Mattea Romani

Claire Carrion

Frédéric Fernandez

Laurent Intertaglia

David Pecqueur

Philippe Lebaron

Raphaël Lami
email: raphael.lami@obs-banyuls.fr

High bacterial diversity in pioneer biofilms colonizing ceramic roof tiles

Keywords: Ceramic roof tiles, Biodeterioration, Pioneer biofilms, Bacterial diversity, Illuminabased High-throughput sequencing

INTRODUCTION

Ceramic tiles are traditionally used as building materials. The first tiles were hand molded with dried earth and had a canal-like shape (semicylindrical) [START_REF] Viollet-Le-Duc | Dictionnaire raisonné de l'architecture française du XIe au XVIe siècle[END_REF]. Currently, ceramic tiles are produced industrially, but the traditional canal-like shape has been retained. The use of these tiles remains extremely widespread. For example, clay tiles cover 74% of French roofs (FFTB, 2015). Tiles are commercially available in a wide range of colors and shapes, presenting very different chemical compositions and being produced by very diverse manufacturing processes [START_REF] Fassier | Interactions entre l'environnement et la terre cuite[END_REF]. Similar to many other types of building materials (bricks, glazed tiles, stones, and concrete) [START_REF] Saiz-Jimenez | Biodeterioration: An Overview of the State-of-the-Art and Assessment of Future Directions[END_REF][START_REF] Warscheid | Biodeterioration of stone: a review[END_REF], ceramic tiles are exposed to rain, wind, hail, snow, frost, substantial temperature variations, pollution, and other extreme environmental conditions [START_REF] Coutinho | Biological colonization and biodeterioration of architectural ceramic materials: An overview[END_REF]. Therefore, ceramic tiles undergo natural degradation, including biodedeterioration [START_REF] Guiamet | Biodeterioration of funeral sculptures in La Recoleta Cemetery, Buenos Aires, Argentina: Pre-and post-intervention studies[END_REF][START_REF] Warscheid | Biodeterioration of stone: a review[END_REF]. The latter corresponds to vital activities that occur on tiles, such as cell proliferation (i.e., colonization by microorganisms and biofilm formation) and production of acids by diverse types of microorganisms, and cause degradation of the material [START_REF] Guiamet | Biodeterioration of funeral sculptures in La Recoleta Cemetery, Buenos Aires, Argentina: Pre-and post-intervention studies[END_REF]. Tiles are particularly prone to biodeterioration because of their high porosity (between 19% and 30%, with pores ranging in size from 6 to 7 µm) that makes them one of the most porous building materials [START_REF] Fassier | Interactions entre l'environnement et la terre cuite[END_REF][START_REF] Gazulla | Relationship between certain ceramic roofing tile characteristics and biodeterioration[END_REF]. This susceptibility of tiles to colonization by organisms is also called bioreceptivity [START_REF] Miller | Bioreceptivity of building stones: A review[END_REF].

The major disadvantage of roof tile biocolonization is the resulting unsightly appearance, as the biofilm can be pigmented and can change the color of the roof [START_REF] Martino | What about biofilms on the surface of stone monuments[END_REF]. For example, dark or green spots may appear, and the roof may turn darker. In the long term, other adverse effects are observed, as biodeterioration weakens ceramic tiles by forming deep cracks and increasing the porosity of the tiles [START_REF] Berdahl | Weathering of roofing materials -An overview[END_REF][START_REF] Coutinho | Biological colonization and biodeterioration of architectural ceramic materials: An overview[END_REF]. This weakness promotes erosion and wear on the roof due to bad weather and favors colonization by larger organisms such as lichens, plants or bryophytes. Tile damage can also lead to loss of structural and thermal insulation, loss of drainage capacity or solar reflectance [START_REF] Berdahl | Weathering of roofing materials -An overview[END_REF]Pena-Poza et al., 2018). Biodeterioration of tiles is therefore the result of biocolonization by a wide variety of micro-and macroorganisms, such as bacteria, fungi, microalgae, plants, bryophytes and lichens [START_REF] Barberousse | Factors involved in the colonisation of building façades by algae and cyanobacteria in France[END_REF][START_REF] Coutinho | Biological colonization and biodeterioration of architectural ceramic materials: An overview[END_REF][START_REF] Fassier | Interactions entre l'environnement et la terre cuite[END_REF][START_REF] Ortega-Calvo | Effect of biological colonization on ceramic roofing tiles by lichens and a combined laser and biocide procedure for its removal[END_REF][START_REF] Warscheid | Biodeterioration of stone: a review[END_REF]. Only the "green part" of the bacterial diversity (i.e. cyanobacteria) present on roof tiles have been extensively studied.

Cyanobacteria were described as primary colonizers of clay tiles [START_REF] Gazzano | Biocidal effect of lichen secondary metabolites against rock-dwelling microcolonial fungi, cyanobacteria and green algae[END_REF] in studies that relied on culture-based diversity inventories [START_REF] Barberousse | Factors involved in the colonisation of building façades by algae and cyanobacteria in France[END_REF][START_REF] Coutinho | Microbial communities on deteriorated artistic tiles from Pena National Palace (Sintra, Portugal)[END_REF][START_REF] Crispim | Algal and cyanobacterial biofilms on calcareous historic buildings[END_REF][START_REF] Hauer | Diversity of cyanobacteria on rock surfaces[END_REF][START_REF] Macedo | Biodiversity of cyanobacteria and green algae on monuments in the Mediterranean Basin: An overview[END_REF][START_REF] Nowicka-Krawczyk | Diversity of an aerial phototrophic coating of historic buildings in the former Auschwitz II-Birkenau concentration camp[END_REF]. Among the most frequent cyanobacterial species identified on building materials were Aphanocapsa sp., Calothrix sp., Chroococcus sp. and Synechococcus sp. [START_REF] Hauer | Diversity of cyanobacteria on rock surfaces[END_REF][START_REF] Macedo | Biodiversity of cyanobacteria and green algae on monuments in the Mediterranean Basin: An overview[END_REF]. In contrast, the diversity of heterotrophic bacteria on building materials remains very poorly studied. To date, only a few works have focused on the cultivable diversity of these bacteria [START_REF] Krakova | Complex bacterial diversity in the white biofilms of the Catacombs of St. Callixtus in Rome evidenced by different investigation strategies: Bacterial diversity in white biofilms[END_REF][START_REF] Urzì | Microbial Diversity in Paleolithic Caves: A Study Case on the Phototrophic Biofilms of the Cave of Bats (Zuheros, Spain)[END_REF], revealing the potential importance of Arthrobacter agilis, Bacillus sp., Paenibacillus sp., Pseudomonas sp. and Rhodococcus sp. [START_REF] Krakova | Complex bacterial diversity in the white biofilms of the Catacombs of St. Callixtus in Rome evidenced by different investigation strategies: Bacterial diversity in white biofilms[END_REF][START_REF] Urzì | Microbial Diversity in Paleolithic Caves: A Study Case on the Phototrophic Biofilms of the Cave of Bats (Zuheros, Spain)[END_REF] as pioneer organisms.

