting of average roughness (Ra in nm) measurements on five boxes of five different sizes for each images.

Stiffness measurements

Results were analyzed using the Data Processing software from JPK Instruments. The stiffness value (k cell in N.m -1 ) measured on cells was determined from the cantilever spring constant (k) and the slope (s) of the linear part of the force curve according to {Arnoldi, 2000 4684 /id}:

Force spectroscopy

For force spectroscopy experiments, the applied force was kept constant at 0.5 nN. To probe cell surface polysaccharides, AFM tips were functionalized with the concanavalin A (ConA) from Canavalia ensiformis (Sigma-Aldrich, L7647) via a dendritip as described in {Jauvert, 2012 5569 /id}.

The coupling with the lectin was made by immersion of the dendritip in 100 µL of ConA solution (100 µg.ml -1 in a 0.1 M carbonate buffer). After 1 hr incubation, 100 µL of NaBH 4 3.5 mg.mL -1 solution was added and incubated 15 min in order to reduce the unreacted groups. Finally, the cantilever bearing the functionalized tip was washed three times and stored in acetate buffer. To analyze the stretching of polysaccharides at the surface of the cell, elongation forces were stretched using the worm-like chain (WLC) model introduced by Bustamante {Bustamante, 1994 5811 /id} which describes the polymer as a curved filament and the force F vs the extension x is given by:

F(x) = k b T/l p [0.25 (1-x/L c ) -2 + x/L c -0.25]
where the persistent length (l p ) represents the stiffness of the molecule, the contour length (L c ) is the total length of the stretched molecule, k b is the Boltzmann constant and T is the absolute temperature. This model has already been successfully used for the stretching of polysaccharides of yeasts especially for S. cerevisiae and S. carlsbergensis {Alsteens, 2008 457 /id} and gives the best fitting of force curves. Blocking control experiments were performed by injecting 100 mM Dmannose solution into the cell surface.

Cell wall isolation and quantification of polysaccharides

Yeast cell walls were extracted and purified from yeast culture of L71 and 69 taken in exponential phase of growth on glucose (OD 600 =1), rehydrated YCW and 'cream' according to the protocol described in {Francois, 2006 4678 /id}. Cell wall polysaccharides mannan, chitin, -(1,3) and -(1,6) glucan in the purified cell walls were determined by a combination of acid and enzymatic hydrolysis recently developed {Schiavone, 2014 457 /id} and quantification of the release sugar monomers (mannose, glucose and N-acetylglucosamine) was determined by High Performance Anionic Exchange Chromatography (HPAEC) with Pulsed Amperometric Detection (PAD) as described in {Dallies, 1998 224 /id}.

Transcriptomic analyses

Three independent biological cultures of industrial strains L71 and L69 were carried out in 50 ml of YPD in a 250 ml shake flasks. Yeast cells (about 10 OD 600 units) were collected at OD 600 =1 by centrifugation (3,000 rpm, 4°C, 2 min), followed by a washing step with 1 ml of sterilized water. The Transcriptome analyses were done in R computing environment (www.R-project.org) using the Limma package {Yang, 2002 2877 /id} (www.bioconductor.org). The estimates used for the foreground and background intensities were the median of pixels intensity. Raw data were imported into R and spot quality weights were performed assigning a weight of 1 or 0 to each spot. Low-quality spots, non-uniform spots, spots with low signal/background ratio or spots with low signal-to-noise ratio and empty or non-validated spots were down weighted. Data were preprocessed by base 2 logarithmic transformation and within-array normalized was performed using the weighted global median (spots with zero weight were not included in the normalization). To achieve consistency of expression values between arrays, normalization across all the microarrays for each strain was performed. After normalization, the expression of a gene was calculated by the median of replicate spots within each microarray. Gene expression data for both strains were pairwise compared using the Limma package {Smith, 2005 5083 /id}. Genes with significant evidence for differential expression were identified with a modified t-test in conjunction with an empirical Bayes method to moderate the standard errors of the estimated log-fold changes. The P-values for the genes of interest were adjusted for multiple testing by the ''BH'' method {Hochberg, 1990 3432 /id}.

Results

Effects of autolysis/drying process on cell wall composition of two industrial yeast strains.

Since it is considered that the interaction cell-environment and sorption capacity to undesirable compounds are dependent on the surface properties of yeasts, which are in turn impacted by the biochemical composition of cell wall {Pradelles, 2008 5792 /id;Nunez, 2008 4953 /id;Armando, 2012 5219 /id}, we aimed at investigating effects of the industrial autolysis/drying process on cell wall composition of two industrial strains. These strains were chosen on the basis of their different winemaking behavior and content of mannoproteins (Lallemand Inc, unpublished data). To evaluate impact of the process, we compared the polysaccharides composition of cell walls purified from autolyzed/dried yeast cells, which are commercialized as "YCW", with those extracted from the same industrial strains that were cultivated under laboratory standard conditions (Yeast Peptone Dextrose medium) and harvested at the exponential phase of growth. The cell wall composition was also determined on packed yeast cells which are provided as 'cream' and which was obtained from batch fermentation in molasses-based medium. Results of this analysis are reported in Table 1. Overall, the polysaccharides content in purified cell wall was between 85 to 90 % of the cell wall dry mass, the remaining 10 to 15 % corresponded to proteins (data not shown). Also, the proportion of -(1,3), -(1,6)-glucan, mannan and chitin in cell wall of the two industrial strains were statistically not different (p value > 0.05) between the three conditions/ treatments investigated.

However, it can be noticed a strain effect on the biochemical composition of the cell wall that was statistically significant. Indeed, strain L71 either in a living, cream or dried stated exhibited two times more chitin (p value < 0.001) and a higher proportion of -(1,6)-glucan in total -glucan than in strain L69. On the other hand, the mannan content of the L69 strain was roughly 20 -25 % higher than in strain L71 (p value < 0.01), which agreed with the fact that this industrial winemaking strain was selected on the basis of its higher production of mannoproteins. This high mannan content of strain L69 could be associated with the fact that this strain has a higher surface hydrophobicity than strain L71 (data not shown) as well as to a tendency to hold cells together in small branched chain during growth and to produce clumps upon rehydration of YCW produced from this strain (data not shown).

Effects of autolysis/drying process on nanomechanical properties of two industrial yeast strains.

In accordance with a previous work {Hernawan, 1995 4965 /id}, rehydrated YCW regained round to ovoid cell shape like a normal living yeast cell (data not shown). This cell shape retention allowed using our immobilization methodology that is based on trapping of single round/ovoid yeast cell into microchambers made in microstructured PDMS stamp by capillary/convective means {Dague, 2011 442 /id}. This methodology is very efficient to readily monitor several isolated cells in liquid environment by AFM. Topographic images using Quantitative Imaging™ mode {Chopinet, 2013 454 /id} of living yeast cells from strain L71 and rehydrated YCW derived from L71 strain is reported in Figure 1 (a and b). For both types of yeast samples, height images illustrated a rather round shape of the immobilized object with an apparent smooth surface. However, a closer inspection of the AFM images from the rehydrated sample revealed the presence of a single hole of 600 nm diameter size curiously localized in the middle of immobilized cell (Fig. S1 in supplementary data), and this feature was obtained in at least 15 % of the analyzed cells. At higher resolution, height images (Fig. 1 c andd) from contact mode clearly showed significant differences of the surface between living and rehydrated sample from L71 strain, as the surface of the latter was severely fractured. In quantitative terms, the roughness of cell surface (determined on 1 µm x 1 µm areas) was around 1.9 ± 0.2 nm for the living cell and increased to 5.1 ± 1.1 nm for the rehydrated YCW sample (Table 2). This surface change could be linked to the ultrastructural changes that was reported by Low Temperature Scanning Electron Microscopy (LSTEM) of a commercial yeast strain after 14 hr of induced autolysis in a model wine medium {Martinez-Rodriguez, 2001 4964 /id}. Figure 1 (e, f) shows adhesion images of a living and rehydrated cell from YCW of L71 strain that were obtained by probing the surface with an AFM tip in QI™ mode. As the tip is made of silicon nitride and the immobilized sample is under an acidic pH (sodium acetate 18 mM at pH 5.2), these conditions most likely favor hydrophobic interactions, which are however relatively weak with both type of cells, and hence agreed with exposure of 'neutral' cell wall carbohydrates.

Similar AFM analyses were carried out with active living yeast cells and rehydrated YCW of L69 strain. The topographic images taken at low resolution (Fig 2, a andc) did not show difference in both types of cell samples immobilized in PDMS (Fig. 2). At higher resolution, some discretional dots could be visualized that were rather larger in rehydrated YCW (Fig 2, b andd). In addition, we found a hole in immobilized rehydrated samples of YCW in about at least 15 % of the AFM images, like it was seen for strain L71 (see Fig. S1 supplementary data). The force curves acquired in QI mode were also analyzed to obtain adhesion images. This analysis revealed adhesives patches that were not present in L71 strain and which became apparently bigger in the sample from rehydrated YCW (compared Fig. 2 a, e, i for active yeast cells with c, g and k for rehydrated YCW). On average, the patches from the rehydrated YCW had a mean area of 156663.4 ± 3904.4 nm 2 (0.016 ± 0.004 µm 2 ) and a diameter of 141 ± 32 nm (measured from 56 patches on 7 different cells with Image J software). Additional details of the morphology and mechanical properties of these patches are reported in Figure 3 and -40 nm (Fig. 3A). These protuberances strongly interacted with the AFM tip (Fig. 3B). Example of adhesion force is schematically illustrated by force-distance curves taken from 3 different patches (Fig. 3B). On the contrary, these patches exhibited lower stiffness than the ground surface of the rehydrated YCW sample. Recently, similar observation was made with other Candida albicans cells that also harbored nanodomains at its cell surface represented by hydrophobic patches that were likely due to high expression the ALS1 encoding cell surface protein (Alsteens et al., 2010;Formosa et al., 2014, Nanomedicine). Taken together, these results provide some clues about the surface properties of L69 strain which were distinct from those of L71 strain.

