Sébastien R David

Agathe Jaouen

Dris Ihiawakrim

Valérie A Geoffroy
email: valerie.geoffroy@unistra.fr

Biodeterioration of asbestos cement by siderophore-producing Pseudomonas

Keywords: asbestos cement, Pseudomonas, pyoverdine, iron, weathering

Since the ban on the use of asbestos due to its carcinogenic properties, the removal of asbestos cement, representing the major asbestos-containing waste, has proven to be a challenge in most industrial countries. Asbestos-containing products are mainly disposed of in landfills and have remained untreated. Bioremediation involving bacteria previously reported the ability of Pseudomonas aeruginosa to release iron from flocking asbestos waste through a siderophore-driven mechanism. We examined the involvement of siderophore-producing Pseudomonas in the biodeterioration of asbestos cement. Iron and magnesium solubilization were evaluated by specific siderophore-producing mutants. The absence of one of the two siderophores affected iron extraction, whereas equivalent dissolution as that of the control was observed in the absence of siderophore. Both pyoverdine and pyochelin biosynthesis was repressed in the presence of asbestos cement, suggesting iron bioavailability from the waste.

We compared the efficiency of various pyoverdines to scavenge iron from asbestos cement waste that revealed the efficiency of all pyoverdines. Pyoverdines were efficient in iron

Introduction

Asbestos comprises a family of natural compounds that have been intensively used for many purposes, such as heat-insulating boards, plastic and spun products, tar, and asbestos cement (AC), due to their insulating, chemical, and mechanical properties. Exposure to asbestos fibers by inhalation results in pleural calcification, pulmonary fibrosis, pleural, lung and peritoneal mesothelioma, and ovarian cancer. The toxicity of asbestos is typically attributed to the presence of the transition metal iron as a catalyst of the Fenton reaction, leading to the generation of free radicals and reactive oxygen species (ROS) [START_REF] Kamp | Asbestos causes DNA strand breaks in cultured pulmonary epithelial cells: role of ironcatalyzed free radicals[END_REF][START_REF] Toyokuni | Mechanisms of asbestos-induced carcinogenesis[END_REF][START_REF] Valko | Redox-and non-redoxmetal-induced formation of free radicals and their role in human disease[END_REF]. Consequently, asbestos has been banned since the beginning of the 80's in many countries, but some still use asbestos, such as Russia, India, China, and Brazil. Asbestos removal from buildings, its main area of application, generates a vast amount of asbestos-containing waste (ACW). ACW management is obliged to follow current safety recommendations, which can include rendering it inert by heating to 1,300 to 1,500°C. However, the most widely used technique is disposal in landfills, although this does not provide a definitive solution to health risks [START_REF] Paolini | Asbestos treatment technologies[END_REF]Spasiano and Pirozzi, 2017). Moreover, damage to landfill storage sites could potentially release asbestos fibers into the surrounding environment. Thus, alternative treatments are required to alter asbestos fibers and convert this waste into non-harmful compounds. Various treatment technologies have been investigated, such as thermal, chemical, hydrothermal, and mechanochemical approaches, which differ in terms of cost, energy consumption, and management of the endproducts [START_REF] Paolini | Asbestos treatment technologies[END_REF]Spasiano and Pirozzi, 2017). Bioremediation strategies have also been explored, as asbestos consists of minerals originating from soil, with a worldwide distribution. Indeed, Asbestos consist of fibrous silicate minerals belonging to amphibole or serpentine families. Amphiboles include five types of minerals (actinolite, anthophyllite, tremolite, amosite (brown asbestos), and crocidolite (blue asbestos)), whereas serpentine consists of only chrysotile (white asbestos). Among the six species, chrysotile, crocidolite, and amosite have the most commercial applications, with chrysotile representing 95% of world production, as it is the most common type found in many asbestos-containing products [START_REF] Kanarek | Mesothelioma from Chrysotile Asbestos: Update[END_REF]. Chrysotile is a magnesium phyllosilicate, which may carry iron as an integral part of the chemical structure, depending on soil composition. Indeed, substitution can occur between Si 4+ and Mg 2+ , which can be replaced by Fe 3+ and Fe 2+ , respectively [START_REF] Bernstein | Health risk of chrysotile revisited[END_REF][START_REF] Virta | Asbestos: Geology, mineralogy, mining, and uses[END_REF]. The structure of chrysotile, which consists of a double layer comprised of a silicate sheet and brucite (Mg(OH)2), is sensitive to acid attack, whereas it is well known that basic solutions have no effect.

Iron and magnesium are two essential elements present in asbestos that are required by many microorganisms. Bacteria are well-known to interact with minerals through various mechanisms [START_REF] Ferret | Siderophore-promoted dissolution of smectite by fluorescent Pseudomonas[END_REF][START_REF] Kraemer | Iron oxide dissolution and solubility in the presence of siderophores[END_REF], amongst which the use of siderophores is a common strategy to overcome iron deprivation, as iron is poorly bioavailable in aerobic circumneutral environments. Siderophores are highly Fe(III)-specific ligands [START_REF] Neilands | Microbial iron compounds[END_REF] present in significant amounts in soil [START_REF] Hersman | Growth of Pseudomonas mendocina on Fe(III) (Hydr)Oxides[END_REF]. Fluorescent Pseudomonas are soil bacteria that produce the siderophore pyoverdine (PVD), which is a green fluorescent compound produced in large quantities under iron starvation [START_REF] Cocozza | Siderophore production and associated characteristics in rhizosphere and non-rhizosphere fluorescent pseudomonads[END_REF].

PVD is composed of three distinct structural components i) a dihydroxyquinoline chromophore, which confers the color and the fluorescence of the molecule, ii) a peptide chain, comprised of 6 to 12 amino acids, bound to its carboxyl group, and iii) a small dicarboxylic acid (or its monoamide) connected to the NH2-group [START_REF] Budzikiewicz | Siderophores of the Pseudomonadaceae sensu stricto (fluorescent and non-fluorescent Pseudomonas spp.)[END_REF][START_REF] Budzikiewicz | Siderophores of fluorescent Pseudomonads[END_REF][START_REF] Budzikiewicz | Characterization of the chromophores of pyoverdins and related siderophores by electrospray tandem mass spectrometry[END_REF][START_REF] Demange | Bacterial siderophores : structure and NMR assignment of pyoverdins Pa, siderophores of Pseudomonas aeruginosa ATCC 15692[END_REF][START_REF] Fuchs | Structural studies of pyoverdins by mass spectrometry[END_REF].

Pseudomonas aeruginosa is a ubiquitous fluorescent Pseudomonas species [START_REF] Goldberg | Pseudomonas: global bacteria[END_REF] known to produce, aside from PVD, a second siderophore, called pyochelin (PCH), a lowmolecular-weight thiazoline derivative [2(2-o-hydroxy-phenyl-2-thiazolin-4-yl)-3methylthiazolidine-4-carboxylic acid] that has a lower affinity for iron (Ka = 10 28.8 M -2 ) [START_REF] Brandel | Pyochelin, a siderophore of Pseudomonas aeruginosa: Physicochemical characterization of the iron(III), copper(II) and zinc(II) complexes[END_REF] than PVD (Ka = 10 32 M -1 ) [START_REF] Albrecht-Gary | Bacterial iron transport: Coordination properties of pyoverdin PaA, a peptidic siderophore of Pseudomonas aeruginosa[END_REF]. Siderophoreproducing Pseudomonas can mobilize iron from various minerals, such as Fe(III)(hydr)oxide [START_REF] Hersman | Growth of Pseudomonas mendocina on Fe(III) (Hydr)Oxides[END_REF] or aluminosilicate clay minerals [START_REF] Ams | Siderophore production by an aerobic Pseudomonas mendocina bacterium in the presence of kaolinite[END_REF] by Pseudomonas mendocina. Furthermore, [START_REF] Ferret | Siderophore-promoted dissolution of smectite by fluorescent Pseudomonas[END_REF] showed that PVD and PCH, produced by P.

