Sophie Drouillard

Laurent Poulet

Eric Marechal

Alberto Amato

Laurine Buon

Mélanie Loiodice

William Helbert

Structure and enzymatic degradation of the polysaccharide secreted by Nostoc commune

Keywords: Nostoc commune, Polysaccharide structure, NMR, Enzymatic degradation

Noctoc commune is a cyanobacterium living in various and extreme environments. Its ability to survive in desert, on ice or high altitude is explained by its exceptional metabolism and its capacity to resist to desiccation. N. commune cells are embedded in a gelatinous matrix made of polysaccharides which fixes water and participates in maintaining the cells in hydrated conditions. The structure of the polysaccharide of N. commune harvested in Saint Martin d'Uriage (France) and the oligosaccharides obtained after its enzymatic degradation were determined. The repeating unit of the main chain is a tetra-saccharide: [→4)-

branched at position 6 of a glucose residue by a -linked pyruvated glucuronic acid residue. About 30% of the Xylp residues were branched with a Xylf residue. Comparison of this structure with the polysaccharides secreted by other Nostoc species and strains suggest a strong selection pressure on the structure in agreement with its important biological role.

Introduction

The Nostoc genus groups very diverse species of filamentous cyanobacteria that form microscopic or macroscopic colonies embedded in a gelatinous matrix. The colonies may adopt various morphologies: sheet-like (N. commune), hair-like (N. flagelliform) or spheric morphology (N. pruniform, N. sphaeroides) as a function of the species and their growth conditions. Nostoc sp. have been collected and used for food and medicine for centuries in many places in the world. Currently, Nostoc flagelliform ("facai") in China and Nostoc pruniform ("llullucha") in South America are nowadays probably the most used for their nutritive properties as diet complement [START_REF] Dodds | The ecology of Nostoc[END_REF][START_REF] Gao | Chinese studies on the edible bluegreen alga, Nostoc flagelliforme: a review[END_REF][START_REF] Qui | Distribution and ecology of the edible cyanobacterium Ge-Xian-Mi (Nostoc) in rice fields of Hefeng County in China[END_REF]Sand-Jensens, 2014). These microorganisms may be found in various and extreme, terrestrial and aquatic environments. They tolerate the freezing temperature of Antarctic (e.g. Nostoc commune) [START_REF] Davey | The effects of freezing and desiccation on photosynthesis and survival of terrestrial antarctic algae and cyanobacteria[END_REF][START_REF] Novis | Annual carbon fixation in terrestrial populations of Nostoc commune (Cyanobacteria) from an Antarctic dry valley is driven by temperature regime[END_REF], high temperature of desert (e.g. Nostoc flagelliform; [START_REF] Gao | Chinese studies on the edible bluegreen alga, Nostoc flagelliforme: a review[END_REF][START_REF] Hu | Extracellular carbohydrate polymers from five desert soil algae with different cohesion in the stabilization of fine sand grain[END_REF] and sun radiation found in the altitude of Andes [START_REF] Fleming | Characterization of cyanobacterial communities from highelevation lakes in the Bolivian Andes: high-elevation Andes Cyanobacteria[END_REF]. The exceptional capacity of Nostoc spp. to live in poor habitat and to adapt to extreme environments is explained by their metabolisms and their capacity to resist to desiccation [START_REF] Dodds | The ecology of Nostoc[END_REF][START_REF] Sand-Jensen | Ecophysiology of gelatinous Nostoc colonies: unprecedented slow growth and survival in resource-poor and harsh environments[END_REF]. As other cyanobacteria, Nostoc spp. can fix carbon by photosynthesis and they developed strategies to incorporate atmospheric nitrogen. To face desiccation, cyanobacterial cells differentiate into akinete cells (resting-state cells of cyanobacteria) which germinate when conditions become more favorable [START_REF] Perez | Clear differences in metabolic and morphological adaptations of akinetes of two Nostocales living in different habitats[END_REF].

The gelatinous matrix embedding the cyanobacterial colonies is mainly composed of a high molecular weight polysaccharide forming a highly viscous solution. The physicochemical properties of the polysaccharide and its capacity to retain water is correlated to the structural properties of the macromolecule. Composition analyses of the secreted polysaccharide conducted on numerous species and strains of Nostoc revealed that some neutral monosaccharide residues (glucose: Glc; galactose: Gal; xylose: Xyl) and uronic residues (glucuronic acid:GlcA) are very often present. Other pentose residues such as arabinose (Ara) and ribose (Rib), and organic groups (lactate, pyruvate) also found in the composition increase the complexity of the polysaccharides [START_REF] Huang | Studies on polysaccharides from three edible species of Nostoc (cyanobacteria) with different colony morphologies: comparison of monosaccharide compositions and viscosities of polysaccharides from field colonies and suspension cultures[END_REF][START_REF] Brüll | Studies of polysaccharides from three edible species of Nostoc (cyanobacteria) with different colony morphologies: structural characterization and effect on the complement system of polysaccharides from Nostoc commune[END_REF][START_REF] Pereira | Complexity of cyanobacterial exopolysaccharides: composition, structures, inducing factors and putative genes involved in their biosynthesis and assembly[END_REF].

The first structure of the polysaccharides secreted by two strains of Nostoc commune collected in Asia were determined by Helm and co-workers (2000) and Brüll and co-workers (2000). The structures showed a similar tetrasaccharide repeating unit of the main chain made of [4) -D-Glc (14) -D-Gal (14) -D-Glc (14) Xyl (1] carrying an uronic residue (glucuronic acid: GlcA, or nosturonic acid: pyruvated-GlcA) and a deoxy residue (Ara, Rib). The structure of the polysaccharides, and more especially the decoration of the main chain, was found to change with growth conditions [START_REF] Brüll | Studies of polysaccharides from three edible species of Nostoc (cyanobacteria) with different colony morphologies: structural characterization and effect on the complement system of polysaccharides from Nostoc commune[END_REF].

The exact biological role of the matrix polysaccharide is not well understood but it probably participate in the survival of Nostoc spp. by retaining water in dry environment and by participating in the rewetting of the akinete cells prior they germinate when environmental conditions are favorable.

Assuming that the biological role in the survival of Nostoc cells is driven by the structural features of the macromolecules, we hypothesized that their chemical characteristics were probably conserved in the Nostoc commune species and may be shared with other species of the genus Nostoc. In this context, we have analyzed the polysaccharide structure of a strain of Nostoc commune collected in Europe. With the help an enzyme catalyzing the degradation of the Nostoc commune polysaccharides, recently identified through the systematic screening of distantly related carbohydrate active enzymes [START_REF] Helbert | Discovery of novel carbohydrate-active enzymes through the rational exploration of the protein sequences space[END_REF], we have produced a series of oligosaccharides helping in the resolution of the chemical structure of the polysaccharide.

Material and methods

DNA extraction

Frozen sheet-like Nostoc commune were grinded to fine powder in liquid nitrogen. The sample was suspended in 500µl of lysis buffer (250 mM Tris pH 8.2, 100 mM EDTA, 2% SDS, 100 mM NaCl) and was warmed for 30 minutes at 90°C. During this step, the suspension was vortexed for 30 seconds every 10 minutes. Then, 500 µL of phenol:chloroform:isoamyl alcohol (25:24:1) were added to the sample and mixed vigorously to obtain a homogeneous suspension (at least 3 minutes with vortex). In order to separate the cell debris and the aqueous solution containing the nucleic acids, the samples were centrifuged 10 minutes at 17,000 × g at 4°C. The upper phase was transferred to a new tube and 300 µL of chloroform:isoamyl alcohol (24:1) were added. The sample was vigorously mixed by inverting the tubes followed by centrifugation for 10 min at 13,000 × g at 4°C aiming at separating the two phases. The upper aqueous phase was transferred in a new tube and 30µL of 3M Na-acetate and 1.5 volumes (750 µL) of absolute ethanol were added to precipitate the DNA. Precipitation was conducted for at least 20 minutes at -20°C. DNA salts were pelleted by centrifugation (10 min. at 13,000 × g at 4°C). The supernatant was discarded and the pellet was washed twice with 500 µL of 70% ethanol. Washing steps were done by adding ethanol to the pellet and centrifuging 5 minutes at 13,000 × g at 4°C. After the second ethanol washing, the supernatant was carefully removed without disturbing the pellet and it was air dried for 30-60 minutes. The pellet was then resuspended in TE buffer. Purity and quantity were controlled with a NanoDrop spectrophotometer.

PCR amplification and sequencing

100 ng of the purified DNA was used as template for PCR amplification. CYA359F and CYA781R [START_REF] Boutte | Testing of primers for the study of cyanobacterial molecular diversity by DGGE[END_REF] primer pair was used to amplify a 427nt fragment of the 16S rRNA gene. The PCR reactions were performed in 25 µL total volume, containing 5 µl 5X Phusion HF buffer, 200 µM (final concentration) dNTPs, 0.5 µM of each primer, 0.5 units of BioLabs Phusion® High-Fidelity DNA Polymerase (M0530). A Biorad T100™ Thermal Cycler was used for PCR reactions with the following cycles: a desaturation step at 98°C for 30 seconds, a gradient from 45 to 60 °C for the annealing (5 temperatures) and an elongation of 15 seconds at 72°C. The PCR products were loaded on a 0.8% agarose TAE gel electrophoresis working for 30 minutes at 100V. The DNA fragment was visualized on a UV/white light transilluminator. All the tested annealing temperatures produced an amplification with a clear band at the expected size. Because no band of no specific amplification was observed on the gel, PCR products were purified using a Macherey-Nagel NucleoSpin® Gel and PCR Clean-up kit following the manufacturer's instructions. Purified PCR products were sent out to Eurofins Genomics for sequencing in both 5' and 3' directions using the amplification primers.

Sequences were visualized and assembled in BioEdit Sequence Alignment Editor [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF] and blasted against GenBank. All the top hit sequences were downloaded and further analyzed.

Polysaccharide extraction and purification

Small pieces of Nostoc commune were suspended in distilled water at 100°C for 1h. After removing the water and rinsing twice the N. commune fragments with fresh water, the sample was treated at 70°C for 6 h by a bleaching solution which consisted of 1 volume of 1.7% aqueous NaClO 2 and 1 volume of acetate buffer (pH 4.9), completed with 3 volumes of distilled water. The sample was washed with distilled water by repeated centrifugation and finally resuspended in 3% (w/v) KOH solution overnight at room temperature. The insoluble fraction was discarded by centrifugation. The polysaccharide was precipitated by adding an equal volume of ethanol to the supernatant. The pellet was washed by repeated centrifugation with water/ethanol solution (1/1, v/v). The polysaccharide was finally suspended in water and lyophilized.