Probing mannans polysaccharides at the cell surface using an AFM tip functionalized with

Concanavalin A.

To go deeper in the analysis of the process effect on cell surface properties and illustrate differences in cell surface between the two industrial strains, we employed the single molecule force spectroscopy (SMFS) using an AFM tip functionalized with ConA. This molecule is a lectin protein that has high affinity to α-mannosyl residues, with an unbinding force which corresponds to the rupture of a single lectin-mannose has been estimated in the range of 60 pN {Alsteens, 2008 457 /id}. Figure 4 shows results of distribution, adhesion and flexibility of mannans at the surface of a living and YCW rehydrated from L71 strain. Adhesion force maps (Fig. 4, b and f) obtained with 1024 force-distance curves recorded with the ConA tip showed a relatively homogenous distribution of mannans polysaccharides across the surface of the rehydrated L71 sample, whereas this distribution was apparently less regular for the living cell. However, the adhesion frequency which represent the percentage of single or multiple unbinding forces was in the range of 25 % for both types of yeast samples (Fig. 4, c and g) with a mean value of unbinding forces around a maximum of 60 pN (58 ± 21 pN for the living cell and 65 ± 38 pN for the rehydrated sample, this difference being statistically not significant with a calculated p value > 0.05 ) in accordance with a previous report {Alsteens, 2008 457 /id}. Moreover, this binding interaction was blocked at 92.5 % by addition of excess of mannose solution onto the immobilized samples (see Fig. S2, in supplementary data). This result indicated that the adhesion force merely originates from specific lectin-mannans interactions. However, the 7.5% residual adhesion suggested some unspecific interactions. We also noticed that bud scars in both type of yeast samples were not adhesive (data not shown). An additional property of the mannan polysaccharides that can be obtained using this SMFS approach is the rupture distances which define the distance needed to retract the conA tip from its binding with the mannan polysaccharides. These data can be informative on the flexibility and extension of these macromolecules. As can be seen in Fig. 4 (d andh), the rupture distances recorded for both types of samples was comprised between 0 and 250 nm, with a slight wider distribution towards larger lengths for the rehydrated sample suggesting that the polysaccharides chains in this sample were slightly more adhesive and extended than in living cells.

We then carried out a similar experiment with strain L69. Results of this SMFS analysis using

ConA tip are reported in Figure 5. It can be seen that, like for the previous L71 strain, adhesion force map from the rehydrated YCW of L69 strain was more homogeneous than that of a living cell.

However, longer rupture distances (up to 350 nm) were recorded on the living cell of L69 strain as compared to those with living L71 cells (compare Fig. 4d and Fig. 5d), suggesting difference in the stretching of polysaccharides between these two strains. In addition, longer rupture distances in L69 than in L71 strain were consistent with higher adhesion and presence of patches at the cell surface of the L69 strain. Alternatively or complementary to this explanation, it is possible that L69 strain expressed more and/or different mannoproteins than L71 strain. Also, on average, the rupture distance obtained with living cell of L69 was apparently longer than that of the rehydrated YCW of the same strain (Fig. 5 d andh).

Transcriptomic analyses to infer differences in cell surface properties between the two industrial strains.

To seek for a molecular explanation of the difference between cell surface properties between the two industrial strains, we carried out a genome-wide scale expression analysis using DNA microarrays. On a global view, we obtained 392 differentially expressed genes between L69 versus L71 strain, that were distributed into 175 upregulated and 217 downregulated genes (see table S1 and S2 in supplementary material). Major differences between L69 versus L71 strains were found in the downregulation of several genes implicated in sulfate and methionine metabolism as well as in a relatively increased expression of genes with yet unclear function. We looked more carefully to genes implicated in cell wall biogenesis and assembly to find out potential differentially expressed genes that could be linked to difference in cell surface properties between the two strains.

As reported in Table 2, strain L69 was characterized by higher expression levels of genes encoding mannoproteins. In particular, FLO11 encoding a mucin-like protein that belongs to the flocculation gene family {Lo, 1996 164 /id;Van Mulders, 2009 5797 /id} was strongly upregulated in L69 strain.

Since this protein has a highly hydrophobic character {Karunanithi, 2010 5795 /id}, this can explain the hydrophobic adhesions recorded by AFM (Fig. 3). In addition, the relative higher expression of YHR213w encoding a putative flocculin may further contribute to this hydrophobicity property as well as to the presence of patches at the cell surface of L69 strain. There was however some cell wall encoding genes whose expression was slightly more expressed in strain L71 relative to L69 strain, such as FLO5 encoding a lectin-like cell wall protein, but this differential expression can be considered as insignificant to account for difference in adhesion properties between the two strains.

Discussion

Combination of biochemical analyses and AFM technology allows us to observe that packaging yeast cells as "YCW" by autolysis and drying process significantly affected the global surface properties of the cells, in spite of the fact that the biochemical composition of the cell wall was barely altered by this packaging process. This result reinforces our previous finding showing no direct relationship between the polysaccharides composition of cell wall and its bionanomechanical properties {Dague, 2010 5052 /id;Pillet, 2014 5726 /id;Formosa, 2013 5624 /id}. Also, the finding that cell wall composition remained barely unchanged after autolysis and drying process is at variance to other reports that indicated some loss of -glucan and mannans during this process {Cavagna, 2010 4950 /id;Giovani, 2007 4954 /id}. However, the discrepancy can be explained by the difference in the autolysis process. In our study, it is an industrial process that consisted in an induced autolysis at high temperature for 20 h, whereas other studies were related to yeast autolysis during wine fermentation that lasted several days and was done at lower temperature {Martinez-Rodriguez, 2009 455 /id;Alexandre, 2006 456 /id}. As compared to living or active yeast cells, the main effect of autolysis/drying process was to increase the roughness of the cell surface, which was visualized as a change from a relatively smooth to wrinkled structure. It remains to evaluate whether this modification has some impact in the sorption capacity of the yeast cells.

In a previous work, we showed that the mode of cultivation and medium composition influenced the cell wall composition {Aguilar-Uscanga, 2003 1148 /id}, while other studies demonstrated differences in cell wall composition among different yeasts species {Nguyen, 1998 432 /id}. Here, we furthermore showed that even for a same yeast species that is endowed with same genome, the biochemical composition of the cell wall and its cell surface organization can be dramatically different. These differences can be explained in part by difference in gene expression levels. Indeed, we found that the transcript levels of FLO11 encoding a flocculin and of YHR213w that is suggested to code also for a flocculin, were relatively much higher in L69 than in L71 strain. In addition, several patches in the nanometer size were detected at the cell surface of L69 strain by AFM that resembled nanodomains identified at the cell surface of the pathogenic yeast C. albicans (Alsteens et al., 2010;Formosa et al., 2014). In C. albicans, these nanodomains were attributed to the presence of massive amount of ALS1 encoding adhesins which forms amyloid due to seven residues sequence enriched in -branched amino acids (Ile, Thre, Val) that are predicted to form intramolecular -sheet like interactions or amyloids {Lipke, 2012 449 /id}. Recently, it was found that the sequence of the S. cerevisiae Flo11 protein presents such amyloid forming motifs VVSTTV and VTTAVT that can lead to partial -aggregation {Ramsook, 2010 5812 /id}. Therefore, the higher expression for this flocculin encoding gene in L69 strain may account for the formation of the nanodomains, for the difference in the stretching properties of the mannoprotein of this strain as compared to L71 strain, as well as for the tendency of L69 cells to form aggregates (data not shown).

Furthermore, the higher content of mannans measured in L69 strain can be also explained by a higher level in Flo11 which is known to be a very highly glycosylated protein as well as by other GPIanchored mannoproteins, since the expression of their corresponding genes was found increased as compared to strain L71. On the other side, the reduced levels of -(1,6) glucan and chitin in L69 strain were not associated with reduced expression levels of the main genes related to their biosynthesis or regulation {Lesage, 2006 3212 /id}, suggesting that these differences between the two strains would be at the post-transcriptional regulation. For chitin, it is well established that chitin synthase 3 (CSIII) encoded by CHS3 is responsible for > 90 % of the chitin in exponential growing yeast and that this protein is regulated at the post-translational level, which implicated notably an endocytic process mechanism that retrieves CSIII from chitosomes to plasma membrane (review in {Orlean, 2012 5610 /id}). Regulation of -(1,6)-glucan is also very complex and takes place along the secretory pathway because several of the proteins implicated in the synthesis of this polymer were localized in the ER, Golgi or plasma membrane. Thus, the synthesis of -glucan can be regulated at the level of any of these enzymatic steps during this process.