aeruginosa, release structural iron from smectite. Asbestos weathering related to bacterial and fungal siderophores is a common mechanism that has been previously described [START_REF] Daghino | Fungal weathering and implications in the solubilization of metals from soil and from asbestos fiber[END_REF][START_REF] Rajkumar | Biotechnological applications of serpentine soil bacteria for phytoremediation of trace metals[END_REF]. Aspergillus tubingensis and Coemancia reversa, two filamentous fungi isolated from a Rajasthan Indian mine, efficiently remove iron from asbestos fibers (Bhattacharya et al., 2016a). These isolates are able to produce siderophores and were considered to be potential bioremediators for asbestos, as previously demonstrated for Fusarium oxysporum [START_REF] Daghino | Inorganic Materials and Living Organisms: Surface Modifications and Fungal Responses to Various Asbestos Forms[END_REF][START_REF] Martino | Physical and biochemical interactions of soil fungi with asbestos fibers[END_REF], Verticillium leptobactrum, and Aspergillus fumigatus [START_REF] Daghino | Bioweathering of chrysotile by fungi isolated in ophiolitic sites[END_REF]. [START_REF] Bhattacharya | Bacterial weathering of asbestos[END_REF] showed a decrease in iron content from asbestos by several Rajasthan mine bacterial isolates identified as Gram-positive and -negative bacteria, with two Gram-positive siderophore producers (Bhattacharya et al., 2016b). Bacterial weathering of asbestos has been recently investigated. Several studies focusing on raw asbestos demonstrated that certain Grampositive and Gram-negative bacterial isolates decrease the iron content of asbestos rocks [START_REF] Bhattacharya | Bacterial weathering of asbestos[END_REF], whereas bacterial metabolites, such as organic acids and ligands produced by another Gram-positive bacterium, Bacillus mucilaginosus, promote the dissolution of serpentine [START_REF] Yao | Serpentine dissolution in the presence of bacteria Bacillus mucilaginosus[END_REF]. Bhattacharya et al. (2016b) suggested that the observed iron dissolution from fibers probably correlates with siderophore production by the mine bacterial isolates. Several studies have focused on desferrioxamine, the siderophore produced by actinomycetes. [START_REF] Mohanty | Siderophore-mediated iron removal from chrysotile: Implications for asbestos toxicity reduction and bioremediation[END_REF] showed that iron can be removed from chrysotile raw asbestos by desferrioxamine, similarly to the findings of [START_REF] Chao | Effect of long-term removal of iron from asbestos by desferrioxamine B on subsequent mobilization by other chelators and induction of DNA single-strand breaks[END_REF], who observed the same for amphibole fibers, such as crocidolite and amosite. We previously showed the involvement of the bacterial siderophores produced by P. aeruginosa, PVD and PCH, in the weathering of chrysotile, crocidolite, and amosite raw fibers, resulting in iron depletion from the fibers (David et al., 2020b). These findings have provided new insights into the bacterial degradation of asbestos but further studies are still required to understand how these materials can be transformed into a non-toxic form when the fibers are mixed in a matrix inside the waste. The matrices can be very different, depending on the ACW, consisting of plastic, tar, gypsum, or cement, and the percentage of asbestos fibers can also vary, from 10 to more than 90%. In addition, the fibers can be free or embedded in the matrices, which can potentially impede the biodeterioration process. Thus, the challenges of waste degradation are highly specific for each type of waste. The bacterial alteration of flocking asbestos waste (FAW) has been recently investigated. Indeed, David et al. (2020a) showed the ability of P. aeruginosa to use FAW as iron and magnesium nutrient sources, with iron removal linked to a siderophore-driven mechanism. In addition, iron removal from FAW by PVDs has been demonstrated, showing varying efficiency depending on the PVD structure (David et al., 2020c). AC materials have been widely used in pipelines, flat sheets, corrugated roof sheeting, and insulation boards, which represent 80% of the world production of asbestos [START_REF] Bordebeure | Déchets amiantés[END_REF][START_REF] Dériot | Le drame de l'amiante en France[END_REF][START_REF] Douguet | Effets sur la santé des principaux types d'exposition à l'amiante[END_REF]Fiche toxicologique amiante, 2018). These materials contain a ratio corresponding to approximately 10% asbestos fibers embedded in 90% cement [START_REF] Douguet | Effets sur la santé des principaux types d'exposition à l'amiante[END_REF], leading to compact compounds, in contrast to FAW, which contains 90% free fibers. [START_REF] Wasserbauer | Nitrifying bacteria on the asbestos-cement roofs of stable buildings[END_REF] showed that nitrifying bacteria, such as Nitrosomonas and Nitrobacter, increase the porosity of AC roofs and reduce their strength due to the production of nitrous and nitric acid.

Moreover, slime-forming bacteria and heterotrophic aerobic bacteria lead to the corrosion of the inside surface of AC pipes of water distribution systems due to acid production resulting from bacterial metabolism [START_REF] Wang | Biodeterioration of asbestos cement (AC) pipe in drinking water distribution systems[END_REF][START_REF] Wang | Bacteriological challenges to asbestos cement water distribution pipelines[END_REF]. [START_REF] Balducci | Process for treating an asbestos containing material[END_REF] also demonstrated that lactic-acid production by lactic bacteria causes the efficient dissolution of the cement phase, while Spasiano et al. (2017[START_REF] Spasiano | Dark fermentation process as pretreatment for a sustainable denaturation of asbestos containing wastes[END_REF][START_REF] Spasiano | Sustainable bio-hydrothermal sequencing treatment for asbestos-cement wastes[END_REF] showed that dark fermentation can degrade the cement phase together with a partial dissolution of brucitic layer of chrysotile fibers of AC wastes thanks to volatile fatty acids metabolized during the process [START_REF] Race | Simultaneous treatment of agro-food and asbestos-cement waste by the combination of dark fermentation and hydrothermal processes[END_REF] .

We aimed to investigate the involvement of siderophore-producing bacteria in AC biodeterioration and examine the role of PVD and PCH in mineral dissolution. The induction of the PVD-and PCH-biosynthetic pathways was measured in the presence of AC waste.

Given the low bacterial requirement for iron and the release of iron from AC, which would repress siderophore production, we also tested the supplementation of several representative PVDs to AC to determine their ability to scavenge iron and selected the most efficient. The best PVDs were used in long-term weathering experiments to determine whether complete degradation could be achieved. A better understanding of the bacterial weathering of AC could lead to its definitive conversion into non-harmful compounds and the development of an eco-compatible alternative technology, avoiding the continual disposal of such waste in landfills.

Materials and Methods

Asbestos waste preparation

The AC used in this study was obtained from the Center for Studies and Training in Asbestos and Chemical Safety (CEFASC ENVIRONNEMENT) and consisted of chrysotile associated with a cement matrix. Scanning Electron Microscopy (SEM) was performed on AC waste as described in [START_REF] Talbi | Virtuous cycle of destruction and total recycling of pure asbestos and asbestos-containing waste[END_REF]. This analysis method was coupled with Energy Dispersive X-ray (EDX) to determine the chemical composition of the samples. Matrix composition was 48 % CaO, 24.3 % SiO2, 11.7% MgO, 8 % Na2O; 2.3% Al2O3, 1.3 % Fe2O3

and the fiber composition was 57.9 % MgO, 42.1 % SiO2. The Mg/Si ratio measured in the fiber found in the roof tile range from 1.0 to 1.4, which is very close to the Mg/Si ratio of pure chrysotile. The diameter of the fibers was approximately 0.05 to 0.50 µm. Due to the grinding, a heterogeneity in fiber length was obtained ranging from 0.5 µm to 500 µm.

For laboratory scale experiments, AC was ground for 10 min at 500 rpm in a planetary ball mill (Pulverisette 7, Fritsch). Asbestos samples (0.2 g) were sterilized by autoclaving (121°C for 20 min) and incubated at 70°C for 14 days before experiments for complete sterilization of the material. Samples were washed with 20 mL sterile casamino acids medium (CAA) without magnesium (CAA-Mg) before each experiment to remove free iron and magnesium.

Bacterial strains and growth conditions

The bacterial strains used in this study are listed in Tables 1 and2. Strains were stored in LB-20% glycerol at -80°C. Pseudomonas cells were grown routinely overnight at 30°C in Luria Bertani medium (LB) with shaking (220 rpm). CAA (composition in g.L -1 : low-ironcasamino acids, 5.0; K2HPO4.3H2O, 1.46; MgSO4.7H2O, 0.25) was used as iron-restricted medium and sterilized using 0.2-µm polyethersulfone membrane filter units (Stericup Merck).

For iron and magnesium-depleted cultures, cells were harvested from a LB pre-culture (24 h incubation) and washed once with sterile CAA medium without magnesium. Cultures were incubated for 24 h at 25°C with shaking (200 rpm) [START_REF] Geoffroy | Siderotyping of Antarctic fluorescent Pseudomonas strains[END_REF]. For bacterial suspension, cells were washed once with sterile CAA medium without magnesium. Cell density was adjusted to an OD600 of 0.1, corresponding to 1-2.10 8 CFU.ml -1 , and diluted to obtain a started inoculum between 6.10 4 and 1.10 5 CFU.ml -1 .

Pyoverdine production

PVDs used in this study are listed in Table 2. First, 1 mL overnight LB culture of Pseudomonas strains was centrifuged (3 min at 9,871 x g). For iron starved cells, irondeficient CAA medium was used. The pellet was washed twice with 1 ml sterile CAA medium, suspended in 1 ml sterile CAA medium, used to inoculate 15 mL of sterile CAA medium, and the suspension incubated at 25°C with shaking (220 rpm). After 24 h of growth, 7.5 mL of the culture was transferred to an Erlenmeyer flask containing 2 L sterile CAA medium. After 48 h of incubation at 25°C with shaking (220 rpm), the culture was centrifuged at 2,664 x g for 40 min. The supernatant was filtered twice with glass microfiber filters (Whatman, GF/C) and once with a nitrocellulose filter (0.45 μm porosity) [START_REF] Meyer | The Fluorescent Pigment of Pseudomonas fluorescens: Biosynthesis, Purification and Physicochemical Properties[END_REF]. The PVD concentration was determined by measuring the OD400 using a Specord 205 spectrophotometer (Analytik Jena). When needed, the PVD concentration was adjusted with PVD purified according to the method described thus far. Siderophorecontaining supernatant was loaded, after acidification (pH 6.0) onto an XAD-4 column, washed with two volumes of purified water, and eluted with one volume of 50% ethanol. The eluate was concentrated under vacuum on a rotary evaporator and lyophilized.