Monosaccharide analysis

The molar ratio of the monosaccharides was determined according to [START_REF] Kamerling | Characterization by gas-liquid chromatography mass spectrometry and proton-magnetic-resonance spectroscopy of pertrimethylsilyl methyl glycosides obtained in the methanolysis of glycoproteins and glycopeptides[END_REF] modified by [START_REF] Montreuil | Glycoproteines. In Carbohydrates Analysis: A Practical Approach[END_REF]. The EPS was hydrolysed with 3 M MeOH/HCl at 110°C for 4 h, followed by re-N-acetylation with Ac 2 O overnight at room temperature. The methyl glycosides were converted to their corresponding trimethylsilyl derivatives. Separation and quantification of the per-O-trimethylsilyl methyl glycosides were performed using gas-liquid chromatography (GLC) on an Agilent system equipped with a HP-5 ms capillary column (Agilent 0.25 mm x 30 m). The trimethylsilyl derivatives were analysed using the following temperature program: 120°C180°C at 3°C/min, 180°C200°C at 2°C/min, 200°C for 5 min.

Methylation analysis

Glycosyl linkage positions were determined as described in [START_REF] Hakomori | A rapid permethylation of glycolipid, and polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl sulfoxide[END_REF]. Hydroxyl groups were methylated using the lithium dimethylsulfonyl as the anion and methyl iodide in Me 2 SO [START_REF] Blakeney | Methylation of carbohydrates with lithium methylsulphinyl carbanion[END_REF]Kvernheim, 1987). After two methylation steps, the permethylated products was carboxylreduced by LiBD4 in absolute ethanol for 2h at 80°C [START_REF] Linnerborg | Structural Studies of the O-Antigenic Polysaccharide from Escherichia Coli O167[END_REF]. Excess of borodeuteride was decomposed with 10% acetic acid. After evaporation, the permethylated and carboxyl-reduced products were hydrolysed in 2M trifluoroacetic acid (TFA) for 2h at 120°C, then reduced with NaBD 4 in an NH 4 OH solution for 30min at 80°C, and finally acetylated with 200µL of 1-methyl imidazole and 2mL acetic anhydride for 10min at room temperature. GC-mass spectrometry (MS) was performed on an Agilent instrument fitted with a Supelco SP2380 capillary column (0.25 mm× 30 m). The temperature program was 150 °C for 2 min, 150 °C → 240 °C at 3 °C/min, 240 °C for 5 min. Ionisation was carried out in electron impact mode (EI, 70 eV). All analyses were conducted in triplicate.

Preparation of the Nostoc polysaccharide hydrolase

Nostoc polysaccharide hydrolase was overexpressed and purified by affinity chromatography as previously reported in Helbert and co-workers (2019). Briefly, the gene encoding the enzymes was cloned in pHTP1 (NZYtech, Portugal) vector harboring a His 6 -tag at the N-terminal position. The enzyme was overexpressed in E. coli BL21(DE3) pLysS strain grown in NZY auto-induction LB media (NZYtech, Portugal). After a freeze-thaw cycle, the protein was purified by affinity using a nickel agarose affinity resin (Ni-NTA resin, Qiagen) loaded on poly-prep® chromatography columns (Bio-Rad 731-1550). The His 6 -tagged recombinant enzyme was eluted with increasing amounts of imidazole (20 mM, 50 mM, 150 mM, 300 mM and 500 mM). The purity of the fractions was estimated by 10% SDS-PAGE analysis.

Enzymatic degradation and purification of oligosaccharides

Enzymatic degradations were carried out by incubating 10 mL of purified polysaccharides (1% w/v in 20 mM Tris-HCl pH 7.5, 20 mM NaCl) with 500 µL of purified enzyme (0.60µg.µL -1 ) at 25°C. End-products were purified on a semi-preparative size-exclusion chromatography system which consisted of a Knauer pump (pump model 100), a HW40 Toyopearl column (120 x 16 mm; Tosoh Corporation), a refractive index detector (Iota 2, Precision Instruments) and a fraction collector (Foxy R, Teledyne Isco 1) mounted in series. Elution was conducted at a flow rate of 0.4 mL/min at room temperature using 100 mM (NH 4 ) 2 CO 3 as the eluent. The fractions containing the oligosaccharides were collected and freeze-dried.

NMR

Carbon-13 and proton NMR spectra were recorded with a Bruker Avance 400 spectrometer operating at a frequency of 100.618 MHz for 13 C and 400.13 MHz for 1 H. Samples were solubilized in D 2 O at a temperature of 293 K for the oligosaccharides and 353 K for the polysaccharide. Residual signal of the solvent was used as internal standard: HOD at 4.85 ppm at 293 K and 4.25 at 353 K. 13 C spectra were recorded using 90° pulses, 20,000 Hz spectral width, 65,536 data points, 1.638 s acquisition time, 1 s relaxation delay and between 8192 and 16,834 scans. Proton spectra were recorded with a 4006 Hz spectral width, 32,768 data points, 4.089 s acquisition times, 0.1 s relaxation delays and 16 scans. The 1 H and 13 C-NMR assignments were based on 1 H-1 H homonuclear and 1 H-13 C heteronuclear correlation experiments (correlation spectroscopy, COSY; heteronuclear multiple-bond correlation, HMBC; heteronuclear single quantum correlation, HSQC). They were performed with a 4006 Hz spectral width, 2048 data points, 0.255 s acquisition time, 1 s relaxation delay; 32 to 512 scans were accumulated.

Results and discussion

Identification of the Nostoc commune strain

The Nostoc commune strain was collected at Saint Martin d'Uriage (France, 45°09'53.7"N 5°51'18.9"E). The macroscopic colonies have dark green gelatinous sheet-like morphology. After extraction, 16S RNA was sequenced and compared with Nostoc 16S RNA sequences available in Genebank. The sequence showed >98% identity with Nostoc commune strains isolated in Europe (AB113665.1, France; AY577536.1, Czech Republic, strain EV1-KK1; KY283050.1, Russia, strain ACSSI 030), Asia (AB933330.2, Japan, strain KU006; EU178142.1, China, strain AHNG0605; KY129708.1, India, strain BHU81) and North America (HQ877826.1, Mexico, strain 257-16). The 16S RNA analyses demonstrated that the cyanobacterial colonies collected share the same molecular characteristic of Nostoc commune strains.

Table 1. Methylation analysis of the matrix polysaccharide secreted by Nostoc commune. The percentage of the main linkages observed were estimated by measuring the surface area of the signal.

Residue

Partially Methylated Alditol Acetate Deduced Linkage

Peak surface area (%) Glucose 1,4,5-Tri-O-acetyl-1-deuterio-2,3,6,-tri-O-methyl-D-glucitol 4)-Glcp-(1 22.7 Glucose 1,4,5,6-Tetra-O-acetyl-1-deuterio-2,3,-di-O-methyl-D-glucitol 4,6)-Glcp-(1 24.1 Xylose 1,3,4,5-Tetra-O-acetyl-1-deuterio-2-O-methyl-D-xylitol 3,4)-Xylp-(1 7.7 Xylose 1,4,5-Tri-O-acetyl-1-deuterio-2,3 di-O-methyl-D-xylitol 4)-Xylp-(1 8.8 Xylose 1,4-Di-O-acetyl-1-deuterio-2,3,5-tri-O-methyl-D-xylitol Xylf-(1 1.8 Galactose 1,4,5-Tri-O-acetyl-1-deuterio-2,3,6-tri-O-methyl-D-galactitol 4)-Galp-(1 18.1 Glucuronic acid 1,5,6-Tri-O-acetyl-1,6,6-trideuterio-2,3,4-tri-O-methyl-D- glucitol GlcAp-(1 1.5

Characterization of the matrix polysaccharide of Nostoc commune

Composition analysis using gas chromatography, after the complete hydrolysis of the polysaccharide and trimethylsilyl derivatization of the products, revealed glucose (Glc), galactose (Gal), xylose (Xyl) and glucuronic acid (GlcA) (% molar ratio Glc/Gal/Xyl/GlcA 1/0.48/0.90/0.14) (Figure S1).

Traces of mannose (% molar ratio Glc/Man 1/0.08) were also detected in the purified polysaccharide.

Methylation analyses (Table 1) revealed that the Glcp residues is mainly involved in 1,4-and 1,4,6linkages; the Galp residue participate in 1,4 linkages and the Xylp residue is bound by the position 1, 3 and 4. Terminal GlcpA was also observed suggesting its branching on the main chain. In addition, we detected a derivative presenting the same elution time than the 1,4-di-O-acetyl-1-deuterio-2,3,5-tri-Omethyl-D-xylitol (Figure S2A) which is present in low amount in the xylose standard (Figure S2B). The fragmentation products of the derivatives were compared (Figure S3) and validated the occurrence of a terminal xylofuranoside (T-Xylf) involved in a side chain.

Carbon and proton NMR recorded on the polymer revealed the occurrence of six different residues as illustrated by the heteronuclear 1 H/ 13 C chemical shift correlation (HSQC) presented in Figure 1. The proton correlation systems observed in the homonuclear 1 H/ 1 H chemical shift correlation (COSY, TOCSY), the determination of the chemical shifts and coupling constants (Table 2) confirmed the occurrence of glucose, galactose, glucuronic acid and xylose residues expected from chemical analyses. Table 2: 1 H and 13 C NMR chemical shifts recorded on the secreted polysaccharide of Nostoc commune.

Analyses of the enzymatic degradation products of the N. commune polysaccharide

To deepen the structural characterization of the polysaccharide, we took advantage of our recent discovery of an endo-acting enzyme able to cleave the polysaccharides in oligosaccharides [START_REF] Helbert | Discovery of novel carbohydrate-active enzymes through the rational exploration of the protein sequences space[END_REF]. This new enzyme which is the first member of the glycoside hydrolase family GH160 to cleave the linkage between the galactose (sub-site -1) and a glucose (sub-site +1) residues giving oligosaccharide 214 series with galactose at the reducing end and glucose at the non-reducing end. 215 Enzymatic degradation of the polysaccharide was monitored by size exclusion chromatography and oligosaccharides obtained after complete digestion of the macromolecule were collected (Figure 2A).

Composition of the oligosaccharide fractions was determined (Figure S5) and their structure characterized by NMR (Figure 2B). The peak eluting at 507 min contained two oligosaccharides which could be identified by collecting different fractions of this peak. Based on composition analyses, 13 C and 1 H NMR spectra, the smallest oligosaccharide was found to have a degree of polymerization of DP=5 (elution time 510 min) with galactose at the reducing end and glucose at the non-reducing end as expected.

