Diogo Simas

Bernardes Dias

Lizeth Y A Jaramillo

Douglas Guedes
email: douglas.guedes.ferreira@gmail.com

Robert Duran
email: robert.duran@univ-pau.fr

Anne Carbon
email: anne.carbon@univ-pau.fr

Luiz C Bertolino
email: lcbertolino@cetem.gov.br

Ulrich Vasconcelos
email: u.vasconcelos@cbiotec.ufpb.br

Márcia T S Lutterbach
email: marcia.lutterbach@int.gov.br

Eliana F C Sérvulo
email: eliana@eq.ufrj.br

Cristiana Cravo-Laureau
email: cristiana.cravo-laureau@univ-pau.fr

Diogo S B Dias
email: diogodias@eq.ufrj.br

Assessment of acid mist on mortar biodeterioration simulating the wall of Jardim da Princesa, the National Museum of Rio de Janeiro, Brazil

Keywords: National Museum of Rio de Janeiro, bioreceptibility, biodeterioration, acid mist

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

INTRODUCTION

Ancient and modern monuments are prone to deterioration caused by variable interaction of biotic and abiotic factors. They are composed by a wide range of materials of different texture and structure, features determining their ability to resist weathering [START_REF] Miller | Bioreceptivity of building stones: A review[END_REF][START_REF] Mapelli | Mineral-microbe, interactions: biotechnological potential of bioweathering[END_REF][START_REF] Saddique | Effect of ureolytic bacteria on concrete properties[END_REF][START_REF] Sarró | Biodeterioration of the lions fountain at the Alhambra palace, Granada (Spain)[END_REF]. The occurrence of biodeterioration in historical monuments is multifactorial and not fully understood because it is a multifactorial phenomenon that affect differently according to the type of materials, the location, and the colonization by microorganisms [START_REF] Mihajlovski | Bacterial diversity on rock surface of the ruined part of a French historic monument: The Chaalis abbey[END_REF][START_REF] Perez-Alvaro | Climate change and underwater cultural heritage: Impacts and challenges[END_REF]. Anthropogenic activities accelerate the biodeterioration processes by depositing organic pollutants on material surfaces that stimulates microbial growth [START_REF] Manso | Bioreceptivity evaluation of cementitious materials designed to stimulate biological growth[END_REF][START_REF] Saiz-Jimenez | Deposition of anthropogenic compounds on monuments and their effect on airborne microorganisms[END_REF]. In addition, abiotic conditions, such as marine spray and acid rain, also favor the microbial development increasing the deterioration of materials, including modern materials such as mortar [START_REF] Traversetti | Wind-driven rain as a bioclimatic factor affecting the biological colonization at the archaeological site of Pompeii, Italy[END_REF][START_REF] Shirakawa | Climate as the most important factor determining anti-fungal biocide performance in paint films[END_REF]Mitchell & Gu, 2000).

Bioreceptibility, concept proposed by [START_REF] Guillitte | Bioreceptivity: a new concept for building ecology studies[END_REF], aims to characterize materials according to their ability to be colonized by microorganisms, providing useful information on the resistance of materials to biodeterioration. Several studies have characterized the bioreceptibility of construction materials such as stones [START_REF] Nuhoglu | In Situ Investigation of the biodeteriorative microorganisms lived on stone surfaces of the Sumela Monastery (Trabzon, Turkey)[END_REF], granite [START_REF] Vásquez-Nion | Bioreceptivity index for granitic rocks used as construction material[END_REF] or mortar [START_REF] García-Esparza | A multi-analysis characterization of medieval and vernacular coating mortars in rural Valencia (Spain): An experimental study for a Heritage Action Plan[END_REF][START_REF] Jurado | Recolonization of mortars by endolithic organisms on the walls of San Roque church in Campeche (Mexico): A case of tertiary bioreceptivity[END_REF], including both covered and uncovered surfaces [START_REF] Shirakawa | Biodeterioration of painted mortar surfaces in tropical urban and coastal situation: comparison of four paint formulations[END_REF][START_REF] Gaylarde | Biodeterioration of external architectural paint films e a review[END_REF]. Cement based materials contain organic adjuvants increasing bioreceptibility, i.e. favoring the primary colonization by microorganisms involved in the biodeterioration process (De Muynck et al., 2009). However, further studies are required in order to evaluate the parameters influencing the microbial colonization and development dynamics in cements-based materials, knowledge of paramount importance for the implementation of preservation strategies (Wiktor et al., 2011;[START_REF] Reis-Menezes | Accelerated testing of mold growth on traditional and recycled book paper[END_REF].

Regrettably, little attention has been devoted to the biodeterioration effects on Brazilian heritage monuments. Brazil, a 520-year-old country, own many historical monuments and cities that are strengthened by biodeterioration, particularly context of global changes [START_REF] Gaylarde | Weathering of granitic gneiss: A geochemical and microbiological study in the polluted sub-tropical city of Rio de Janeiro[END_REF][START_REF] Shirakawa | Inhibition of Cladosporium growth on gypsum panels treated[END_REF][START_REF] Lutterbach | A berlinda de aparato do imperador D. Pedro II: identificação de fungos em partes selecionadas e sua relação com biodeterioração e aerobiologia[END_REF]. The present work aimed to evaluate the biodeterioration process on material similar to that found in the walls of the Jardim da Princesa, located in the National Museum of Rio de Janeiro (NM). In a perspective of preservation, the study examined the effect of acid mist on microbial colonization of mortar coated and uncoated coupons. We developed an experimental microbial ecology approach combining elemental and chemical analysis, imagery and biology tools, in order to follow microbial colonization and the effects on the construction materials. The gained information will be useful for the implementation of countermeasure for the preservation of Brazilian cultural heritage monuments.

MATERIALS AND METHODS

Site of study

The National Museum (NM), located in the Quinta da Boa Vista, São Cristóvão neighborhood, (22º 90 '57 "S, 43º 22' 65" W) is the oldest scientific institution in Brazil and the largest museum of natural history and anthropology in Latin America. It was created by King D. João, the 6 th on June 6, 1818, serving as Residence of the Brazilian Imperial Family until 1889. The NM is located in an urban area, surrounded by heavy vehicle traffic. The place is well wooded, however, characterizing a zone that is usually very humid (60-70%).

Biomass collection

The samples were taken from two equidistant points on the northern wall of the Jardim da Princesa in the NM, by means of scraping, with a sterile scalpel (Medbisturi 23) and placed in penicillin bottle with 50 ml reducing solution, composed by NaCl 0.85 g/L, ascorbic acid 0.132 g/L and 4 mL of rezasurin solution 0.0025 g/L [START_REF] Dias | Sessile aerobic microbiota from the wall of the National Museum, Brazil: characterization and quantification[END_REF].

Quantification of seven microbial groups were carried out as follows (Table S1): aerobic and facultative (not "strict anaerobe") heterotrophic bacteria, aerobic and facultative acid producing bacteria, iron-oxidizing bacteria, filamentous fungi, and photosynthetic microorganisms, using spread plate and Most-Probable-Number (MPN) techniques (Genhardt et al., 1994). Significant differences between conditions were determined by analysis of variance (ANOVA).

Biodeterioration experiments

The assay was carried out in a glass reactor (Figure 1), measuring 60 cm wide x 30 cm deep x 40 cm high, was fitted with an artificial lighting system with two 60W tubular fluorescent lamps (Famastil). Fifty-six coupons similar to the composition of mortar found in the wall of the NM were prepared with the mixture of sand (20 g), cement (30 g; commercial cement composed by tricalcium and dicalcium silicate, 10-70%; calcium iron-aluminate, 5-15%; calcium sulfate, 2-10%; tricalcium aluminate, 1-15%; calcium carbonate, 0-5%; magnesium oxide, 0-4%; calcium oxide, 0-0.2%), clay (5 g) and distilled water (13 mL). The coupons were molded into rectangles (4.0 cm long x 2.0 cm wide x 1.0 cm high) and cylinders (1.5 cm in diameter x 1.0 cm in height). Half of the coupons were coated with commercial acrylic paint (Coral, Rio de Janeiro, Brazil).

Biofilm development on the coupons was investigated in triplicate over 100 days (28°C±3°C and humidity ranged 30-50%), under 8 conditions (Table S2, The inocula (50 mL) corresponded of a consortium prepared by mixing equal volumes of suspensions of five isolated groups from the wall samples: aerobic heterotrophic bacteria (10 10 CFU/mL), anaerobic heterotrophic bacteria (10 8 cells/mL), iron-oxidizing bacteria (10 8 CFU/mL), total fungi (10 6 CFU/mL) and photosynthetic organisms (10 5 Cells/mL). The consortium was inoculated on the coupon surface at a rate of 0.25 mL/cm 2 in 3 applications at day 0, 7 and 14.