Expression of the pyoverdine and pyochelin pathways in the presence of AC

Expression of the biosynthetic enzymes for siderophore production was analyzed using Pseudomonas aeruginosa PAO1 strains with the biosynthetic enzymes for PCH and PVD, PvdJ (David et al., 2020a) and PchA [START_REF] Cunrath | A cell biological view of the siderophore pyochelin iron uptake pathway in Pseudomonas aeruginosa[END_REF], tagged with the fluorescent protein mCherry. Bacterial suspensions (20 mL) prepared in CAA medium without magnesium, as described in paragraph 2.2, were incubated with AC samples. After 40 h of incubation at 25°C with shaking (220 rpm), the number of bacteria was determined by serial dilution-plating on LB-agar plates and incubation at 30°C for 24 h. For the expression analysis, the samples were gently centrifuged for 5 min at 67 x g to separate the bacteria from the asbestos fibers. Bacterial cells (100 μL) were dispensed into individual wells of a 96-well plate (Greiner, PS flat-bottomed microplate). mCherry fluorescence (excitation/emission wavelengths: 570 nm/610 nm) was measured in a Tecan microplate reader (Infinite M200, Tecan). Assays were performed in triplicate.

Asbestos dissolution by Pseudomonas and pyoverdines

The involvement of PVD and PCH in iron and magnesium removal from AC was evaluated using the wildtype Pseudomonas aeruginosa PAO1 strain [START_REF] Stover | Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen[END_REF] and three siderophore mutants: i) a PVD-deficient strain (ΔpvdF) [START_REF] Hoegy | Stereospecificity of the siderophore pyochelin outer membrane transporters in fluorescent Pseudomonads[END_REF], ii) a PCHdeficient strain (ΔpchA) [START_REF] Cunrath | A cell biological view of the siderophore pyochelin iron uptake pathway in Pseudomonas aeruginosa[END_REF], and iii) a PVD-and PCH-deficient strain (ΔpvdFΔpchA) [START_REF] Gasser | Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore-antibiotic conjugates development[END_REF]. Bacterial suspensions (20 mL) prepared in CAA medium without magnesium, as described in paragraph 2.2, were added to AC samples. Controls without bacterial cells were included. After 40 h of incubation at 25°C with shaking (220 rpm), the number of bacteria was determined by serial dilution-plating on LB agar plates and incubation at 30°C for 24 h [START_REF] Ferret | Siderophore-promoted dissolution of smectite by fluorescent Pseudomonas[END_REF]. The assays were gently centrifuged for 5 min at 67 x g to separate the bacterial cells from the asbestos fibers. Bacterial cells (10 mL) were centrifuged 10 min at 9,871 x g and the supernatants filtered (0.22 μm porosity). Bacterial pellets were mineralized by washing them once with ultrapure water and drying at 50°C for 48 h. Cells were mineralized by incubation in 77 μL 65% (v/v) HNO3 for 48 h at room temperature [START_REF] Ferret | Siderophore-promoted dissolution of smectite by fluorescent Pseudomonas[END_REF]. The volume was brought to 5 mL with ultra-pure water to obtain 1% HNO3 and the samples filtered through a membrane with a 0.22 μm filter unit. The iron and magnesium content were measured. Assays were performed in triplicate.

To study the effect of various PVDs on the dissolution of iron from AC, 20 mL of PVD-containing supernatants adjusted to 100 µM at pH 7 were incubated with the materials.

The kinetics of iron dissolution from AC were followed for 24 h with regular sampling of the solution at 0, 2, 4, 8, 12, and 24 h. After 24 h of incubation at 30°C with shaking (220 rpm), the assays were centrifuged 30 min at 9,871 x g to recover the supernatants. Two additional cycles were repeated for 48 h and 96 h under the same conditions to prolong biodeterioration.

The kinetic samples and cycle supernatants were filtered (0.22 µm porosity) to eliminate the material from the solutions and the iron content measured. Each assay was performed in five replicates.

Long-term weathering of AC by PVD-containing supernatants of P. syringae and P.

aeruginosa was performed by renewal cycles at 30°C with shaking (220 rpm). For each renewal, 20 mL PVD-containing supernatant was added to the samples. Twenty cycles of 24 hours were realized in the presence of 100 µM PVD-containing supernatant. After each cycle, the assays were centrifuged 30 min at 9,871 x g and the supernatants filtered (0.22 µm porosity). The iron content was measured after each cycle and analysis of the fibers was performed by transmission electron microscopy at the end of the experiment. Assays were performed in five replicates.

Determination of Fe and Mg content

We determined iron content using the bathophenanthroline method, a sensitive colorimetric method (adapted from Bauman et al., 2019) for determining ferrous iron content.

Total iron was determined by converting ferric iron to ferrous iron using thioglycolic acid.

Each sample was analyzed in triplicate. Twenty microliters of sample was dispensed into each well of a 96-well plate (Greiner, PS flat-bottomed microplate), to which were added 40 μL saturated sodium acetate (5.5 M) (Sigma-Aldrich), 80 μL cold distilled water, 10 μL of a thioglycolic acid solution diluted 10 times in water (Sigma-Aldrich), and 10 μL bathophenanthroline (5 mg.mL -1 ) (Sigma-Aldrich). After shaking, microplates were stored overnight at 4°C. Absorbance was measured at 535 nm in a Tecan microplate reader (Infinite M200, Tecan) and iron concentration was determined by comparing the values to those of a standard reference curve.

The calmagite method was used to measure magnesium content (ELITech magnesium calmagite kit). Magnesium forms a colored complex with calmagite. EGTA (Ethylene Glycol Tetraacetic Acid) was used to eliminate any calcium interference. Each sample was analyzed in triplicate. The reactive solution consisted of one volume of reagent 1 (1 mol.L -1 2-Methyl-2-Amino-1-Propanol and 215 μmol.L -1 EGTA) and one volume of reagent 2 (300 μmol.L -1 calmagite). Three microliters of sample was dispensed into each well of a 96-well plate (Greiner, PS flat-bottomed microplate) and 300 μL reactive solution added. After shaking and 30 s of incubation, absorbance was measured at 500 nm in a Tecan microplate reader (Infinite M200, Tecan) and the magnesium concentration determined by comparing the values to those of a standard reference curve.

STEM-EDX of asbestos fibers

TEM (Transmission Electron Microscopy) images were recorded with a JEOL 2100 microscope, with a 200-kV potential applied to a LaB6 filament as an electron source.

Resolution of the TEM was 0.21 nm. TEM mapping was performed in STEM (scanning transmission electron microscopy) mode (resolution 2 nm), using an SSD-EDX (silicon drift detector-energy dispersive X-ray) spectrometer to determine the chemical composition.

Statistical analysis

Results presented in all figures correspond to the mean values of three to five replicates. Significant statistical differences between values were determined using the Kruskal-Wallis test (R version 1.0.153) followed by a Conover post-hoc analysis. Values with the same letter are not significantly different (p ≥ 0.05).

Results and discussion

Role of pyoverdine and pyochelin in iron and magnesium dissolution from AC

We investigated the role of the P. aeruginosa siderophores PVD and PCH in iron and magnesium removal from AC using P. aeruginosa strains producing both endogenous siderophores (WT), no PVD (ΔPVD), no PCH (ΔPCH), or neither of the two siderophores (2Δ). Bacteria were incubated in iron-minimal CAA medium depleted of magnesium, supplemented or not with AC. In this medium, inoculated with an average started inoculum of 7.10 4 CFU.ml -1 , weak growth occurred for all strains (7.10 6 to 5.10 7 CFU.ml -1 ), whereas more significant growth occurred in the presence of AC (3 to 7.10 8 CFU.ml -1 ) (Supplementary Fig. 1). AC provided sufficient magnesium to sustain bacterial growth equivalent to that in the CAA control with sufficient magnesium. However, no difference between the growth of the WT and mutant strains was observed in the presence of AC. The iron and magnesium measurements presented in Fig. 1 are represented as the concentration of the element measured in the presence of AC subtracted from that measured in the presence of bacteria in CAA-Mg. Iron and magnesium dissolved from the AC was evaluated both in the supernatants and bacteria. Iron was dissolved from the AC in the cultures with WT, ∆PVD, or ∆PCH, with 0.54, 0.33, and 0.17 mg. L -1 respectively, whereas no difference was observed for the 2∆ strain (0.08 mg. L -1 ) relative to the CAA-Mg control (0.13 mg. L -1 ) (Fig. 1A). The theoretical iron content after mineralization is 2.3 mg of Fe for 0.2 g of AC, which represents 117 mg.L -1 .

The evolution of the pH varied from 7 at the beginning of the culture to 8.5 at the end of the cycle which favor the precipitation of iron hydroxydes, leading to a very low iron solubility in those conditions. During the culture, bacterial metabolism synthesize various small organics acids and siderophores that will complex iron and other metals in order to keep them solubilized. In our experiment, the solubility of metal is correlated mainly to the presence of siderophore which will maintain metal in solution thanks to their high affinity. The absence of one of the two siderophores affected iron removal, with a stronger effect in the absence of PCH. There was thus a synergistic effect on iron dissolution in the WT strain in which both siderophores were produced. These results show the involvement of both siderophores in iron dissolution from waste.