After mild acid hydrolysis, allowing the removal of the pyruvate group, the chemical shift of the glucuronic acid residue was shifted from C1/H1: 101.29/5.03 ppm to C1/H1; 102.63/4.57 ppm (Figure 2B, spectra DP5(-)Pyr). Analysis of the correlation system in the COSY (Figure 3A) allowed attributing the chemical shifts to five residues: the galactose located at the reducing end (Gal-C1/H1: 92.27/5.32 ppm and Gal-C1/H1: 96.38/4.67 ppm), the glucose residue at the non-reducing end (Glc1nr-C1/H1: 101.07/4.57 ppm), the glucuronic acid residue decorated by a pyruvate (GlcA-C1/H1: 101.29/5.03 ppm), a second glucose residue (Glc2-C1/H1→Gal: 103.35/4.74 ppm and Glc2-C1/H1→Gal: 103.49/4.73 ppm) and a xylopyranoside residue (Xyl-C1/H1: 103.06/4.49ppm) (Table 3). The linkage between these residues were determined straightforwardly using heteronuclear 1 H/ 13 C chemical shift correlation (HMBC, Figure 3B). Altogether the structure of the oligosaccharide deduced from these analyses was a pentasaccharide made of the tetrasaccharide:

-D-Glc (1 → 4)--D-Xyl (1 → 4)--D-Glc (1 → 4)-/-D-
Gal, branched with a 2,3pyruvated -D-GlcA linked at the position 6 of the internal glucose residue.

We noticed that the two glucose residues observed in the main chain presented very different chemical shifts. The residue Glc1nr-C1/H1: 101.07/4.57 ppm showed a lower C1/H1 chemical shifts than for the residue Glc2 (Glc2-C1/H1→Gal: 103.65/4.74 ppm and Glc2-C1/H1→Gal: 103.49/4.73 ppm).

The resolution of the NMR spectra recorded on the oligosaccharides and, more especially, the carbon/proton coupling observed in heteronuclear single quantum coherence (HSQC) were obvious. The lower Glc1-C1/H1 chemical shifts is explained by its bounding to a xylose residue as previously observed by Helm and co-workers (2000). Calculating of the chemical shifts using the online CASPER program The asymmetric center of the pyruvate group was determined by NOESY (Nuclear Overhauser Effect Spectroscopy) according to the same strategy reported by [START_REF] Severn | The acidic specific capsular polysaccharide of Rhodococcusequi serotype-3 -structural elucidation and stereochemical analysis of the lactate ether and pyruvate acetal substituents[END_REF]. The NOESY spectrum revealed a long distance correlation between the protons of the methyl group of pyruvate (Pyr-CH3= 1.64 ppm) with proton H2 of the glucuronic residues (GlcA-H2=3.52 ppm). No correlation was observed with proton H3 demonstrating the pyruvate group adopted the R configuration.

Composition analyses of the fractions containing higher size oligosaccharides were enriched in xylose residues (Figure S5) suggesting the branching of xylose residues on the main chain as observed for the undigested polysaccharide. The fraction collected at 495 min, contained an oligosaccharide which NMR spectra and chemical shifts recorded in 1 H and 13 C were almost identical to that of the previous DP5 suggesting a very similar structure (Figure 2B, DP6). However, additional signals observed in NMR spectra (Xylf-C1 =101.88 ppm, Xylf-H1 = 5.55 ppm were attributed to an additional residue suggesting the occurrence of an oligosaccharide of DP=6. Comparison and of the DP5 and DP6 spectra, as well as the proton correlation system evidenced by the well-resolved COSY spectra and the 1 H/ 13 C chemical shift correlation highlighted by the HMBC spectra allowed determining straightforwardly the chemical shifts and the coupling constants. The data recorded were in agreement with an -xylofuranoside residue and similar to interpretations reported in previous works [START_REF] Angyal | Hudson's rules of isorotation as applied to furanosides, and the conformations of methyl aldofuranosides[END_REF][START_REF] Kocharova | The structure of the Citrobacter freundii O8a,8b O-specific polysaccharide containing Dxylofuranose[END_REF]. Other

furanoside residues not detected in composition analyses could also be excluded after NMR investigations. Indeed, the expected C4/H4= 84.7 ppm/4.11 ppm of -ribofuranose [START_REF] Helm | Structural Characterization of the Released Polysaccharide of Desiccation-Tolerant Nostoc commune DRH-1[END_REF], the H4 = 3.68 ppm of -mannofuranose [START_REF] Angyal | Hudson's rules of isorotation as applied to furanosides, and the conformations of methyl aldofuranosides[END_REF] or C4/H4= 82 ppm /3.71 ppm of galactofuranoside [START_REF] Katzenellenbogen | Structural and serological studies on the O-antigen show that Citrobacter youngae PCM1505 must be classified to a new Citrobacter O-serogroup[END_REF] were too different to the recorded C4/H4 = 78.47 ppm/4.40 ppm attributed to the -xylofuranoside. Signals of the heteronuclear 1 H/ 13 C chemical shift correlation (HMBC) were too weak to determine unambiguously the linkage of the residue.

The peak eluting at 415 min was a mixture of several oligosaccharides which were not isolated and which sizes were estimated to DP10, 11 or 12, corresponding to different combinations of the DP5 and DP6. Based on the signal attribution of the DP5 and DP6, most of the 1 H and 13 C NMR signals could be ascribed (Table 3). More interestingly, the linkage between the Glc and Gal residues cleaved by the enzymes was determined using heteronuclear 1 H/ 13 C chemical shift correlation (HMBC, Figure 3C). The (1 → 4)--D-Gal (1 → ], systematically branched at position 6 of a glucose residue by a -linked glucuronic acid residue. No decoration of the glucuronic acid residue was reported for the polysaccharide secreted by the Chinese strain [START_REF] Brüll | Studies of polysaccharides from three edible species of Nostoc (cyanobacteria) with different colony morphologies: structural characterization and effect on the complement system of polysaccharides from Nostoc commune[END_REF]. A lactate group was observed in the case of the polysaccharide of the Mongolian origin [START_REF] Helm | Structural Characterization of the Released Polysaccharide of Desiccation-Tolerant Nostoc commune DRH-1[END_REF] instead of the pyruvate group found in our studied polysaccharide. Interestingly, both lactate and pyruvate were identified in the polysaccharides of a strain isolated in Japan [START_REF] Kajiyama | Structure of hetero polysaccharide from desiccation-tolerant terrestrial cyanobacterium Nostoc commune[END_REF] suggesting that derivatization of the glucuronic acid residue by lactate or pyruvate can co-occur in Nostoc commune. The occurrence of only one specific group -pyruvate or lactate -may reflect some adaptation to the environment or are an independent evolution of the strains which could have abandoned one derivatization pathway. Also it can't be excluded that specific modification of the polysaccharides may be caused by biotic interactions such as, for example, co-existing bacterial strains.

Another difference observed is the replacement of the ribose residue found in the Mongolian strain polysaccharide by a xylose residue in our study, adopting in both cases the furano configuration. In a biosynthesis point of view, the replacement a D-ribose by L-xylose its epimer at position 3 residue is not biochemically improbable and could proceed from an evolution of the biosynthetic pathway. Composition analyses of polysaccharides secreted by various strains of N. commune show that in addition to ribose and xylose, other residues such as arabinose, fucose or rhamnose could occur along the polysaccharide chain [START_REF] Huang | Studies on polysaccharides from three edible species of Nostoc (cyanobacteria) with different colony morphologies: comparison of monosaccharide compositions and viscosities of polysaccharides from field colonies and suspension cultures[END_REF][START_REF] De Philippis | Assessment of the potential of Nostoc strains from the Pasteur culture collection for the production of polysaccharides of applied interest[END_REF]. Altogether, its seems that the polysaccharides secreted by Nostoc commune strains isolated in various place in the world have conserved structural characteristic which suggested a selection pressure on the biosynthetic pathway leading to the preservation of mandatory biological and physico-chemical properties. The capacity of the gelatinous substance embedding of the cyanobacterial cells composed of the polysaccharide, associated to another component such as proteins, is probably its capacity to swell/deswell as a function of the amount of water in a reversible way. Implication of the polysaccharide in the defence mechanisms as a mechanical or biological barrier to protect akinete resting cells probably played also a role in maintaining the structural properties of the polysaccharide.

N. commune strain investigated in this study and the strains isolated in the desert of Mongolia (N.

commune DRH-1) were identified using genetic tools. Few other structural data on the polysaccharides secreted by other species of Nostoc were also reported. However, genetic data were lacking and identification of the species was based on morphology considerations. In this context, comparison of the polysaccharide structures in regard to the evolution of Nostoc species is therefore difficult. However, the oligosaccharides obtained after acid hydrolysis of the polysaccharide of a strain identified as Nostoc flagelliforme (Mongolia, [START_REF] Kanekiyo | Isolation of an antiviral polysaccharide, nostoflan, from a terrestrial cyanobacterium, Nostoc flagelliforme[END_REF]

Conclusion

The structure of the polysaccharide secreted by the strain of N. commune isolated in Saint Martin systematically branched by a -linked glucuronic acid, presented lactate and pyruvate derivatives according to the origin of the Nostoc commune strain. Also, we noticed that a mannose or methylated mannose glucose residue was found in place of a glucose residue of the main chain. The structure of N.

flageliforme polysaccharides showed was similar to N. commune suggesting a common biosynthesis pathway ancestor. The important role of the polysaccharide in the growth and survival of the cyanobacterial cells supposes that strong alteration of modification of the structure and, therefore, the associated physico-chemical properties will be detrimental to the cells. The conservation of the polysaccharide structure of the world-wide spread N. commune strains probably reflects a strong selection pressure on the structure of the polysaccharides.

Figure 1 .

 1 Figure 1. Heteronuclear 1 H/ 13 C chemical shift correlation (HSQC) spectrum of the polysaccharide secreted by Nostoc commune. The spectrum was recorded at 353 K

Figure 2 .

 2 Figure 2. Purification and characterization of the enzymatic degradation products of N. commune polysaccharide. A) Size exclusion chromatography of the degradation products. Inset: the structure of the oligosaccharides determined by NMR analyses. B) 1 H NMR spectra of the oligosaccharides isolated after purification by chromatography. The spectra were recorded at 293 K

Figure 3 .

 3 Figure 3. Analyses of the oligosaccharides by NMR. A) COSY spectrum of DP5. B) Detail of the HMBC spectrum of DP5. C) Detail of the HMBC spectrum of DP10, 11, 12. The spectra were recorded at 293 K.