All these organisms colonize tiles by forming biofilms, which allow bacteria to grow under extreme conditions, such as those that occur on the tops of roofs [START_REF] Flemming | Biofilms: an emergent form of bacterial life[END_REF][START_REF] Flemming | The biofilm matrix[END_REF][START_REF] Mayer | The role of intermolecular interactions: studies on model systems for bacterial biofilms[END_REF]. The classic pattern of biofilm development is as follows: biofilm growth is initiated from planktonic (i.e., free-living) bacteria [START_REF] Siboni | Conditioning film and initial biofilm formation on ceramics tiles in the marine environment[END_REF], which adhere to a surface and form microcolonies before the development of a mature biofilm that is embedded in extracellular matrix (ECM), composed notably of polysaccharides, proteins and extracellular DNA [START_REF] Flemming | The biofilm matrix[END_REF][START_REF] Molin | Gene transfer occurs with enhanced efficiency in biofilms and induces enhanced stabilisation of the biofilm structure[END_REF]. The environment created by the ECM allows not only improved tolerance and resistance to stress factors, such as the presence of antibiotics, heavy metals or biocides in the environment, but also improved resistance to desiccation.

Moreover, the ECM improves the supply and storage of nutrients via the capture of resources by sorption and degradation of these resources by extracellular enzymes in the surrounding environment. Therefore, in biofilms, organisms have access to nutrients that they could not use outside these biofilms and to microenvironments that are very favorable for their growth [START_REF] Flemming | The biofilm matrix[END_REF]. Thus, the formation of a mature biofilm from a pioneering biofilm leads to colonization by macro-organisms and is a crucial step in biodeterioration processes.

However, there is a distinct lack of studies that focus on the crucial initial steps of pioneer biocolonization, which determines the conditions for further steps of biofilm development.

Indeed, a large majority of current studies focus on advanced cases of biodeterioration, with visible colonization and numerous green or dark spots. Thus, mostly mature biofilms have been studied on building materials, with a focus on the algal diversity present on these surfaces [START_REF] Coutinho | Microbial communities on deteriorated artistic tiles from Pena National Palace (Sintra, Portugal)[END_REF][START_REF] Martino | What about biofilms on the surface of stone monuments[END_REF][START_REF] Hallmann | Molecular diversity of phototrophic biofilms on building stone[END_REF][START_REF] Krakova | Complex bacterial diversity in the white biofilms of the Catacombs of St. Callixtus in Rome evidenced by different investigation strategies: Bacterial diversity in white biofilms[END_REF][START_REF] Ma | The community distribution of bacteria and fungi on ancient wall paintings of the Mogao Grottoes[END_REF][START_REF] Maresca | Analysis of bacterial communities in and on concrete[END_REF][START_REF] Tomaselli | Biodiversity of photosynthetic microorganisms dwelling on stone monuments[END_REF][START_REF] Urzì | Microbial Diversity in Paleolithic Caves: A Study Case on the Phototrophic Biofilms of the Cave of Bats (Zuheros, Spain)[END_REF][START_REF] Warscheid | Biodeterioration of stone: a review[END_REF]. Even though some studies have focused on the diversity of pioneer cyanobacteria present on building materials [START_REF] Barberousse | Factors involved in the colonisation of building façades by algae and cyanobacteria in France[END_REF][START_REF] Hallmann | Molecular diversity of phototrophic biofilms on building stone[END_REF], the overall diversity of bacteria has hardly been studied [START_REF] Krakova | Complex bacterial diversity in the white biofilms of the Catacombs of St. Callixtus in Rome evidenced by different investigation strategies: Bacterial diversity in white biofilms[END_REF][START_REF] Urzì | Microbial Diversity in Paleolithic Caves: A Study Case on the Phototrophic Biofilms of the Cave of Bats (Zuheros, Spain)[END_REF] by either culture-based or culture-independent approaches.

To fill this gap, this study aims to characterize the existence of a pioneering biofilm on new and few exposed ceramic roof tiles (i.e. clay tiles), with a focus on heterotrophic bacterial colonization during the early colonization stages. These stages occur before the appearance of green or dark spots, before the biofilms become visible on the tiles and cause cracking, and before the increase in porosity promotes increased colonization by other organisms. To fully depict the pioneering bacterial diversity of clay tile biofilms, we combined imagery and culture-based investigations with state-of-the-art Illumina-based high-throughput sequencing methods based on non-exposure and 5 or 8 months of exposure.

MATERIALS AND METHODS

Sampling strategy.

Pioneer biofilms were sampled on 3 different types of commercial ceramic roof tiles that are commonly available in the French market and were named T1, T2 and T3 in this study. These tiles were sampled without exposure (new commercial products) and after slight outdoor exposure for 5 or 8 months at two commercial and industrial exhibition sites located in France. The site of exposure (site n°1) was located in Aquitaine. At this location, tiles were exposed with a north orientation, for 8 months. The second site of exposure (site n°2) was located in Burgundy. At this location, tiles were exposed with a south/southwest orientation, for 5 months. The environmental data to characterize both sites were also collected (Table 1) The T1 tiles had a black engobe with water-repellent coating; the T2 tiles had a red engobe with a water-repellent coating; and the T3 tiles had a pale red engobe but no waterrepellent coating. These three different types of tiles were chosen to know (i) if the chemical composition and the color of the tile could affect the bacterial colonization. For example, the color could influence the temperature of the tile, as a black tile will heat up more in summer than a lighter tile. and (ii) if the water-repellent coating protects tiles against biological colonization. This coating is designed to reduce the water permeability of the tile and therefore, by reducing the water available to microorganisms, might slow down biological colonization [START_REF] Urzì | Evaluation of the efficiency of water-repellent and biocide compounds against microbial colonization of mortars[END_REF].

Biofilms from all the types of tiles (either new or after slight exposure to the outdoors) were collected in triplicate (i.e. tree tiles of each type were sampled), and for each tile, 4 15 cm x 10 cm areas were sampled. First, the tiles were gently washed with distilled water before sampling. Then, the tiles were sampled using 2 different methods. (i) Two areas on each tile were scraped with a sterile scalpel [START_REF] Urzì | Microbial Diversity in Paleolithic Caves: A Study Case on the Phototrophic Biofilms of the Cave of Bats (Zuheros, Spain)[END_REF], producing a biofilm and tile powder that were transferred into a sterile tube containing 10 ml of saline water (9 g.L -1 NaCl). This suspension was then mixed for 15 s at maximum speed. (ii) Two other areas on each tile were sampled using a sterile swab previously soaked with a buffer solution (saline water (9 g.L -1 NaCl) + 1% Triton X-100), a methodology adapted from [START_REF] Grice | Topographical and temporal diversity of the human skin[END_REF] This swab was rubbed for 30 s on the tile and placed in a sterile tube with 5 mL of saline water (9 g.L -1 NaCl). The samples were then vortexed for 30 s at a maximum speed and stored at 4°C until use.

Negative controls preparation

For 2.3 to 2.7 experiments, negative controls were prepared with sterile tiles. These ones were autoclaved and systematically included in the experimental procedures. For all fluorescent microscopic approaches, an unstained sample was systematically observed for each type of tile to confirm the specificity of the selected fluorescent probes.

Scanning Electron Microscopy and Energy Dispersive X-ray spectrometry.