Induction of the PVD and PCH pathways was followed by measuring the fluorescence of mCherry fused to PvdJ as an indicator of expression of the PVD biosynthetic pathway and PchA for that of the PCH biosynthetic pathway. There was weak induction after 40 h of incubation in CAA-Mg medium (1.10 -7 and 3.10 -8 U.A. fluorescence/UFC.mL -1 for PvdJ and PchA respectively), which correlated with weak bacterial growth, as shown before. The CAA control showed optimal induction for both siderophore pathways (1.1 10 -5 and 4.2 10 -6 U.A.

fluorescence/UFC.mL -1 for PvdJ and PchA, respectively), whereas supplementation with AC resulted in strong repression for both PVD and PCH (4. 10 -7 and 1.10 -7 U.A.

fluorescence/UFC.mL -1 for PvdJ and PchA respectively) (Fig. 2). These results are in accordance with those for iron solubilization shown in Fig. 1A, which repressed the production of both siderophores. Moreover, these results are also consistent with the WT siderophore production measured in the previous experiment. Indeed, AC supplementation decreased PVD and PCH production (6.5 µM and 9.7 µM, respectively) relative to that of the positive control in CAA (112.4 and 82.4 µM respectively) (Data not shown).

David et al. (2020b) studied mineral interactions with siderophore-producing

Pseudomonas for the main asbestos species, chrysotile and amphiboles. Results showed direct evidence of the involvement of the bacterial siderophores PVD and PCH on fiber weathering for the three tested asbestos species, chrysotile, crocidolite, and amosite. Such a processes of bio-weathering could lead to a reduction in asbestos toxicity due to iron removal [START_REF] Daghino | Soil Fungi Reduce the Iron Content and the DNA Damaging Effects of Asbestos Fibers[END_REF][START_REF] Martino | Soil Fungal Hyphae Bind and Attack Asbestos Fibers[END_REF] and may represent an alternative bioremediation strategy to solve this environmental problem. However, these studies focused only on raw asbestos and rocks. David et al. (2020a) recently investigated these new bio-weathering perspectives on asbestos waste. The same results were successfully obtained for FAW, with iron removal linked to a siderophore-driven mechanism. Moreover, new insights into asbestos waste management were obtained, with magnesium and iron being an elemental source to sustain bacterial growth. Despite the highly different matrix composition and texture of AC from that of FAW, our results demonstrate the ability of Pseudomonas to use AC as a nutrient source to acquire the necessary iron and magnesium required for bacterial metabolism. [START_REF] Yao | Serpentine dissolution in the presence of bacteria Bacillus mucilaginosus[END_REF] suggested that ligands and organic acids, such as carboxylic acids or alcohols, metabolites produced by Bacillus mucilaginosus, could be responsible for the increased mineral dissolution of serpentine. The resulting interaction with this soil bacterium dissolved magnesium from the brucite portion of the chrysotile, inducing an effective bioweathering process and degradation of the serpentinic mineral. In our study, if the magnesium contained in the asbestos assay (0.2 g) would be dissolved completely, it would represent 7.2 mg of magnesium corresponding to 360 mg.L -1 . In our experiment, we measured 0.95 to 1.86 mg.L -1 which is far lower than the magnesium content that could be released from asbestos fibers. However, there were no significant differences in magnesium solubilization between any of the strains and the control (Fig. 1B), confirming previous results obtained for FAW weathering, in which siderophores did not appear to influence magnesium dissolution (David et al., 2020a). Bacterial organic acids have already been shown to act primarily on rocks and therefore allow better accessibility to siderophore-specific chelation [START_REF] Ahmed | Siderophores in environmental research: roles and applications[END_REF]. This synergistic effect may play an important role in altering asbestos.

Efficiency of various pyoverdines in iron extraction from AC

Our results showed the siderophore pathways of P. aeruginosa to be repressed in the presence of AC. We studied the effect of PVD on iron removal from AC waste by incubating various PVDs, differing in their structure, with the material and measuring the dissolved iron after various times of incubation. PVDs belong to a large family that contains more than a hundred structures. Little data is available related to the specificity of each PVD towards iron and more generally to metal complexation. Indeed, PVDs are well-known to chelate iron but have also been shown recently to chelate other metals [START_REF] Braud | New insights into the metal specificity of the Pseudomonas aeruginosa pyoverdine-iron uptake pathway[END_REF][START_REF] Schalk | New roles for bacterial siderophores in metal transport and tolerance[END_REF].

This may be beneficial if, for example, the asbestos came from a nickel-rich soil. Indeed, varying efficiency of iron removal from FAW by various PVDs has already been demonstrated (David et al., 2020c). It is thus important to understand the effect of PVD on AC waste to extend the bioremediation process, as AC represents most of the waste collected.

PVDs bind to Fe 3+ in a 1:1 stoichiometry (Albrecht-Gary et al., 1994) and structural

differences correspond to variations in the length, nature, and presence of cyclic rings in the peptide chain, as well as the radical R [START_REF] Meyer | Siderotyping of fluorescent pseudomonads: Characterization of pyoverdines of Pseudomonas fluorescens and Pseudomonas putida strains from Antarctica[END_REF]. We selected 10 PVDs from a bank of more than 100 and measured the kinetics of iron extraction from AC after adjusting them to a final concentration of 100 µM to determine the efficiency and optimal time of incubation.

Assays were carried out with PVD-containing supernatants, since previous experiments showed the same iron extraction efficiency between purified PVDs and PVD-containing supernatants from chrysotile (David et al., 2020b). We measured the dissolved iron of the samples after 0, 2, 4, 8, 12, and 24 h. All tested PVDs dissolved iron within the first hours of incubation relative to the CAA medium (Fig. 3A). Within the first 2 h, the dissolution pattern distinguished two groups of PVDs, with one group showing rapid iron removal followed by a decrease between 2 and 4 h of incubation, probably linked to the binding of PVD to the fibers, as previously observed (Data not shown). The other group showed time-dependent dissolution for 24 h. After 2 h of incubation, the PVD from P. syringae dissolved 2.6 mg.L -1 and P. putida 90.51 2.3 mg.L -1 , whereas the concentration for the others varied from 0.9 to 1.9 mg.L - 1 . At the end of the experiment, P. syringae was still the most efficient (3.1 mg.L -1 ), whereas iron dissolution reached a plateau after 24 h for P. putida 90.51, with 2.2 mg.L -1 . Our results

show that certain PVDs exhibit the same dynamics as the siderophore desferrioxamine, as the study of [START_REF] Mohanty | Siderophore-mediated iron removal from chrysotile: Implications for asbestos toxicity reduction and bioremediation[END_REF] showed an iron extraction equilibrium at 24 h for raw chrysotile fiber weathered by desferrioxamine. Iron removal from AC was faster than that from FAW (David et al., 2020c), which can be explained by the granulometry of the waste, which was ground, thus increasing the capacity for surface exchange between the PVDs and asbestos, improving the extraction and accessibility of iron release. At the end of the experiment, 50% of the siderophore was in a PVD-Fe complex. Thus, the reaction involved sufficient apo-PVD to extract iron, indicating that a limiting factor could be the time of contact.

We therefore continued the exposure with renewal cycles to 48 and 96 h with the PVD-containing supernatants. The PVD supernatant was maintained at a final concentration of 100 µM. We had already noticed the greater effect of the three PVDsyringae, PVDaeruginosa and PVDmonteilii at 24 h, corresponding to the end point of the kinetic study, with no statistical differences between the three conditions (Fig. 3B). However, PVDsyringae and PVDaeruginosa tended to be more efficient than the other tested PVDs, although the efficiency was not statistically different (PVDfluorescens 13525 and PVDmosselii for 48 h and PVDfluorescens 13525 and PVDlini for 96 h). At 48 h, the dissolved iron in the presence of PVDsyringae and PVDaeruginosa was 2.7 and 2.8 mg.L -1 , respectively, whereas lower concentrations (1.7 to 2.1 mg.L -1 ) were extracted for the others. For the renewal cycle of 96 h, PVDsyringae and PVDaeruginosa resulted in iron removal of 2.9 and 3.2 mg.L -1 versus 1.3 to 1.7 mg.L -1 for the other PVDs. At the end of the renewal cycles, the siderophore treatment dissolved a total of 172 µg iron for PVDsyringae and PVDaeruginosa and 96 µg for the less efficient PVDmandelii versus 2 µg for the medium control. Interestingly, the efficiency of the PVDs varied depending on the type of ACW.

Indeed, we previously showed that PVDmandelii was the most efficient for iron removal from FAW (David et al., 2020c), whereas it showed poor activity with AC. The affinity constant is well known for the well-studied PVDaeruginosa but there is little available data for the other PVDs. Our unpublished data lead us to believe that the affinity constant between PVDs is not sufficiently different to explain our results. However, the 3D structures vary between PVDs and there may be structures that can more easily access the structural iron present in the layers of chrysotile. Longer exposure did not increase the iron extracted but the efficiency was almost constant, even though the quantity of apo-PVD remained sufficiently high (Fig. 3B).

Therefore, the limiting factor is the availability of iron from the fibers and matrix.