In conclusion, our combined biochemical and biophysical analyses of living-active and autolyzed cells confirmed, on the one hand that for a given yeast strain, changes in biomechanical properties of the cell surface caused by the process packaging are not linked to changes in cell wall composition. On the other hand, the significant difference in a particular and specific cell wall component between yeast strains could result in important difference in the biophysical properties of the surface between these strains. L71. The living cell was from an exponential culture of L71 strain on YPD medium, whereas the rehydrated cell were from autolysed/dried sample of L71 strain obtained as "YCW" and rehydrated according to manufacturer's recommendation as described in Material & Methods. The lived cell was from an exponential culture of L69 strain on YPD medium, whereas rehydrated cells were from "YCW" of strain L69 rehydrated according to manufacturer's recommendation as described in Material & Methods. Culture YPD* 1.9 ± 0.2 1.7 ± 0.2 autolysis/drying 5.1 ± 1.1 6.4 ± 1.4

Legend to figures:

Supplementary data:

The yeast strain L69 and L71 (Lallemand Inc collection) were cultivated in shake in flask under laboratory condition (Yeast Peptone Dextrose medium, 30°C) or obtained as 'cream' or 'YCW'

(autolysis/drying process (24h at 55°C). Surface roughness was calculated on AFM high resolution images of 1 x 1 µm on a total of 15 cells taken from three independent biological replicates for each condition. *Differential genes expression between L69 and L71 strain were retained on the basis of fold change < 0.6 at p-value <0.01

  cell pellets were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. RNA extraction, quantification and labelling were carried out as described in {Alkim, 2013 5685 /id}. Labeled cDNA were hybridized on Agilent glass slides microarrays, which bear the whole www.biocompare.com/ProductDetails/760330/S-cerevisiae-Saccharomycescerevisiae-Whole-Genome.html). Hybridization was carried out in an automatic hybridization chamber (Agilent Technologies, Wilmington, USA) for 17 hr at 65°C. The hybridization signals were detected by scanning using Innoscan 900 laser Scanner (Innopsys Instruments), and transformed to numerical values using Feature Extraction V.11.5.1.1. The microarrays hybridization and processing were carried out at the Biochips Platform of Toulouse (http://biopuce.insa-toulouse.fr).

Figure

  Figure S2 in supplementary data. From a cross-section taken through several patches (see the green

Figure 1 :

 1 Figure 1: Cell surface topography of a living cell and rehydrated sample from strain L71

Figure 2 :

 2 Figure 2: Cell surface topography of a living cell and rehydrated cell from strain L69

Figure 3 :

 3 Figure 3: Detailed cell surface analysis identified adhesive patches in rehydrated cell from strain

Figure 4 :

 4 Figure 4: Mapping mannan polysaccharides on the surface of a living and rehydrated cell from

Figure 5 :

 5 Figure 5: Mapping mannan polysaccharides on the surface of a living and rehydrated cell from

Figure S1 :

 S1 Figure S1: AFM images on rehydrated cell reveal the presence of a hole

Figure S2 .

 S2 Figure S2. Interaction of conA-tip with surface polysaccharides is abolished by excess of mannose

  L69 and L71 (Lallemand SAS collection) were cultivated in shake in flask under laboratory condition (Yeast Peptone Dextrose medium, 30°C) or obtained as 'Cream' or 'YCW' (autolysis/drying process (24 h at 55°C). The amounts of chitin, β-1,3-glucan, β-1,6-glucan and mannans in the cell wall are mean values ± SD obtained from three independent biological replicates, each made 2 times. Statistical comparisons in the cell wall composition between strains for each condition were made by one-way analysis of variance followed the Tukey's comparison test (XLstat software), for which p values were obtained indicated by an asterisk. *is meaning p-value < 0.05, **p-value <0.01 and ***p-value <0.001.

Table 1 :

 1 Cell wall composition of industrial strain L71 and L69

	cell wall	Culture YPD	Cream		Autolysis/drying
	polysaccharides	Strain L71	Strain L69	Strain L71	Strain L69	Strain L71	Strain L69
				% of cell wall dry weight
	-(1,3)-glucan	30.5 ± 6.0	36.2 ± 5.5*	34 ± 2.3	33.7 ± 5.0	32 ± 3.4	33.3 ± 4.0
	-(1,6)-glucan	24.4 ± 5.4	16.7 ± 1.7**	22.5 ± 5.5	17.3 ± 3.6**	22.5 ± 5.5	15.1 ± 2.7

Table 2 :

 2 Effect of autolysis/drying process on cell surface roughness

	Condition		Roughness (nm)
	/treatment	Strain L71	Strain L69

Table 3 :

 3 Difference in expression levels of genes related to cell wall biogenesis between L69 versus L71 strain

	ORF	Gene	Biological function	Fold change
	YER011W	TIR1	Cell wall mannoprotein	1.9
	YGR166W	KRE11	-1,6-glucan biosynthesis	2.0
	YHR213W	YHR213W Pseudogenic fragment, similar to flocculin	7.7
	YIR019C	FLO11	flocculin, GPI-anchored cell surface glycoprotein	11.4
	YJR151C	DAN4	Cell wall mannoprotein, similar to TIR1, TIR2, TIR3, and TIR4	1.7
	YLR037C	PAU23	Cell wall mannoprotein, similar to TIR1, TIR2, TIR3, and TIR4	1.5
	YLR194C	YLR194C	Unknown function and unknown phenotype	1.8
	YOR382W	FIT2	GPI-anchored cell wall mannoprotein	3.1
	YOR383C	FIT3	GPI-anchored cell wall mannoprotein	4.1
	YER096W	SHC1	Sporulation-specific activator of Chs3p (chitin synthase III)	0.5
	YGR279C	SCW4	Soluble cell wall protein with similarity to -glucanases	0.6
	YHR211W	FLO5	flocculin, lectin-like cell wall protein	0.5
	YKL163W	PIR3	Member of PIR family protein, cell wall structural protein	0.5
	YLR300W	EXG1	Exo--1,3-glucanase	0.4
	YOR010C	TIR2	Putative cell wall mannoprotein	0.5
	YOR140W	SFL1	Suppressor of flocculation	0.7

Table S1 :

 S1 Classification of genes whose expression was higher in strains L69 compared to L71 into MIPS categories*

Upregulated genes classified according to MIPS Functional Classification (459 categories)

	MIPS category	Category	up-regulated genes in the given category	number of genes category in MIPS	% genes category in each	% genes in this category genes upregulated / total of	p-value	In category from Cluster
		siderophore-iron transport					8E-07	ARN1 ARN2 FET3 FRE4 ENB1 FRE3
	transport facilitation	drug/toxin transport	20	1038	1.9	11.4	3E-06	FLR1 AZR1 ARN1 ARN2 PDR11 QDR1 ENB1 YRM1 PDR10
		ABC transporters					2E-03	ARB1 PDR11 ENB1 PDR10 PXA1
		fermentation					7E-06	ADH7 AAD3 AAD4 AAD6 ALD2 AAD15 ATF1
		tetracyclic and pentacyclic
		triterpenes (cholesterin, steroids and hopanoids)					1E-05	ERG28 ERG25 ERG11 ERG7 ERG3 ERG27 ERG6 HMG1
	metabolism & energy	metabolism C-compound and carbohydrate metabolism	41	1881	2.2	23.0	3E-04	GPI18 ADH7 AAD3 AAD4 YEL047C AAD6 ATF2 PHO12 FLO11 KTI12 GAL80 ALD2 PGM3 YNR071C YNR073C CSI2 AAD15 ATF1
		metabolism of phenylalanine					6E-04	AAD3 AAD4 AAD6 AAD15
		metabolism of tyrosine					8E-04	AAD3 AAD4 AAD6 AAD15
	stress response	detoxification by export	9	554	1.6	5.0	3E-03	QDR1 YRM1 FLR1 AZR1 ARN1 ARN2 YLR046C ENB1 SSU1
								PAU8 YAR064W YBL044W PSY4 YBL055C APD1 YBR242W HBN1 RNQ1 YCR051W
								YDR056C RTN1 FDC1 YEL057C YER077C AIM11 YER137C YFL051C PAU11 YGR017W
	unclassified proteins	unclassified proteins	60	1393	4.3	34.0	6E-03	ECL1 HGH1 PAU12 YHL042W YHR078W YHR213W YIL086C YIL096C YJL213W YJR039W YJR056C YJR115W DAN4 YKL047W COS9 YLL056C PAU18 PAU23 AVL9 YLR194C YLR326W YLR346C YMR007W YMR178W YMR181C YMR265C RSN1 YNL022C TCB2
								YNL095C YNL193W YOL166C YOR012W IRC11 YOR342C YOR385W YOR390W YPL068C
								YPL279C YPR071W
	regulation of
	metabolism and protein	protease inhibitor	2	253	0.8	1.1	9E-03	RAD23 TFS1
	function
	others	in several non-enriched categories	43			25.0

Table S2 :

 S2 Classification of genes whose expression was lower in strains L69 compared to L71 into MIPS categories*

Downregulated genes classified according to MIPS Functional Classification (459 categories)