(

  http://www.casper.organ.su.se/casper/; Lundborg and Widmalm, 2011) gave Glc1-C1/H1= 101.94/4.59 ppm and Glc2-C1/H1 = 104.48/4.65 ppm which values were close to those recorded.

  correlation between the Gal-C1 and Glc-H4 observed in the spectrum, demonstrated unambiguously the -D-Gal (1→4)-D-Glc linkage. The linkage of the xylofuranoside residue was also well identified in the HMBC spectra and revealed the residue was bound to the xylopyranoside residue of the main chain by (1,3) linkage. The repeating unit of the polysaccharide deduced from the detailed analyses of the oligosaccharide and the the unmodified polymer was: 3.4 Dispersion and diversity of Nostoc commune polysaccharides After an exhaustive survey of the literature, the structures of the polysaccharides secreted by Nostoc strain were presented in Figure 4. The polysaccharide secreted by the Nostoc strain isolated at Saint Martin d'Uriage (France, Europe) showed very similar structural characteristic with those secreted by several Asian strains (Figure 4). The polysaccharide of Nostoc commune strains isolated in Mongolia, China or Japan shared a tetra-saccharide repeating unit:[→ 4) -D-Glc (1 → 4)--D-Xyl (1 → 4)--D-Glc

  suggested also a conserved carbohydrate backbone with the same pentasaccharide sequence. Nostoc commune can form filament depending on growth condition and it is then difficult to discriminate some Nostoc commune var flagelliforme with true Nostoc flagelliforme. Structure of reported Nostoc sp. polysaccharides presented in Figure4suggested that the repeating unit made of a penta-saccharide seemed common probably reflecting a common biosynthesis template which evolved in the Nostoc lineages.

Figure 4 .

 4 Figure 4. Comparison of the structures of the polysaccharides secreted by Nostoc sp. strains isolated in various places in the world.

  d'Uriage (France) was resolved. Comparison of our results with previously characterized structure of polysaccharide of N. commune strains found in Europe and Asia suggested the conservation of a pentasaccharide template reflecting a conservation of the biosynthesis pathway. The building block made of the tetrasaccharide [→4)--D-Glc (1→4)--D-Xyl (1→4)--D-Glc (1→4)--D-Gal (1→],

Figure S2 :

 S2 Figure S2: Gas chromatogram recorded on the permethylated residues of the Nostoc commune polysaccharide (A) and the xylose standard residues (B).

Figure

  Figure S3: (A) Fragmentation product of the 1,4-di-O-acetyl-1-deuterio-2,3,5-tri-O-methyl-D-xylitol eluting at 9.468 min in the gas chromatogram of the xylose derivatives standard (Figure S3A). (B) Fragmentation products obtained on the derivatives eluting at 9.419 in the gas chromatogram recorded on the N. commune polysaccharide (Figure S3B)

Table 3 :

 3 1 H and 13 C NMR chemical shifts of the oligosaccharides obtained after enzymatic degradation of the N. commune polysaccharide. Only the chemical shifts of the DP6 and longer oligosaccharides which were different from those of DP5 and depyruvated DP5 were reported in the table. a : Glc2→Gal/ Glc2→Gal values.

	Sugar Residue		1	2	3	4	5	6 (6a,6b) pyr (CH3, C, CO)
	DP5
	Glc-1 -D-Glcp-(1→	1 H	4.57	3.36	3.53	3.47	3.52	3.98, 3.78
		13 C	101.07	72.79 75.41 69.58	75.90	60.70
	Xylp →4)--D-Xylp-(1→	1 H	4.49	3.36	3.65	3.88 4.16, 3.49
		13 C	103.06	73.08 73.64 76.20	62.88
	Glc-2 →4,6)--D-Glcp-(1→	1 H	4.74/4.73 a	3.44	3.66	3.72	3.73	4.37, 4.01
		13 C 103.35/103.49 a 73.45 74.06 77.99	73.48	68.07
	Pyr-GlcA -D-(2,3Pyr)-GlcpA-(1→	1 H	5.03	3.52	3.76	3.76	4.01	no	1.64
		13 C	101.29	75.59 80.72 78.47	70.57	175.33	22.56, 108.94, 175.99
	Gal →4)--D-Galp	1 H	5.32	3.95	4.01	4.26	4.19	3.87, 3.77
		13 C	92.27	68.69 69.58 78.47	69.96	61.15
	Gal →4)--D-Galp	1 H	4.67	3.62	3.82	4.19	3.79	3.87, 3.77
		13 C	96.38	72.07 73.16 77.25	74.47	61.29
	Depyruvated DP5
	Glc-1 -D-Glcp-(1→	1 H	4.59	3.34	3.54	3.47	3.52	3.98, 3.78
		13 C	101.09	72.78 75.48 69.55	75.93	60.67
	Xylp →4)--D-Xylp-(1→	1 H	4.55	3.39	3.65	3.90 4.17, 3.44
		13 C	102.91	73.01 73.74 76.40	62.87
	Glc-2 →4,6)--D-Glcp-(1→	1 H	4.75	3.44	3.66	3.77	3.73	4.35, 3.98
		13 C 103.50/103.59 a 73.47 74.06 77.86	73.47	67.95
	GlcA -D-GlcpA-(1→	1 H	4.57	3.42	3.56	3.52	3.77	no
		13 C	102.63	72.83 75.48 71.82	76.33	175.81
	Gal →4)--D-Galp	1 H	5.33	3.94	4.01	4.29	4.19	3.88, 3.78
		13 C	92.28	68.70 69.55 78.44	69.84	60.92
	Gal →4)--D-Galp	1 H	4.67	3.62	3.82	4.23	3.79	3.88, 3.78
		13 C	96.39	72.10 73.17 77.36	74.31	61.10
	DP6
	Xylp →3,4)--D-Xylp-(1→	1 H	4.54	3.44	3.82	3.88 4.16, 3.49
		13 C	103.02	72.26 79.87 76.20	62.68
	Xylf -D-Xylf-(1→	1 H	5.55	4.23	4.40	4.40	3.82
		13 C	101.88	76.87 74.90 78.47	60.54
	DP10, 11, 12
	Glc-1 →4)--D-Glcp-(1→	1 H	4.59	3.39	3.81	3.70	3.64	3.98, 3.78
		13 C	100.88	72.80 76.00 76.87	74.91	60.68
	Gal →4)--D-Galp-(1→	1 H	5.47	4.00	4.01	4.22	4.11	3.87, 3.77
		13 C	99.80	68.88 69.72 78.07	71.16	61.04

Acknowledgments

This work was supported by the French National Research Agency (Grant ANR-14-CE06-0017). W.H., E.M and A.A have received support from the Glyco@Alps Cross-Disciplinary Program (Grant ANR-15-IDEX-02), Labex ARCANE, and Grenoble Graduate School in Chemistry, Biology, and Health (Grant ANR-17-EURE-0003). E.M. and A.A. are supported by Grant Alpalga ANR-20-CE02-0020.


Veronica Mitroi

Véronique Maleval

José-Frédéric Deroubaix

Brigitte Vinçon-Leite

Jean-François Humbert

Veronique Maleval

Jean- François Humbert

What urban lakes and ponds quality is about? Conciliating water quality and ecological indicators with users' perceptions and expectations about urban lakes and ponds quality in urban areas

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

Introduction

As recently shown by [START_REF] Oertli | Review: Toward management of urban ponds for freshwater biodiversity[END_REF], an increasing amount of attention is being paid to urban lakes and ponds (L&Ps) in relation to the expansion of urbanization worldwide. Most of the time, these L&Ps have an anthropogenic origin or are ancient natural habitats that have been strongly modified by urban planning politics. Additionally, these ecosystems also known as 'retention basins' play an important role in urban stormwater management [START_REF] Hassall | The ecology and management of urban pondscapes[END_REF][START_REF] Sender | Diversity of aquatic ecosystems in urban areas -public expectations[END_REF]. Because of their main characteristics (small, shallow, highly artificial shorelines, concentration of contaminants, often hypertrophic tendency), they are usually considered to have less ecological importance than nonurban and natural lentic ecosystems [START_REF] Brucet | Biological assessment of European lakes: Ecological rationale and human impacts[END_REF][START_REF] Mitroi | Artificial aquatic ecosystems[END_REF]. In particular, the levels of biodiversity found in urban L&Ps appears to be lower than those in nonurban area even if, depending on the local conditions and the management of these ecosystems, these ecosystems can reach similar levels of biodiversity than those of nonurban area [START_REF] Céréghino | The ecology of European ponds: Defining the characteristics of a neglected freshwater habitat[END_REF][START_REF] Hill | Urban ponds as an aquatic biodiversity resource in modified landscapes[END_REF][START_REF] Naselli-Flores | Urban lakes: Ecosystems at risk, worthy of the best care[END_REF][START_REF] Oertli | Review: Toward management of urban ponds for freshwater biodiversity[END_REF][START_REF] Williams | Comparative biodiversity of rivers, streams, ditches and ponds in an agricultural landscape in Southern England[END_REF]. Consequently, these L&Ps are increasingly integrated into ecological networks for biodiversity conservation in Europe, such as Natura 2000 and Blue and Green Corridors. Urban L&Ps are also widely recognized by local actors and users as providing a large range of vital ecosystem services for cities [START_REF] Amigues | Evaluer les services écologiques des milieux aquatiques: Enjeux scientifiques, politiques et opérationnels[END_REF][START_REF] Bolund | Ecosystem services in urban areas[END_REF][START_REF] Lundy | Integrating sciences to sustain urban ecosystem services[END_REF]. In addition to their primary hydraulic regulation function, urban L&Ps contribute to climate change mitigation [START_REF] Amigues | Evaluer les services écologiques des milieux aquatiques: Enjeux scientifiques, politiques et opérationnels[END_REF] and provide appealing landscapes [START_REF] Bonnin | Quand la nature s'urbanise. Natures urbanisées [When the nature is going urban[END_REF][START_REF] Boutefeu | La demande sociale de nature en ville : enquête auprès des habitants de l'agglomération lyonnaise[END_REF][START_REF] Hill | Urban ponds as an aquatic biodiversity resource in modified landscapes[END_REF], spaces for physical and recreational activities important to human health and well-being [START_REF] Mishra | The development of a tool for assessing the environmental qualities of urban blue spaces[END_REF][START_REF] Völker | Determining urban open spaces for health-related appropriations: A qualitative analysis on the significance of blue space[END_REF], and social interaction and environmental educational areas [START_REF] Blicharska | Urban ponds for people and by people[END_REF]. Due to the coexistence of these numerous ecological and social services provided by urban L&Ps, the managers of these ecosystems must deal with conflicts of use and incompatibilities between the water quality criteria needed for these various uses (e.g. a protected bird nesting area deteriorates the water quality, which has consequences for recreational activities). They also need to consider the particular urban characteristics and pressures [START_REF] Schueler | Watershed Protection Techniques, SPECIAL ISSUE: Urban Lake Management[END_REF] and the diversity of meanings associated with small ponds in urban landscapes [START_REF] Mitroi | Artificial aquatic ecosystems[END_REF]. Based on interdisciplinary research conducted on 49 urban L&Ps in the Ile-de-France area, we choose to focus on three lakes that are representative of the wide variety of situations encountered, to explore the relationships between (i) their characteristics in terms of integration in the urban landscape, water quality and ecological status, and (ii) the perceptions by various actors (visitors, managers, etc.) of the general state of these ecosystems and of their usefulness and (iii) the related management practices. We discuss how different knowledge and evaluation methods of urban lake quality (developed by scientists, managers and users) can enlighten L&P management to strengthen the synergies between ecological conservation goals, multiple social uses and amenities in an urban context. To address conflicts of use and incongruencies between various goals, the current paper suggests that the management of urban lakes should be envisaged not at the scale of each individual pond but rather at territorial scales including a few L&Ps that can accomplish various functions according to their ecological status.