The asbestos fibers contained in FAW and AC consist of chrysotile. The amount of iron solubilized from AC (3 mg.L -1 ) was higher than that from FAW (1 mg.L -1 ). These results

were confirmed by mineralization of each type of waste, which showed that the amount of iron in AC (12 mg of iron per waste gram) is higher than in FAW (5 mg of iron per waste gram). FAW contains 90% chrysotile, whereas AC contains only 10%. Thus, part of the iron necessarily comes from the cement matrix of AC, which appears to also be bioavailable.

Long-term weathering of AC by pyoverdines

Short-term experiments showed that iron can successfully be extracted from AC after each renewal with a PVD solution. Iron still appeared to be bioavailable after three renewal cycles, raising the question of how many cycles could still be performed to reach the limit of extraction. We conducted a long-term AC-weathering experiment by renewing the apo-PVD solution every 24 h for 20 cycles. We performed this experiment using PVDsyringae and PVDaeruginosa, given their previously observed equivalent iron release. Not surprisingly, iron was poorly dissolved in the CAA medium (0.01 to 0.5 mg.L -1 ), as already shown. Both PVDs dissolved iron from AC throughout the 20 cycles, with a steady decrease from T24-1 to T24-11 cycles from 3.4 to 1.6 mg.L -1 for PVDaeruginosa and 3.0 to 1.4 mg.L -1 for PVDsyringae (Fig. 4).

The rate of dissolution then remained stable from T24-11 to T24-20 (the end of the renewals) at 1.0 mg.L -1 iron. The cumulative iron amount removed from waste was 0.75 mg solubilized by PVDaeruginosa and 0.65 mg by PVDsyringae at the end of the experiment. Thus, such a weathering process could be pursued for some time, as iron was still being extracted after 20 cycles. This batch experiment was not continued, given that it is time consuming, but this process provides interesting perspectives for a bioremediation strategy and the development of eco-friendly asbestos treatment, avoiding the continuous disposal of landfills with ACW.

The same experiments carried out on FAW showed that PVD extraction was limited to 16 renewal cycles with 100 µM PVD. When the exposure time was increased to 96 H, dissolution was higher for seven cycles and the extraction limit was rapidly attained (David et al., 2020c). For the sampling used in our assay, PVD-mediated degradation resulted in extraction of 34 and 27% of the iron from AC by PVDaeruginosa and PVDsyringae, respectively, whereas the extraction of 12% for FAW was measured with PVDaeruginosa and PVDmandelii. This higher efficiency may be due to the grinding of AC waste, as already discussed for the kinetics experiments. Considering the heat-energy consumption of grinding, the effectiveness of this biological treatment should be tested on ungrinded samples to develop an ecological process. Several studies have focused on siderophore dissolution mechanism for asbestos fibers. [START_REF] Mohanty | Siderophore-mediated iron removal from chrysotile: Implications for asbestos toxicity reduction and bioremediation[END_REF] suggested that the siderophore concentration could have an effect on the efficiency of iron extraction. Indeed, at low concentrations, most of the siderophore may be adsorb onto the asbestos, as already demonstrated for other minerals, such as PVD on smectite [START_REF] Ferret | Siderophore-promoted dissolution of smectite by fluorescent Pseudomonas[END_REF]. Increasing the siderophore concentration would allow better siderophore surface coverage and may increase iron removal. Renewal cycles primarily dissolve the iron from the matrix, the iron from the fiber surface, and that in the brucite layer, with later access to the silica layer, for which iron may be slow to be released, as suggested by [START_REF] Mohanty | Siderophore-mediated iron removal from chrysotile: Implications for asbestos toxicity reduction and bioremediation[END_REF] and David et al. (2020c). Further studies would provide a better understanding of iron dissolution and confirm these hypotheses that suggest a prolonged incubation time and higher siderophore concentrations.

Analysis by STEM-EDX of the chrysotile fibers resulting from the long-term weathering of AC (Fig. 5) showed a large decrease of iron content for each PVD treatment. In AC waste, iron can be present in both chrysotile fibers and the cement matrix. Our results (Fig. 3) already demonstrate the bioavailability of iron from AC and these results confirm that iron is released from the fiber structure, as well as that of the matrix, thus confirming the active removal of iron driven by PVD. The magnesium content also decreased after treatment with PVDaeruginosa. This can be explained by a synergistic effect between iron and magnesium dissolution. Indeed, iron extraction from chrysotile fibers may destabilize the brucite layer and thus allow the release of magnesium. However, the magnesium content did not decrease after treatment with PVDsyringae, probably linked to the heterogeneity of iron substitution in fibers. However, given the heterogeneity of iron substitution in fibers, the iron extraction does not necessarily affect the dissolution of magnesium as observed with PVDsyringae treatment.

Conclusion

AC is a worldwide concern due to the considerable amount of waste generated as a consequence of asbestos removal. We aimed to investigate the role of siderophore-producing

Pseudomonas in AC bio-weathering and to evaluate the efficiency of various PVDs to release iron from AC waste. Pseudomonas was able to use AC as a nutrient source for its required iron and magnesium for bacterial metabolism, as already demonstrated for FAW. We clearly showed the involvement of the siderophores PVD and PCH in iron dissolution from AC waste. Indeed, the absence of one of the two siderophores significantly reduced the iron removal from asbestos waste. The interaction of the bacteria with AC waste resulted in repression of the siderophore biosynthetic pathways, demonstrating the bioavailability of iron.

Moreover, all 10 PVDs selected from PVD diversity were able to dissolve a significant amount of iron from AC, but with differences in efficiency depending on the PVD. Overall, PVDaeruginosa and PVDsyringae extracted the highest amount of iron from AC waste. Long-term treatment of AC by these two PVDs showed a large decrease in iron content from the chrysotile fiber structure, with no extraction limit after 20 days of renewal cycles. In AC waste, crocidolite and amosite can also be found in low amounts, in addition to chrysotile fibers. Given the ability of PVDs to progressively extract iron from both amphibole and chrysotile fibers, the biodeterioration of ACW by siderophore-producing Pseudomonas could contribute to the development of the eco-friendly management of asbestos waste to reduce asbestos-related environmental and health problems together with the disposal of ACW in landfills. Table 1. Strains and plasmids used in this study.

Figure and table legends

Table 2. List of pyoverdines used, with their molecular mass, isoelectrofocusing characteristics, and peptide composition.

Pyoverdine and pyochelin are important in iron removal from asbestos cement.

Pyoverdines exhibited various weathering efficiency in iron extraction.

No extraction limit reached after pyoverdine alteration cycles.

Asbestos waste weathering by pyoverdine decrease Fe content in fibers.

Pyoverdines could be used in bioremediation process of asbestos waste.

Highlights

Table 1. Strains and plasmids used in this study.

Strains

Relevant characteristics References

Pseudomonas aeruginosa PAO1 Wild-type strain [START_REF] Stover | Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen[END_REF] ∆pvdF PAO1; ∆pvdF [START_REF] Hoegy | Stereospecificity of the siderophore pyochelin outer membrane transporters in fluorescent Pseudomonads[END_REF] ∆pchA PAO1; ∆pchA [START_REF] Cunrath | A cell biological view of the siderophore pyochelin iron uptake pathway in Pseudomonas aeruginosa[END_REF] ∆pvdF∆pchA PAO1; ∆pvdF∆pchA [START_REF] Gasser | Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore-antibiotic conjugates development[END_REF] pvdJmcherry PAO1; pvdjmcherry (David et al., 2020b) pchAmcherry PAO1; pchAmcherry [START_REF] Cunrath | A cell biological view of the siderophore pyochelin iron uptake pathway in Pseudomonas aeruginosa[END_REF] Table Table 2

In color online

Fig. 1 .

 1 Fig. 1. Concentration of iron (A) and magnesium (B) dissolved from asbestos cement after 40 h in the presence of casamino acids medium without magnesium (CAA-Mg) as a control or inoculated with the wildtype Pseudomonas aeruginosa PAO1 strain (WT), a pyoverdinedeficient strain (∆PVD), a pyochelin-deficient strain (∆PCH), or a pyoverdine-and pyochelindeficient strain (2∆). Elements were measured in the bacterial cells (pellet) and supernatants. Error bars indicate the standard errors of the means of three replicates. Bars with the same letter are not significantly different (p ≥ 0.05, Kruskal-Wallis test, three replicates).

Fig. 2 .

 2 Fig. 2. Fluorescence of PvdJ-mCherry and PchA-mCherry after 24-h growth of Pseudomonas aeruginosa PAO1 in casamino acids medium with (CAA) or without magnesium (CAA-Mg), in the presence or absence of asbestos cement (FIBRO). Error bars indicate the standard errors of the means of three replicates. Bars with the same letter are not significantly different (p ≥ 0.05, Kruskal-Wallis test, three replicates).

Fig. 3 .

 3 Fig. 3. Concentration of iron dissolved from asbestos-cement waste in the presence of 100 µM of various pyoverdine-containing supernatants (SN) during a 24-h kinetics experiment (a) and after renewal cycles to 48 h and 96 h (b). Histogram bars of the 24 h timepoint (b) correspond to the endpoint of the kinetics experiment. Error bars indicate the standard errors of the means of five replicates. Bars with the same letter are not significantly different (p ≥ 0.05, Kruskal-Wallis test, five replicates).

Fig. 4 .

 4 Fig. 4. Iron removal from asbestos-cement waste after 24-h renewal cycles in the presence of 100 µM (T24-1 to T24-20) Pseudomonas syringae or Pseudomonas aeruginosa pyoverdinecontaining supernatants (SN). Error bars indicate the standard errors of the means of five replicates.