In order to monitor the climatic conditions of the system, a hygrometer (INCOTERM ® ) was used, and a humidifier (Multitoc Health) coupled with a thermostat (ATMAN, AT-100) were used to humidify the system. In order to simulate the composition of rainfall in the city of Rio de Janeiro, an acid mist was prepared containing: NaNO3 (1607 μg/L), CaSO4.2H2O (5281 μg/L), K2SO4 (1570 μg/L), NH4Cl (1118 μg/L), NaCl (3680 μg/L), NaOH (212 μg/L), pH 5.12 [START_REF] Hernández | Chemical characterization and anticorrosion properties of corrosion products formed on pure copper in synthetic rainwater of Rio de Janeiro and São Paulo[END_REF]. The control mist was made with sterilized distilled water, pH = 6,0 ± 0,5.

Microbial quantification was carried out in the one hundred day of incubation. Aerobic heterotrophic bacteria, iron-oxidizing bacteria and total fungi were determined using spread plate technique and photosynthetic microorganisms, facultative heterotrophic bacteria, aerobic and facultative acid producing bacteria were estimated using MPN technique.

2.4 Composition and structure of materials by X-Ray Fluorescence (XRF) and Scanning Electron Microscopy (SEM)

The oxides composition of the wall and coupons were determined by X-ray fluorescence (XRF) spectrophotometry (WDS, AXIOS Panalytical). Both samples, from wall and coupons were prepared in a VANEOX automatic press (20 mm mold, P = 20 ton and t = 30s), using boric acid 1:0.5 (2 g of the sample to 1.0 g of boric acid) as binder. The results are semi-quantitative expressed in %, calculated as 100% standardized oxides. The determination of the loss by calcination (LBC) of the samples was done in Leco TGA-701 equipment. First heating ramp 10°C/min at 25-107°C (for moisture removal), second ramp 40°C/min at 107-1000°C (for LBC determination). The test was completed after 3 identical weighing sequences. The measurement was performed under normal air conditions.

The images of the coupons were performed by Scanning Electron Microscopy (SEM, TM3030 PLUS). The samples had been previously metallized with gold/carbon with argon as the entrainment gas, using BAL-TEC (SCD 005, Sputter Coater), with a 30 mA current for 150 seconds under pressure between 10 1 -10 2 Bar.

Microbial molecular analysis

DNA extraction

Biofilm samples for molecular analysis were collected from coupons, using sterile scalpel, and suspended in 50 mL sterile reducing solution (NaCl 0.85 g/L, ascorbic acid 0.132 g/L and 4 mL/L of rezasurin solution at 0.0025 g/L) in Penicillin bottles. 10 mL of solution was filtered through a membrane (0.2 µm Supor® Membrane, Microfunnel Filter Unit, Life Sciences). Membranes were cut before DNA extraction using UltraClean Soil DNA Isolation Kit (MOBIO) according to the manufcturer's instructions.

DNA amplification, purification and Terminal Restriction Fragment Lenght

Polymorphism (T-RFLP) analyses 16S rRNA genes were amplified using Bacteria primers 63F (5′-CAGGCCTAACACATGCAAGTC-3′) and 1387R (5′-GGGCGGWGTGTACAAGGC-3′) [START_REF] Marchesi | Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA[END_REF]. 18S rRNA genes were amplified using universal eukaryotic primers Euk1 (5′-CTGGTTGATCCTGCCAG-3′) and Euk516R (5′-ACCAGACTTGCCCTCC-3′) [START_REF] Sogin | Structural diversity of eukaryotic small subunit ribosomal RNAs: evolutionary implications[END_REF]. The reaction mix (25 µL final volume) contained 12.5 µL of AmpliTaq Gold 360 Master Mix 2x (Life Technologies), 0.2 µM of each primer and 5 µL of DNA template. PCR conditions were as follows: initial denaturation (95°C for 10 min) followed by 35 cycles of denaturation (95°C for 30 s), annealing (58°C for 45 s for Bacteria 16S rRNA; 56°C for 45 s for 18S

rRNA gene) and extension (72°C for 1 min). PCR products were purified using an Illustra GFX 96 PCR Purification Kit (GE Healthcare) and digested with 3 U of Alu1 and Rsa1 (New England Biolabs) for 16S rRNA and with 3 U of Alu1 and HaeIII (New England Biolabs) for 18S rRNA, in a total volume of 10 μL at 37 °C for 3 h. T-RFLP analysis was performed as previously described (Stauffert et al., 2009). Datasets were constructed using a minimum peak height of 100 fluorescence units. T-RFLP profiles were aligned by identifying and grouping homologous fragments and normalized by calculating relative abundances of each terminal restriction fragment (T-RFs) from height fluorescence intensity, using T-REX software [START_REF] Culman | T-REX: software for the processing and analysis of T-RFLP data[END_REF].

In order to compare bacterial and eukaryotic communities (T-RFLP patterns data)

according to treatment and time, two-dimension nonmetric multidimensional scaling ordination (NMDS) based on Bray-Curtis distances was performed using Primer 6 software (Primer E, Plymouth, UK software). The significant differences between groups were tested with an analysis of similarities (ANOSIM) and similarity percentages test (SIMPER) performed with Primer 6.

High-throughput sequencing and data analysis

The bacterial and eukaryotic diversity of 36 samples was analyzed by Illumina MiSeq sequencing. MiSeq sequencing was performed at the GeT platform (Toulouse, France).

The primers used were 104F (GGCGVACGGGTGAGTAA) [START_REF] Wang | Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies[END_REF] and 530R (CCGCNGCNGCTGGCAC) [START_REF] Weisburg | 16S ribosomal DNA amplification for phylogenetic study[END_REF] for amplification of 16S rRNA gene, and EUK1A (CTGGTTGATCCTGCCAG) and 516R

(ACCAGACTTGCCCTCC) [START_REF] Sogin | Structural diversity of eukaryotic small subunit ribosomal RNAs: evolutionary implications[END_REF] for amplification of 18S rRNA gene. PCR conditions included10-min heating step at 95 °C followed by 30 cycles of 95°C -30 s, 568°C -30 s, 72°C -45 s, and final extension at 72°C for 10 min for Bacteria and 10 min heating step at 95°C followed by 35 Cycles of 95°C -45 s, 56°C -45 s, 72°C -1 min, and final extension at 72°C for 10 min for Eukaryote.

QIIME2 software (Quantitative Insights Into Microbial Ecology) was used to analyze gene sequence reading of 16S rRNA and 18S rRNA (Bolyen et al., 2018). The taxonomy assignment (97% similarity) was performed comparing reference sequences to SILVA (v132) database of known 16S and 18S rRNA genes [START_REF] Quast | The SILVA ribosomal RNA gene database project: improved data processing and web-based tools[END_REF]. The alpha-diversity analysis (Shannon-Weaver, Simpson, Pielou's eveness and Good's coverage) was performed on rarefied count data using the apha_diversity.py script in Qiime2. Differential analysis and barplots were performed with SHAMAN (SHiny application for Metagenomic Analysis, shaman.c3bi.pasteur.fr) [START_REF] Quereda | Bacteriocin from epidemic Listeria strains alters the host intestinal microbiota to favor infection[END_REF].

The complete dataset was deposited in the NCBI Sequence Read Archive (SRA) database (SUB7892718). It is available under the Bioproject ID PRJNA655939.

RESULTS

Characterization of coupons and wall

The composition of coupons was similar to that of the National museum (NM) wall, with the same composition of nine oxides (Table S3), with only a difference in the percentage of Al2O3.

Characterization of biodeterioration

The impact of biodeterioration on the surface of the coupons and the formed biofilm were characterized by SEM observations after 100 days of incubation (Figure 2). The coated surfaces (conditions 2-CCW, and 4-CCA) showed smoother surfaces than uncoated conditions (conditions 1-CUW and 3-CUA), which presented roughness and deposits of inorganic and organic matter, dust and other debris (Figure 2). Noteworthy, hyphae were observed in uncoated condition treated with acid mist (condition 7-MUA) associated with the greenish and orange color as shown in Figure 3 and observed by optical microscopy (data not shown), probably due to the development of photosynthetic microorganisms and by the activity of iron-oxidizing bacteria, respectively.

Biofilms of coated conditions were less dense compared to the uncoated coupons (conditions 5-MUW and 7-MUA). In addition, the coupons in condition 5 (MUW)

showed the bulkier structure and less adherent than coupons in condition 7 (MUA). The presence of microorganisms altered the porosity of the material, as revealed by grooves observed in the surface of coupons. The coated coupons submitted to water mist (condition 6-MCW) presented depigmentation, despite the lower microbial development. Cracks were observed in the surface of coated coupons (conditions 6-MCW and 8-MCA), suggesting microbial activity.