Theoretical background

Challenges for defining and managing urban L&P

Despite the numerous studies that have been performed by various disciplines (such as hydrology, ecology, microbiology, limnology, geography) on urban and peri-urban L&Ps, our knowledge of their functioning and management remains limited [START_REF] Birch | Shallow urban lakes: A challenge for lake management[END_REF][START_REF] Céréghino | The ecology of European ponds: Defining the characteristics of a neglected freshwater habitat[END_REF]EPCN, 2008). These ecosystems are subjected to multiple anthropogenic pressures and to management strategies that can dramatically change their ecological state (e.g. control of flows, dredging, etc.) [START_REF] Mitroi | Artificial aquatic ecosystems[END_REF]. Another difficulty in producing relevant knowledge about these ecosystems is disciplinary fragmentation (i.e. each discipline adresses a particular aspect and develops its own quality indicators) and management fragmentation. For example, management responsibility for urban lakes is frequently shared by various municipal services and institutions, which are in charge of a particular use or function [START_REF] Birch | Shallow urban lakes: A challenge for lake management[END_REF][START_REF] Blicharska | Urban ponds for people and by people[END_REF][START_REF] Wagner | Incompatibility of common lake management objectives[END_REF]. Incompatibilities in the management priorities of these various actors comprise a major issue of urban L&P conservation [START_REF] Wagner | Incompatibility of common lake management objectives[END_REF][START_REF] Oertli | Pond conservation: from science to practice[END_REF]. Finally, [START_REF] Brucet | Biological assessment of European lakes: Ecological rationale and human impacts[END_REF] have shown that managers are confronted with a great variety of expert criteria to assess the water quality and ecological status of these ecosystems, and that there is a need for the development of cost-effective diagnostic tools able to disentangle the effects of complex multiple pressures and their mitigation. The main regulatory framework for the assessment of water bodies in Europe -Water Framework Directive (WFD) -is minimally relevant for assessing artificial urban L&Ps, either because most of them are too small (i.e. only L&Ps larger than 50 ha are monitored, according to WFD procedures), or because they are too artificial and strongly impacted by urban pressures and uses. The WFD defines indicators of 'good ecological state' (i.e. biological quality, physico-chemical quality and hydromorphological criteria) and threshold values independently of any reference to social uses [START_REF] Carré | The Water Framework Directive's "percentage of surface water bodies at good status": unveiling the hidden side of a "hyperindicator[END_REF]; however, managers still try to refer to this regulatory framework when defining their management objectives [START_REF] Steyaert | The European water framework directive: How ecological assumptions frame technical and social change[END_REF]. If these 2 V. MITROI ET AL. indicators are pertinent for evaluating sanitary risks and ecological status (such as eutrophication), then they are less appropriate for considering the diversity of social uses and amenities of urban ponds. Consequently, institutional actors in charge of the management of urban L&Ps face numerous questions that are related to the multiplicity of valuing and evaluation criteria that can be deployed to characterize L&P quality. For example, how is the ecological state of urban L&Ps affected by their urban characteristics? Should they be considered and managed as important 'spots of urban nature' even when their ecological and sanitary quality is very poor? How they can integrate into their management plans, the criteria used by residents and users of urban ponds to appreciate the overall quality of these ecosystems?

Social perceptions of L&P quality and users' aesthetic preferences

Usually located within parks or surrounded by green belts and vegetation, urban L&Ps are considered as 'green areas' and 'blue areas providing important amenities for cities' habitants' [START_REF] Mishra | The development of a tool for assessing the environmental qualities of urban blue spaces[END_REF][START_REF] White | Blue space: The importance of water for preference, affect, and restorativeness ratings of natural and built scenes[END_REF]. Compared with terrestrial green ecosystems in urban areas (i.e. forests, parks and gardens), very few investigations have been achieved on the way in which urban L&Ps are perceived by urban residents and users. Nevertheless, the literature informs on their aesthetic appeal [START_REF] Gao | Public perception towards river and water conservation practices: Opportunities for implementing urban stormwater management practices[END_REF][START_REF] Hassall | The ecology and management of urban pondscapes[END_REF][START_REF] Sender | Diversity of aquatic ecosystems in urban areas -public expectations[END_REF], their positive impact on well-being and health [START_REF] Higgins | Urban freshwaters, biodiversity and human health and wellbeing: Setting an interdisciplinary research agenda[END_REF][START_REF] Völker | Determining urban open spaces for health-related appropriations: A qualitative analysis on the significance of blue space[END_REF] and their contribution to sense of place and neighborhoods' identity [START_REF] Decamps | How a riparian landscape finds form and comes alive[END_REF]. Although they are generally highly appreciated in urban landscapes, some negative aspects are also identified as being related to urban L&Ps, such as unwanted and invasive species or cyanobacteria blooms that affect people's ability to enjoy water bodies [START_REF] Brisson | Social construction of cyanobacteria blooms in quebec: A matter of perceptions and risk management[END_REF]. The aesthetic and visual preferences of users (neighbors, visitors, etc.) are important issues to be taken into consideration for the definition of conservation scenarios of urban water bodies. The theory of cultural sustainability suggests that when ecosystems and landscapes are perceived as attractive, people are more likely to have sustainable behaviors over the long term [START_REF] Decamps | How a riparian landscape finds form and comes alive[END_REF][START_REF] Nassauer | The appearance of ecological systems as a matter of policy[END_REF][START_REF] Nassauer | Monitoring the success of metropolitan Wetland restorations: Cultural sustainability and ecological function[END_REF]. This propensity for ecosystem pro-conservation attitudes is directly related to their appearance:

When the appearance of the landscape elicits human perceptions and behaviors that maintain its ecological health, the landscape could be described as culturally sustainable; a landscape that people enjoy or are proud of is more likely to be culturally sustainable. [START_REF] Nassauer | Monitoring the success of metropolitan Wetland restorations: Cultural sustainability and ecological function[END_REF] In the field of urban water, public perception and aesthetic values are indeed considered as key issues for the sustainable management of urban wetlands [START_REF] Kaplowitz | Michigan residents' perceptions of wetlands and mitigation[END_REF][START_REF] Nassauer | Monitoring the success of metropolitan Wetland restorations: Cultural sustainability and ecological function[END_REF], river restorations [START_REF] Junker | Aesthetic preferences versus ecological objectives in river restorations[END_REF] and urban stormwater management practices [START_REF] Apostolaki | The social impacts of stormwater management techniques[END_REF][START_REF] Gao | Public perception towards river and water conservation practices: Opportunities for implementing urban stormwater management practices[END_REF]. Nevertheless, there is no conclusive evidence about the concordance between the public perception of the quality of aquatic ecosystems and their ecological richness (or water sanitary quality, as judged by experts). Some landscape ecology investigations have shown that what people enjoy as the appearance of nature may have little inherent relationship with its ecological quality measured according to species richness or abundance criteria [START_REF] Dallimer | Biodiversity and the feel-good factor: Understanding associations between self-reported human well-being and species richness[END_REF][START_REF] Hassall | The ecology and management of urban pondscapes[END_REF]. Nevertheless, few studies have shown that even if people may not explicitly recognize (or quantify) biological diversity, the subjective perceptions of species diversity may reflect objective measures. The presence of charismatic species (e.g. kingfishers, dragonflies) considered by experts in ecological inventories is very positively interpreted by people [START_REF] Mcginlay | Do charismatic species groups generate more cultural ecosystem service benefits?[END_REF] because it allows them to connect with biodiversity in a tangible or emotional way [START_REF] Higgins | Urban freshwaters, biodiversity and human health and wellbeing: Setting an interdisciplinary research agenda[END_REF]. If some concordances can be established between the visible (macro-) biodiversity and social appeal of urban L&Ps, then the relation between water quality and social perception of the quality of these ecosystems is even less investigated and difficult to capture. This is not surprising if we consider that water quality refers in a large part, to various invisible contaminants (i.e. micropollutants, heavy metals, fecal bacteria, etc). Other visual criteria, such as water color and turbidity can nevertheless be shared indicators between expert and profane grids of water quality lectures. Some interesting citizen science experiences have confirmed that citizens can inform cyanobacteria blooms on lakes and shallow waterbodies in relevant ways [START_REF] Castilla | Quantification of phytoplankton bloom dynamics by citizen scientists in urban and peri-urban environments[END_REF][START_REF] Mitroi | Can participatory approaches strengthen the monitoring of cyanobacterial blooms in developing countries? Results from a pilot study conducted in the Lagoon Aghien (Ivory Coast)[END_REF], thereby complementing the institutional monitoring of the trophic status of these aquatic systems [START_REF] Hadj | Getting the full picture: Assessing the complementarity of citizen science and agency monitoring data[END_REF]. In addition to the aesthetic and visual criteria, other factors play a role in the public perception of hardly transformed ecosystems such as the health benefits related to the possibilities offered by these water bodies to enjoy nature or to provide recreational activities (angling, swimming, boating) [START_REF] Junker | Aesthetic preferences versus ecological objectives in river restorations[END_REF]. This is about the 'functional level of landscape' [START_REF] Nassauer | The appearance of ecological systems as a matter of policy[END_REF][START_REF] Nassauer | Monitoring the success of metropolitan Wetland restorations: Cultural sustainability and ecological function[END_REF], which refers to the accessibility to infrastructures for walking and recreational activities or the possibility of visually enjoying related landscapes. The public safety feeling that emerges in these landscapes also play a role in their appreciation by users. This is directly related to the social frequentation of L&P surroundings as open public places that can easily be occupied by unwanted populations with unappropriated behaviors (i.e. noisy, potentially dangerous, etc.). Finally, a place that is well cared for (i.e. trash-free water and shorelines and infrastructures that are well maintained) contributes to the security and well-being that a water body can provide [START_REF] Nassauer | Monitoring the success of metropolitan Wetland restorations: Cultural sustainability and ecological function[END_REF]. Because these last features have little to do with the water quality or ecological value of a lake they are often neglected by managers; however, they are very important indicators for urban L&P users. Not addressing social perceptions about L&P quality may lead to a clash between public perception and public action concerning urban L&P conservation.