Fig. 5 .

 5 Fig. 5. STEM images and STEM mapping of chrysotile fibers from asbestos-cement waste after 24-h renewal cycles in the presence of casamino acids medium (A and B), Pseudomonas syringae (C and D), or Pseudomonas aeruginosa (E and F) pyoverdine-containing supernatants (SN). Large images obtained from the combination of the distributions of Mg, Si, and Fe, with analysis areas (A, C and E). Atomic ratios of Mg/Si and Fe/Si of various areas (B, D and F) and total area (H). Mass percentage of magnesium, silicon, and iron after renewal cycles with casamino acids medium, Pseudomonas syringae or Pseudomonas aeruginosa pyoverdine-containing supernatants (G).

Figure

  Fig. 1. Concentration of iron (A) and magnesium (B) dissolved from asbestos cement after 40 h in the presence of casamino acids medium without magnesium (CAA-Mg) as a control or inoculated with the wildtype Pseudomonas aeruginosa PAO1 strain (WT), a pyoverdinedeficient strain (∆PVD), a pyochelin-deficient strain (∆PCH), or a pyoverdineand pyochelindeficient strain (2∆). Elements were measured in the bacterial cells (pellet) and supernatants. Error bars indicate the standard errors of the means of three replicates. Bars with the same letter are not significantly different (p ≥ 0.05, Kruskal-Wallis test, three replicates).

FigureFigureFigure

  Figure Click here to access/download;Figure;Figure 2.pdf Texte

Figure

  Figure Click here to access/download;Figure;Figure 5.pdf Texte

.

  List of pyoverdines used, with their molecular mass, isoelectrofocusing characteristics, and peptide composition.

	Species	Siderovars	MM	Number of	Pyoverdine peptide chain	References
				isoforms and
				pHi values
	P. mandelii	SB8.3	1 046	3	9,0/8,8/7,6	Ala-Lys-Thr-Ser-AOHOrn-cOHOrn	(Budzikiewicz
							et al., 2014)
	P. fluorescens	CHA0	1 287	3	8,5/7,5/5,1	Asp-FOHOrn-Lys-(Thr-Ala-Ala-FOHOrn-Lys)	(Wong-Lun-
							Sang et al.,
							1996)
	P. putida	12633	1 336	3	4,6/4,2/4,0	Asp-εLys-OHAsp-Ser-Thr-Ala-Glu-Ser cOHOrn	(Persmark et
							al., 1990)
	P. mosselii	Lille 17	-	3	9,0/7,4/5,2	ND	(Dabboussi et
							al., 2002)
	P. lini	B10/PL9/	989	3	7,5/5,2/4,6	εLys-OHAsp-Ala-aThr-Ala-cOHOrn	(Teintze et al.,
		7SR1/A225					1981)
	P. putida	90-51	1 234	3	7,3/7,0/5,3	Asp-εLys-OHAsp-Ser-Gly-aThr-Lys-cOHOrn	(Sultana et
							al., 2000)
	P. fluorescens	13525	1 160	3	8,7/7,3/7,1	Ser-Lys-Gly-FOHOrn-(Lys-FOHOrn-Ser)	(Hohlneicher
							et al., 2014)
	P. monteilii	Lille 1	1 291	2	7,3/4,6	Asp-Lys-AcOHOrn-Ala-Ser-Ser-Gly-Ser-	(Meyer et al.,
						cOHOrn	2008)
	P. aeruginosa	PAO1	1 333	2	8,8/7,0	Ser-Arg-Ser-FOHOrn-(Lys-FOHOrn-Thr-Thr)	(Briskot et al.,
							1989)
	P. syringae	Syr	1 123	2	5,0/4,0	εLys-OHAsp-Thr-(Thr-Ser-OHAsp-Ser)	(Jülich et al.,
							2014)

Time (h) SN P. mandelii SB8.3 SN P. putida 90.51 --SN P. lini A225 CAA control SN P. fluorescens CHA0 SN P. fluorescens 13525 SN P. putida 12633 SN P. monteilii Lille 1 SN P. mosselii Lille 17 SN P. aeruginosa PAO1 SN P. syringae 19310

Acknowledgements

This work was supported by the French Environment and Energy Management Agency (ADEME), the French R&D plan for asbestos removal (PRDA) and SOMEZ (Mediterranean Company of Zeolites). The authors would like to acknowledge the help of the Institute Charles Gerhardt Montpellier (ICGM) for asbestos grinding.

Supplementary Material

Click here to access/download;Supplementary Material;Supplementary Figure 1.pdf

Texte

Graphical Abstract

In color online

Pseudomonas

Graphical Abstract


domain: spi nlin phys chim

text:
Soukaina Ben Salk

Reetu Raj Pandey

Phi Hq Pham

Di Zhou

Dominique Vignaud

Emiliano Pallecchi
email: emiliano.pallecchi@univ-lille.fr

Peter J Burke

Henri Happy
email: henri.happy@univ-lille.fr

Physical and electrical characterization of bubble free transferred single crystal graphene

Keywords: monocrystalline graphene, electrochemical delamination, bubble-free transfer, Raman spectroscopy, defect-free, electrical characterization

Monocrystalline graphene is desirable for practical applications since the large-scale polycrystalline graphene grown by chemical vapor deposition is limited in terms of performance. The transfer of graphene film from the metallic growth substrate to a host substrate, which is generally required for practical applications, can further damage the graphene by inducing strain, defects and wrinkles or by leaving contamination such as polymer residues. In this work, we have investigated the influence of the transfer process on the monocrystalline graphene in terms of quality, morphology and electrical properties by analyzing the data obtained from optical microscopy, scanning electron microscopy, Raman spectroscopy and electrical characterizations. The influence of copper oxidation on graphene prior to the transfer is also discussed. Our results show that the "Bubble-free" electrochemical delamination transfer is an easy and fast transfer technique suitable for transferring large single crystals graphene with an almost defect free surface. Moreover, Raman spectroscopy investigation reveals that the Cu surface oxidation modify the strain of the graphene film. We have observed that graphene laying on unoxidized Cu is submitted to a biaxial strain in compression, while graphene on Cu oxide is submitted to a biaxial strain in tension. However, after graphene was transferred to a host substrate, these strain effects disappeared leaving a homogeneous graphene on the substrate. Finally, we used the transferred single crystal graphene on silicon oxide substrate to fabricate transmission line method (TLM) devices. These electrical measurements show low contact resistance ~150 Ω.µm, which confirms the homogeneity and good quality of transferred graphene. Therefore, electrochemical delamination process can be used to transfer large single crystal graphene in a fast and reliable way.

Introduction

Graphene with its extraordinary properties has dragged attention among the researchers to investigate its technological applications as graphene has high electrical and thermal conductivities, flexibility and stability. [START_REF] Cao | Elastic straining of free-standing monolayer graphene[END_REF]- [START_REF] Pandey | Tuning the electrical property of a single layer graphene nanoribbon by adsorption of planar molecular nanoparticles[END_REF] Chemical vapor deposition (CVD) on copper has been broadly used to synthesize large-area and high-quality graphene. [START_REF] Ren | The global growth of graphene[END_REF] Recently, the synthesis of large-area monocrystalline graphene has aroused interest among researchers to avoid the grain boundaries, responsible for graphene performance degradation.

Because the majority of applications requires the as-grown graphene to be transferred to a host substrate, it is important to study how large monocrystalline domains behave during this process. Indeed, the transfer of graphene can have a considerable impact on its physical and electrical properties. [START_REF] Ma | Transfer Methods of Graphene from Metal Substrates: A Review[END_REF][10]- [START_REF] Li | Large-Area Graphene Single Crystals Grown by Low-Pressure Chemical Vapor Deposition of Methane on Copper[END_REF] The transfer steps may damage the graphene film causing cracks, wrinkles and polymer residues and thus, reducing the carrier mobility of graphene, a parameter of the upmost importance for electronic applications. [START_REF] Yu | Control and characterization of individual grains and grain boundaries in graphene grown by chemical vapour deposition[END_REF] Several methods have been developed to transfer the graphene from metal to the host substrates. The most common transfer technique involves the wet etching of the metal layer used as a catalyst template for the graphene growth, typically copper for monolayer graphene. This technique is reliable and well-studied [START_REF] Deokar | Towards high quality CVD graphene growth and transfer[END_REF]- [START_REF] Li | Large-Area Synthesis of High-Quality and Uniform Graphene Films on Copper Foils[END_REF], but has the disadvantage of a slow etching process, and destroying the copper film is non environmentally friendly. To overcome these limitations, remarkable progress has been made in the development of transfer techniques that avoid the etching of growth substrate. The most used method is the so-called electrochemical delamination transfer technique [START_REF] Ghosh | Extremely High Thermal Conductivity of Graphene: Prospects for Thermal Management Applications in Nanoelectronic Circuits[END_REF], [START_REF] Pham | Controlling Nucleation Density While Simultaneously Promoting Edge Growth Using Oxygen-Assisted Fast Synthesis of Isolated Large-Domain Graphene[END_REF]. Using this technique, few minutes are sufficient to separate the graphene layer from the growth substrate, compared to many hours required by the wet etching transfer method. Furthermore, this transfer technique significantly reduces metal consumption and the associated environmental pollution, and allows the reuse of the metal substrate, , [START_REF] Gao | Repeated growth and bubbling transfer of graphene with millimetre-size single-crystal grains using platinum[END_REF], [START_REF] Wang | Electrochemical Delamination of CVD-Grown Graphene Film: Toward the Recyclable Use of Copper Catalyst[END_REF] which is especially promising for large-scale, low-cost production.