After scraping off the biofilms, coated coupons presented more visual alterations than uncoated coupons, with numerous spots over all the surface. In addition, whitening was observed in the coupons exposed to acid mist (condition 8-MCA), suggesting growth of fungal mycelium and/or presence of iron-oxidizing bacteria. On the other hand, the color of the paint changed to bright yellow, quite different from the coupons not exposed to the acid mist (condition 6-MCW).

The description of deterioration aspects and the quantification of microbial groups are summarized in Table 1. Under all inoculated tested conditions, heterotrophic bacteria were dominant, with a prevalence of aerobic communities. Also, the numbers of aerobic acid producing bacteria were similar under all conditions, with values around 10 2 cells/cm 2 . The largest biomass was observed on the uncoated coupons (conditions 5-MUW and 7-MUA). The paint was not inhibiting microbial growth, except that of the facultative acid-producing and photosynthetic microorganisms (conditions 6-MCW and 8-MCA) for which the number of cells/cm 2 was reduced by at least 10 3 units (cells/cm 2 ) in comparison to the uncoated coupons (ANOVA, p-value < 0.001) irrespective of the atmospheric treatment (Table 1, conditions 5-MUW and 7-MUA). The presence of coating (condition 6-MCW) inhibited also the development of the iron-oxidizing bacteria (10 3 cells/cm 2 , Table 1) (ANOVA, p-value < 0.001) and the total fungi (10 3 CFU/cm 2 ) (ANOVA, p-value < 0.001) but the inhibition effect was suppressed in presence of acid mist (condition 8-MCA).

Microbial communities structure and composition

Bacterial and eukaryotic community structures were monitored by T-RFLP based on 16S and 18S rRNA genes analyses for all incubation conditions at the end of the experiment (Figure 4). The T-RFLP analysis revealed that the bacterial communities were structured according to the incubation condition after 100 days of incubation (Figure 4A), the impact of coating (ANOSIM R= 0.833) on bacterial community structure was more important than the acid mist effect (R=0.289). The eukaryotic community structure (Figure 4B) was not affected by the acid mist (R=0.130), whereas the impact of coating by paint (R=0.389) has been observed. These results suggest that paint coating contributed to the selection of specific microbial species resulting in the dominance of some microbial groups.

In order to determine the impact of coating and acid mist on the microbial assemblages in the biofilms developed in coupons, bacterial and fungal communities composition were characterized by high throughput sequencing of 16S and 18S rRNA genes respectively. For bacterial community analysis, a total of 485567 16S rRNA gene reads were obtained for the 12 samples. The obtained valid reads after preprocess filtering and normalization were reduced to 6983 sequences for each sample, clustered into 126 amplicon sequence variants (ASVs). For eukaryotic community, 267103 18S rRNA gene reads were obtained. After filtering and normalization, and focusing only in Fungi, 4599 sequences were obtained for each sample, clustered into 23 ASVs. Good's coverage values, nearly 100% in each sample, indicated that the main bacterial and fungal diversity were assessed.

The alpha diversity indexes [Shannon (H'), Simpson (D) and equitability] varied according to the treatment for bacterial and fungal communities (Table 4). H' and D were higher for uncoated conditions, particularly when submitted to acid mist.

Furthermore, microbial communities from coated conditions were dominated by some microorganisms, as indicated by equitability index (Pielou's eveness).

The dataset consisted of 5 distinct bacterial phyla: Actinobacteria, Chloroflexi, Cyanobacteria, Firmicutes and Proteobacteria. Among the most abundant genus in uncoated coupons with water mist (condition 5-MUW, Figure 5A), Microbacterium was the most abundant (comprising 40.1 % of the classified community), followed by Brevundimonas (34.9 %), Roseomonas (10.6 %), Skermanella (6.5 %) and Methylobacterium (4.5 %). In uncoated coupons submitted to acid mist (condition 7-MUA) a modification of the bacterial community composition was observed (Fig. 5A)

showing the dominance of Cyanobacteria belonging to Leptolyngbya (38.8 %) and Nostoc (5.4 %) genus. A decrease of the abundance of Microbacterium (6.7 %) and the detection of Micromonospora (7.6 %), both belonging to Actinobacteria, was also observed. Proteobacteria dominated the bacterial communities of coated coupons (conditions 7-MUA and 8-MCA), with Methylobacterium (Fig. 5A) as the most abundant genus in both water and acid mist conditions (conditions 7-MUA and 8-MCA, 81.2 % and 91.1 % respectively).

Fungal communities were represented by two phyla, Ascomycota and Basidiomycota.

Uncoated coupons (conditions 5-MUW and 7-MUA) were dominated by the class Tremellomycetes (68.8 % and 69.8 % respectively) followed by Sordariomycetes (31.2 % and 30.1 % respectively) irrespective of the mist condition (Figure 5B). Coated coupons (conditions 6-MCW and 8-MCA, Figure 5B) were dominated by Sordariomycetes (97.3 % and 89.4 % respectively). At the ASVs level, for the two fungal dominant classes (Figure 5C), data analysis showed that conditions (coat and mist) had an impact on the distribution of ASVs abundances but not on the composition.

DISCUSSION

Biodeterioration of mortar

The composition of coupons was overall similar to that of the National museum (NM)

wall supporting that coupons can be used to simulate NM wall. However, a difference in the aluminium oxide content was observed. Aluminium toxic effects on microorganisms has been showed (Piña & Cerventes, 1996) as well as tolerance capacities [START_REF] Piotrowska-Seget | Metal-Tolerant Bacteria Occurring in Heavily Polluted Soil and Mine Spoil[END_REF][START_REF] Ahmed | Destruction of Cell Topography, Morphology, Membrane, Inhibition of Respiration, Biofilm Formation, and Bioactive Molecule Production by Nanoparticles of Ag, ZnO, CuO, TiO2, and Al2O3 toward Beneficial Soil Bacteria[END_REF]. The concentration of Al2O3 observed in the NM wall (400 ppm) can be considered as high and inhibitory for microbial growth (Piña & Cerventes, 1996). This suggests that microorganisms colonizing the wall are tolerant and able to be adapted and grow in the coupons, result obtained in the present study support this hypothesis. Moreover, calcination loss showed differences, probably due to the presence of organic matter on the wall from the paint, and/or microorganisms, exopolysaccharides and other particles deposited by wind and rain [START_REF] Hartmann | Distinct soil microbial diversity under long-term organic and conventional farming[END_REF].

The impact of biodeterioration of mortar samples was greater on the surface in uncoated coupons than on the coated coupons. The uncoated coupons were more susceptible to bioreceptibility due to their structural features that favor microbial development as previously described [START_REF] Silva | Cobertura antifúngica à base de nanopartículas contendo piocianina[END_REF][START_REF] Viana | Pseudomonas aeruginosa and pyocyanin act negatively on the establishment of Enterobacteriacea biofilm[END_REF].

In addition, the uncoated coupons also showed thicker biofilm and microbial morphological diversity, showing heterogeneous development, compared to the coated coupons, suggesting the influence of paint on microbial growth and biofilm development, without complete inhibition. These observations are in agreement with several studies showing that external painted surfaces support a diverse microbial population (for review, see [START_REF] Gaylarde | Biodeterioration of external architectural paint films e a review[END_REF]. Furthermore, a previous study showed that microbial activity had promoted material surface deterioration, resulting in an increase in the porosity and a decrease in its resistance and subsequent crumbling [START_REF] Papida | Enhancement of physical weathering of building stones by microbial populations[END_REF], as well as desquamation was reported as result of volume change, matrix penetration and release of microbial metabolites such as organic and inorganic acids [START_REF] Coutinho | Microbial communities on deteriorated artistic tiles from Pena National Palace (Sintra, Portugal)[END_REF].

The positive effect of acid mist on the biofilm adhesion has been observed irrespective of the presence of paint, suggesting that acid mist promote microbial growth and consequently, material biodeterioration. These observations corroborate the hypothesis that acid mist causes more damage to the material than humidity caused by the water mist, because it keeps the surface moister beyond the acids act on surface deterioration.

These observations are in accordance with a study that demonstrated the impact of atmospheric conditions on the degree of material deterioration [START_REF] Gil | Indoor atmospheric corrosion of electronic materials in tropical-mountain environments[END_REF].

Additionally, microbial influence was greater than that of the atmosphere in the process of surface deterioration, due to production of acids and other metabolites [START_REF] Pitzurra | Microbial growth and air pollution in carbonate rock weathering[END_REF].