Case study

The Ile-de-France region is one of the most populated metropoles in Europe and the most urbanized areas of France (IAURIF, 2016). The urban area was extended through a suburbanization process initiated in the late 1960s, which included the construction of new cities (so-called villes nouvelles). Many artificial ponds and lakes were created as a result of the sand and gravel extraction used for new urban planning during the 1970s and 1980s. These water bodies were integrated into the urban landscapes for different assigned functions such as stormwater outlets, urban landscape, and recreational activities (e.g. sailing, swimming, fishing, etc.). Currently, the Ile-de-France region contains approximately 1,000 L&Ps, 250 of which have an area >5 ha [START_REF] Catherine | Design and application of a stratified sampling strategy to study the regional distribution of cyanobacteria (Ile-de-France, France)[END_REF]. The ecological and sanitary states of these L&Ps, which are subject to various and combined anthropogenic pressures and pollution, are very little known and represent a real challenge in regard to understanding their ecological functioning. Local authorities, national agencies, and user associations (e.g. fishermen, naturalists and local residents) acknowledge urban ponds as increasingly important elements of the urban landscape and human wellbeing and lately as important 'biodiversity spots' to preserve.

Material and methods

Study sites

To better understand the diversity of the ecological state of L&Ps in Ile-de-France, 49 lakes in the Ilede-France region were monitored between 2011 and 2013. These 49 lakes were representative of the 250 lakes in the Ilede-France region displaying a >5 ha surface by their hydrogeological and limnological characteristics and by the various land-uses and population densities around these lakes [START_REF] Catherine | Design and application of a stratified sampling strategy to study the regional distribution of cyanobacteria (Ile-de-France, France)[END_REF]. Among these 49 lakes, we chose to focus on three lakes located on the outskirts of Paris (Créteil: 11 km from Paris, 48°46′ N 02°27′ E; Enghien-les-Bains, 13 km from Paris, 48°58′ N 02°18′ E and Saint-Quentin-en-Yvelines, 24 km from Paris, 48°47′ N 02°01′ E). These lakes displayed contrasting characteristics in terms of history, landscape evolution, watershed size, shoreline artificialization and land-use profiles in the 1 km area around the lake, which reflect their diversity. For example, there is a green area around the lake of St-Quentin-en-Yvelines, while in Enghien-les-Bains, there is just a very small green area in the southeastern part of the lake. Finally, in Creteil, the western part of the lake is green while the eastern part is very urbanized (Table 1).

Digital orthophotograph interpretation

The landscape evolution related to the three selected lakes during the past 65 years, was assessed using digital orthophotographs within a 1-km area from the lakeshore. This distance was determined through a compromise between ecological criteria (i.e. lake catchments) and landscape criteria (i.e. the visual perceptions of users). This analysis was based on four aerial missions conducted between 1949 and 2014. The oldest photographs were georeferenced from the orthophotograph of 2014. Using MapInfo software, we built a database and produced twelve descriptive land use maps for each lake at different time periods, which generated land use evolution maps for each of the studied lakes. These maps were then used to quantify the evolution of land use between 1949-1950 and 1971-1972; between 1971-1972 and 1991-1992, and finally between 1991-1992 and 2014. The interpretation of the orthophotographs was completed with some bibliography concerning the evolution of the three concerned cities and the initial landscape of the lakes.

Constructing a regional reference framework for the ecological status of urban lakes

The data used for the assessment of the lake ecological status were based on three sampling campaigns performed during the summers of 2011-2013 on 49 lakes of the Ile-de-France region. These data were used to set up a regional frame of reference for the ecological status of the lakes in Ile-de-France [START_REF] Mitroi | Évaluer la qualité des plans d'eau urbains. Vers des pratiques de recherche et de gestion transdisciplinaires et régionalisées. [Evaluate the quality of urban water bodies. Towards transdisciplinary and regionalized research and management practices[END_REF]. The ecological quality of the three lakes considered in this article is therefore presented not only in a comparative perspective between the three lakes, but also in reference to this regional framework. The ecological indicators used for the assessment of the ecological status of the lakes are the result of a consultation process between the researchers and L&P managers and their operational needs. These indicators allow us to characterize (i) the trophic status of the water bodies, (ii) their microbiological quality and (iii) their level of contamination by anthropogenic contaminants. Twenty parameters considered by [START_REF] Carr | Water quality index for biodiversity technical development document[END_REF] to reflect the ecological functioning of water bodies, were used to estimate four indices: the water quality index (WQi), the microbiological index (Mi), the trace metals index (TMi), and the polycyclic aromatic hydrocarbons index (PAHi) (Table 2). The value of these indices for each lake was calculated as the average of the deviations from the guideline value for all selected parameters. Thus, an index could reach a value of 100 only if the entire set of parameters included in the index indicated a good status for the water body. Finally, the global quality index (GQi) of each water body was calculated as the average of the different quality indices (WQi, Mi, TMi, PAHi). Similarly, this index could only be set to 100 if all the different quality indices had a maximum value (i.e. good status). In addition to their contribution to a global characterization of water bodies, some indices were also used to evaluate whether a water body was suitable for human activities. For example, the microbiological index (Mi) was used to assess whether a water body was suitable for recreational activities, particularly swimming.

Sociological approaches for the assessment of the representation of uses and lake quality

A sociological survey was conducted on various social actors (neighbors, visitors, planners, and managers), to identify public perceptions [START_REF] Barnetta | Influence of recreational activity on water quality perceptions and concerns in Utah: A replicated analysis[END_REF][START_REF] Gao | Public perception towards river and water conservation practices: Opportunities for implementing urban stormwater management practices[END_REF] of the global quality of the three lakes and their 'naturalness'. We conducted 26 interviews with lake managers from the Ile-de-France region, and 159 questionnaires were administered during the summer of 2013 to users of the three selected lakes (Enghien-les-Bains: 41; Saint-Quentin-en-Yvelines; 43; and Créteil: 75). The survey was performed 'au fil de l'eau' by varying the days of the week and the hours of the day to cover a wide and diverse panel of participants. The panel of users was relatively well balanced by age but women were generally overrepresented (Table 3). We used questionnaires with both closedended questions (with a single possible answer between a few already codified) and open-ended questions (where people could express themselves by using their own words). All data were statistically treated by using specific sociological methods for qualitative and quantitative codification and analyzed with XLStat software.

Results

Urban profiles of lakes -landscape and uses

Concerning the urban patterns, the strong densification of urban areas is apparent for each of the three lakes during the studied period , but the urbanization timelines and ratios between built and green areas of the shorelines are very different (Table 4):

• Built at the end of the eighteenth century, Lake Enghien (36 ha) is a historical lake that was landscaped by the construction of a dam on the marshy area of the Montmorency Valley. In the first half of the nineteenth century, the place became a leisure destination for the Parisian bourgeoisie [START_REF] Neu | Enghien-les-Bains nouvelle histoire[END_REF] and residential buildings began to be built on the lakeshore. In 1949, the urbanization level of this space was already high (65.8%) and consisted mainly of individual residential housing (44%). The city continued to expand and become denser until the 1970s with the development of collective housing. By 1972, nearly 85% of the 1-kilometer surrounding area was already built. The remaining green and woodland areas (approximately 10% in 2014) are mainly situated on private properties that are not accessible to the public.

• For Creteil Lake, cartographic data indicate that this lake (40.8 ha) was landscaped in 1976 as part of the Park of General Interest of 23 ha dedicated to recreational activities for the 'ville nouvelle ' de Créteil, which was built between 1950and 1972. Massive urbanization started in 1950and continued between 1972and 1991. The eastern part of the lakeshore is strongly urbanized and occupied by collective housing and administrative buildings, while the western bank has preserved green spaces and leisure establishments.

• Lake Saint-Quentin-en-Yvelines was originally built in the seventeenth century inside a complex hydrological network meant to supply water to the fountains of Versailles. Located 24 km southwest of Paris, the lake area remained very rural until 1971, when the construction of the new town of Saint-Quentin-en-Yvelines began. This new city led to the disappearance of important agricultural land (from 59% to 11%), but the landscape around the lake remained very 'green' since a significant part of the agricultural land was converted into green and wooded areas (19.4% in 1949-50.1% in 1992). An outdoor recreational space of 600 ha was created in 1969 around Lake Saint-Quentin-en-Yvelines, which became an important leisure location for the new city as well as for the entire region.

The users of Lake Creteil are mostly locals (only 16 of 80 come from neighbor municipalities), while in Enghien-les-Bains and Saint-Quentin, more than half of the users are inhabitants of neighbor cities, including Paris. We also found some differences between the three lakes regarding the frequency of visits and indirectly, the degree of familiarity of the users with the lakes. While in Creteil there are mainly regular users (up to 40% visit several times per week), in Saint-Quentin, the majority of users (40%) visit the lake only two to four times in their lifetime, while only 30% frequent the lake more regularly (one or more times per month). In Enghien, people's presence on the lake's banks is very diverse, ranging from daily attendance for those living in the city to less frequent visits for those living in neighbor districts.

Despite different landscape patterns and degrees of urbanization in the surrounding areas of these lakes, all of them have experienced a similar evolution toward a diversification of their original landscape functions. For instance, they all provide stormwater storage and recreational functions for riparian populations, in addition to landscape and urban biodiversity functions.

Water quality and ecological status of the three lakes

Based on the quality indices, none of the three lakes can be considered to have a good ecological status, but for different reasons. The weak GQi of Lake Enghien-les-Bains is mainly due to the high degree of contamination by PAHs, while Lake Creteil is mostly downgraded by the WQi and TMi indices and Saint-Quentin is primarily downgraded by the WQi index (Table 5). Compared to the 49 lakes monitored in the Ile-de-France region, Enghien-le-Bains is the most degraded on the basis of the GQi (and mostly due to PAHi), while Créteil and Saint-Quentin-en-Yvelines lakes are characterized by better GQi values though they display the lowest values for WQi (Table 5).

When considering these characteristics, it appears that the lake with the lowest GQi (Enghien-les-Bains) is the shallowest and the smallest of the three lakes and the lake with the most urbanized direct watershed within its 1-km area (90%). In contrast, the lake with the best water quality (Saint-Quentin-en-Yvelines) is the largest lake with the less urbanized watershed within its 1-km area (35%).

Public perceptions of the naturalness and quality of urban ponds

Among the large number of social uses that were identified for the three lakes (boating, swimming, fishing, observation of nature, etc.), relaxation/evasion is the most often cited use for the three lakes (Figure 1A). Although it is difficult to precisely quantify the use of 'relaxation', it appears that most people visit the lakes' banks primarily to 'relax', 'get some fresh air' and 'to escape from urban pressures' (35% in Saint-Quentin, 60% in Enghien and 70% in Créteil). These answers are coherent with users' vision about the main function of urban lakes, which provide relaxing places where they can escape urban pressure (Figure 1B). Lakes and the surrounding green areas are mainly seen as places to play sports, go on outings with children, have a picnic, or engage in other social activities.