SEM and EDS were conducted to characterize the presence of a pioneering biofilm on the studied tiles by direct observations and analyses of elementary composition (carbon in particular). For each type of tile, a small sample (the same as used for fluorescence microscopy) was studied, at the University of Montpellier, Platform MEA, using a SEM (FEI Quanta FEG 200) instrument at an operational acceleration voltage of 15kV and under low vacuum (3.76e -1 Torr). Images were obtained using the backscattered electron detector (BSED). Chemical analysis was done using Energy Dispersive X-ray spectrometry (EDS), aluminium (Al) was labelling in yellow, silicon (Si) in red (the two major components of tile) and carbon (C) was labelled in green.

Samples staining

Samples staining was conducted to detect the presence of microbial DNA and biofilm matrix in the studied pioneering biofilms using fluorescent macroscopy, confocal laser scanning microscopy (CLSM) and two-photon microscopy. For each type of tile, a small sample (around 2 cm x 2 cm) was labeled with SYTO 9™ (DNA intercalant, green, Invitrogen™) and FilmTracer™ SYPRO™ Ruby Biofilm Matrix Stain (Polysaccharide labelling, red, Invitrogen™). Tile samples were immersed in the FilmTracer™ SYPRO™ and incubated for 30 minutes, in the dark at room temperature. After incubation, tile samples were rinse 3 times with ultrapure water. Then, tile samples were incubated in the dark for 15 min with SYTO 9 (10µM) at room temperature and rinse 3 times with TBS [START_REF] Domenech | Evidence of the presence of nucleic acids and β -glucan in the matrix of non-typeable Haemophilus influenzae in vitro biofilms[END_REF].

Fluorescence microscopy and macroscopy

All samples were preliminary observed under a CLSM (Confocal SP8-X Leica) with 10X or 20X magnification [START_REF] Neu | Investigation of Microbial Biofilm Structure by Laser Scanning Microscopy[END_REF][START_REF] Schlafer | Confocal microscopy imaging of the bio fi lm matrix ☆[END_REF] (Data not shown). Samples were then observed under a CLSM (confocal LSM880 Zeiss) with a 20X air objective, a fluorescent macroscope (AZ100 Nikon) with a 4X air objective and a zoom 8X, and a twophoton microscope (MVX10 FV1000, Olympus) using a 25X water immersion objective.

Moreover, CLSM was associated with surface imaging of the tile by using the reflected light on the ceramic. Images were analyzed with Fiji software [START_REF] Rueden | ImageJ2 : ImageJ for the next generation of scientific image data[END_REF][START_REF] Schindelin | Fiji : an open-source platform for biological-image analysis[END_REF].

Evaluation of bacterial density by flow cytometry.

Flow cytometry was used to measure bacterial cells abundances in the sampled pioneering biofilms. For each type of tile, one 15 cm x 10 cm area was scraped with a sterile scalpel into a sterile tube containing 1mL of saline water (9 g.L -1 NaCl) filtered at 0.2µm. Samples were filtered at 20µm (to remove the larger particles that could clog the cytometer). 200µL of bacterial cells were stained with 1µL of SYBR® Green I (1:100 dilution in DMSO, Lonza) and were incubated at least 15 min in the dark at room temperature [START_REF] Hammes | Flow-cytometric total bacterial cell counts as a descriptive microbiological parameter for drinking water treatment processes[END_REF].

Briefly, cells were counted by flow cytometry (CytoFlex, Beckman Coulter) for 60 sec at a flow rate of 60 µL.min -1 , at the Oceanological Observatory of Banyuls sur Mer, Platform BIOPIC. Polycarbonate beads (1.002 μm, Polyscience Europe) were used to normalize cell fluorescence and light scattering values. Cell density was calculated by removing the blank (saline water filtered at 0.2µm).

In-situ analysis of dehydrogenase activity.

This technique aims to characterize the presence of viable cell in the sampled pioneering biofilms. These assays were performed only on T2 and T3 tiles, due to the black engobe of T1. Briefly, analysis of dehydrogenase activity (revealing cell viability [START_REF] Gong | Short Communication Dehydrogenase Activity in Soil : a Comparison betweeb the TTC and INT Assay Under Their Optimum Conditions[END_REF] was performed on unexposed tiles following the protocol described by [START_REF] Warscheid | A rapid method to demonstrate and evaluate microbial activity on decaying sandstone[END_REF] Tile samples were covered by PBS [START_REF] Thundyil | Evidence That the EphA2 Receptor Exacerbates Ischemic Brain Injury[END_REF] containing 0.2% of Tetrazolium Red (2,3,5-triphenyl tetrazolium chloride) and incubated in the dark for 24h at 25°C. After incubation, samples were dried 1h at 60°C and pictures were taken [START_REF] Warscheid | A rapid method to demonstrate and evaluate microbial activity on decaying sandstone[END_REF].

Another negative control was done, tile samples were incubated in PBS only for 24h in the dark at 25°C.

Isolation of bacterial strains by culture-based techniques.

To depict the bacterial diversity colonizing clay tiles, we conducted isolation and characterization of bacterial strains. The isolation of bacterial strains on tiles followed routine and previously described protocols used to isolate strain on building materials [START_REF] Puškárová | Microbial communities affecting albumen photography heritage: a methodological survey[END_REF][START_REF] Urzì | Effects of biocide treatments on the biofilm community in Domitilla's catacombs in Rome[END_REF]. Briefly, for each sample, 100 µL of the solution collected previously was inoculated on Petri dishes containing R2A culture medium (BD Difco) [START_REF] Puškárová | Microbial communities affecting albumen photography heritage: a methodological survey[END_REF][START_REF] Urzì | Effects of biocide treatments on the biofilm community in Domitilla's catacombs in Rome[END_REF] with 100 mg.L -1 cycloheximide [START_REF] Urzì | Effects of biocide treatments on the biofilm community in Domitilla's catacombs in Rome[END_REF] and 4 mg.L -1 benomyl [START_REF] Lage | Bringing Planctomycetes into pure culture[END_REF] or solid BG11 culture medium (BG11 50× (Sigma Aldrich) supplemented with 15 g.L -1 agar) [START_REF] Rippka | Generic assignments, strain histories and properties of pure cultures of cyanobacteria[END_REF][START_REF] Urzì | Effects of biocide treatments on the biofilm community in Domitilla's catacombs in Rome[END_REF] with 100 mg.L -1 cycloheximide [START_REF] Urzì | Effects of biocide treatments on the biofilm community in Domitilla's catacombs in Rome[END_REF]. Both culture media were supplemented with sterile red ceramic roof tile powder (15 g.L -1 ) or left untreated. Cultures were maintained at 25°C in the dark for cultures on R2A and under continuous light for cultures on solid BG11 until isolation and identification.

Identification of isolated microbial strains.

After, bacterial strains were isolated on solid medium, a single colony was transferred to a liquid culture medium (same media as the solid culture without tile powder) and identified using routine protocols based on sequencing of the 16s rRNA genes [START_REF] Blanchet | Quorum sensing and quorum quenching in the mediterranean seagrass Posidonia oceanica microbiota[END_REF].

Molecular identification of heterotrophic bacteria started with the preparation of a cellular pellet that was obtained from 1 mL of liquid culture after centrifugation (10 000×g, 3 min).

Then, the genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega) following the manufacturer's instructions for gram-positive bacteria.