	MIPS category	Category	down-regulated genes in the given category	number of genes in category MIPS	% genes category in each	% of genes in this category / genes downregulated total of	p-value	In category from Cluster
	cell cycle and DNA	DNA topology	14	1012	1.4	6.5	7E-06	YBL113C YEL077C YRF1-2 YRF1-3 YIL177C YJL225C YLL067C YRF1-6 YRF1-7 YPR204W
	progressing	somatic / mitotic recombination					4E-03	YRF1-2 YRF1-3 YRF1-6 YRF1-7
	communication	G-protein mediated signal transduction cAMP/cGMP mediated signal transduction	6	234	2.6	2.8	5E-03 6E-03	GPB2 STE18 STE4 PDE1 RAS1 SFL1
		sulfate assimilation					3E-10	MET10 MET3 MET5 MET14 MET1 MET22 MET16
		purine
		nucleotide/nucleoside/nucleobase					2E-07	ADE1 ADE5,7 ADE3 MTD1 ADE13 IMD3 ADE4 ADE2 SER1
		anabolism
		biosynthesis of methionine					2E-05	CBF1 MET14 MET2 MET22
		metabolism of methionine					5E-04	MET32 MET3 MET1 MET17 MET16
	metabolism	degradation of glycine	42	1514	2.8	19.4	6E-04	GCV3 GCV1 SHM2
		biosynthesis of serine					1E-03	SER33 SHM2 SER1
		biosynthesis of homocysteine					1E-03	MET10 STR3 MET5
		biosynthesis of leucine					2E-03	LEU2 LEU1 BAT1
		tetrahydrofolate-dependent C-1-transfer					8E-03	ADE3 MTD1 SHM2
		C-1 compound catabolism					1E-02	GCV3 GCV1
	protein binding	NAD/NADP binding	7	1049	0.7	3.2	1E-04	GPD1 MET10 ADE3 SER33 MET5 MTD1 GPD2
	transport facilitation	sulfate/sulfite transport	2	1038	0.2	0.9	3E-03	OAC1, SUL2
	others	in several non-enriched categories	143			67.3

Acknowledgements

We thank Henri Durand and Anne Ortiz-Julien from Lallemand Inc. for critical discussions during this work. This work was supported in part by a grant n°10051296 from Region Midi Pyrénées to JMF and an ANR Young Scientist project ANR-11-JSV5-001-01, n° (SD) 30 02 43 31 to ED. ED is researcher at the Centre National de la Recherche Scientifique (CNRS) and MS is supported by a grant from Lallemand SAS (Cifre fellowship).


MS Cécile Formosa

MS Marion Schiavone

PhD Anita Boisrame

PhD Mathias L Richard

PhD Raphaël E Duval

PhD Etienne Dague
email: edague@laas.fr

Multiparametric imaging of adhesive nanodomains at the surface of Candida albicans by Atomic Force Microscopy

Candida albicans is an opportunistic pathogen. It adheres to mammalian cells through a variety of adhesins that interact with hosts ligands. The spatial organization of these adhesins on the cellular interface is however poorly understood, mainly because of the lack of an instrument able to track single molecules on single cells. In this context, the Atomic Force Microscope (AFM) makes it possible to analyze the force signature of single proteins on single cells. The present study is dedicated to the mapping of the adhesive properties of C. albicans cells. We observed that the adhesins at the cell surface were organized in nanodomains composed of free or aggregated mannoproteins. This was demonstrated by the use of functionalized AFM tips and synthetic amyloid forming/disrupting peptides. This direct visualization of amyloids nanodomains will help in understanding the virulence factors of C. albicans.

Background

The yeast Candida albicans has emerged as a major public health problem these last two decades. This opportunistic pathogen causes a wide range of infections from surface infections, to mucosal and blood-stream infections [START_REF] Gow | Importance of the Candida albicans cell wall during commensalism and infection[END_REF] . Whereas mucosal infections are common and occur in healthy organisms, blood-stream infections are observed only in immunocompromised patients and are life-threatening. This type of infections, also known as candidaemia, can develop into disseminated candidiasis when the infection spreads to internal organs, leading to high mortality rates [START_REF] Poulain | Candida albicans , plasticity and pathogenesis[END_REF] . In order to colonize and subsequently to disseminate in the blood stream C. albicans needs to adhere to different substrates. This first stage of infection [START_REF] Naglik | Candida albicans interactions with epithelial cells and mucosal immunity[END_REF] is mediated by adhesins that are found on the surface of the yeast cell wall. Many of these adhesins are mannoproteins, and among them, the adhesin family identified as having a major role in host cell attachment is the Als (Agglutinin-like Sequences) family [START_REF] Hoyer | The ALS gene family of Candida albicans[END_REF] .

The Als were initially reported as having homologies with the proteins responsible for auto-agglutination in the baker yeast Saccharomyces cerevisiae. Eight Als have been identified, they all are primarily involved in host-pathogen interactions [START_REF] Hoyer | Discovering the secrets of the Candida albicans agglutinin-like sequence (ALS) gene familya sticky pursuit[END_REF] . It was found that there were amyloid-forming sequences in the Als adhesins of Candida albicans [START_REF] Ramsook | Yeast Cell Adhesion Molecules Have Functional Amyloid-Forming Sequences[END_REF] . Amyloids are insoluble fibrillar protein aggregates whose core consists in crystalline arrays of identical sequence in many molecules of the amyloid protein [START_REF] Sawaya | Atomic structures of amyloid cross-β spines reveal varied steric zippers[END_REF][START_REF] Garcia | A Role for Amyloid in Cell Aggregation and Biofilm Formation[END_REF] . Cells expressing the Als proteins can rapidly aggregate, and the aggregation has amyloid-like properties. Like amyloid formation, aggregation ability propagates through the adherent cell population and depends on conformational changes of the Als protein. This transition of the conformational state to an aggregative state of the proteins is characterized by the formation of hydrophobic nanodomains on the entire surface of the cell [START_REF] Rauceo | Global Cell Surface Conformational Shift Mediated by a Candida albicans Adhesin[END_REF] .

A few papers written by Lipke's team were dedicated to the direct visualization of these nanodomains using fluorescent dyes such as thioflavin T or 8-anilino-1-naphtalene-sulfonic acid (ANS) [START_REF] Ramsook | Yeast Cell Adhesion Molecules Have Functional Amyloid-Forming Sequences[END_REF][START_REF] Garcia | A Role for Amyloid in Cell Aggregation and Biofilm Formation[END_REF][START_REF] Rauceo | Global Cell Surface Conformational Shift Mediated by a Candida albicans Adhesin[END_REF] . Another technique that can be used to visualize these nanodomains is Atomic Force Microscopy (AFM). AFM has recently emerged as a valuable tool to study the surface of living cells [START_REF] Müller | Atomic force microscopy: a nanoscopic window on the cell surface[END_REF] , and especially pathogenic cells [START_REF] Alsteens | Atomic Force Microscopy: A New Look at Pathogens[END_REF] . This technology has been used by Alsteens et al. to image the formation and propagation of nanodomains in living yeast cells [START_REF] Alsteens | Force-induced formation and propagation of adhesion nanodomains in living fungal cells[END_REF] and also to unfold amyloid proteins from the yeasts surface using Single Molecule Force Spectroscopy [START_REF] Beaussart | Single-Molecule Imaging and Functional Analysis of Als Adhesins and Mannans during Candida albicans Morphogenesis[END_REF][START_REF] Alsteens | Unfolding Individual Als5p Adhesion Proteins on Live Cells[END_REF][START_REF] Alsteens | Unzipping a Functional Microbial Amyloid[END_REF] . To this end, the authors functionalized AFM tips with antibodies targeted against the Als protein directly or against an epitope tag present in the Als protein. These studies allowed the authors to localize the adhesive nanodomains caused by the aggregation of Als proteins at the surface of living yeast cells, and to unravel the structure of the Als proteins studied by stretching.

In our study, we used AFM as an imaging tool to visualize and localize adhesins nanodomains at the surface of living wild-type Candida albicans cells. Using recent developments in the AFM technology, we have imaged and quantified at the same time the nanomechanical properties, the adhesiveness (force and nature of the interaction), the size and the thickness of the nanodomains [START_REF] Chopinet | Imaging living cells surface and quantifying its properties at high resolution using AFM in QI TM mode[END_REF][START_REF] Dufrêne | Multiparametric imaging of biological systems by force-distance curve-based AFM[END_REF] , at high resolution. The data collected showed that these nanodomains are localized differently at the surface of the cell, depending on the structures featured by the cells (bud scars, buds). We also showed that there were degrees of adhesiveness, depending on whether the amyloid proteins had totally aggregated (hydrophobic nanodomains) or not, and that these degrees of aggregation were directly correlated to the stiffness of the yeast cell wall. Finally, using force measurements and amyloid forming or inhibiting peptides, we showed that Als proteins (probably among others) were participating to these nanodomains.

Methods

Yeasts growth conditions

Candida albicans (from ABC Platform® Bugs Bank, Nancy, France) was stocked at -80°C, revivified on Yeast Peptone Dextrose agar (Difco, 242720-500g) and grown in Yeast Peptone Dextrose broth (Difco, 242820-500g) for 20 hours at 30°C under static conditions.

Sample preparation for AFM experiments

Yeast cells were concentrated by centrifugation, washed two times in acetate buffer (18 mM CH 3 COONa, 1 mM CaCl 2 , 1 mM MnCl 2 , pH = 5.2), resuspended in acetate buffer, and immobilized on polydimethylsiloxane (PDMS) stamps prepared as described by Dague et al [START_REF] Dague | Assembly of live microorganisms on microstructured PDMS stamps by convective/capillary deposition for AFM bio-experiments[END_REF] .

Briefly, freshly oxygen activated microstructured PDMS stamps were covered by a total of 100 µL of the solution of cells and allowed to stand for 15 minutes at room temperature. The cells were then deposited into the microstructures of the stamp by convective/capillary assembly.