If we consider the degree of urbanization of the 1-km areas surrounding the lakes, it is interesting to see that the more the surrounding environment is urbanized, the more the landscape function is appreciated (Figure 1B). In Lake Enghien, which is the most urban of the study, more than 20% of the users consider the lake to have a major landscape function for the city while for the lake of Saint-Quentin-en-Yvelines, which is located in a green area outside the residential neighborhoods, the landscape function is not perceived as important (only 5% of the answers). On the other hand, the lakes of Saint-Quentin-en-Yvelines and Creteil are perceived as having an important ecological function of nature protection (24% and 22% respectively), while in Enghien, only 15% of the respondents cited this function (Figure 1B).

Even if a larger proportion of the respondents in Creteil considered that the lake was artificial, there was no significant difference (chi-2 test) concerning this criterion of the natural/artificial lake character (Figure 2A) when comparing the respondents from the three lakes. In the same way, there was no significant difference (chi-2 test) in their responses concerning the ecological state of the lakes, even if those of Creteil provided the lowest proportion of the good state (Figure 2B). Finally, no significant difference (chi-2 test) was detected in regard to the age and gender of the respondents concerning these two criteria.

Users were asked to freely name three main criteria they employed to evaluate the 'naturalness' and the 'ecological state' of the lakes. For 'naturalness', they evoked a patrimonial relationship with the lakes, referring to the 'social origin' of these water bodies. The 'social origin', meaning that the lakes were landscaped through the process of urbanization, largely dominated the biophysical (e.g. the aspect of the banks and level and aspect of the water) and morphological (e.g. size) criteria (Figure 3). This is less the case for Lake Saint-Quentin, where many users come from other cities of the region and therefore know less about the social history of the lake. The degree of greening/artificiality of the lake's shoreline was the second criterion mentioned by users to evaluate the naturalness of the lake. Other criteria do not seem to be important in their assessment of naturalness, excepted the size of the lake for users in Enghien-les-Bains.

Concerning the criteria used to assess the lake ecological quality, the most important for users were the 'cleanliness' and 'maintenance of the lake' (the water and its surroundings) (Table 6). When users mentioned 'water appearance', this primarily referred to hygiene (e.g. no trash or pollution residue) or the healthy aspect of the water (e.g. 'clean water', 'no algae', 'transparent water'). The natural aspect of the lake and its environment (e.g. the presence of vegetation, birds, fish, etc.) was ranked in third position. However, the relationship between these two major categories of judgment ('cleanliness' and 'natural appearance') differed according to the surrounding environment. For Saint-Quentin, which is located in a vegetal environment, users considered the 'natural appearance' to be a more important criterion than 'cleanliness', which was the first criterion for the users of Enghien and Créteil, which are located in a more urban environment. For many users of Saint-Quentin, the 'green and blue' image of the area is seen as a guarantee of biodiversity and hence a criterion by which to assess lake quality. For the users of Creteil and Enghien, which are located in a much more urbanized landscape, the criterion of 'leisure facilities' was much more important than the 'natural appearance' and refers to the presence of leisure facilities, accessibility, walking paths, etc. Surprisingly, pollution was a very secondary concern for users of the three lakes.

The key criteria used by managers for the assessment of the ecological quality of lakes were very different from those of users. Managers mainly mentioned 'water quality', followed by 'biodiversity'. Landscape and visual criteria, including those of cleanliness and maintenance of lakes and shorelines were rarely mentioned by managers.

Main threats identified by actors

As shown in Figure 4, the main threat reported by the users for the ecological quality of the lakes consisted in 'the other users' (50% of respondents), while managers identified users as the second major degradation factor (23% of respondents). The idea that too many users are a threat to lake quality is associated with users who exhibit problems related to cleanliness, insecurity and the deterioration of the 'social ambiance'. This can be linked to very high visitation levels (especially in Créteil and Saint-Quentin), particularly during weekends and sports or cultural events. For managers, 'urbanization and associated pollution' was the first major degradation factor (39%), while this criterion was the second one for the users (32%) (cf. Figure 4). These perceptions of the threatening factors were directly related to the actions that are considered necessary to improve or conserve lake quality. Users considered that the most important action is to better control users. First, they proposed more education-, information-and awareness-raising measures for all categories of users. Second, they requested action to better regulate and control user practices including better supervision and the limitation of users (e.g. number of visitors, boats, 'selective' access, contraventions for noncompliant activities). Another type of action frequently cited by users concerned the 'maintenance' of water bodies and their banks. Other improvements concerned the necessity to assign additional spaces for nature and protect the environment from urban pressure. A similar importance (approximately 7%) was given to water quality, whether through dredging or filtering, decanting or purification.

Discussion

The impact of the urban environment on L&P quality -ecological indicators and public expectations

The effects of urbanization on the evolution of urban L&Ps are quite contradictory when considering both ecological (and water quality) indicators and public expectations. The ecological indicators suggest that the urbanization of the 1-km surrounding area of the L&Ps is an important source of pollution pressures for these ecosystems (e.g. nutrients, PAHs, heavy metals, microbes). If the size of the watershed and its degree of urbanization is considered a good proxy for assessing the nutrient and pollution fluxes to the L&Ps [START_REF] Catherine | Projecting the impact of regional land-use change and water management policies on lake water quality: An application to Periurban lakes and reservoirs[END_REF], several studies have shown that the land use in the area adjacent to a lake (between 500 m and 1 km) has a more significant impact on the water quality and the trophic status of these lakes than does the whole catchment land use [START_REF] Akasak | Effects of land use on aquatic macrophyte diversity and water quality of ponds[END_REF]. As suggested by our data, this could be because the degree of urbanization in the 1-km area is frequently more important than that in the whole catchment.

In contrast, the sociological investigation of public perceptions of urban L&P quality shows that they are highly appreciated by users, regardless of their ecological status or urbanization degree in the 1km surroundings. This outcome is in accordance with recent works on users' perceptions of urban nature and blue spaces, which show that users tend to adapt their expectations and quality evaluation criteria according to the uses they envisage and to their urban location (manicured urban park, green area, residential area) [START_REF] Junker | Aesthetic preferences versus ecological objectives in river restorations[END_REF][START_REF] Sender | Diversity of aquatic ecosystems in urban areas -public expectations[END_REF][START_REF] White | Blue space: The importance of water for preference, affect, and restorativeness ratings of natural and built scenes[END_REF]. For example, in the particular case of the lake of Enghien-les-Bains (a very urbanized and residential water body), users do not expect high level of biodiversity but rather aesthetic characteristics for the urban landscape and assets for open space activities. In contrast, for users of the lake of Saint-Quentin (located in a green area outside the city), the lake's quality is mainly judged on environmental criteria, including the presence of birds and vegetal varieties and the green aspect of the shoreline. This adaptation of quality criteria to the surrounding landscape probably explains why, from the users' point of view, the evaluation of the quality in the Enghien and Saint-Quentin lakes appears to be very similar. These results must be interpreted since we know that we focused our survey on direct users of these green and blue spaces instead of on all residents living around these lakes, which could have more negative perceptions or weak interest levels for them.

In terms of management objectives, the fact that no significant relationship was found between the ecological quality of these lakes and the social perception of their quality and wellbeing amenities opens up new considerations about the possible pathways of bridging together social and ecological targets in management practices. If managers focused their actions on the protection/restoration of a good ecological state, then their choices must also include users' perceptions and expectations to be able to mutually assure a diversity of uses and a good ecological functioning of these urban lakes.

Of course, we are aware that these data obtained on the three lakes are probably nonrepresentative of the great diversity of the situations that could be encountered in the urban L&Ps of the Ile-de-France area. However, during the data presentation of our findings to the managers of numerous of these L&Ps, which was performed at the end of the research program, most of the managers shared our analyses built on the data recorded from the three lakes. In the same way, one potential bias in this study concerns the fact that in this study, we were not able to consider people who choose to avoid these urban L&Ps because they have a negative point of view about them. This omission could lead to an overrepresentation of people who have a good opinion about the quality of these ecosystems.

Defining management objectives within a diversity of meanings

The first consideration is about the acknowledgement of the diversity of meanings encompassed by urban L&P quality. Frequently, managers have the tendency to refer to standardized quality indicators and threshold values to evaluate urban L&P quality. Even when not concerned with the WFD institutional monitoring, managers are often attached to the idea of 'reference status', because standardized indicators help them set quality objectives and define action plans. Consequently, they tend to homogenize the quality management objectives to get closer to 'good ecological status' standards [START_REF] Carré | The Water Framework Directive's "percentage of surface water bodies at good status": unveiling the hidden side of a "hyperindicator[END_REF][START_REF] Steyaert | The European water framework directive: How ecological assumptions frame technical and social change[END_REF]. Moreover, in the framework of ecosystem services approaches, a better ecological quality of lakes could allow for the maximization of ecosystem services, but this is not sufficient to prevent the existence of conflicts of use and some incompatibilities between the various uses and the water quality required for them [START_REF] Bolund | Ecosystem services in urban areas[END_REF][START_REF] Lin | Lake watershed management: Services, monitoring, funding and governance[END_REF][START_REF] Lundy | Integrating sciences to sustain urban ecosystem services[END_REF].

This approach eludes the incoherencies between various management objectives [START_REF] Blicharska | Urban ponds for people and by people[END_REF][START_REF] Wagner | Incompatibility of common lake management objectives[END_REF] and fails to address the problem of trade-offs within various ecosystem services [START_REF] Janssen | Shifting states, shifting services: Linking regime shifts to changes in ecosystem services of shallow lakes[END_REF][START_REF] Schueler | Watershed Protection Techniques, SPECIAL ISSUE: Urban Lake Management[END_REF]. In some shallow lakes located in Ile-de-France, it has been found that the bird population is an important source of nutrients that contributes to the eutrophication of lakes and affects their capacity to provide other ecosystem services, such as recreational activities (i.e. that are disturbed for example by cyanobacterial blooms due to the eutrophication) or stormwater storage (i.e. that necessitates a different management of water level than the one appropriated to bird nesting needs). This situation shows that for each urban lake, managers are inevitably confronted with choices of quality criteria and quality objectives that should be explicitly discussed to clarify which ecological and social functions can be privileged and which functions will be affected or impossible to accomplish.

From a diversity of meanings to a diversity of water bodies at the regional scale

To address the conflicts between management objectives and the incompatibilities between the multiple expected ecosystem services, we propose that management practices need to evolve from a focus on each individual L&P to a consideration of the management of urban L&Ps at a larger territorial scale. Despite the small size of numerous urban L&Ps and the 'bad' quality values obtained by some of them, all these water bodies provide social and cultural amenities and are rich in terms of biodiversity when they are considered all together. Following recent research results on the biodiversity richness of small pond networks [START_REF] Hill | Urban ponds as an aquatic biodiversity resource in modified landscapes[END_REF][START_REF] Oertli | Review: Toward management of urban ponds for freshwater biodiversity[END_REF][START_REF] Williams | Comparative biodiversity of rivers, streams, ditches and ponds in an agricultural landscape in Southern England[END_REF], we consider that at the neighborhood and city scales, it may be interesting to maintain a diversity of L&Ps with various trophic states via a 'pondscape approach' [START_REF] Boothby | Pond conservation: Towards a delineation of pondscape[END_REF][START_REF] Hassall | The ecology and management of urban pondscapes[END_REF] focused on the management of lake networks rather than individual entities.