Molecular identification of cyanobacterial strains started with the preparation of a cellular pellet, which was obtained from 1 mL of liquid culture and resuspended in 10 µL of ultrapurified water. The genomic DNA was then extracted using the WB120028 CloneSaver card in 96-well format (FTA card technology, Whatman). After incubation of the CloneSaver card for 1 hour at room temperature, a small disc was punched out and washed 3 times for 5 min with TE buffer (Whatman).

PCR amplification of (i) bacterial 16s rRNA gene fragments was performed using the primer pair 27F

(5'-AGRGTTTGATCMTGGCCTCA-3'; 10 µM) and 1492R

(5'-TACGGYTACCTTGTTAYGACTT-3'; 10 µM) and (ii) cyanobacterial 16s rRNA gene fragments was performed using the primer pair 106F (5'-CGGACGGGTGAGTAACGCGTGA-3'; 10 µM) and 1313R (5'-CTTCACGTAGGCGAGTTGCAGC-3'; 10 µM). Briefly, the PCR protocol consisted of a denaturation step of 5 min at 95°C; 30 cycles of denaturation (95°C for 15 s), annealing (55°for bacterial 16S and 60°C cyanobacterial 16S for 15 s), and elongation (72°C for 15 s); and a terminal elongation step of 10 min at 72°C. All details of this protocol were previously published in [START_REF] Leboulanger | Microbial diversity and cyanobacterial production in dziani dzaha crater lake, a unique tropical thalassohaline environment[END_REF] All DNA amplification reactions were checked by migration of a genomic DNA aliquot on a 1% agarose gel. Then, the PCR products were purified with the Agencourt AMPure XP Kit following the manufacturer's instructions. The sequencing reaction was performed using the BigDye Terminator technology (Nimagen) with the following protocol: 30 cycles of denaturation (95°C for 10 s), annealing (50 or 60°C for 5 s depending on the previous PCR), and elongation (55°C for 2 min 30 s). The PCR products obtained were cleaned using the Agencourt CleanSeq Kit following the manufacturer's instructions. Sanger sequencing was carried out with the AB3130xl 16 capillary Sanger sequencer (Applied Biosystems) on the Bio2Mar platform (http://bio2mar.obs-banyuls.fr/fr/index.html).

The 16s rRNA gene sequences were cleaned on Snap Gene Viewer 3.3.3 software, and phylogenetic affiliation was assessed on EZBioCloud (http://ezbiocloud.net/). A phylogenetic tree was constructed in MEGA7 by following the K2+G+I model [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets[END_REF]. The phylogenetic tree was constructed using FigTree1.4.3 software (http://tree.bio.ed.ac.uk/software/figtree/). All sequences of the identified strains have been submitted to GenBank and have accession numbers MK207390 to MK207433 (Table S1) and maintained in the Banyuls Bacterial Culture Collection (BBCC).

Evaluation of the biofilm-forming capacity of isolated and identified strains.

A pre-culture of each identified strain was performed in 6mL of R2B. When the cultures reached an optical density (O.D.) of 0.8. Each strain was deposited in a 96-well microplate in triplicate, diluted 1/100 in R2B; as well as a negative control containing only R2B. The 96well microplate was incubated for 48 hours at 25°C and in the dark. After 48 hours of incubation, the O.D. at 640nm was measured to control bacterial growth. A crystal violet assay was performed to identify strains able to form biofilm, following protocols described in [START_REF] Blanchet | Quorum sensing and quorum quenching in the mediterranean seagrass Posidonia oceanica microbiota[END_REF] To achieve this goal, the culture medium was gently removed from the 96-well microplate to avoid damaging the biofilm, and the wells were rinsed with PBS. Then, crystal violet (0.2%) was added to the wells and the microplate was incubated for 15 minutes in the dark. Afterwards, the wells were rinsed 4 times with ultra-pure water, before adding a discoloration solution (10% acetic acid, 50% ethanol). Finally, the O.D. was measured at 540nm. Strains with O.D. > 1.5 were considered to form a high level of biofilm (++), those with 1.5 ≥ O.D. ≥ 0.5 were considered to form biofilm (+), and those with O.D. < 0.5 were considered as non-biofilm forming strains (-).

Extraction of total DNA for Illumina MiSeq-based high-throughput sequencing.

Tubes containing samples (scraped or swabbed) were centrifuged (15 min, 10 000×g, 25°C), and then, the supernatant was removed. The pellet was resuspended in the lysis solution of the Quick DNA Fungal/Bacterial Miniprep Kit (Zymo Research) and transferred into the BashingBead lysis tubes from the kit. The kit was used in accordance with the manufacturer's instructions with the addition of beta-mercaptoethanol to the genomic lysis buffer for a final dilution of 0.5% (v/v) as recommended. The manufacturer's instructions were followed with the exception of the first step, where vortexing was replaced with the use of the FastPrep-24 5G homogenizer (2 times, 45 s, 6 m.s -1 ) (MP Biomedicals) [START_REF] Coutinho | Microbial communities on deteriorated artistic tiles from Pena National Palace (Sintra, Portugal)[END_REF][START_REF] Jroundi | Molecular Tools for Monitoring the Ecological Sustainability of a Stone Bio-Consolidation Treatment at the Royal Chapel, Granada[END_REF]. Finally, the extracted DNA was quantified with a Quantus fluorometer (Promega) using the Picogreen Low program.

Identification of bacterial communities with Illumina-based high-throughput sequencing.

Illumina-based high-throughput sequencing of 16s rRNA gene fragments was performed using MR DNA (Shallowater, TX, USA; http://www.mrdnalab.com). Briefly, the V4 variable region of the 16s rRNA gene was amplified with the PCR primers 515R/806F, which included a barcode on the forward primer. A 30-cycle PCR was conducted using the HotStarTaq Plus

Master Mix Kit (Qiagen, USA) under the following conditions: 94°C for 3 min, followed by 28 cycles of 94°C for 30 s, 53°C for 40 s and 72°C for 1 min, after which a final elongation step at 72°C for 5 min was performed. After amplification, PCR products were checked by 2% agarose gel electrophoresis to determine whether amplification had been successful and measure the relative intensities of the bands. Multiple samples were pooled together in equal proportions based on their molecular weights and DNA concentrations. Pooled samples were purified using calibrated Ampure XP beads. Then, the pooled and purified PCR products were used to prepare the DNA library by following the Illumina TruSeq DNA library preparation protocol. Sequencing was performed on a MiSeq following the manufacturer's guidelines.

The obtained FASTQ files (BioProject ID: PRJNA506643, accession numbers: SAMN10465566 to SAMN10465585) were analyzed with QIIME (1.9.1) [START_REF] Caporaso | Using QIIME to analyze 16S rRNA gene sequences from microbial communities[END_REF], USEARCH v11 [START_REF] Edgar | Accuracy of taxonomy prediction for 16S rRNA and fungal ITS sequences[END_REF][START_REF] Edgar | Search and clustering orders of magnitude faster than BLAST[END_REF] and the Brazilian Microbiome Project scripts [START_REF] Pylro | Data analysis for 16S microbial pro fi ling from different benchtop sequencing platforms[END_REF]. Using QIIME, the mapping files were validated to ensure correct formatting.

The barcodes were extracted from the FASTQ files, and those files were demultiplexed.