The results of microbial quantification are consistent with the SEM observations that showed a larger biofilm development on uncoated coupons. It has been reported that the paint on materials (coated) impact in microbial growth [START_REF] Gaylarde | Biodeterioration of external architectural paint films e a review[END_REF]. Most of microorganisms in our study have not been totally inhibited, in the exception of photosynthetic microorganisms (condition 6-MCW). This observation was in contradiction with several studies that reported the presence of Cyanobacteria on painted surface, particularly in tropical and subtropical countries [START_REF] Shirakawa | Biodeterioration of painted mortar surfaces in tropical urban and coastal situation: comparison of four paint formulations[END_REF][START_REF] Gaylarde | Algae and cyanobacteria on painted surfaces in Southern Brazil[END_REF]. It was also reported that fungi constitute the major biomass on painted surfaces in Latin America [START_REF] Gaylarde | A comparative study of the major microbial biomass of biofilms on exteriors of buildings in Europe and Latin America[END_REF].

It is important to highlight that the presence of acrylic paint (condition 6-MCW) also inhibited the development of two groups: iron-oxidizing bacteria and the total fungi and the inhibition effect was suppressed in the presence of acid mist (condition 8-MCA).

Overall, the acid mist has favored microbial development, especially on the coated coupons (Table 1, condition 5-MUW vs condition 7-MUA and condition 6-MCW vs condition 8-MCA). The same observation was previously demonstrated by some studies [START_REF] Flemming | Relevance of microbial extracellular polymeric substances (EPSs)--Part I: Structural and ecological aspects[END_REF], [START_REF] Costerton | Microbial biofilms[END_REF].

Microbial communities structure and composition

The culture-based approach used in the present study allow to obtain diverse microbial communities in the different conditions. The composition of these microbial communities is representative of the microbial community observed in situ (data not shown). Indeed, the in situ community was mainly dominated by Cyanobacteria (46%, relative abundance), Proteobacteria (20%, relative abundance) and Actinobacteria (17%, relative abundance), also observed as main bacterial phyla in coupons from the biodeterioration experiment. The fungal in situ community was dominated by the phylum Basidiomycota, as observed in coated coupons. The comparison of environmental and culture-derived microbial communities showed that the diversity of culture-derived communities reflected selectivity of the culture media and/or the culture condition, as previously described [START_REF] Pédron | Comparison of environmental and culturederived bacterial communities through 16S Metabarcoding: a powerful tool to assess media selectivity and detect rare taxa[END_REF].

The five phyla observed in the present study have been found dominant in recent works evaluating the biodeterioration of cultural heritage monuments (Lepinay et al., 2018;[START_REF] Adamiak | First evaluation of the microbiome of built cultural heritage by using the Ion Torrent next generation sequencing platform[END_REF][START_REF] Chimienti | Profile of microbial communities on carbonate stones of the medieval church of San Leonardo di Siponto (Italy) by Illumina-based deep sequencing[END_REF]. Among these phyla, Actinobacteria have been identified as dominant in biofilms of historic monuments [START_REF] Adamiak | First evaluation of the microbiome of built cultural heritage by using the Ion Torrent next generation sequencing platform[END_REF][START_REF] Mihajlovski | Bacterial diversity on rock surface of the ruined part of a French historic monument: The Chaalis abbey[END_REF]. It is well known that they inhabit more effectively stone and mural paintings, by their ability to use various nitrogen and carbon sources [START_REF] Sterflinger | Bio-susceptibility of materials and thermal insulation systems used for historical buildings[END_REF]. Surprisingly, in the present study these microorganisms were impacted by the acid mist. In fact, these microorganisms are known to play a crucial role in the decomposition of organic compounds and environmental pollutants, and thus expected to resist and adapt to the acid mist. It is a premise that the atmospheric condition inhibits both the size and diversity of the biofilm microbial community (Mitchell & Gu, 2000).

The acid mist promoted the development of Cyanobacteria on uncoated coupons.

Cyanobacteria are known to support the growth of heterotrophic bacteria and fungi by being a nutrient source, thus constituting a key element in the biofilm formation and maintenance. Cyanobacteria have been described to be involved in biodeterioration of cultural heritage monuments [START_REF] Dyda | Application of metagenomic methods for selection of an optimal growth medium for bacterial diversity analysis of microbiocenoses on historical stone surfaces[END_REF][START_REF] Herrera | The importance of atmospheric effects on biodeterioration of cultural heritage constructional materials[END_REF] and to be dominant in biofilms in different climatic sites [START_REF] Gaylarde | A comparative study of the major microbial biomass of biofilms on exteriors of buildings in Europe and Latin America[END_REF].

Additionally, Proteobacteria dominated the bacterial communities of coated coupons, indicating their resistance to coating and supporting their implication on material biodeterioration (Lepinay et al., 2018;[START_REF] Mihajlovski | Bacterial diversity on rock surface of the ruined part of a French historic monument: The Chaalis abbey[END_REF][START_REF] Jurado | Recolonization of mortars by endolithic organisms on the walls of San Roque church in Campeche (Mexico): A case of tertiary bioreceptivity[END_REF].

Concerning fungal diversity, the uncoated coupons were dominated by

Tremellomycetes and Sordariomycetes, whereas coated coupons were dominated by Sordariomycetes. These observations suggested that fungi affiliated to this class are adapted to the presence of paint. Our results also support the impact of paint and acid mist in the fungal community composition (e.g. TRFLP analysis, Figure 4B) showing stronger impact of paint than acid mist in the fungal community composition. Fungi affiliated to the Sordariomycetes class have been identified as common colonizers of paint biofilms in Brazil [START_REF] Shirakawa | Climate as the most important factor determining anti-fungal biocide performance in paint films[END_REF][START_REF] Shirakawa | Fungal colonization and succession on newly painted buildings and the effect of biocide[END_REF] and recently identified in mortar coatings of a historical building [START_REF] Guerra | Biodeterioration of mortar coating in historical buildings: Microclimatic Characterization, material, and fungal community[END_REF]. Furthermore, they have been described as involved in concrete deterioration [START_REF] Wei | A yeast-based rapid prototype platform for gene control elements in mammalian cells[END_REF].

The use of paint reduced the fungal population. Some antimicrobial additives found in paints, such as titanium dioxide (Amorim et al., 2020), zinc pyrithione [START_REF] Reeder | Zinc pyrithione inhibits yeast growth through copper influx and inactivation of iron-sulfur proteins[END_REF], ammonium-aqueous solution, ethane 1,2-diol and kaolin (Chukwujike and Igwe, 2019) act to prevent fungal adsorption and consequently avoid the establishment of biofilm. The paint used in the present work, one of the most sold in Brazil, is composed by antifungal, antialgal and antibacterial additives, according to manufacturers.

CONCLUSIONS

Our results indicated that microbial deterioration will always occur, even at different degrees, irrespective of the coating and moisture condition. The experimental approach applied in the present work is proposed as a strategy to address bioreceptibility. This approach has the advantage to be less time-consuming than in situ studies, not destructive, using lab controlled systems with the possibility of extrapolation to the real ecosystem.

Wiktor, V., Grosseau, Ph., Guyonnet, R In situ -2.0 ± 0.3 x 10 4 1.0 ± 0.1 x 10 3 1.0 ± 0.2 x 10 3 5.0 ± 0.1 x 10 4 5.0 ± 0.1 x 10 2 5.0 ± 0.2 x 10 3 1.0 ± 0.1 x 10 3

Inoculum -1.0 ± 0.1 x 10 10 -9.0 ± 0.2 x 10 7 1.0 ± 0.1 x 10 8 -2.0 ± 0.1 x 10 6 6.0 ± 0.1

x 10 4 1-CUW Not significant --- --- --- --- --- --- --- 2-CCW Not significant --- --- --- --- --- --- --- 3-CUA Not significant --- --- --- --- --- --- --- 4-CCA Not significant --- --- --- --- --- --- ---

5-MUW

Soft bulkier structure and robust biofilm 5.7 ± 0.2 x 10 6 1.8 ± 0.1 x 10 2 6.0 ± 0.3 x 10 4 1.9 ± 0.1 x 10 6 1.4 ± 0.1 x 10 4 1.6 ± 0.1 x 10 6 9.4 ± 0.5 x 10 4 6-MCW Presence of cracks on surface coating, less biofilm and depigmentation 4.8 ± 0.2 x 10 4 7.5 ± 0.4 x 10 1 3.5 ± 0.3 x 10 3 2.5 ± 0.1 x 10 3 5.0 ± 0.1 x 10 -1 1.9 ± 0.1 x 10 3 NG 7-MUA Adherent bulkier structure, large number of hyphae. Robust biofilm. Greenish and orange colonies.

1.2 ± 0.1 x 10 7 1.8 ± 0.1 x 10 2 1.0 ± 0.1 x 10 5 5.6 ± 0.2 x 10 6 5.6 ±0.2 x 10 3 7.0 ± 0.4 x 10 4 5.6 ± 0.2 x 10 5 8-MCA Presence of cracks on surface coating and paint changed to bright yellow, less biofilm and whitening. Greenish and orange colonies.