Images were recorded in acetate buffer in Quantitative Imaging TM mode with MLCT AUWH (Bruker) cantilevers (nominal spring constant of 0.01 N/m). The applied force was kept at 1.5 nN for imaging and at 0.5 nN for force spectroscopy experiments. The loading rate for imaging was of 2 500 000 pN/s (acquisition frequency of the force curves is of 25 Hz) and for force spectroscopy of 75 000 pN/s (acquisition frequency of the force curves is of 1.25 Hz). For imaging and force spectroscopy, we used an AFM Nanowizard III (JPK Instruments, Berlin, Germany). The cantilevers spring constants were determined by the thermal noise method [START_REF] Hutter | Calibration of atomic-force microscope tips[END_REF] . For all the results presented in this study, silicon nitride AFM tips were bare, expect in the case of figure 4g (lower panel), where a functionalized AFM tip has been used.

AFM tips functionnalization

The functionalized tips were produced according to a french patent of the authors described later in sensors and actuators [START_REF] Jauvert | Probing single molecule interactions by AFM using bio-functionalized dendritips[END_REF] . Briefly, AFM tips were functionalized with dendrimers presenting CHO functions able to covalently link with NH 2 functions of proteins. These dendritips were then incubated with the lectin Concanavalin A (Sigma, L7647-100MG, 100µg/mL) for 1 hour, before being used for force spectroscopy experiments.

Results analysis

All results were analyzed using the Data Processing software from JPK Instruments. The stiffness values measured on cells were determined from the slope of the linear portion of the raw deflections versus piezo displacement curves, according to: with s the experimentally accessible slope of the compliance region reached for sufficient loading forces. In this model, the experimental setup can be represented by two linear springs, one is the AFM's cantilever, and the other is the cell envelope exhibiting an effective spring constant. It is then possible to calculate the effective spring constant kcell of the cell envelope from the observed slope s of the force curve and the known spring constant k of the cantilever [START_REF] Arnoldi | Bacterial turgor pressure can be leasured by atomic force microscopy[END_REF] .

Results

Candida albicans cells display localized adhesiveness

Thanks to our innovative method to immobilize cells into PDMS stamps [START_REF] Dague | Assembly of live microorganisms on microstructured PDMS stamps by convective/capillary deposition for AFM bio-experiments[END_REF] , and using Quantitative Imaging TM mode [START_REF] Chopinet | Imaging living cells surface and quantifying its properties at high resolution using AFM in QI TM mode[END_REF] , we were able to image and quantify the adhesive properties of single C. albicans cells at the same time. Figure 1a shows a budding yeast cell; on the corresponding adhesion image (Figure 1b), we can see that only the bud, and not the mother-cell, presents adhesives patches. This original result is surprising as non-budding cells are highly adhesive (see below). This result seems to indicate that the mother-cell cell wall changes during the budding process. As for the cell in figure 1c, this cell displays two bud scars, a common feature at the surface of yeast cells, which are not adhesive whereas the rest of the cell is. This type of distribution of the adhesion on yeast cells has already been seen using immunofluorescence with antibodies targeted against surface proteins of C. albicans. Coleman et al. for example showed that the Als1 protein was expressed all over C. albicans cells, with the exception of bud scars [START_REF] Coleman | Heterogeneous distribution of Candida albicans cell-surface antigens demonstrated with an Als1-specific monoclonal antibody[END_REF] . The comparison of our results to these data suggests then that the adhesions probed by AFM might be due to surface proteins, such as Als1 in the case of the cell presenting bud scars, but perhaps also others adhesins.

C. albicans cell wall adhesins are able to aggregate into nanodomains

As showed before [START_REF] Rauceo | Global Cell Surface Conformational Shift Mediated by a Candida albicans Adhesin[END_REF][START_REF] Alsteens | Force-induced formation and propagation of adhesion nanodomains in living fungal cells[END_REF] , the proteins expressed at the surface of C. albicans cell wall are able to aggregate, and to form nanodomains. However, these nanodomains have not been yet characterized at the nanoscale, nor were imaged at high resolution. In fact, these nanodomains have specific adhesive properties that can be mapped using AFM in the Quantitative Imaging TM mode. High resolution (256 pixels²) adhesive images are presented in Figure 2. It shows adhesive nanodomains, at the surface of a living wild-type C. albicans cell. On the cell presented in this figure (Figure 2a), the corresponding adhesion image shows very distinct adhesive nanodomains that were probed with bare AFM tips. These nanodomains are homogeneously distributed all over the cell here, which does not present any morphological features such as buds or bud scars. When zooming into small areas on top of the cell (white squares on figure 2b), we could measure the area of each nanodomain. On this cell and on another one showed in Figure 3a, 60 nanodomains areas were measured; the values obtained plotted on figure 2e shows that nanodomains have an average area of 0.09 ± 0.03 µm². This corresponds to an average diameter of 170 nm, which confirms the nanoscale of these nanodomains. Some of the nanodomains are also higher than the rest of the cell wall. When the whole cell is imaged, it is not visible; however, specific analysis of the Figure 3d and the graphic 3h representing the topography of the cell surface revealed nanodomains that had a different height compared to the rest of the cell. The cross-sections taken along the blue line showed the height of a nanodomain of 20 nm. Once again, this confirmed the nanoscale of the nanodomains at the surface of C. albicans.

Different nanodomains have different nanomechanical properties

Adhesion is measured as the rupture force recorded when retracting the tip from the surface, when approaching and pulling with the tip on the cell wall, thus AFM makes it possible to measure nanomechanical properties of living cells. Here we choose to use an analysis based on the Hooke model which considers the coupled cantilever / cell wall as a spring. The stiffness values measured on cells were determined from the slope of the linear portion of the raw deflections versus piezo displacement curves, according to: with s the experimentally accessible slope of the compliance region reached for sufficient loading forces. Indeed, the most interesting result in this study is the correlation that can be directly made between the adhesiveness of the nanodomain, and its stiffness. Nanodomains on figure 3e (adhesion map) circled in red were found on the stiffness image (figure 3f) circled in black; they correspond to the zones where the stiffness of the cell wall is increased, to 13.4 ± 0.3 nN/µm. As for less adhesive nanodomains, they do not present any difference in stiffness from the rest of the cell, and are 12.4 ± 0.2 nN/µm. The 3D-view of the adhesion, mapped with the stiffness (figure 3g) illustrates this clear correlation; the more adhesive the nanodomain is, the stiffer it is.

Another fascinating point is that for the more adhesive nanodomains, the retract force curves present typical hydrophobic adhesions [START_REF] Dague | Chemical force microscopy of single live cells[END_REF][START_REF] Alsteens | Direct measurement of hydrophobic forces on cell surfaces using AFM[END_REF][START_REF] Dague | High-Resolution Cell Surface Dynamics of Germinating Aspergillus fumigatus Conidia[END_REF] , with adhesions occurring immediately when the tips is retracted from the surface. Force curves from the other, less adhesive, nanodomains presented retract adhesions resembling to proteins unfolding, occurring several nanometers after the tip withdrawal. Therefore it seems that the nanodomains are of 2 different natures. There is a class of nanodomains, hydrophobic, higher and stiff, and another class displaying proteins unfolding properties, as soft as the rest of the cell wall. What is the molecular nature of these 2 types of nanodomains, and are they correlated?

Understanding the adhesive properties of the 2 nanodomains classes

To answer the previous question, we monitored the retract force curves recorded on the nanodomains (Figure 4). We found the same correlation as in figure 3; the force curves recorded on an adhesive nanodomains presented hydrophobic retract adhesions, whereas the force curves recorded on a less adhesive nanodomain presented protein, unfolding like, profiles. In order to determine the nature of these last unfoldings, we probed the surface of C. albicans cells with an AFM tip functionalized with Concanavalin A (ConA), a protein that interacts with yeast mannoproteins, such as surface adhesins. The resulting force curves (figure 4g) showed retract adhesions displaying unfoldings of different lengths, but with a similar profile. We also observed condensed spikes with adhesion forces between 0 and 50 pN. This value was consistent with specific interactions between ConA and mannoproteins [START_REF] Alsteens | Structure, cell wall elasticity and polysaccharide properties of living yeast cells, as probed by AFM[END_REF] .

In a previous study conducted in 2009 [START_REF] Alsteens | Unfolding Individual Als5p Adhesion Proteins on Live Cells[END_REF] by Alsteens et al., adhesins (Als5) were unfolded from the surface of live S. cerevisiae cells overexpressing this protein. The retract force curves obtained in this study show high similarity with the ones we obtain here with functionalized AFM tips, with the presence of serin-threonin rich segments (condensed pikes on figure 4g). We can therefore, based on this comparison with the data of the literature, conclude that the less adhesive nanodomains at the surface of live C. albicans are composed of free adhesins, and maybe of Als proteins. However, since all adhesins (like Als, Hwp1, Eap1, Rbt1 etc) are mannoproteins, we cannot, at this stage, make a statement on which adhesins are unfolded here.

As for the second type of nanodomains, the hydrophobic ones, our hypothesis is that they are composed of the same proteins as the less adhesive ones. In fact, adhesins (like Als) display amyloids sequences located on a domain of the protein called T, that enable them to change their conformation [START_REF] Ramsook | Yeast Cell Adhesion Molecules Have Functional Amyloid-Forming Sequences[END_REF] and to aggregate into amyloid nanodomains. And when this phenomena is started, it propagates to the whole cell [START_REF] Rauceo | Global Cell Surface Conformational Shift Mediated by a Candida albicans Adhesin[END_REF] . We therefore made the hypothesis that the adhesive nanodomains are in fact amyloid nanodomains, made of Als proteins.