Using USEARCH v11 and the scripts published by the Brazilian Microbiome Project, quality filtering based on the characteristics of each sequence was performed to remove any lowquality or ambiguous reads. Then, the files were concatenated and dereplicated. Chimeras were identified and removed; the sequences were denoised, and the zero-radius operational taxonomic units (ZOTUs) were defined with the unoise3 command from USEARCH v11 software [START_REF] Edgar | UNOISE2 : improved error-correction for Illumina 16S and ITS amplicon sequencing[END_REF]. Then, a representative sequence of each ZOTU based on 97% similarity of the 16s rRNA gene sequence, named operational taxonomic units (OTU), was selected and taxonomically assigned based on the Silva 132 database [START_REF] Quast | The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools[END_REF][START_REF] Yilmaz | The SILVA and "all-species Living Tree Project (LTP)" taxonomic frameworks[END_REF]. The representative sequences were aligned and filtered before the production of a table of OTUs, and sequences affiliated with chloroplasts and mitochondria were removed from the table according to previously published studies [START_REF] Bokulich | Antibiotics, birth mode, and diet shape microbiome maturation during early life[END_REF]. Bacterial communities were analyzed for taxonomic composition using the command summarize_taxa_trough_plots.py. Alpha diversity indexes (observed richness, Chao 1, and Shannon diversity index) were calculated and statistically analyzed with the Phyloseq R package (1.26.0) on RStudio (1.1.463) with R 3.5.0. Venn diagrams were created from the table.biom (obtained during bioinformatic data processing) corresponding to each category of interest (tile type and exposure) and using the following website:

http://bioinformatics.psb.ugent.be/webtools/Venn/. Samples were processed according to tile type and weathering step.

In addition, in order to extract sub-specific information from our dataset, the FASTQ files were also analyzed with the R package DADA2 (Divisive Amplicon Denoising Algorithm)

(1.10.0) [START_REF] Callahan | DADA2 : High-resolution sample inference from Illumina amplicon data[END_REF]. This methodology rely on a statistical modeling approach to denoise raw sequencing reads and to infer the abundance of "biological variants". First, the set of Illumina-sequenced paired-end FASTQ files from which the barcodes/adapters, were removed by the sequencing platform were demultiplexed and split by sample with QIIME

(1.9.1) before being analyzed using the online pipeline published by the authors of the DADA2 R package (https://benjjneb.github.io/dada2/tutorial.html). The end product of this pipeline is an amplicon sequence variant (ASV) table, a higher-resolution table compared to the OTU table previously obtained, and which records the number of times each amplicon sequence variant was observed in each sample. In this approach, the taxonomic assignation process was based on the Silva 132 database. Finally, the obtained data were represented with the phyloseq (1.26.0) and ggplot2 (3.1.0) R packages on RStudio (1.1.463). All data from the DADA2 analysis are presented as supplementary data and supplementary figures S3 to S6.

RESULTS

Evaluation of the presence of a pioneering biofilm

The presence of a pioneering biofilm was evidenced combining different imaging techniques on unexposed tiles (Figure 1). First, under SEM, dark spots could be seen (white circle and arrows, Figures 1A, 1B, SEM). They appeared blurred, like a matrix (Figure 1B, SEM). With retro-diffused imaging, these dark spots appeared richer in light elements than the rest of the tile, probably due to the presence of carbon and organic matter. EDS was achieved to confirm that dark spots were effectively richer in carbon than the rest of the tile (Figures 1C, 1D, EDS). In this sense, these dark spots appeared to be the first evidence of the presence of a pioneering biofilm on all sampled tiles. Then, several fluorescent microscopy approaches were combined (CLSM and two-photon microscopy) to observe the presence of cells and matrix on those tiles (Figure 1) and to confirm the existence of such pioneering biofilms . On tiles, clusters of cells were shown up in green (SYTO9) (Figure 1E, fluorescent macroscopy).

The observed biofilms were very thin and sparse. Nevertheless, CLSM of the fluorescent dye associated with reflection imaging of the tile allowed to show more precisely the location in a 3D-scale of the cell clusters in green (SYTO9) (Figures 1F,CLSM). Cells were also frequently observed in the anfractuosity of the tile (Figures 1F, CLSM). Finally, two-photon microscopy combining DNA dye in green (SYTO9) and matrix dye in red (FilmTracer™ SYPRO™) evidenced that clusters of cells were surrounded and embedded in a matrix (Figures 1G,1H, twophoton microscopy). Also, the plot Z-axis profiles of SYTO 9™ staining (Figure 1I) and FilmTracer™ SYPRO™ staining (Figure 1J) showed that the maximum of fluorescence was located at 46µm from the surface of the tile for the SYTO9 and that the thickness of the matrix ranged between 10 and 126µm. Those profiles reinforced the images evidencing that cells were embedded in a biofilm matrix. These techniques were completed by TTC assays and flow cytometry measures (Supp. Data).

Evaluation of biofilm growth

For each type of tile, total extracted DNA was quantified (Figure 2). Total extracted DNA corresponded all DNA present on the tiles and therefore to the biofilm as a whole, including heterotrophic bacteria but also all other colonizing microorganisms. A significant increase in the amount of DNA collected was systematically observed after the exposition of tiles. For T1 tiles, an average of 97 pg.cm -2 (±88) and of 688 pg.cm -2 (±378) of DNA were extracted before and after 8 months exposure, respectively. Similarly, for T2 tiles and T3 tiles, respectively 7.5 pg.cm -2 (±4.6) and 5.7 pg.cm -2 (±6) of DNA were extracted before exposure, while 149.8 pg.cm -2 (±3.3) and 143.6 pg.cm -2 (±33.1) of DNA were extracted after 5 months exposure. In addition, the capacity of isolated strains to form biofilm was evaluated using a classical crystal violet-based assay (Table 2). It revealed that 12 of the 41 isolated and tested strains were able to form a large biofilm. Two of these strains were isolated on unexposed tiles; 2 strains were isolated on tiles exposed 5 months and 8 strains isolated on tiles exposed 8 months (Table 2).

3.3 Bacterial diversity among cultivated and isolated strains from unexposed tiles (new tiles).

Culturable bacterial diversity from unexposed tiles (new tiles).

A total of 9 strains were isolated from new and unexposed tiles and identified based on their 16s rRNA gene sequences (Table 3). Only bacteria present on the T1 tiles could be isolated, and all of these bacteria were affiliated with the phylum Firmicutes under the genus Bacillus.

The potential species identified were B. safensis (100.00% and 99.88% 16s rRNA similarity with the closest known isolate), B. circulans (100.00%) and B. simplex (99.88%).

Culturable bacterial diversity from exposed tiles.

More bacterial strains (37 strains) were isolated, cultured and identified from the exposed tiles than from the unexposed ones (Table 3). These strains were assigned to 5 different phyla, including Firmicutes (8 strains), Cyanobacteria (2 strains), Bacteroidetes (1 strain), Proteobacteria (8 strains) and Actinobacteria (18 strains). In addition, there were some common potentially identified species between the different types of tiles, including Bacillus simplex (2 strains), Rhodococcus corynebacterioides (4 strains), Arthrobacter agilis (2 strains)

and Mesorhizobium alhagi (4 strains). All these strains presented at least 98.58% 16s rRNA gene similarity with the closest known isolate. As noted above, 2 strains of phototrophic microorganisms (cyanobacteria) were isolated on T1 tiles and identified as being closely related to the cyanobacterial clone 3GSG3R_K29 (accession number: JX127188, 99% 16s rRNA gene similarity) [START_REF] Hallmann | Molecular diversity of phototrophic biofilms on building stone[END_REF].