In this framework, the key issue of this approach will be to know how to improve the coordination of social uses and amenities with the various ecological statuses of urban L&Ps. Instead of considering that a single lake can answer to all ecological and social quality criteria, we suggest that a diversity of lakes with various ecological statuses can better support a large diversity of social uses and amenities. Consequently, the water quality targets for each urban lake will be different based on the accordance between their intended uses (recreation, water supply, flood control, biodiversity, etc.) and their trophic state (Figure 5).

For example, eutrophic lakes could be oriented toward environmental and educational uses (e.g. natural areas dedicated to bird resting and nesting, mini-natural parks landscaped for environmental observation, etc.), while lakes displaying a good microbiological quality could be primarily dedicated to recreational uses (e.g. swimming, boating, etc.). As a matter of consequence, lake management should prioritize restoration actions on the ponds where they can obtain the best results in terms of water quality. This will particularly concern the control of pollutant inputs (organic and inorganic).

Such a territorial approach should also permit the avoidance of the application of remedial methods to try to maintain some uses in aquatic ecosystems, regardless of the water quality [START_REF] Humbert | The suitability of chemical products and other short-term remedial methods for the control of cyanobacterial blooms in freshwater ecosystems[END_REF]. For example, short-term solutions based on the application of chemicals (copper sulfates, hydrogen peroxide, etc.) and/or on the use of ultrasonic device are frequently used to kill cyanobacteria, which are dangerous for humans and consequently disturb recreational activities in water bodies. Knowing that cyanobacterial blooms are mainly due to eutrophication, the application of short-term solutions is frequently chosen by managers rather than sustainable solutions for the control of nutrient loads, which take time to have visible effects. Knowing also that there are many uncertainties about the environmental impacts of these short-term solutions and on their applications in the field, a territorial management approach to urban L&Ps should permit a better adequation between water quality and the uses that are required by these ecosystems [START_REF] Humbert | The suitability of chemical products and other short-term remedial methods for the control of cyanobacterial blooms in freshwater ecosystems[END_REF].

The choice of the most appropriate uses for each ecosystem should not be considered to be definitive; a revision of these uses should be periodically performed based on the evolution of the water quality and on the users' capacity to adapt their practices to a given environment.

Of course, this territorial management approach raises many questions, including: the relevant territorial unit (which is not necessarily the watershed scale), the likely acceptance of management choices concerning the use of spatial organization, the possible obstacles for its implementation, and the difficulty of capturing and dealing in an inclusive way with various stakeholders' expectations and values [START_REF] Chan | Where are cultural and social in ecosystem services? A framework for constructive engagement[END_REF]. Moreover, a water body that offers the possibility of specific uses does not have the same value if it is part of the 'reference territory' of an individual (i.e. close to his or her home, work, or usually used for accessing services and leisure) or if it is outside of this territory. From a territorial and land-based perspective, uses are quite incommensurable, and when a spatial variable is introduced, we cannot determine the equivalence of a use in two distinct places. Thus, the users' capacity to adapt their practices and evaluation criteria from one environment to another must be taken into account.

For all these reasons, the territorial distribution of ecosystem services should be balanced between different lakes based on their specific quality and particular urbanization conditions (i.e. nearby water bodies with similar access conditions). In any case, this reorganization of water body ecosystems services must consider the following factors together: the sociohydraulic constraints (related to the history of urban development), the ecological quality of the lakes (measured according to the parameters of the environmental regulation), and the social demand as expressed by various users and managers.

Conclusion

Organizing the multifunctionality of urban L&Ps is a core issue for the management of their ecological quality. Many urban planning schemas worldwide include the creation of small urban ponds [START_REF] Boix | Pond research and management in Europe: "small is beautiful[END_REF]EPCN, 2008;[START_REF] Oertli | Review: Toward management of urban ponds for freshwater biodiversity[END_REF] that are intended to provide various urban and ecological functions. This article suggests that a more integrative management of the existing and future urban L&Ps and water bodies (such as Stormwater Drainage Utility System (SDUS) must be organized at the city (or neighborhood) scale, including various waterbodies. This article shows that managing urban shallow L&P quality is not only about improving the ecological and water quality indicators of each lake but also better coordinating the various ecological statuses of L&P and their social uses (ecosystem services, benefits, values and expectations). Consequently, to provide the various uses and services that are expected from urban L&P, it is necessary to conserve or promote a diversity of ecosystems regarding their ecological status.

As shown in our paper, public perceptions about lake quality can provide some interesting understandings of the role of urban L&P, their expected functions and quality indicators. Despite the fact that users' valuations of urban L&P quality are mainly based on aesthetic aspects (i.e. the 'natural' and 'green' appearance) rather than on clearly established ecological values in terms of biodiversity for example, users' preferences are not necessarily incompatible with ecological quality objectives. We show that user expectancies can be an important driver of L&P preservation, mainly via blue-green landscape conservation, which can provide important well-being amenities within cities. This advocates for the preservation of the vegetal coverage of lake shorelines and of the green surrounding areas, which are strongly appreciated by the public and will have a positive impact on biodiversity and water quality by reducing anthropic urban pressures.


Youssef Berro

Romain Masse

Marianne Balat-Pichelin
email: marianne.balat@promes.cnrs.fr

Regenerating metal fuels through the carbothermal reduction of magnesia and alumina using concentrated solar energy

Introduction

The need for sustainable clean fuels, caused by the depletion of fossil fuels and global warming, makes it necessary to develop novel processes to meet growing demand, especially in the critical transport sector [1]. In this context, metal fuels are promising zero-carbon energy-carrying materials that can be directly burned in a metal-fueled combustor [2]. The regeneration of those fuels using renewable energy sources, through the reduction of the combustion product oxides, provides the sustainability of the combustion/reduction cycles [3]. A previous exergy analysis proved that operating under low vacuum is economically advantageous when the heat required for the reduction is supplied using concentrated solar energy [4]. Therefore, this study investigates the vacuum-assisted carbothermal reduction of magnesia and alumina using concentrated solar energy aiming to improves the metal fuels production and thus the combustion/reduction cycle efficiency.

Experimental Set-up

Solar experiments are performed in the Sol@rmet reactor [START_REF] Puig | AIP Conference Proceedings[END_REF] placed at the focus of a 1.5 kW solar furnace at Odeillo (France) consisting of a tracking heliostat and a parabolic mirror (Fig. 1) that concentrates the solar radiation up to 15000 suns. The reaction temperature is controlled by opening the shutter placed between the heliostat and the parabola, and is measured using a monochromatic pyrometer. A primary pump provides the vacuum and the reactor pressure is set to 190-840 Pa by adjusting the flow rate of the argon carrier gas (0.5-5 NL min -1 ). Oxide powders and charcoal reducing agents were manually or mechanically milled with various binders (PVA, starch, bentonite) and metal catalysts (Fe, Ni, Fe-Ni). C/MgO and C/Al 2 O 3 pellets are formed through dry pressing at a molar ratio of 1.25 (carbon excess) and 3 (stoichiometry) respectively. The produced metal powders were collected on a porous stainless-steel filter installed on the exit tube (m filter ) and on the reactor walls (m reactor ). Those powders were analyzed by XRD to determine their chemical composition (metal purity), and by granulometry and SEM measurements to determine their size and structural morphology. Whereas, the gases (CO and CO 2 ) production was observed all along the reaction time using the Xstream infrared gas analyzer. The performance of the reduction is determined by computing the metal yield according to the following equation, where is the maximum metal quantity that can be produced based on the initial oxide content. Fig. 1: Experimental set-up and photo of the concentrated solar system and the Sol@rmet reactor.

Results and Discussion

Solar carbothermal reduction of magnesia

Based on numerical simulations performed using ANSYS-CFX software, we confirmed by solar experiments that a double Ar entry from the top and bottom parts of the reactor will create a swirl circulation allowing a better purge of the products and thus a higher Mg yield by around 15% as show in Table 1. A mechanical milling of the C/MgO powders prevents the existence of magnesia agglomerates and increases the C/MgO contact thus increasing the reduction yield to 84.7%. The effect of the charcoal type is investigated showing that birch charcoal gives a yield three times higher than using chimney charcoal and proving that a minimal fixed carbon content of 85% is required. Using 5% starch -5% bentonite binder allows reaching a Mg yield of 96% of highly pure agglomerates of submicron Mg particles and crystals and this catalytic effect can be attributed to the prevention of the MgO sintering. Metal catalysts (Fe, Ni, Fe-Ni) proved to have a negative effect as the yield decreases to around 75% when 2% Fe is added. We assume that iron catalyst accelerates the reaction at the beginning thus decreasing the C content and promoting the MgO sintering.

Parameters and conditions during the solar experiments

yield (%) purity (%) Previous results using Sol@rmet reactor (one Ar entry, 900 Pa, 5 Nl min -1 ) [START_REF] Puig | [END_REF] 50.0 97 Progressive temperature increase, one Ar entry, 900 Pa, 3.5 Nl min -1 52.0 87 Double entry: 0.5 Nl min -1 from bottom entry and 3 Nl min -

Solar carbothermal reduction of alumina

The main problem encountered during the carbothermal reduction of alumina is the formation of unwanted Al-oxycarbide by-products. Solar experiments are performed at various pressures (190-840 Pa) and by slowly or quickly increasing the temperature to optimize the conditions limiting the formation of those byproducts. Results proved again the beneficial effect of mechanical milling as the yield improves by around 15% while the bentonite effect becomes adverse as it accelerates the beginning of the reaction, promoting the formation of by-products. At 840 Pa, a maximum yield of 75% is reached when the temperature is gradually increased, obtaining 85% purity of nano-and micro-sized Al particles. Decreasing the pressure to 285 Pa, by reducing the argon flow, allows to obtain 77% Al yield and to prevent the formation of Al 2 OC (91% Al purity). However, an additional decrease of the pressure to 190 Pa is disadvantageous (43% Al yield) as most of the produced powders deposit in the reactor where they can be easily oxidized (40% Al 2 O 3 ).

Conclusions

The amelioration of the reaction conditions and parameters during the vacuum-assisted solar carbothermal reduction of magnesia and alumina allows to reach higher metal yields of highly pure metal powders making their use as recyclable fuels in combustion/reduction cycles more attractive.

Table 1 :

 1 Comparison of the Mg yield and purity obtained after various solar MgO reduction experiments.

	1 from upper entry	68.0	87
	Mechanical milling of C/MgO powders	84.7	93
	C/MgO + 5% starch + 5 % bentonite	95.8	96
	C/MgO + 5% starch + 5 % bentonite + 2% Fe	77.1	91