From adhesins to amyloid nanodomains: the role of Als proteins

To verify this hypothesis, and according to the literature on Als proteins, we synthesized a peptide exhibiting the same sequence as the one of the T domain of the Als1/3/5 proteins. We then put this peptide in the presence of the cells in order to trigger the amyloids formation. We also synthesized the same peptide, but with a mutation on one amino acid (V326N peptide), in order to obtain a peptide that inhibits the formation of the amyloid nanodomains [START_REF] Garcia | A Role for Amyloid in Cell Aggregation and Biofilm Formation[END_REF] . Since Als3 is only expressed on the surface of hyphae, we will only be able to generate or destroy the amyloid formation of Als 1 and 5. The results presented in figure 5 showed a cell before and after adding the mutated peptide. We can clearly see on these adhesion images the loss of general adhesion and of two nanodomains at the center of the cell. It seems like the mutated peptide disrupted the amyloids at the surface of the cell. And the other way around, when cells were incubated with the amyloid forming peptide, we could observe the formation of the nanodomains at the surface of the cells, as it is showed on the adhesion images on local areas on top of C. albicans cells in figure 5c, d ande. These results allow us to conclude that the proteins at the origin of the nanodomains are then mannoproteins, more specifically Als1 or 5, or both, that form amyloids.

Discussion

We show in this study that wild-type live C. albicans cells exhibit extraordinary adhesive properties. In the case of budding cells, placed in acetate buffer at 25°C for 2h, we observed that the mother cell is not adhesive and that only the bud presents adhesive nanodomains. On the contrary, we show that non budding cells are covered by adhesive nanodomains, in the same experimental conditions. This illustrates the amazing plasticity of this species [START_REF] Odds | Candida and candidiosis. A review and bibliography[END_REF][START_REF] Poulain | Candida albicans , plasticity and pathogenesis[END_REF] able to grow as a commensal or as a pathogen [START_REF] Gow | Importance of the Candida albicans cell wall during commensalism and infection[END_REF][START_REF] Gow | Candida albicans morphogenesis and host defence: discriminating invasion from colonization[END_REF] , in all the parts of the intestinal track, but also on the vaginal mucosa, as unicellular budding cells or as filamentous hyphae. Moreover its cell wall is permanently remodeled as a reaction to its environment (temperature, pH, dissolved O 2 , ions, interacting surface/cells/bacteria) what makes it challenging to reproduce the experimental conditions inducing a certain cell wall phenotype. We then demonstrate that the molecules at the origin of these adhesions could aggregate into nanodomains, which can be probed at high resolution using a suited AFM mode, QI TM . These nanodomains are different in terms of level of adhesiveness, which is a property directly correlated to their stiffness and to the hydrophobic state or not of the molecule at the origin of these nanodomains. We then went further in the study, using functionalized AFM tips, and were able to determine that the less adhesive nanodomains were formed by mannoproteins that can interact specifically with Concanavalin A. These mannoproteins are able to aggregate to form the adhesive nanodomains because they have amyloid properties as we showed in figure5.

Amyloid aggregation is a primitive [START_REF] Greenwald | Biology of Amyloid: Structure, Function, and Regulation[END_REF] and very stable [START_REF] Perczel | Dead-End Street of Protein Folding: Thermodynamic Rationale of Amyloid Fibril Formation[END_REF] protein folding and a common structural motif. It is a cross β-sheet quaternary structure that usually auto-aggregates as fibrils. It has been, first, associated with neurodegenerative diseases like Alzheimer, Parkinson, or Creutzfeldt-Jakob diseases. However it is more and more unclear if the amyloids lesions are the cause or a consequence of the disease. Amyloid aggregates are now described as functional proteins assembly and can be found from bacteria to humans [START_REF] Fowler | Functional amyloid--from bacteria to humans[END_REF] . In microorganisms, amyloid has been described as a functional coat [START_REF] Gebbink | Amyloidsa functional coat for microorganisms[END_REF] . It constits in curli (E. coli), chaperons (Streptomyces) or hydrophobins (Aspergillus etc); all of these proteins are implicated in adhesion to the host and in the invasion, infection process. It is now well known that adhesins (and especially Als) of C. albicans have amyloid-forming sequences [START_REF] Ramsook | Yeast Cell Adhesion Molecules Have Functional Amyloid-Forming Sequences[END_REF][START_REF] Otoo | Candida albicans Als Adhesins Have Conserved Amyloid-Forming Sequences[END_REF] and that these proteins form domains involved in cell aggregation or biofilm formation [START_REF] Garcia | A Role for Amyloid in Cell Aggregation and Biofilm Formation[END_REF] . Nevertheless the characterization, structure and properties of the amyloids adhesive nanodomains remain unclear.

In this work we measured for the first time the nanoscale size of amyloid domains (average area of 0.09 µm 2 ) at the surface of live C. albicans cells. The domains are of 2 different classes. Some present the characteristic of individual proteins whereas the others are hydrophobic, stiffer than the rest of the cell (13.4 ± 0.3 nN/µm compared to 12.4 ± 0.2 nN/µm), and are slightly protruding. It means that there is a state modification from soluble proteins into insoluble proteins, which is a characteristic of amyloid structures. This transformation is dependent on the proteins concentration and can only occur when the protein density exceeds a threshold. The roles of the two classes of domains are probably different. On one hand we could hypothesize that the hydrophobic nanodomains were involved in the cell adhesion to abiotic hydrophobic surfaces or to cell membrane as it is known that membrane binding is an inherent property of amyloid aggregates [START_REF] Sparr | Islet amyloid polypeptide-induced membrane leakage involves uptake of lipids by forming amyloid fibers[END_REF][START_REF] Gellermann | Raft lipids as common components of human extracellular amyloid fibrils[END_REF] . Amyloid aggregation is also a way to store proteins, in a limited space and to sort them when required. This has been demonstrated for hormones in secretory granules 39,40 . Thus C. albicans may store some adhesins for the subsequent invasion phases. On the other hand the protein like domains may be responsible for specific adhesion to

Figure 1 .

 1 Figure 1. Localization of the adhesive properties of C. albicans cells. (a) Height image (z-

Figure 2 .

 2 Figure 2. Imaging of the adhesive domains of C. albicans cells in acetate buffer, at 25°C for

Figure 3 .

 3 Figure 3. Nanomechanics of the adhesive domains of C. albicans cells. (a) Height image (z-

Figure 4 .

 4 Figure 4. Adhesion force curves of C. albicans adhesive domains. (a) Height image (z-range =

Figure 5 .

 5 Figure 5. Imaging of the adhesive domains of C. albicans cells treated with Als1, 3, 5p

  39. Maji SK, Perrin MH, Sawaya MR, Jessberger S, Vadodaria K, Rissman RA, et al. Functional Amyloids As Natural Storage of Peptide Hormones in Pituitary Secretory Granules. Science. 2009;325:328-332. 40. Dannies PS. Concentrating hormones into secretory granules: layers of control. Mol Cell Endocrinol. 2001;177:87-93.

fibronectin and other extra cellular proteins of the matrix. It seems rational that several adhesins, brought together, would be more efficient in a binding process than a single adhesion. This finding has to be added to C. albicans plasticity 28 and participate to explain its remarkable adaptation and pathogenicity.

However, there are still many things to explore on the cell wall of C. albicans, and future work will be dedicated to exploring the changes appearing on the mother cell during the budding process.

This work was supported by an ANR young scientist program (AFMYST project ANR-11-JSV5-001-01 n° SD 30024331) to ED. CF and MS are respectively supported by a grant from "Direction Générale de l'Armement" (DGA) and from Lallemand SAS.


H Trari

O Kerdjoudj

Mohamed Arous

Amara

S Bensaadi

N Nasrallah

A Amrane

M Trari

H Kerdjoudj

O Arous

M Amara

Dialysis and photo-electrodialysis processes using new synthesized polymeric membranes for the selective removal of bivalent cations

Keywords: photoelectro-dialysis; Membrane, Polymer, FeAl2O4, Metals

Dialysis and photo-electrodialysis processes using new synthesized polymeric membranes for the selective removal of bivalent cations

Introduction

The rapid industrial development, depleting energy resources and growing concern for the environment safety have put an increasing demand to enhance the efficiency of existing technologies. The exploration of new sources of raw materials and the development of novel alternatives that allow the water treatment as well as the recuperation of valuable and toxic materials has become an obligation.

In this context, membrane technology provides solutions to some of these problems [1] and the ions exchange technology allows the concentration of metals. Novel membranes have been developed by introducing new compounds and/or by modifying their surface yielding the separation between anions or cations with mixed valences [2][3][4][5]. A new generation of ion exchange membranes can be prepared by changing the hydrophilic and/or the hydrophobic balance. These types of membranes permit the separation between ions of the same valence are today reconsidered from the point of view of their possible substitution by new strategy using synthetic membranes. Recently, a novel type of liquid membrane called polymer inclusion membrane (PIM) was developed to provide metal ion transport with high selectivity, as well as an easy setup [START_REF] Bartsch | ACS Symposium Series[END_REF]. PIMs offer a great potential for industrial separations over other membrane types. Amara et al. studied previously a combination between membrane selectivity and photo-electrochemistry using commercial cation exchange membrane (noticed CRA) and Cadmium Sulfate (CdS) as photo electrode for the separation and recovery of some metallic ions (Cu 2+ , Zn 2+ and Ni 2+ ) in aqueous medium [START_REF] Amara | [END_REF]. The authors observed that illuminated n-CdS electrode leads to an increase of the transport flux at least 10 times more than that obtained in the dark. A polymeric membrane obtained by the combination of cellulose triacetate and poly-4-vinylpyridine in the presence of n-Bi2S3 as photoelectrode has been used by the same authors showing an important affinity either for anionic species or cationic species depending on the composition of the solution The semiconductor is an essential element of photo electrochemical (PEC) device and in order to approach the problem systematically, we have chosen an oxide with a new band structure. Among oxides, the spinel FeM2O4, where M is commonly a 3d metal, have not FeAl2O4 is a mixed oxide normal spinel, where one eighth of the tetrahedral sites is occupied by Fe 2+ cations and one half of the octahedral sites are occupied by Al 3+ cations. Sometimes, depending on the synthetic process, the Fe 2+ cations can also occupy octahedral sites [START_REF] Russo | Mossbauer Spectroscopy Applied to Magnetism and Materials Science[END_REF].