3.6 ± 0.2 x 10 6 1.3 ± 0.1 x 10 2 5.1 ± 0.2 x 10 4 1.4 ± 0.1 x 10 6 3.1 ± 0.05 x 10 0 3.6 ± 0.2 x 10 5 3.1 ± 0.1 x 10 0 Ah: Aerobic heterotrophic bacteria; Ap-a: aerobic acid-producing bacteria; Ib: Iron-oxidizing bacteria; Ah-f: facultative heterotrophic bacteria;

  Condition 1-CUW: Absence of microorganism, uncoated coupons and water mist; Condition 2-CCW: Absence of microorganism, coated coupons and water mist; Condition 3-CUA: Absence of microorganism, uncoated coupons and acid mist; Condition 4-CCA: Absence of microorganism, coated coupons and acid mist; Condition 5-MUW: Presence of microorganism, uncoated coupons and water mist; Condition 6-MCW: Presence of microorganism, coated coupons and water mist; Condition 7-MUA: Presence of microorganism, uncoated coupons and acid mist; Condition 8-MCA: Presence of microorganism, coated coupons and acid mist).

Figure 1 .

 1 Figure 1. Glass reactor (A) and top view of the arrangement of the coupons (B).

Figure 2 .

 2 Figure 2. SEM image of the uninoculated (A) and inoculated (B) mortar coupons after 100 days of incubation. Conditions 2, 4, 6 and 8 represented coated mortar and 1, 3, 5 and 7 represented uncoated. Treatment with acid mist were used in conditions 3, 4, 7 and 8.

Figure 3 .

 3 Figure 3. Image of mortar coupons at the beginning of the experiment (left, uncoated; right, coated), after 100 days of incubation and after scrapping off the biofilms. Conditions 2, 4, 6 and 8 represented coated mortar and 1, 3, 5 and 7 represented uncoated. Treatment with acid mist were used in conditions 3, 4, 7 and 8.

Figure 4 .

 4 Figure 4. Bacterial (A) and Eukayotic (B) communities structures. Non-metric multidimensional scaling analysis (Bray-Curtis) based on T-RFLP profiles from 16S and 18S rRNA genes from biofilm developed in inoculated coupons under different conditions: uncoated coupons (C5 in green, condition 5-MUW), coated coupons (C6 in blue, condition 6-MCW), uncoated coupons under acid mist (C7 in cyan, condition 7-MUA) and coated coupons under acid mist (C8 in red, condition 8-MCA). The vectors connect the triplicates. The percentages indicate the average similarity (SIMPER) of samples in each group (condition).

Figure 5 .

 5 Figure 5. Relative abundance of bacterial community at the genus level (A), fungal community at the class level (B) and ASVs distribution of the two dominants classes of Fungi (Sordariomycetes and Tremellomycetes) (C) in coated (conditions 5 and 7) and uncoated (conditions 6 and 8) mortar treated with water mist (conditions 5 and 6) and acid mist (conditions 7 and 8).

Table 1 .

 1 .,Garcia-Diaz, E., Lors, C. (2011) Accelerated weathering of cementitious matrix for the development of an accelerated laboratory test Deterioration and microbial density found in coupons after 100 days, in the in situ and in the inoculum samples. The values are

	of	bio-deterioration.	Materials	and	Structures.	44,	623-40.
	https://doi.org/10.1617/s11527-010-9653-1.

Table 2 .

 2 Alpha-diversity indexes of bacterial and fungal communities from the cultured mortar treated with water mist (conditions 5 and 6) and acid mist (conditions 7 and 8) Mean values and standard deviation of triplicates of each condition. ASVs, Amplicon

	Microbial	Condition	# of ASVs	H'	D	Equitability
	group
	Bacteria	5 (uncoated)	21.00±1.70	2.68±0.60	0.75±0.14	0.61±0.15
		6 (coated)	13.00±3.60	1.13±0.25	0.36±0.09	0.31±0.05
		7 (uncoated)	42.30±9.10	3.28±0.27	0.82±0.05	0.61±0.05
		8 (coated)	14.30±5.50	1.09±1.28	0.29±0.36	0.27±0.28
	Fungi	5 (uncoated)	7.00±0.00	1.90±0.37	0.65±0.15 0.68±0.13
		6 (coated)	7.33±2.08	1.26±0.04	0.49±0.01	0.44±0.07
		7 (uncoated)	9.67±2.52	2.09±0.44	0.68±0.12	0.65±0.14
		8 (coated)	7.67±0.58	1.24±0.72	0.42±0.27	0.42±0.24

Sequence Variants; H', Shanon-Weaver's index; D, Simpson's index; Equitability, Pielou's eveness.

ACKNOWLEDGEMENTS

The English text of this paper has been revised by Sidney Pratt, Canadian, MAT (The Johns Hopkins University), RSAdip -TESL (Cambridge University).

FORMATTING OF FUNDING SOURCES

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES -Fellowship Program number 99999.008278/2014-08).

Ap-f: facultative acid-producing bacteria; F: Total fungi; Ph: Photosynthetic microorganisms. *(CFU/cm 2 ). † (MPN cells/cm 2 ); NG: no-growth.


domain: qfin spi

text:
Benoît Lécureux
email: benoit.lecureux@entpe.fr

Adrien Bonnet
email: adrien.bonnet@vedecom.fr

Ouassim Manout

Jaâfar Berrada

Louafi Bouzouina

This paper : "Acceptance of Shared Autonomous Vehicles: A Literature Review of stated choice experiments" (

Keywords: Shared Autonomous vehicles, Acceptance, Discrete choice, Mode choice, Stated preferences, Attitudes, Literature review

Automated mobility has always conveyed fantasies about its ability to meet future mobility needs and challenges. If research is still debating the when, where and how of this mobility disruption, there seems to be a consensus on its advent. Meanwhile, the investigation of the demand for this mobility is of critical importance. Stated preference surveys are a common and powerful tool to foresee this demand. This paper tackles the question of the acceptance of shared autonomous vehicles (SAVs) by reviewing most significant published papers on travel demand and travel surveys for SAVs. Given the recent developments in the last three years, the paper updates and complements previous literature reviews.

In contrast with previous research, this paper shows that the impact of various factors on the intention to use SAVs is still controversial. This includes age, gender, income or car ownership. We identify most consensual and controversial effects and correspondingly suggest future research tracks to address some the identified gaps.

Introduction

Shared autonomous vehicles (SAVs) are likely to impact existing mobility services and induce new mobility practices [START_REF] Narayanan | Shared autonomous vehicle services: A comprehensive review[END_REF]. However, 10 years after the publication of the first papers on the acceptance of autonomous vehicles (AVs), considerable uncertainty still surround the adoption and use of SAVs. In this research, we address the following question: did researchers reach a consensus on SAVs' acceptance factors? We assume that despite the abundant literature on AVs, the study of its acceptance factors is, to some degree, still speculative and a consensus on their nature and magnitude is yet to be reached. The aim of this paper is to compare most studied AV acceptance factors in the economic literature and to investigate whether a scientific consensus has been reached or not and what future research is still needed to reach a consensus.

There have been two major literature reviews on the acceptance of AVs [START_REF] Becker | Literature review on surveys investigating the acceptance of automated vehicles[END_REF][START_REF] Gkartzonikas | What have we learned? A review of stated preference and choice studies on autonomous vehicles[END_REF]). Both reviews focus on survey methodologies and explanatory variables of AV acceptance. In particular, [START_REF] Gkartzonikas | What have we learned? A review of stated preference and choice studies on autonomous vehicles[END_REF] review stated-preferences (SP) for AVs by including attitudes. Their review highlights the lack of well-established theories that account for attitudinal dimensions.

In this paper, we update the state of the art on the acceptance of autonomous mobility services by reviewing most significant papers on this subject. 65% of the reviewed papers were published after 2019, i.e. the publication year of the latest literature review on AVs' acceptance. Most importantly, this review evaluates the degree of consensus on the effect of various SAVs' acceptance factors. Unlike [START_REF] Becker | Literature review on surveys investigating the acceptance of automated vehicles[END_REF] and [START_REF] Gkartzonikas | What have we learned? A review of stated preference and choice studies on autonomous vehicles[END_REF], our review focuses on SAVs and especially on three types: (1) autonomous shuttles or buses (AS) that operate like conventional buses; (2) robo-taxis (RT); and (3) ride-sharing robo-taxis (RS RT). The focus on SAVs is motivated by the fact that AVs are most likely to be first introduced in shared mobility services [START_REF] Narayanan | Shared autonomous vehicle services: A comprehensive review[END_REF]. We also address some of the limitations identified by [START_REF] Gkartzonikas | What have we learned? A review of stated preference and choice studies on autonomous vehicles[END_REF] and discuss most used theoretical models in psychology that consider the acceptance of the AV technology.