In this study, large isolated graphene monocrystals of millimeter scale dimensions (~5mm) have been synthesized on polycrystalline Cu foil [START_REF] Pham | Controlling Nucleation Density While Simultaneously Promoting Edge Growth Using Oxygen-Assisted Fast Synthesis of Isolated Large-Domain Graphene[END_REF]. After the growth, an annealing step was applied on some of the graphene-covered copper films to promote Cu oxidation.

Afterwards, monocrystals of graphene, with and without post-growth oxidation, were transferred to Si/SiO2 substrate using an optimized electrochemical delamination method.

SEM and optical microscopy were used to analyze the morphology and quality of the material before and after transfer. Micro-Raman spectroscopy has been used to investigate how the oxidation of the copper underneath the graphene affects the graphene properties, by studying the strain and doping of the graphene films. Finally, transmission line measurement (TLM) structures were fabricated using the transferred graphene. Measurements performed on 6 TLM patterns, fabricated on a graphene single crystal, reveals a relatively low value of the contact resistance (~ 150 Ω.µm) which is attributed to the good quality of the transferred graphene and the fabrication process. The results presented in this work provide a path towards large-scale high-quality monocrystalline graphene transfer, which could contribute to the development of graphene-based electronic devices.

Experimental

This section briefly reminds the CVD synthesis of graphene on copper foils and details the transfer process of graphene samples.

Growth of large-size single crystal graphene

The synthesis of millimeter size monocrystal graphene -first reported in Ref. [START_REF] Wu | Crystal Structure Evolution of Individual Graphene Islands During CVD Growth on Copper Foil[END_REF] begins with an electrochemical polishing of the copper to improve the surface quality and thus decrease the nucleation density. The crystal growth is carried out inside enclosed copper pockets, to reduce the spreading of carbon and to slow the nucleation rate. The growth consists of two phases: a low methane flow is first employed during the nucleation phase to induce a low nucleus density, and the size of the crystals is enlarged from the nucleus in the second phase at an increased methane flow, as schematized in Fig. S1 in the supplementary information. Next, the copper pocket is unfolded and the as-grown sample (Fig. S2.a) is exposed to an intense flash light to identify the graphene crystals (Fig. S2.b). Latterly, the sample is annealed on a hot plate (250°C for 15 min) in order to oxidize the copper surface and to improve the visual contrast of graphene monocrystals, the size of which reaching 5 mm (see Fig. S3).

Electrochemical delamination transfer process of graphene (Bubble-free transfer)

Firstly, thin layer of 5% PMMA (polymethyl methacrylate) diluted in anisole was spin-coated onto graphene on one side of the Cu foil (3000 rpm, 1000 rpm/s, 10 s). Next, the sample (PMMA/graphene/Cu) was cured at 90°C for 30 min with very slow heating and cooling rates (30min), in order to prevent cracks in graphene caused by the strain induced in PMMA resist. Since the graphene was grown on both sides of the copper foil, the graphene on the back-side was removed by O2 plasma etching (2min, 50W RF power) before being subjected to delamination.

Latterly, for electrochemical delamination "Bubble-free", PMMA/graphene/Cu stacks were immersed gradually in KOH solution (40 mmol/L) and a two-electrode system held at a potential of -2.7V. A continuous voltage was then applied between the cathode (the polymer/graphene/Cu sample) and anode (glassy carbon rod) in an electrolytic cell. KOH was used as the electrolyte solution and the decomposition of H2O causes the generation of H2 bubbles at the Cu-graphene interface: 2H2O (l) → H2 (g) + 2OH (l) H2 bubbles help to separate graphene from the Cu surface. However, the uncontrolled production of hydrogen bubbles can cause mechanical damage in the graphene film. The complete delamination occurs within few minutes (less than 20 min). The graphene/PMMA film was then thoroughly rinsed (10 times) in deionized water to ensure the removal of residual KOH.

Finally, the graphene/PMMA film was transferred to Si/SiO2 substrate and dried at 90°C for 30 min with very slow heating and cooling rates (30 min) in order to remove the water trapped underneath the graphene. The complete delamination and transfer process of graphene is presented in supporting figures S4 and S5 and the detailed optimization of the electrochemical delamination "Bubble-free" process is given in Supplementary Fig. S6.

Scanning electron microscopy (SEM) images of the samples were taken with a Zeiss Ultra 55 SEM, equipped with a high efficiency lens. Raman spectrum were obtained with a confocal micro-Raman LabRam HR spectrometer (Horiba Jobin-Yvon) using an excitation laser (473 nm) focused with 100x objective.

Results and discussion

In this section, we present the detailed analysis of the bubble-free transfer process on the morphology and on the physical and electrical properties of the synthesized monocrystal of graphene.

Morphological and structural characterization of the as-grown single-crystal graphene

The synthesized graphene crystals were fully characterized before proceeding with the transfer. Two techniques were used, SEM and Raman spectroscopy as they can provide the local information on the structural uniformity and homogeneity of graphene samples. The SEM allows to image all the peculiarities of graphene, for example the shape of the synthesized crystals, grain boundaries and the presence of holes and discontinuities in the sheet of graphene. Fig. 1.a is an inverted contrast SEM image of a graphene sample after post-growth annealing in air. The hexagonal shape and the lack of observable grain boundaries suggest that the synthesized graphene is monocrystalline, which is confirmed by selected area electron diffraction (SAED) as reported in [START_REF] Pham | Controlling Nucleation Density While Simultaneously Promoting Edge Growth Using Oxygen-Assisted Fast Synthesis of Isolated Large-Domain Graphene[END_REF]. Fig. 1.c is a magnification of the area framed in Fig. 1.b, where we identify the three different contrasts which are pointed out by red, blue and black circles. The black circle refers to the oxidized copper substratefree of grapheneand the red and blue circles correspond to the areas covered by the graphene film. In order to determine the source of the contrasts presented on the surface of the hexagonal domain, the areas marked by black, blue and red circles, and the edge of the graphene crystal (indicated by a green cross) have been studied by Raman spectroscopy. Fig. 2.a presents the typical Raman spectra corresponding to the different areas pointed in the SEM image of the Fig. 1.c with the respective colors. As expected, graphene peaks (G at ~1580 cm -1 and 2D at ~2700cm -1 ) are present in all areas except the black ones corresponding to the substrate. The blue spectrum in Fig. 2.a is the typical spectrum of a high-quality graphene monolayer on copper with I2D/IG ~2. The red and green spectra also feature the G and 2D peaks, but have extra peaks between 900 cm -1 and 1500 cm -1 as represented in Fig. 2.c. It should be noticed that in the case of the red area, none of these peaks corresponds to the D peak (1349 cm -1 ), a signature of structural defects in the graphene. The D peak is only observed at the edge of graphene (green cross in SEM image of the Fig. 2.a).

Thus, the absence of the D peak away from the edge of graphene as well as the presence of G and 2D peaks in areas pointed by the red and blue circles with an intensity ratio I2D/IG > 2 indicates that the graphene crystal is monolayer, homogeneous and of high quality. In Fig. 2.b, peaks below 900 cm -1 are attributed to copper oxides (CuO and Cu2O) [START_REF] Karle | Synthesis and evaluation of new copper ketoiminate precursors for a facile and additive-free solution-based approach to nanoscale copper oxide thin films[END_REF]. These copper oxide peaks appear in the red and green spectra in Fig. 2.a and are absent in the blue area.

These oxides are therefore the source of the contrast observed in the different areas of the graphene monolayer. The contrast is observed over the entire surface covered by graphene showing that the oxidation under graphene is not homogeneous. We can thus consider that the graphene crystal lays on two different substrates, copper and copper oxides.

Morphological characterization of single-crystal graphene on SiO2 after transfer

The electrochemical delamination method described above was used to transfer the isolated large crystal graphene samples with and without air post-growth annealing. The optical images in Fig. 3 show the same graphene single crystals before and after transfer, confirming that the electrochemical transfer yields a homogeneous layer of graphene without tears or holes. This also demonstrates that the transfer process maintains the structure of graphene and should be achieved without damage.. To further analyze the oxidation effects of graphene crystals after post growth annealing, SEM investigations were carried out as shown in Fig. 4. Before transfer, the copper oxidation under graphene is non homogeneous as pointed out by the red arrows in Fig. 4.b which show areas where graphene stands on unoxydized copper. However, after the graphene transfer of this crystal, the surface of graphene looks uniform, keeping its original shape with no damage as seen in Fig. 4.c-d. This confirms that the transfer process does not depend on the oxidation of the Cu surface. The graphene layer after transfer remains homogeneous even though the oxydized Cu layer below the graphene was not. Analysis of the optical and SEM images shows that the "bubble-free" electrochemical delamination transfer process provides a homogeneous layer of graphene.