Diversity of bacterial phyla and species identified by Illumina-based high-throughput

sequencing on unexposed tiles.

Diversity of the bacterial phyla identified on unexposed tiles.

The bacterial diversity observed on all the unexposed tiles was very similar regardless of the type of sampled tile (Figure 3). Indeed, the 3 types of tiles were largely colonized by Proteobacteria, which represented between 40 and 70% of the total observed OTU (97% of 16s rRNA gene similarity) diversity (Figure 3). The Alphaproteobacteria class was dominant and represented more than 50% of the Proteobacteria colonizing tiles (Supplementary Figure S1). The OTUs affiliated with Actinobacteria represented between 8 and 15% (on average 11.3 +/-3.7); Firmicutes were between 9 and 13% (on average 11.5 +/-2.2); and

Bacteroidetes were between 3 and 12% (on average 7.2 +/-4.3) (Figure 3). Among the slight differences that were observed between the different types of collected tiles, notably, it was possible to identify some sequences related to Deinococcus-Thermus (also named [Thermi])

from both T2 and T3 tiles (8% of the OTUs). In contrast, only 0.5% of the OTUs were identified as Deinococcus-Thermus from T1 tiles.

Diversity of bacterial species identified on new (unexposed) tiles.

Our results show that the unexposed new tiles were colonized by large consortia of phylogenetically diverse bacteria, with 97% 16s rRNA gene similarity. A total of 145 OTUs (97% of 16s rRNA gene similarity) were identified on T1 tiles, 294 OTUs on T2 tiles and 290

OTUs on T3 tiles. Very few OTUs represented more than 1% of the total number of sequences. In fact, only 19 clustered OTU sequences represented more than 1% of the sequences for T1 tiles, 17 OTUs for T2 tiles and 20 OTUs for T3 tiles (Figure 4). Nevertheless, these few OTUs represented a large proportion of the bacterial diversity present on unexposed tiles: 70% of the bacteria identified on T1 tiles, 55% on T2 tiles and 68% on T3 tiles.

Only 8 OTUs belonging to 3 different phyla were common to the 3 types of tiles. These OTUs (Gammaproteobacteria). Notably, these 8 OTUs represented a large proportion of the bacterial diversity on the colonized tiles, constituting between 26% and 41% of the bacterial diversity identified on the 3 types of tiles.

Diversity of bacterial phyla and species identified by Illumina-based high-throughput

sequencing on exposed tiles.

Diversity of the bacterial phyla identified on tiles exposed to the outdoors.

The exposed tiles also showed colonization by similar major bacterial phyla (Figure 3) despite exposures at different sites and under different climatic conditions (Table 1). Site n°1 is located in an area of oceanic climate with reduce marine influences. This site is very humid. Site n°2 is located in an area of semi-continental climate, a less humid area but with a lot of fog in winter and heavy rainfall in spring (the seasons during which the tiles were exposed) (Table 1). All the exposed tiles were mainly colonized by Proteobacteria (between 45 and 57% of all OTUs (97% of 16s rRNA gene similarity) (on average 51 +/-5.8), Actinobacteria (between 6 and 14%, on average 11 +/-4.3) and Bacteroidetes (between 8 and 13%, on average 8.7 +/-3.4). After exposure, the T1 tiles showed a much lower percentage of Firmicutes than the unexposed tiles (9% versus 0.2%). The T1 tiles also exhibited significant colonization by cyanobacteria (7.5% of total OTUs), whereas these bacteria constituted only between 0.3 and 1.1% of all the OTUs observed on the T2 and T3

tiles. Despite differences in exposure conditions, compositions and colors, the tiles appear to be colonized by very similar bacterial phyla. Alphaproteobacteria was present in abundance (approximately 50% of the Proteobacteria) on all the types of tiles. However, this proportion varied slightly from tile to tile (on average 30% +/-7). The abundance of Gammaproteobacteria did not vary much (on average 21% +/-6). Deltaproteobacteria were present at a very low percentage (between 0.2 and 0.7%, on average 0.4% +/-0.3).

Diversity of bacterial species identified on tiles exposed to the outdoors.

Similar to unexposed tiles, the short-term exposed tiles were also colonized by large bacterial consortia. On T1 tiles, 201 OTUs (97% of 16s rRNA gene similarity) were identified, 323 OTUs on T2 tiles and 364 OTUs on T3 tiles. Of all these OTUs identified, only a few constituted more than 1% of the total sequences: 17 OTUs on T1 tiles, 17 on T2 tiles, and 20 on T3 tiles (Figure 5). However, as seen before exposure to the outdoors, these few OTUs represented a large proportion of the diversity, constituting 82% of the total diversity present on T1 tiles, 52% on T2 tiles and 48% on T3 tiles. Among these abundant OTUs, Hymenobacter sp. (Bacteroidetes) and Sphingomonas sp. (Alphaproteobacteria) were common to all the types of tiles. Furthermore, a substantial proportion of Cyanobacteria on T1 tiles was reported (Figure 5A), comprising exclusively OTUs affiliated with Chroococcidiopsaceae (also known as Chroococcidiopsidaceae).

Comparison of bacterial diversity between unexposed and exposed tiles.

Alpha diversity indexes (Chao1, Shannon and observed richness) were calculated for the different types of tiles and exposure conditions studied (Table 4). Bacterial diversity was confirmed to be high on all the types of tiles studied and under all exposure conditions. The total observed richness was between 150 and 353 (on average 261 +/-80); the Chao1 index was between 265 and 423 (on average 343 +/-68); and the Shannon index (H) was between 5.4 and 7.2 (average: 6.1 +/-1.0) (Table 4). However, no significant difference could be shown between any type of diversity index and any type of tile or exposure condition (nonparametric t-test using Monte Carlo permutations to calculate the p-value; p-value > 0.05). However, Bray-Curtis based nMDS (Non-metric multidimensional scaling) analysis of our different samples showed two distinct groups: tiles exposed 0 and 5 months on one side and tiles exposed 8 months on the other (Supplementary Figure S3). In the long term, the bacterial diversity on tiles could therefore change.

When considering the total observed diversity in our samples, we identified 452 different OTUs (97% of 16s rRNA gene similarity). A large fraction of this diversity did not vary substantially from sample to sample. In fact, a total of 181 OTUs were common to all sample types examined in this study, representing 40% of all the observed OTUs (Figure 6). New unexposed tiles shared 92% of their OTUs with exposed tiles (after 5 months and 8 months).

Unexposed T1 tiles presented 145 identifiable OTUs, while a total of 201 OTUs were identified on exposed tiles (Figure 7A). Among these OTUs, 121 were shared between unexposed and exposed tiles, representing a large proportion of the total diversity. In fact, unexposed T1 tiles shared 83% of their diversity with exposed T1 tiles. The exposed T1 tiles therefore shared 60% of their diversity with the unexposed T1 tiles. After exposure, Firmicutes, Acidobacteria and Armatimonadetes presented decreased proportions compared to the whole community, while Bacteroidetes, Cyanobacteria and Deinococcus-Thermus exhibited increased abundances, as described previously.