Methodology

The literature review is based on a systematic search of relevant papers using Google Scholar, Scopus, Web of Science and a list of keywords, including "autonomous vehicles, shared autonomous vehicles, stated preferences, methodology, determinants, attitudes, acceptance" and their most common synonyms following the methodology of [START_REF] Wee | How to Write a Literature Review Paper?[END_REF]. The backward and forward snowballing strategy is used to identify most significant papers on the subject. The selection criteria are: papers published in peer-reviewed journals; papers that include, but are not limited to: autonomous buses, autonomous shuttles, robo-taxis or shared robo-taxis; papers that are empirical and that examine the travel mode choice of SAVs; papers that use the choice experiment method for their data collection. This selection resulted in 29 papers. Most of these papers were published after the last literature review by [START_REF] Gkartzonikas | What have we learned? A review of stated preference and choice studies on autonomous vehicles[END_REF].

Results

Review of survey methodologies

We analyze and classify the selected papers according to: survey administration type, survey area, survey design and the type of discrete choice model. 90% of the reviewed surveys are conducted online. The average number of respondents is 1,140 (min 106 -max 4,625). 55% of case studies are conducted in urban areas and 26% on a national level. The remaining part (19%) are difficult to classify or don't specify the survey area. Little attention has been paid to the acceptance of SAVs in rural and suburban areas. Regarding the choice experiments, SAVs are often compared to: private AVs (15 papers), private conventional cars (12), public transport (9), cycling (10), walking (9), regular taxi (1), carpooling (2) or airplane (1). Regarding the experimental design of choice experiments, the mean number of attributes per each travel alternative is 3 (min 2 -max 6). Given the novelty of the subject and the absence of reliable knowledge, 48% of the surveys rely on the straightforward orthogonal design, 29% on the Bayesian design, and 19% on the efficient design [START_REF] Walker | D-efficient or deficient? A robustness analysis of stated choice experimental designs[END_REF]. The used discrete choice models are: mixed logit (39% of papers), multinomial logit (28%), ordered models (17%), hybrid model (11%).

In the following section, we first review the influence of the most studied acceptance factors (control variables). Second, we analyze and comment on four sets of variables of interest. Finally, we discuss the specific case of psychosocial factors.

Control variables

Gender (reference male): is controlled almost systematically in the reviewed papers with 27 occurrences. The effect of gender on the intention to use the three types of SAVs is not consensual: 11 papers find a positive effect of being male, 11 papers find a non-significant effect and 5 other papers find a negative effect.

Age (reference old): is one of the most studied variables in this review with 25 papers including it. It is most often negatively associated with the intention to use RTs or RS RTs. 12 papers find a negative effect, 8 papers find no significant effect and 5 others find a positive effect of age (being old) on the intention to use RTs or RS RTs. The effect of age on the intention to use AS using choice experiments is yet to be studied.

Trip cost: is controlled in 22 papers and has a systematically negative effect on the intention to use SAVs.

Travel time: has systematically a significant negative effect on the intention to use SAVs. The only study that reports a positive effect is [START_REF] Haboucha | User preferences regarding autonomous vehicles[END_REF]. The authors find that SAVs' travel time is positively perceived by potential users due to the fact that those who make longer trips tend to favor AVs over other alternatives.

Income: is examined in 17 of the reviewed papers. 9 of these find no significant effect of income on the intention to use SAVs. 7 papers find a positive effect of high income and only 1 study shows a negative effect.

Waiting time: is controlled for in 16 studies with almost always a negative effect. Only [START_REF] Kontar | Autonomous vehicle adoption: use phase environmental implications[END_REF] did not find a significant effect of this variable on the intention to use SAVs. The authors explain this finding by the fact that the waiting times of the choice experiment were too low to influence mode choice.

Household size: is studied in 13 papers. It has often a non-significant effect (8 out of 13) on the intention to use RTs and RS RTs. In 3 and 2 papers, the size of households has a positive, negative effect, respectively.

Car ownership: car dependency has a negative impact on the intention to use the RTs or RS RTs (7/11 papers). The other 4 papers show no significant effect.

Education (ref. high education): is included in 10 out of 29 papers. It has often a positive effect, i.e. a high level of education tends to increase the intention to use SAVs (8 of 10 papers). The other 2 papers show no significant effect.

Residential location (ref. urban area): is included in 9 papers. In 2 papers this variable has a positive effect. In 4 papers, it has a negative effect. In the rest of paper (3), it has no significant effect.

Driving license (ref. own a license): is included in 9 papers. It has a non-significant effect in 5 papers and a negative effect in 3 papers and a positive one in 1 paper.

Trip purpose: is studied in 8 papers. The intention to use SAVs is higher for commuting than for leisure or shopping (4 papers out of 8). In 3 papers, no significant effect for the trip purpose is found. Only [START_REF] Guo | When and why do people choose automated buses over conventional buses? Results of a context-dependent stated choice experiment[END_REF] find a negative effect of the commuting trip purpose. This might be explained by the fact that commuters prefer modes that are reliable.

Access/egress time: are studied in only 3 papers and they systematically have a negative effect on the intention to use all forms of SAVs.

It is noteworthy that for most control variables, there is little or no data on their effect on the acceptance of autonomous shuttles. Most research focuses on the case of robot-taxis.

Factors of the intention to use SAVs

4 dimensions are often presented as key drivers of the adoption of SAVs. Attitudes, benefits of SAVs, preference for shared and flexible services and current mode choice.

Attitudes [START_REF] Becker | Literature review on surveys investigating the acceptance of automated vehicles[END_REF] and [START_REF] Gkartzonikas | What have we learned? A review of stated preference and choice studies on autonomous vehicles[END_REF] consider attitudes as key drivers of the adoption of SAVs. Four of them were identified as primary factors : environmental concerns, safety and trust in AVs, tech-savviness and passion for driving.

Environmental concerns are tested in 5 papers (out of 27). This attitude positively influences the intention to use SAVs in 80% of the cases. Pro-environment concerns positively influence the intention to use RTs in 3 out 4 papers, and RS RTs in 1 paper. [START_REF] Yap | Preferences of travellers for using automated vehicles as last mile public transport of multimodal train trips[END_REF] note that attitude toward AV sustainability is the most important attitudinal factor of the intention to use AVs as a last mile service. This finding is in contradiction with other findings showing that security concerns are the most impactful [START_REF] Nazari | Shared versus private mobility: Modeling public interest in autonomous vehicles accounting for latent attitudes[END_REF]). It should be noted that in [START_REF] Yap | Preferences of travellers for using automated vehicles as last mile public transport of multimodal train trips[END_REF], the AV sustainability latent construct is defined by only two items. For [START_REF] Webb | Will people accept shared autonomous electric vehicles? A survey before and after receipt of the costs and benefits[END_REF], the influence of the environmental attitude can be amplified if information on SAV's environmental benefits is provided. [START_REF] Haboucha | User preferences regarding autonomous vehicles[END_REF] find a contextual effect of the environmental attitude on the use of AVs. The authors reveal that the effect of environmental attitudes on SAVs' acceptance is significant in Israel, but not in the USA. [START_REF] Nazari | Shared versus private mobility: Modeling public interest in autonomous vehicles accounting for latent attitudes[END_REF] find a correlation between green travel patterns and socio-demographic variables. This correlation is positive for young men with high education and negative for rich people and those with children.

Security/safety concerns and trust are studied in a total of 5 papers each. Safety concerns influence negatively the intention to use SAVs in 100% of the cases, while trust in AVs influences it positively. As regards the different types of SAVs, safety concerns are studied in the case of RTs, RS RTs and AS in 2, 2 and 1 paper out 5, respectively. Trust in AVs is tested for RTs, RS RTs and AS in 3, 1 and 1 paper, respectively. [START_REF] Nazari | Shared versus private mobility: Modeling public interest in autonomous vehicles accounting for latent attitudes[END_REF] confirm that safety concerns are a primary factor to the adoption of SAVs that is more important, for example, than being interested in Mobility On Demand (MOD) or having a green travel pattern. [START_REF] Yap | Preferences of travellers for using automated vehicles as last mile public transport of multimodal train trips[END_REF] also identify safety as a major attitudinal determinant of the intention to use SAVs. [START_REF] Smith | Attitudes and Perceived Usefulness of Autonomous Shuttles: an On Board Survey[END_REF] argue that the main safety concern is related to system tampering that might compromise the safety of passengers. Nevertheless, [START_REF] Lavieri | Modeling individuals' willingness to share trips with strangers in an autonomous vehicle future[END_REF] find that despite the importance of privacy in the acceptance of RS RTs, delays associated with sharing a ride (due to detours for example) are more detrimental to their use than sharing a ride with strangers.