FeAl2O4 provides an excellent combination of physical and chemical properties. It has high ductility (fracture-mechanical strength) and flexibility against cracking and spalling.

However, reports on this spinel compound are scarce especially those dealing with its magnetic behavior [START_REF] Smith | [END_REF][START_REF] Tristan | [END_REF][24].

The present work deals with two attractive aspects, energetic (solar energy) and environmental based on the membrane selectivity for metal ions. New polymeric membranes based on a mixture of CTA and poly-electrolytes plasticized by tris ethylhexyl phosphate were synthesized. The membranes (CTA + poly-electrolyte + plasticizer) were characterized by Fourier Transform Infrared (FTIR), scanning electron microscopy (SEM) and Thermogravimetric Analysis (TGA). As application, the photo electro-dialysis of solutions containing copper, zinc and nickel was studied; thereby, opening new pathways to fabricate hybrid systems dedicated to environmental protection. The membrane was combined with a photoelectrode FeAl2O4, with the aim of increasing the photon-to current yield by minimizing the loss of electron/hole (e -/h + ) pairs by recombination. FeAl2O4 has environmentally friendly characteristics and absorbs over the whole solar spectrum. Moreover, it has a conduction band of 3d parentage with a high reducing capacity able to reduce the M 2+ ions into their elemental states.

Materials and Methods

Chemicals

Copper (II) nitrate, zinc (II) nitrate, nickel (II) nitrate, chloroform, cellulose triacetate (CTA), polyvinylpyrrolidone (PVP), polyethylenimine (PEI) and polyethylenglycol (PEG) were analytical grade reagents purchased from Fluka. Tris ethylhexyl phosphate (TEHP) (GC ≥ 98%) was a product of Merck Company. The aqueous solutions were prepared by dissolving the different reagents in bi-distilled water (~ 1 M Ω cm).

Membrane preparation

PIMs were prepared using the same procedure described by Sugiura et al. Briefly, 0.2 g of CTA and 0.1 g of polyelectrolyte were dissolved in 20 mL of chloroform and stirred for 4 h. Then, 0.1 mL of TEHP was added to the mixture and maintained 2 h under vigorous stirring. The homogeneous solution obtained was poured into a 9 cm diameter glass petri dish and left to evaporate slowly during 24 hours. The resulting membrane was extracted, after evaporation of solvent, by addition of water and dried at 40°C.

Characteristics of CMS commercial membrane:

The physical and chemical characteristics of the commercial membrane used in dialysis are reported in the Appendix.

Synthesis of the semi-conductor FeAl 2 O 4

The spinel FeAl2O4 was developed by the nitrate route, Fe(NO3)2.9H2O (Merck, 99%) and Al(NO3)3.9H2O (Merck, 99.5%) were accurately weighed, dissolved in a nitric solution (volumetric ratio HNO3 (65 wt. %)/H2O = 1/3) and slowly evaporated on a sand bath. The amorphous precipitate was denitrified on a magnetic hot plate. The powder was homogenized in an agate mortar and sintered at 950°C; the phases were identified by X-ray diffraction (XRD) using Cu K anticathode. For photo electrochemical (PEC) characterization, the powder was cold pressed under 5 kbar into 13 mm diameters discs of 1 mm thickness and sintered at 900 °C. The density of the sample was found to be 90% of the theoretical bulk value and the mechanical properties were acceptable.

Characterization of FeAl 2 O 4

FeAl2O4 exhibited a brick red color with homogeneous morphology. The XRD pattern was free of the starting oxides and revealed the existence of a pure spinel phase. All peaks were indexed in a cubic symmetry (space group Fd3m) with a lattice constant of 0.8441 nm in agreement with the JCPDS card N° 22-1012.

The size crystallite (~ 40 nm) is evaluated from the full with at half maximum (β, rad) of the most intense (311) XRD spectrum, according to the empirical relationship [START_REF] Nuffield | X-ray Diffraction Methods[END_REF].

   cos 94 . 0  d (1)
Λ (= 1.54178 Å) is the wavelength of the copper anticathode and θ (rad) the diffraction angle.

The specific surface area (Ssp) of the oxide is defined by the ratio of the surface of the crystalline particle assumed to be spherical.

With:

r 3 r 3 4 . r 4 m S S exp 3 exp 2 sp        (2)
d 6 S exp sp   (3)
Where ex is the experimental density (kg/m 3 ) and r the particle radius (m).

The particle of photo-catalyst was equivalent to a sphere of radius r. According to the calculations of the lattice parameter a, it was found that the solid has a cubic spinel structure.

The density of the photo-catalyst was calculated from the following relationship:

3 exp a . N M 8 V M    (4)
N is the Avogadro's number, and M the molecular weight and V the volume of the unit cell.

The corresponding results are collected in Table 1.

Analysis

The FTIR spectra were recorded with a Perkin-Elmer spectrometer (Spectrum One).

The morphologies of the different membranes were determined by SEM analysis using a JEOL JSM 6301F-FEG. The thermo-gravimetric analyses (TGA) were achieved using a TGA Q500, TA Instrument, programmed from 50 to 600°C at a rate of 10˚C/min under nitrogen atmosphere.

The M 2+ concentration was determined by atomic absorption using a Perkin-Elmer 2380 spectrometer (air/acetylene flame). The wavelengths used for the determination of Cu, Zn and Ni were 324.8, 213.9 and 232 nm, respectively. A digital pH meter Cyberscan 500 was used to accurately measure the pH.

Closed cell device

Figure 1 shows schematically the principle of the closed cell device with the capacity of the different compartments. Three independent experiments were realized to determine the metallic ions concentration; the experimental standard deviation was determined to be ± 5%.

Photo-electrodialysis using (CTA + PEI + TEHP) membrane noted M1

Photo-electrodialysis was performed under visible light using a tungsten lamp (200 W, Osram). The polarization of the photo-electrode, when connected to the inert counter electrode (stainless steel) generates an electric field through the cell containing the membranes. The same cell and the same membrane M1 were used. The open cell used in this work is illustrated in Figure 2.

Results and discussion

Physical characterization of the synthesized membranes

The characteristics of the membrane synthesized from CTA polymer and polyelectrolytes (PEI, PEG and PVP) are gathered in Table 2. As the plasticizer molecules are hydrophobic, the location of TEHP on the surface and in the volume of the modified membranes changes the contact angle which is an indicative parameter of the wetting character of the material. The results indicate that all synthesized membranes were amorphous PIMs whose physical properties were modified. The plasticizer TEHP is hydrophobic and hence caused the elevation of the contact angle of the synthesized membranes (θ= 46° in the case of hydrophilic CTA membrane and θ= ~ 76° in the case of hydrophobic CTA-TEHP membrane). This value decreased in the case of (CTA+ PEI+ TEHP) and (CTA+ PVP+ TEHP) membranes because PEI, PVP and PEG are hydrophilic polyelectrolytes.

FTIR characterization

Figures 3a-c show the FTIR spectra of the three synthesized membranes. The main feature of the spectrum of (CTA + PEI + TEHP) membrane was the absorption band around 1032 cm -1 corresponded to stretching modes of symmetric and asymmetric C-O single bonds respectively. Less intense bands at 2960-2932 and 2870 cm -1 can be ascribed to C-H bonds, while the wide band observed at 3475 cm -1 was due to the N-H bond stretching modes. The spectra of (CTA + PEG + PVP + TEHP) and (CTA + PVP + TEHP) membranes showed the same bands with further ones at 1659 and 1665 cm -1 , assigned to the stretching modes of C=C bonds of PVP and PEG.

Characterization by SEM

SEM characterization is used to assess the effect of additives on the membranes morphology [28-30]. SEM images of M1, M2 and M3 membranes are given as surface section (1 µm) in Figures 4a-c, respectively.

From SEM images, it can be observed that the morphologies of the (polymer + polyelectrolyte + plasticizer) membranes had a uniform surface and appeared dense with no apparent porosity. We remark also that the hybrid membranes exhibited still homogeneous sponge microstructures. These observations confirm the results of the contact angle.

Characterization by thermo-gravimetric analysis (TGA)

Thermal gravimetric analysis (TGA) is a method of thermal investigation in which changes in physical and chemical properties of materials are measured as a function of increasing temperature (with constant heating rate). TGA is commonly used to determine selected characteristics of materials that exhibit either mass loss or gain due to decomposition, oxidation, or loss of volatiles component (such as solvent and plasticizer).

The TGA experiments were done using TA Instrument equipment at a scanning speed of 10 °C min -1 . Figures 5a-c show the TGA thermal behaviors of M1, M2 and M3 membranes, respectively. (CTA + PVP + TEHP) membrane degraded also in two steps (Figure 5c). The first one at 186 °C corresponded to the volatilization of TEHP. The second step, at 323 °C, represented a degradation of CTA and PVP polymers. The results obtained with M2 and M3 confirmed the presence of specific interactions between the different polymers.