The unexposed T2 tiles had 294 identifiable OTUs, while 323 OTUs were identified on the exposed tiles (Figure 7B). Of these OTUs, 230 were shared between the exposed and unexposed tiles. Indeed, the unexposed T2 tiles shared 78% of their diversity with the exposed T2, and the exposed T2 tiles shared 71% of their diversity with the unexposed T2 tiles. After exposure, Acidobacteria, Actinobacteria, Bacteroidetes and Deinococcus-Thermus represented a decreased proportion of the community as a whole, while the proportion of Verrucomicrobia increased, as described above.

On unexposed T3 tiles, 290 OTUs were identified, while 364 OTUs were identified on exposed T3 tiles (Figure 7C). Of these OTUs, 248 were common to both exposed and unexposed tiles, representing a large proportion of the total diversity. In fact, the unexposed T3 tiles shared 86% of their diversity with the exposed T3 tiles. After exposure, Betaproteobacteria, Bacteroidetes and Deinococcus-Thermus represented a decreased proportion of the community as a whole, while the proportion of Actinobacteria increased.

DISCUSSION

Biological colonization of clay roof tiles is a problem for both building owners and tile manufacturers. Disruption of these biofilms constitutes an important scientific challenge and developed and voluminous patches. Combining different and complementary technical approaches, (CLSM, fluorescent macroscopy, two-photon microscopy, SEM, EDS, TTC assays, flow cytometry) we revealed that this pioneering biofilm was present and composed of viable cells. Indeed, we observed dark spots using SEM composed of carbon, as revealed by an EDS analysis. Complementary observations based on fluorescent macroscopy, confocal microscopy and two-photon microscopy highlighted the presence of DNA and of a thick matrix as dyed using green and red fluorescent compounds. An heterogeneity (in terms of fluorescence and aspect) of the observed matrix was also found. Such heterogeneity could be explained by the high diversity of colonizing bacteria and the diversity of biofilm producers, which was confirmed by testing the capacity of isolated strains to produce (or not) biofilm using a classical crystal violet assay. Nevertheless, all our data collectively depict the presence of a pioneering biofilm on new and few exposed tiles.

Our study revealed that the ceramic roof tiles were colonized by a wide diversity of heterotrophic bacteria. From a total of 452 different OTUs identified (97% of 16s rRNA gene similarity), 379 OTUs were detected and identified on unexposed tiles, and 411 OTUs were detected on tiles exposed to the outdoors for 5 or 8 months. All the major phyla were found to be colonizers of ceramic roof tiles. In addition, a high subspecific diversity was detected with a total of 3 943 ASVs or "biological variants". Moreover, the diversity indexes (Chao1, Shannon (H), observed OTUs) were quite high, even on new tiles, also revealing the importance of bacterial diversity during the first steps of clay tile biological colonization.

Interestingly, this significant bacterial diversity was detected on all the tiles studied.

Our study clearly indicates that Sphingomonas sp. and the genus 1174-901-12 related to the Beijerinckiaceae (assigned by BLAST as uncultured bacterial RNA by 16S rRNA partial sequencing; clone: B0610R003_F24 (accession number: AB657476)) were the most common OTUs found on all the types of studied tiles during the early steps of tile colonization.

Interestingly at the subspecific-level, the ASVs assigned to the genus 1174-901-12 (Beijerinckiaceae) was the most commonly found ASVs on all the types of tiles. To the best of our knowledge, the family Beijerinckiaceae has never been previously identified on tiles via culture-based methods, which was not the case for the bacterial genus Sphingomonas depicted in [START_REF] Coutinho | Biological colonization and biodeterioration of architectural ceramic materials: An overview[END_REF]. We propose here the use of these microorganisms as prokaryotic models of pioneering bacteria for the study of biofilm development on building materials. Our result provides new insights into the importance of bacterial diversity in the development of biofilms responsible for tile biodeterioration.

Interestingly, the observed total bacterial specific and subspecific diversity was relatively similar between tiles despite differences in exposure conditions (as revealed by

Table 1) and tile composition, as shown by the calculated diversity indexes. Indeed, the same phyla were detected on tiles at rather similar percentages, with a predominance of Proteobacteria, which represented approximately 50% of the bacterial diversity on each type of tile. Taxonomic analysis at the OTU level (97% of 16s rRNA gene similarity) revealed that the tiles were mainly colonized by Alphaproteobacteria, representing approximately 30% of the bacterial diversity on each tile. Also, an important pattern of tile colonization is that heterotrophic bacteria predominate over Cyanobacteria. However, cyanobacterial colonization was slightly different between tiles. Indeed, this phylum represented less than 2% of the bacterial diversity on most of the tiles studied, except on T1 tiles, where cyanobacteria represented approximately 7.5% of the total bacterial diversity. Interestingly, T1 tiles were exposed from summer (extreme conditions: high temperatures and high UV intensity, no rainfall) to winter (low UV intensity, frost), with more unfavorable growth conditions compared to T2 and T3 tiles, which were exposed from winter to spring (higher temperature, medium UV intensity, rainfall but no extreme conditions). Such observations indicated that the "green" portion of tile biodeterioration should not lead to the exclusion of the very significant diversity of non-green organisms when modeling pioneering biofilms.

Our study also provided new insights into the bacterial diversity of colonized clay tiles after only a few months of outdoor exposure. Interestingly, the T2 and T3 tiles presented almost identical colonization profiles either in OTUs and ASVs based analyses, comprising 45-55% Proteobacteria, 13-19% Firmicutes, 6-13% Actinobacteria and 6-8% Bacteroidetes. The T1 tiles, which were subjected to different exposure conditions compared to T2 and T3 tiles, presented a slightly different type of colonization profile. Even though the T1 tiles were largely colonized by Proteobacteria, similar to the T2 and T3 tiles, these tiles were colonized by Cyanobacteria and hardly colonized by Firmicutes. Interestingly, the T2 and T3 tiles were distinguished by differences in coating, i.e., with the presence and the absence, respectively, of a water-repellent cover and two different types of engobes. However, the T2 and T3 tiles were subjected to exactly the same type of outdoor exposure. Our results show that these 2 types of tiles shared 276 OTUs from a total of 323 OTUs and 364 OTUs, respectively. This result indicates that these tiles shared approximately 80% of the colonizing bacterial diversity. Additionally, the diversity indexes were not significantly different between these 2 types of tiles. Thus, the chemical composition of ceramic tiles did not appear to have an important impact on the colonizing bacterial diversity. Further sampling and comparison will have to be conducted to confirm this important observation. In addition, after exposure, the T2 and T3 tiles showed colonization by original bacteria such as SAR11 and Vibrio sp. which are aquatic bacteria. Previous studies have shown that this type of bacteria has already been found in the atmosphere, fog or rainwater [START_REF] Dueker | Coupling of fog and marine microbial content in the near-shore coastal environment[END_REF][START_REF] Gandolfi | Unravelling the bacterial diversity in the atmosphere[END_REF][START_REF] Hu | Bacteria in atmospheric waters : Detection , characteristics and implications[END_REF][START_REF] Woo | Temporal variation in airborne microbial populations and microbially-derived allergens in a tropical urban landscape[END_REF]. The presence of these original bacteria could therefore be explained by the heavy rainfall in April and May on site 2. Similarly, the presence of SAR11 on unexposed T1 and T3 tiles could be explained by its storage in a wet or rainy area.