Pro-technology attitudes are studied in 5 papers. They positively influence the intention to use SAVs in 100% of the cases. Pro-technology attitudes have been tested for RTs, RS RTs and AS in 2, 1 and 2 papers, respectively. S. [START_REF] Wang | Attitudes towards privately-owned and shared autonomous vehicles[END_REF] and [START_REF] Haboucha | User preferences regarding autonomous vehicles[END_REF] find that those with high interest in technology are more likely to use AVs. [START_REF] Wicki | How technology commitment affects mode choice for a self-driving shuttle service[END_REF] decomposes interest in technology as acceptance, competence and control. Acceptance is highly significant and positive whereas competence and control are not [START_REF] Wicki | How technology commitment affects mode choice for a self-driving shuttle service[END_REF]. [START_REF] Winter | Taking The Self-Driving Bus: A Passenger Choice Experiment[END_REF] confirm this finding in the case of AS.

Passion for driving is studied in 3 papers. It does not influence the intention to use RTs in 66% of the cases (2 out 3 papers) and influences it negatively in 1 case. This attitude was not studied in the case of AS. [START_REF] Gkartzonikas | What have we learned? A review of stated preference and choice studies on autonomous vehicles[END_REF] find that driver are more likely to prefer greater control of their vehicle and less likely to use an AV. [START_REF] Becker | Literature review on surveys investigating the acceptance of automated vehicles[END_REF] suggest to interact passion for driving with traffic conditions as drivers might be interested in being chauffeured in traffic jams. S. [START_REF] Wang | Attitudes towards privately-owned and shared autonomous vehicles[END_REF] note no significant relationship between driving enjoyment and attitudes towards AVs, and suggest that the benefits of driving are balanced by the perceived benefits of AVs.

Benefits Perceived benefits of AVs are explored in 3 papers. These benefits include reducing driver fatigue, environmental-friendliness, increased accessibility of travel for non-drivers and traffic regulation. They positively influence the intention to use SAVs in 100% of the cases. They are studied in the case of RTs and RS RTs in 2 and 1 paper, respectively. The effect of perceived benefits is often assessed at a national level through online surveys (S. [START_REF] Wang | Attitudes towards privately-owned and shared autonomous vehicles[END_REF][START_REF] Wu | Analysis of consumer attitudes towards autonomous, connected, and electric vehicles: A survey in China[END_REF]). In the USA and China, the perceived benefits of SAVs are found to positively impact their acceptance over the purchase of a private AVs. [START_REF] Gkartzonikas | What have we learned? A review of stated preference and choice studies on autonomous vehicles[END_REF] note that safety benefits of AVs are widely expected. Due to its reduced perceived usefulness, [START_REF] Winter | Taking The Self-Driving Bus: A Passenger Choice Experiment[END_REF] find a negative effect of the presence of a steward on board of AS, despite the increased safety brought by this presence.

Preferences for shared and flexible services Preference for shared services is tested in 1 paper. It positively influences the intention to use SAVs [START_REF] Webb | Will people accept shared autonomous electric vehicles? A survey before and after receipt of the costs and benefits[END_REF]. People who already use public transport/ride-sharing are more likely to accept to use SAVs.

Interest for Mobility-On-Demand (MOD) is tested in 2 papers. It positively influences the intention to use SAVs in 100% of the cases. It is tested for RTs, and RS RTs (1 paper each). In a study including, among others, private AVs and SAVs [START_REF] Nazari | Shared versus private mobility: Modeling public interest in autonomous vehicles accounting for latent attitudes[END_REF], interest in MOD is positively associated with the intention to use SAVs for commuting. In another research, [START_REF] Winter | Taking The Self-Driving Bus: A Passenger Choice Experiment[END_REF] show that public transit users have a higher intention to use regular autonomous shuttle services than on-demand autonomous services, due to the extra effort involved by MOD planning.

Current mode choice Experience of carsharing is explored in 8 papers. In 5 of them, it positively influences the intention to use SAVs. In the case of RS RTs, 2 papers ( out of 2) find a positive influence on the intention to use this service. This proportion is 3 out of 6 for RTs. Experience of carsharing is not tested for AS. [START_REF] Zhou | Preference heterogeneity in mode choice for car-sharing and shared automated vehicles[END_REF], [START_REF] Kontar | Autonomous vehicle adoption: use phase environmental implications[END_REF], [START_REF] Krueger | Preferences for shared autonomous vehicles[END_REF], and S. [START_REF] Wang | Attitudes towards privately-owned and shared autonomous vehicles[END_REF] find a positive impact of prior carsharing experience on the intention to use SAVs. It is also easier to share a ride if respondents know the person they share the ride with (Abe 2021). [START_REF] Bansal | Assessing public opinions of and interest in new vehicle technologies: An Austin perspective[END_REF] find a significant and negative relationship between carsharing experience and the intention to use SAVs. This is due to the unwillingness of carsharing users to pay more for an equivalent service with AVs.

Experience of public transport (PT) is explored in 6 papers. In 3 of them, it positively influences the intention to use SAVs. In the case of RTs, 2 experiments out of 3 find a positive influence on the intention to use this service. This proportion is 1 out of 2 for RS RTs. Experience with, or regular use of PT in general has a positive impact on the intention to use AVs [START_REF] Krueger | Preferences for shared autonomous vehicles[END_REF][START_REF] Steck | How Autonomous Driving May Affect the Value of Travel Time Savings for Commuting[END_REF][START_REF] Wang | Attitudes towards privately-owned and shared autonomous vehicles[END_REF]. [START_REF] Winter | Taking The Self-Driving Bus: A Passenger Choice Experiment[END_REF] find that experience of PT is non-significant in the case of AS. In addition, the possession of a PT subscription has a positive effect on the intention to use SAVs. [START_REF] Kontar | Autonomous vehicle adoption: use phase environmental implications[END_REF] suggest, however, that those who frequently use PT are significantly less likely to choose an AV, while K. [START_REF] Wang | Factors Affecting the Adoption of Autonomous Vehicles for Commute Trips: An Analysis with the 2015 and 2017 Puget Sound Travel Surveys[END_REF] find that individuals who reside in neighborhoods with adequate public transit services may be less likely to participate in a SAV system for daily commuting.

Car as the main travel mode is tested in 5 papers. It has mostly a non-significant effect (3 papers out of 5). It's found to have a positive effect on the intention to use RTs in 1 paper out of 2, and a negative effect on the intention to use RS RTs in 1 paper out of 3. Unlike [START_REF] Gkartzonikas | What have we learned? A review of stated preference and choice studies on autonomous vehicles[END_REF] who assume that frequent drivers are more likely to prefer control of the vehicle, [START_REF] Kontar | Autonomous vehicle adoption: use phase environmental implications[END_REF], [START_REF] Krueger | Preferences for shared autonomous vehicles[END_REF], and K. [START_REF] Wang | Factors Affecting the Adoption of Autonomous Vehicles for Commute Trips: An Analysis with the 2015 and 2017 Puget Sound Travel Surveys[END_REF] find that driving control is non-significant for SAVs' adoption in metropolitan areas. [START_REF] Etzioni | Modeling Cross-National Differences in Automated Vehicle Acceptance[END_REF] determine in a cross national survey that car usage makes respondents more reluctant toward AV services in general.

Results' summary

For each variable, Figure 1 and Table 1 evaluate the consensus degree of the reviewed papers. The consensus degree is measured as equations 1 and 2, where Total(+), Total(-) and Total(NS), are the total number of studies with a positive, negative and non-significant effect.

CD = max(T otal(+); T otal(-))

T otal(+) + T otal(-)

By design, 0.5 ≤ CD ≤ 1. CD = 1 indicates a total consensus on the effect of the factor, i.e. either positive or negative effect. CD = 0.5 indicates a no consensus on the effect of the factor, i.e. 50% positive and 50% negative. In between, the consensus is not yet reached. In figure 1 and table 1, the color of the variables shows the consensus degree. Darkest green, darkest red, indicates the most consensual variables with a positive, negative effect on SAVs' acceptance, respectively (CD ≥ 0.9). Lightest green, lightest red, indicates a weak consensus with a positive, negative effect, respectively (0.6 < CD < 0.9). Gray corresponds to a CD ≤ 0.6 or non-consensual effect.

The consensus degree is completed by the statistical significance indicator (SI):

SI = T otal(N S) T otal(+) + T otal(-) + T otal(N S) (2)
We assume that if SI ≤ 0.1, i.e. less than 10% of studies indicate a non-significant effect, the variable is assumed significant. If 0.1 < SI < 0.5 the variable is assumed to be moderately significant. If SI ≥ 0.5 the variable is set to be non-significant.