Based on

Transport of M 2+ ions using synthetic membranes

All synthesized membranes were used for the selective transport of bivalent ions M 2+ , the three membranes contained the same basis polymer (CTA) and the same plasticizer (TEHP); the difference was the polyelectrolyte nature (PEI, PEG, PVP).

Amount of metal transferred (titration in Compartment 2).

The cell used for Dialysis experiments consisted of three compartments, made of Teflon with a maximum filling volume of 100 mL, separated by the synthesized membranes. The feed compartment contained a mixture of M 2+ ions (= Cu 2+ , Zn 2+ and Ni 2+ ) at a concentration of 10 -2 mol L -1 of the metal salt (3.3310 -3 mol L -1 for each metal); the second compartment contained thiourea CS(NH2)2 and H2SO4 (0.5 mol L -1 ); the third compartment was filled with only H2SO4 (0.5 mol L -1 ) solution. The use of thiourea should enhance the selectivity according to a chelation effect toward metals [START_REF] Said | [END_REF]. It is soluble in water but not in organic solvents and presents basic properties due to the electron distinct pairs of sulfide and nitrogen atoms. These electron pairs give thiourea a complexation capacity especially towards heavy metals. According to the Lewis structure, the coordination can be affected by the nitrogen atoms or sulfur atoms. -c show the variation of the M 2+ concentrations in the first receiving compartment according to the dialysis duration. We note that the efficiency and the selectivity of the transferred M 2+ ions depend on the membrane nature.

Figures 6a

In the case of the membrane M1, the transferred amount displays the following order: Ni 2+ > Zn 2+ > Cu 2+ . The amount of Cu 2+ transferred to the second compartment appeared insignificant and this can be due to the two main reasons: i) Cu 2+ competes with Ni 2+ and Zn 2+ which is not at all in favor of this latter ii) Ni 2+ is fully and rapidly transferred from the second to the third compartment. The same findings were also observed with the two other membranes. In the case of Zn 2+ , the concentration in the compartment 2 decreased during 3 h, suggesting that it continues to be transferred to the third compartment. The beginning of the Ni 2+ and Zn 2+ transfer through the membrane M2 was observed after 2 h of dialysis.

Amount of metal transferred (titration in compartment 3)

Figures 7a-c illustrate the variation of concentrations in the compartment 3 using the three synthesized membranes M1-M2-M3. It can be seen that Cu 2+ failed to cross the three membranes. This can be explained by the low selectivity for Cu 2+ and the binding within the membrane through complexation with the nitrogen atoms of PEI and PVP polymers. The presence of Zn 2+ was detected in the compartment 3 using the different synthetic membranes. This is due to the fact that Zn 2+ is not or weakly complexed by the polymers of M1, M2 and M3 membranes. The Ni² + was not detected in the compartment 3, this is due to the fact that Ni² + is complexed by thiourea in the second compartment.

Transport of M 2+ ions using a commercial membrane (titration in the compartments 2 and 3)

For a comparative purpose, a commercial membrane-type cation exchange, CMS, was considered in the same experimental conditions, i.e. the same cell and same solutions. We note that this commercial membrane behaves in a quite similar manner for the three ions.

Photo-electrodialysis through the M1 membrane in the open cell

The M1 membrane showed good ability in the separation of the three ions and hence was chosen for the photo-electrodialysis. The photo-electrode FeAl2O4 was placed in the first compartment while the inert steel counter electrode in the third compartment; therefore, the cell worked in a short circuit configuration.

Effect of the concentration of the transferred metal

Based on Figure 9, note that a significant amount of M 2+ ions was transferred through the membrane M1 using FeAl2O4 as photoelectrode. Concerning the metal transfer and similarly to the trend observed in the absence of the photoelectrode, whatever the experimental conditions Cu 2+ did not react favorably with the membrane M1. Zn 2+ migration from the first to the second compartment was very effective, since 33% (1.210 -3 mol L -1 ) of the initial amount was transferred to the second compartment after 3 h, without taking into account the transferred amount to the third compartment and that adsorbed by the two membranes. A concentration of 610 -4 mol L -1 was found in the third compartment at the end of the experiment. This shows the high selectivity of the membrane M1 in terms of photoelectrodialysis regarding Zn 2+ . Ni 2+ and Zn 2+ moved relatively at low rate, before a sharp increase in the concentration was recorded in the third compartment after 3 h. It is worth noting that the composition of the membrane M1 (consisting of PEI and CTA in addition to the plasticizer) is likely to fix these metals to form stable amine complexes according to the sequence: Cu > Ni > Zn. This is due to the saturation of the membrane by the metal-carrier complex molecules and the decomplexation at the strip interface is presumably the ratelimiting step in such process. Fujimori [33] revealed the existence of copper and nickel complex with PEI in the stoichiometry 3.2 and 3.7 for PEI:Cu and PEI:Ni, respectively. This is due to the geometry of the complex (square planar in the case of copper and octahedral for nickel). These complexes are more stable than the PEI:Zn. In Table 3 are reported the overall formation constants of the three complexes [34]. As it can be observed, the stabilities are involved in the order: PEI:Cu > PEI:Ni > PEI:Zn.

The best transfer for the three metal ions was obtained at a current density of 0.78 mA cm -2 and an initial concentration of 10 -2 mol L -1 .

The effect of the potential

The use of the semi-conducting photoelectrode is considered as a promising tool to generate an electrical gradient field to improve ions transfer. Indeed, the junction electric field generated under illumination substantially increases the diffusion flux of ions. Figure 10 shows the values of the generated potential over time, measured in parallel with a high impedance voltmeter (10 9 ) via the two electrodes; it increased during the first 100 minutes and then remained almost stable.

Variation of the conductivity and the pH

Along with determining the potential through the cell, both the conductivity and pH of the solutions of all compartments were monitored and the results are gathered in Figure 11.

Three distinct zones are visible in the graph of the conductivity.

The first zone corresponds to the decrease of the conductivity over time up to 3 h of dialysis in the first compartment (feed compartment) can be explained by the movement of ions from the solution of a compartment to the inside of the membrane. The second zone corresponds to the second movement of ions or exchange that occurs into the membrane between its protons and ions M 2+ of the solution. In the third zone, a slight decrease of the conductivity was observed, due to the hydrogen evolution, which was observed at the end of the experiment.

The H2-liberation can be mainly attributed to the reactions of reduction of protons at the electrodes.

Conclusion

The possibility of using a less energy-consuming process for wastewater treatment was examined in this work. New polymeric membranes were implemented and tested in processes using ion exchange materials for the treatment of solutions of hydro-metallurgical industries; the ion exchange membrane is an essential part of the process. The membrane was successfully polymerized from cellulose triacetate noted (CTA) modified by poly-electrolytes and plasticized by (TEHP).

The obtained results showed that a developed membrane-based PEI was not selective with regard to Cu 2+ but had a high affinity for Ni 2+ transfer, followed by Zn 2+ . The transferred amount of Zn 2+ was higher during photo-electrodialysis than in simple dialysis, so the developed PEI based membrane becoming more selective to Zn 2+ under illumination. The best transfer for the three metal ions was obtained at a current density of 0.78 mA cm -2 and an initial concentration of 10 -2 mol L -1 . It has also been shown that the commercial membrane was selective to zinc in the closed cell, by cons in the open cell it was selective for copper.

This study may be continued by examining the nature of the medium (pH, conductivity, temperature, etc. ...). Further efforts should be directed toward the determination of the nature of interactions between polymer and poly-electrolytes by the use of other materials and analytical tools as well. Other applications may also be planned for other objectives (biological, medical, environmental, …etc).

  Most of the conventional separation and concentration processes used in hydrometallurgy, like precipitation, ion exchange on resins or liquid-liquid extraction [7-10],

  photocatalysis because of the pH-insensitivity of the electronic bands and optical properties permitting the exploitation of the solar spectrum [19].

  received a great interest although they possess a forbidden band with d-d photo transitions and have relatively large absorption coefficients [20].

Figure 5a ,

 5a the following observation can be made: (CTA + PEI + TEHP) membrane was thermally degraded in three steps. The first one at 155 °C represented the volatilization of TEHP. The second and third steps, at 280 and 390 °C, represent a degradation of polyethylenimine (PEI) and cellulose triacetate (CTA) respectively. It must be noted that the volatilization of TEHP started before its boiling point (215 °C) meaning that TEHP has interacted with CTA and PEI polymers. (CTA + PEG + PVP + TEHP) membrane degraded in two steps (Figure 5b). The first one at 180°C represented the volatilization of TEHP. The second step, at 350 °C, represented a degradation of different polymers (CTA, PEG, PVP).

Figure 8

 8 Figure 8 show the amounts of metal transferred in the compartments 2 (a) and 3 (b) using the commercial CMS membrane. A change in the concentration was observed after 3 h of dialysis. Time-courses of the amount of metal transferred show that Ni 2+ and Cu 2+ were the most transferred ions using CMS commercial membrane in comparison with the three synthesized membranes.

Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 7 .Figure 8 .Figure 9 .Figure 10 .

 234578910 Figure 2. Transport cell scheme: A: Synthetic membrane; B: FeAl2O4 electrode; C: platinum electrode

Table 1 : Physical Parameters of FeAl 2 O 4

 1

	Oxyde	ρexp (g/cm 3 )	Ssp	d
	FeAl2O4	3.83	8.584	0.1825