Our cultivation approaches revealed the presence of bacteria that were previously identified in various studies. Indeed, the strains Arthrobacter agilis, Bacillus simplex, Rhodococcus corynebactroides and Rhodococcus facians, which were identified based on their 16s rRNA gene sequences, were previously isolated from the walls of Paleolithic caves [START_REF] Urzì | Microbial Diversity in Paleolithic Caves: A Study Case on the Phototrophic Biofilms of the Cave of Bats (Zuheros, Spain)[END_REF], catacombs [START_REF] Krakova | Complex bacterial diversity in the white biofilms of the Catacombs of St. Callixtus in Rome evidenced by different investigation strategies: Bacterial diversity in white biofilms[END_REF][START_REF] Urzì | Microbial Diversity in Paleolithic Caves: A Study Case on the Phototrophic Biofilms of the Cave of Bats (Zuheros, Spain)[END_REF], ceramic tiles [START_REF] Coutinho | Biological colonization and biodeterioration of architectural ceramic materials: An overview[END_REF], and bricks [START_REF] Coutinho | Biological colonization and biodeterioration of architectural ceramic materials: An overview[END_REF]. Such observations provide support and strength to our sampling methods. From a wider perspective, this knowledge of the pioneering bacterial diversity of colonized tiles provides insights for identification of new and innovative targets to combat the early biofilms that develop on clay roof tiles. The most abundant bacteria identified in this study, such as Sphingomonas sp. or members related to the Beijerinckiaceae, are known to exhibit very diverse physiological activities that could affect tile deterioration process. These bacteria are able to grow under a wide range of conditions, exhibiting growth between 15 to 55°C and at pH values between 3 and 14 [START_REF] Rosenberg | The Prokaryotes -Alphaproteobacteria and Betaproteobacteria[END_REF]. Many Sphingomonas species are known to produce exopolysaccharide compounds that are involved in biofilm formation [START_REF] Pollock | Gellan-related polysaccharides and the genus Sphingomonas[END_REF] and are also known to degrade xenobiotic and recalcitrant compounds [START_REF] Bending | Infield spatial variability in the degradation of the phenyl-urea herbicide isoproturon is the result of interactions between degradative Sphingomonas spp. and soil pH[END_REF], leading to potential resistance against some treatments [START_REF] Rosenberg | The Prokaryotes -Alphaproteobacteria and Betaproteobacteria[END_REF]. Sphingomonas species are also known to be closely associated to filamentous cyanobacteria, as they could play a role in nitrogen assimilation [START_REF] Urzì | Microbial Diversity in Paleolithic Caves: A Study Case on the Phototrophic Biofilms of the Cave of Bats (Zuheros, Spain)[END_REF]. Moreover, Beijerinckiaceae-related genera are known to produce acids that could potentially play a key role in the biodeterioration of tiles [START_REF] Rosenberg | The Prokaryotes -Alphaproteobacteria and Betaproteobacteria[END_REF][START_REF] Urzì | Microbial Diversity in Paleolithic Caves: A Study Case on the Phototrophic Biofilms of the Cave of Bats (Zuheros, Spain)[END_REF]. In addition, Staphylococcus, Streptococcus and Corynebacterium were identified among the dominant OTUs. The presence of these bacteria might be due to human manipulations (manufacture, storage, installation of tiles on roofs). Such observation suggests that human manipulation might play an important role in the formation of pioneering biofilms on tiles. Indeed, this type of bacteria has already been found on tiles with much longer exposures [START_REF] Jardak | Evaluation of biofilm-forming ability of bacterial strains isolated from the roof of an old house[END_REF].

Collectively, our results highlight the presence of a pioneer biofilm developing on new and few outdoor exposed tiles. We depict a significant diversity of the bacteria that colonize roof tiles as pioneer organisms. These results provide new insights into the biofilm settlement dynamics that are responsible for roof tile biodeterioration.

TABLES (LEGENDS)

Table 1: Environmental data collected at the sites of tiles exposition.

Table 2: Biofilm-forming capacity of isolated strains based on a crystal violet assay. (++) high biofilm-forming (+) biofilm-forming.

Table 3: Cultured bacterial strains with their identification (based on 16SrRNA gene analysis), isolation medium and origin.

Table 4: Alpha diversity estimates for the 3 types of ceramic roof tiles studied (mean ± standard deviation; number of sequences per sample rarefied to 13 618).

FIGURES (LEGENDS)

  were affiliated with Corynebacterium sp. (Actinobacteria), Staphylococcus sp. (Firmicutes), Streptococcus sp. (Firmicutes), the genus 1174-901-12 related to the Beijerinckiaceae (Alphaproteobacteria), Methylobacterium sp. (Alphaproteobacteria), Sphingomonas sp. (Alphaproteobacteria), Acidiphilum sp. (Alphaproteobacteria), and Acinetobacter sp.

Figure 1 :

 1 Figure 1: Evaluation of the presence of a pioneering biofilm on unexposed tiles: (A, B) SEM

Figure 2 :

 2 Figure 2: Quantity of extracted DNA on each tile and for each type of exposure, in picograms

Figure 3 :

 3 Figure 3: Bacterial diversity at the phylum level observed on the 3 types of ceramic roof tiles

Figure 4 :

 4 Figure 4: Bacterial diversity at the OTU level (97% of 16s rRNA gene similarity) determined by

Figure 5 :

 5 Figure 5: Bacterial diversity on exposed roof tiles at the OTU level (97% of 16s rRNA gene

Figure 6 :

 6 Figure 6: Venn diagram representing shared OTUs (97% of 16s rRNA gene similarity) as a

Figure 7 :

 7 Figure 7: Venn diagrams representing shared OTUs and rings representing bacterial diversity

  Zhang, C., Cui, F., Zeng, G.M., Jiang, M., Yang, Z.Z., Yu, Z.G., Zhu, M.Y., Shen, L.Q., 2015.Quaternary ammonium compounds (QACs): A review on occurrence, fate and toxicity in

	the	environment.	Sci.	Total	Environ.	518,	352-362.
	https://doi.org/10.1016/j.scitotenv.2015.03.007

ACKNOWLEDGMENTS

We thank the imagery core facility of BISCEm (Université de Limoges) and Platinum (Xlim institute) for CLSM imagery, fluorescent macroscopy and the two-photon microscopy. We also thank the imagery core facility of MEA (Université de Montpellier) for SEM imagery and EDS analyses. Also, we acknowledge the BIOPIC platform (Sorbonne Université, Observatoire Océanologique de Banyuls-sur-Mer), for their help in flow cytometry and for the preliminary CLSM imagery assays. The authors also thank Dr. Nyree West and the Bio2MAR platform (Sorbonne Université, Observatoire Océanologique de Banyuls-sur-Mer), for their support in Illumina-based high-throughput sequencing data analysis. This work was funded by the Sorbonne University Grant n°C17/2072-P16/0888

SUPPLEMENTARY MATERIALS (LEGENDS)

Table S1: Accession number and closest relative species corresponded to isolated and identified bacterial strains maintained in Banyuls Bacterial Culture Collection. Supplementary Figure S1: (A,B plot of all samples analyzed in this study using the DADA2 package analysis. Supplementary Figure S7: ASVs representing more than 1% of total sequences at least in one sample, splitted by phylum and type of tile.