Discussion

10 years after the first published papers on the intention to use SAVs, our literature review shows that various factors that are deemed to influence this intention still have a non-consensual effect. In this review, we included factors related to: socio-demographic characteristics of respondents (age, gender, income, to name a few), mode-specific characteristics (travel time, waiting time), contextual characteristics (urban, national), attitudes of respondents (pro-environment, safety concerns, for example), perceived benefits and travel habits. As [START_REF] Gkartzonikas | What have we learned? A review of stated preference and choice studies on autonomous vehicles[END_REF], we find that most studies mainly focus on socio-demographic and travel characteristics. Less attention has been paid to the role of psychosocial factors in the economic literature. We find that the better acceptance of SAVs is among young males, with high income and education (moderate consensus, i.e. 0.6 < CD < 0.9). These results are partly in line with [START_REF] Becker | Literature review on surveys investigating the acceptance of automated vehicles[END_REF] since we show that the effect of gender and age is moderately significant (SI > 0.1) and moderately consensual (i.e. 0.6 < CD < 0.9). Trip cost, travel and waiting times have a negative effect on SAVs' acceptance (high consensus, i.e. CD ≥ 0.9 and high significance, i.e. SI ≤ 0.1). Educational attainment improves SAVs' acceptance (high consensus, i.e. CD ≥ 0.9, moderately significant, i.e. SI > 0.1). Owning a car or a driving license is negatively associated with the intention to use SAVs (moderate consensus, i.e. 0.6 < CD < 0.9, moderately significant, i.e. SI > 0.1), while experience of carsharing or public transportation increase SAVs' acceptance (moderate consensus, i.e. 0.6 < CD < 0.9, moderately significant, i.e. SI > 0.1). Finally, perceived benefits, technology savviness and trust in SAVs boost the acceptance of SAVs (high consensus, i.e. CD ≥ 0.9 and high significance, i.e. SI ≤ 0.1).

The majority of studies focus on RTs and RS RTs. SAVs' acceptance factors have similar effect on both types of RTs (shared or not). This cannot be confirmed in the case of AS due to the scarcity of research on these services (only 18% of reviewed papers).

The lack of consensus or significance on the effect of some important SAVs' acceptance factors may be attributable to various reasons: the nature of study areas, data collection methodologies or statistical models that are different from one study/survey to another. Future research needs to disentangle the effect of differences due to methodological approaches and to spatial/social contexts.

The specific case of psychosocial factors

About 30% of reviewed papers explore the effect of psychosocial factors on the intention to use SAVs. Most of these factors relate to attitudes or social norms. 33% of these papers discuss the psychosocial models behind these factors. The identified psychosocial factors are very heterogeneous and, in some cases, are related to concepts which belong to different theories of behavior. [START_REF] Gkartzonikas | What have we learned? A review of stated preference and choice studies on autonomous vehicles[END_REF] and [START_REF] Jing | The Determinants behind the Acceptance of Autonomous Vehicles: A Systematic Review[END_REF] highlight the need for a clear theoretical framework to produce more robust predictors and demand predictions. A number of recent studies have proposed to fill this gap in the field of psychology.

The most widely used model is the Technology Acceptance Model (TAM) [START_REF] Davis | User Acceptance of Computer Technology: A Comparison of Two Theoretical Models[END_REF]). The TAM predicts the intention to use a technology based on two factors: perceived ease of use and perceived utility. It is often preferred to other models thanks to its specification parsimony [START_REF] Jing | The Determinants behind the Acceptance of Autonomous Vehicles: A Systematic Review[END_REF]). [START_REF] Nordhoff | Interrelationships among predictors of automated vehicle acceptance: a structural equation modelling approach[END_REF] investigate the intention to use AS and find that the constructs of the unified theory of acceptance and use of technology 2 (UTAUT2) model [START_REF] Venkatesh | Consumer Acceptance and Use of Information Technology: Extending the Unified Theory of Acceptance and Use of Technology[END_REF]) are validated by the literature. UTAUT2 adds constructs of different acceptance models: social network influence, facilitating conditions, hedonic motivation, price value and habit.

Other variants of the UTAUT2 model exist: Yuen et al. ( 2020) study a mix of the theory of planned behavior and UTAUT2. The dimensions of the latter are mediated by attitudes toward SAVs. The constructs of the Theory of Planned Behavior [START_REF] Ajzen | Organizational Behavior and Human Decision Processes[END_REF]) (attitude, norms, perceived behavioral control) as well as the perceived ease of UTAUT2 are used as predictors of the intention to use SAVs. [START_REF] Nordhoff | A multi-level model on automated vehicle acceptance (MAVA): a review-based study[END_REF] also design a multi-level model on automated vehicle acceptance (MAVA) based on the unified theory of acceptance and use of technology 3 (UTAUT3).

Conclusion and limitations

Despite the substantial research effort put into the study of autonomous mobility, research devoted to SAVs is still scarce. This literature review focuses on this type of services and updates the state of knowledge with recent publications. It also reviews the main empirical and theoretical papers in psychology that consider the acceptance of SAVs.

This paper focuses on the factors of the intention to use SAVs. It reviews the methodologies used in discrete choice surveys, survey areas, the statistical models used to analyze the data and their specifications. Some effects seem strongly consensual: travel costs and times, educational attainment. On the other hand, other effects remain controversial and need further research: age, gender, income, car ownership, driving license or preferences of rural households. Despite the scarcity of research on the effect of psychosocial factors in the economic literature, there seem to be a consensus on their importance. Further research on this subject is needed. Future work should integrate psychosocial and economic models to better assess the role of attitudes and other psychosocial constructs in the intention to use SAVs. Finally, more emphasis needs to be put on the intention to use SAVs in rural or suburban areas.

This research is subject to several limitations. This review is based on 29 articles published in the economic literature between 2015 and 2021. Papers that do not use the discrete choice experiment method were not included. Further research could expand this set by integrating other survey methodologies to include new variables and strengthen the validity of the ones included in this research. In order to compare different research studies, simplifications have also been made to harmonize their methodologies. An effort has been made, for example, to make consistent the definition of the variables and their attribute levels for all surveys. This harmonization might introduce a bias in our results.

Figure 1 :

 1 Figure 1: Research consensus on the effect of SAVs' acceptance factors. The colors are defined following equation 1

Table 1 :

 1 Main results of the literature review. Colors of the column Variables are defined by equation 1. SI column is defined according to equation 2. Non-significant factors/rows are colored in gray.

	Variable groups Variables		Robo Taxi			Ridesharing			Autonomous			Total		Total SI
							Robo Taxi			Shuttle
	Effect direction		(+)	(NS)	(-) (+)	(NS)	(-) (+)	(NS)	(-) (+) (NS) (-)
	Control	Gender (male)	5	8	4	3	3	1	3			11	11	5	27	*
	variables	Age	5	6	6		2	6				5		12	25	*
		Trip cost			12		1	4			5	0		21	22	**
		Travel time	1	1	11			3			4	1		18	20	**
		Income	4	6	1	2	3		1			7		1	17	NS
		Waiting time		1	8			3			4	0		15	16	**
		Household	2	5	1	1	3	1				3		2	13	NS
		Own a car		4	4			3				0		7	11	*
		Education	5	2		2			1			8		0	10	*
		Place of residence	1	3	2	2	1					3		2	9	NS
		Driving licence		4	2	1	1	1				1		3	9	NS
		Trip purpose	3	2		1	1				1	4		1	8	*
		Access/Egress time			1			1			1	0		3	3	**
	Attitudes	Environmental concern	3	1		1						4		0	5	*
		Pro-technology	2			1			2			5		0	5	**
		Passion for driving		2	1							0		1	3	NS
		Security concerns			3			1			1	0		5	5	**
		Trust in AV	2			2			1			5		0	5	**
	Benefits	Perceived benefits of AV	2			1						3		0	3	**
		Presence of a steward									1	0		1	1	**
	Pref. for shared Interest for MOD	1			1						2		0	2	**
	flexible services	Prefer to share	1									1		0	1	**
		Shuttle (VS on demand)									1	0		1	1	**
	Current	Experience of carsharing	3	2	1	2						5		1	8	*
	mode choice	Experience of PT	2		1	1	1			1		3		1	6	*
		Main mode: car	1	1			2	1				1		1	5	NS

This research is conjointly carried out by projects Sécurité Acceptabilité de la conduite et de la Mobilité autonome (SAM) and Expérimentations de Navettes Autonomes (ENA). This research is supported by Programme d'Investissements d'Avenir (PIA) operated by ADEME, the Pack Ambition International of the Région Auvergne Rhône-Alpes, France (Grant number: 19008027 01-157130. Grant recipient: Ouassim Manout) and the Ministry of the ecological transition, DG-ITM/MINT (Grant number: 20-MINT-2c3-02, Grant recipient: Ouassim Manout)