Characterization by Raman spectroscopy

Raman spectroscopy gives quantitative information on the quality of graphene. It allows to quantify the presence of structural defects, the level of graphene doping following the transfer and the evolution of stresses in the graphene layer. Four samples with and without post-growth annealing were studied and the Raman spectra of one of the sample measured after graphene transfer is depicted on Fig. 5.b. The presence of the G and 2D peaks and the absence of the D peak confirms the good quality of the transferred graphene.

Moreover, Raman measurements were carried out in different areas of the graphene crystal, leading to an average value of the intensity ratio I2D/IG of ~3. This confirms that the graphene is monolayer thick and that it remains homogeneous after transfer. The full width at half maximum (FWHM) is ~ 29 cm -1 , which emphasizes the good quality of the crystal.

By comparing the Raman spectra of graphene before and after transfer (Fig. 5), it is noticed that the positions of the G and 2D peak shift (Table 1) depending on, whether the graphene lays on copper or on copper oxide. The shift of the peaks is directly linked to the strain and doping induced by the substrate [START_REF] Lee | Optical separation of mechanical strain from charge doping in graphene[END_REF] .

Before transfer After transfer

Graphene on Si/SiO2

Strain and doping profiles of graphene

This study is based on a vector decomposition method developed to study the influence of strain and doping on graphene as described in references [START_REF] Lee | Optical separation of mechanical strain from charge doping in graphene[END_REF], [START_REF] Fromm | Looking behind the scenes: Raman spectroscopy of top-gated epitaxial graphene through the substrate[END_REF] . The variation in strain is studied before and after the transfer of graphene, considering the graphene on copper and graphene on copper oxide. Since the oxidation of copper under graphene is nonhomogeneous as described above (section 3.1 and 3.2), the Raman mapping is done on these two areas, with the "graphene on copper" and "graphene on copper oxide" as illustrated in the inset of Fig. 6(a) and in the supporting Fig. S7.

We have represented the frequency of the 2D peak as a function of the frequency of the G peak as shown in Fig. 6. The green cross in Fig. 6 represents the Raman peaks of undoped and unstrained graphene (νG0=1582cm -1 , ν2D0=2707cm -1 ) using an excitation laser at 473 nm.

The blue line (biax. strain) represents a prediction of (νG, ν2D) for an undoped graphene under biaxial stress. The orange line (p-doped) and the pink line (n-doped) represent predictions of (νG, ν2D) for strain-free graphene under p or n charge doping respectively.

The Raman analysis of graphene samples before transfer (Fig. 6.a) shows the graphene laying on copper is under biaxial compressive strain (-0.34±0.03 %), whereas the graphene on copper oxide is subjected to a biaxial tensile strain of (0.3±0.1 %).

The Raman analysis is also done for four samples after graphene transfer (on Si/SiO2) as shown in Fig. 6.b. 2 samples have been submitted to copper oxidation by air annealing after growth of graphene ("with post ox-sample1" and "with post ox-sample2"); and 2 others without any post-growth treatment ("Pristine-sample1" and "Pristine-sample2"). The Raman analysis alone cannot leads to any conclusion regarding the type of doping (n or p). But, from the literature which suggests that graphene transferred on SiO2 is always p-doped [START_REF] Park | Optimized poly(methyl methacrylate)-mediated graphene-transfer process for fabrication of high-quality graphene layer[END_REF], it is reasonable to assume that our samples are p-doped as well. Raman measurements have been obtained on different zones for each sample. The data (illustrated in Fig. 6.b) shows an evolution close to the orange line which represents a of (νG, ν2D) for strain-free graphene with p doping in the range 0 <p <4.10 12 cm -2 . Moreover, it can be seen that the graphene biaxial strain is almost completely relaxed after transfer onto Si/SiO2 as shown in Fig. 6.b: the residual strain stands between -0.1 % and 0.16 % for all the samples. The obtained results are in good agreement with the reported literature [START_REF] Park | Optimized poly(methyl methacrylate)-mediated graphene-transfer process for fabrication of high-quality graphene layer[END_REF], which shows that the monolayer graphene having a ratio 1 < I2D / IG < 2 is highly doped.

Electrical characterization of single-crystal graphene after transfer

The TLM method was used to characterize the contact resistance as well as the sheet resistance of the graphene layer. We used single crystal graphene sample with copper oxidation by annealing in air before transfer. With this copper oxidation, the crystalline graphene samples become visible by eyes. Therefore, it is easy to select crystal for transfer.

The interest of this oxidation step is to be able Ni/Au contacts (with respective thickness of 1.5 nm and 50 nm), and a sheet resistance ρsh =

Conclusion

Large graphene single crystals up to a few millimeters were synthesized on copper substrates, and a reliable graphene transfer method ("Bubble-free transfer") has been developed. The applied potential and controlled generation of hydrogen bubbles play an important role in this electrochemical delamination approach. This transfer method is faster than the Cu wet chemical etching method (a few minutes versus a few hours). The quality of graphene is verified by different comparative pre-and post-transfer characterization approaches, including electrical, SEM and Raman spectroscopy.

We have also studied the impact of an extra post-growth annealing step on the copper oxidation on the large single-crystal graphene. The copper oxidation is non-uniform under the graphene crystals. The graphene on oxidized copper is under biaxial tension, whereas the areas where graphene is laying on non-oxidized copper exhibits biaxial compressive strain with a low doping. Moreover, after graphene was transferred to a host substrate, these strain effects disappear and the graphene doping varys depending on the host substrate. The electrical characterization of devices on a graphene single crystal shows a sheet resistance of ρsh = 797 Ω/square and a contact resistance of 150 Ω.µm with Ni/Au contacts. Thus, the electrochemical delamination transfer process paves a path to easy and fast transfer of graphene maintaining its quality and its electrical properties. zoom-in between 0 and 900 cm -1 of the black spectrum showing copper oxide peaks and (c) zoom-in between 900 cm -1 and 1500 cm -1 for the red and green spectra of panel a.

  to choose easily what sample to use for transfer. The optical image of the device fabricated to measure the contact resistance is shown in the inset of Fig. 7.a. Ohmic contacts from six TLM patterns are obtained by analysing the linear behavior of the I-V curves. The resistances are then extracted and plotted as a function of the separation distance. Linear fitting is showing that variations in measured resistance are proportional to the separation distance between contacts and indicates the homogeneity of the graphene. Thus, we obtained a contact resistance RcW = 150Ωµm using

FiguresFig. 1 :

 1 Figures

Fig. 2 :

 2 Fig. 2: (a) Raman spectra corresponding to areas pointed in Fig. 1.c using the same colors, (b)zoom-in between 0 and 900 cm -1 of the black spectrum showing copper oxide peaks and (c) zoom-in between 900 cm -1 and 1500 cm -1 for the red and green spectra of panel a.

Fig. 3 :

 3 Fig. 3: Images of single crystal graphene, (a-b) optical image sample without annealing before (a) and after bubble-free transfer (b), (c-d) SEM and optical images of large single crystal graphene transferred to SiO2 substrate sample with post-growth annealing before (c) and after transfer (d), SEM images (a, c) and optical images (b, d).

Fig. 4 :

 4 Fig. 4: SEM images of graphene samples with post growth annealing (a-b) before transfer, (b) magnification of red square in panel a, red arrows pointing the unoxidized Cu areas,(c-d) same area after transfer, the graphene is homogeneous.

Fig. 5 :

 5 Fig. 5: Raman spectra of graphene from Fig. 1.c after subtracting the baseline. (a) Red represents an area where graphene is on copper oxide, blue represents an area where graphene is on copper. Both spectra are obtained on the same graphene crystal (b) after transfer on SiO2.

Fig. 6 :

 6 Fig. 6: Correlation between the frequencies of the G and 2D Raman peaks of graphene (νG,ν2D), fromRaman spectra of 4 samples, with and without post-growth annealing. The green cross represents (νG0,ν2D0) for undoped and unstrained graphene. The blue line (biax. strain) represents an estimation of (νG,ν2D) for undoped graphene under biaxial strain, while the orange (respectively pink) dashed line corresponds to unstrained p-doped (n-doped) graphene. Horizontal black lines in panel a refer to the values of biaxial strain. (a) before transfer, (b) after transfer, the green line parallel to the blue one represents the (νG,ν2D)strain dependency for a p-type doping of 4.10 12 cm -2 .

Fig. 7 :

 7 Fig. 7: (a) Plot of resistance with TLM structure for a graphene width of 12 µm, inset is an optical image of a TLM structure (b) SEM image showing a magnification of the framed area in the inset

Table 1

 1

		Graphene	on	Graphene on Copper
		copper		oxide
	νG (cm -1 )	1591  2		1579 ± 3	1590  8
	ν2D (cm -1 )	2730  2		2689  3	2708  10

Comparison of the positions of graphene's G and 2D peaks before and after transfer, from a Raman map of a graphene single crystal obtained from Fig.

6

.

Acknowledgments: Renatech, flagship... We acknowledge support from the Army Research Office through the ARO-(Contract No: W911NF-18-1-0076 and W911NF2010103), National Institutes of Health (Contract No: CA182384), the European Union's Horizon 2020 research and innovation program under phase of the Graphene Flagship GrapheneCore2 785219 and GrapheneCore3 881603 and the French American Cultural Exchange (FACE) Partner University Fund program. This work was also partly supported by the French RENATECH network