George Filioussis

Georgios Bramis

Evanthia Petridou

Nektarios D Giadinis

Laurent-Xavier Nouvel

Christine Citti

Joachim Frey
email: joachim.frey@vetsuisse.unibe.ch

Mycoplasma agalactiae ST35: a new sequence type with a minimal accessory genome primarily affecting goats

Keywords: Mycoplasma agalactiae, Sequence type 35, Goats, Full genome, Contagious agalactia

Background: Mycoplasma agalactiae, causing agent of contagious agalactia, infects domestic small ruminants such as sheep and goats but also wild Caprinae. M. agalactiae is highly contagious and transmitted through oral, respiratory, and mammary routes spreading rapidly in an infected herd.

Results:

In an outbreak of contagious agalactia in a mixed herd of sheep and goats, 80% of the goats were affected displaying swollen udders and loss of milk production but no other symptom such as kerato-conjunctivitis, arthritis or pulmonary distress commonly associated to contagious agalactia. Surprisingly, none of the sheep grazing on a common pasture and belonging to the same farm as the goats were affected. Whole genome sequencing and analysis of M. agalactiae strain GrTh01 isolated from the outbreak, revealed a previously unknown sequence type, ST35, and a particularly small, genome size of 841′635 bp when compared to others available in public databases. Overall, GrTh01 displayed a reduced accessory genome, with repertoires of gene families encoding variable surface proteins involved in host-adhesion and variable antigenicity being scaled down. GrTh01 was also deprived of Integrative Conjugative Element or prophage, and had a single IS element, suggesting that GrTh01 has a limited capacity to adapt and evolve.

Conclusions:

The lack of most of the variable antigens and the Integrative Conjugative Element, both major virulence-and host specificity factors of a M. agalactiae strain isolated from an outbreak affecting particularly goats, indicates the implication of these factors in host specificity. Whole genome sequencing and full assembly of bacterial pathogens provides a most valuable tool for epidemiological and virulence studies of M. agalactiae without experimental infections.

Introduction

Mycoplasma agalactiae is an important pathogen causing contagious agalactia in small ruminants such as sheep, goats and wild Caprinae. Contagious agalactia is a notifiable disease listed by the World Organisation for Animal Health (OIE) due to the important economic losses to the dairy industry and to small ruminants holders [START_REF] Lambert | Contagious agalactia of sheep and goats[END_REF]. The contagious agalactia caused by M. agalactiae includes clinical symptoms of mastitis, arthritis and kerato-conjunctivitis, but M. agalactiae infections occasionally also cause abortions, pneumonia and septicaemia in goat and sheep [START_REF] Tardy | Emergence of atypical Mycoplasma agalactiae strains harboring a new prophage and associated with an alpine wild ungulate mortality episode[END_REF][START_REF] Corrales | Contagious agalactia in small ruminants[END_REF]. M. agalactiae is highly contagious and transmitted through oral, respiratory, and mammary routes. Since the end of the nineteenth century, the disease had been reported to be enzootic in many parts of the world [START_REF] Lambert | Contagious agalactia of sheep and goats[END_REF][START_REF] Kumar | Mycoplasma agalactiae , an etiological agent of contagious agalactia in small ruminants: a review[END_REF][START_REF] Nouvel | Molecular typing of Mycoplasma agalactiae: tracing European-wide genetic diversity and an endemic clonal population[END_REF]. Outbreaks of M. agalactiae infections occur notably in the Mediterranean basin, India, Australia, Turkey, Iran, Mongolia, Nigeria, Senegal and Iraq [START_REF] Kumar | Mycoplasma agalactiae , an etiological agent of contagious agalactia in small ruminants: a review[END_REF]. The spread of contagious agalactia within a population is multifactorial including inadequate herding practices, inefficiency of antimicrobial therapies, and adoption of very few prophylactic measures. In addition, the high potential of M. agalactiae to persist in the host and to some extent in the environment often renders difficult to eradicate the infections. The disease is mostly reported from sheep where it causes highest economic losses mainly due to high morbidity rather than high mortality resulting in losses of milk production throughout the world, but goats are equally affected by M. agalactiae infections [START_REF] Kumar | Mycoplasma agalactiae , an etiological agent of contagious agalactia in small ruminants: a review[END_REF]. Mycoplasma agalactiae possess a family of related surface lipoproteins, named variable protein of M.agalactiae (Vpma) that are prone to high-frequency variation in expression and structure thus modulating the mycoplasma surface structure and accessibility to immune components of the host. This provides M. agalactiae the capability to rapidly adapt to various environmental changes and to evade the host's immune defence. Phase and size variations have been shown to occur in vitro with frequencies estimated at 10E-2 to 10E-5 events/cell/generation [START_REF] Citti | Phase and antigenic variation in mycoplasmas[END_REF]. M. agalactiae, type strain PG2 which was used for most molecular studies, carries a genetic locus containing a cluster of 6 vpma genes (vpmaU though Z), while other M. agalactiae strains such as strain 5632 show up to 18 different vpma genes occurring as two clusters on two different loci of the chromosome. In a given cell, only one vpma gene per locus is expressed, while in the overall population the different Vpma antigens occur at different rates [START_REF] Citti | Phase and antigenic variation in mycoplasmas[END_REF][START_REF] Sirand-Pugnet | Being pathogenic, plastic, and sexual while living with a nearly minimal bacterial genome[END_REF][START_REF] Nouvel | Occurrence, plasticity, and evolution of the vpma gene family, a genetic system devoted to high-frequency surface variation in Mycoplasma agalactiae[END_REF]. The intra-clonal variation is due to the presence of a unique promoter at the vpma loci that is alternatingly placed by a DNA recombination mechanism in front of silent vpma genes, resulting in ON and OFF switching of the various Vpma expression. Vpma phase variation allows M. agalactiae to survive in immuno-competent hosts and to establish persistent infections and cause debilitating disease [START_REF] Rosengarten | Host-pathogen interactions in mycoplasma pathogenesis: virulence and survival strategies of minimalist prokaryotes[END_REF][START_REF] Chopra-Dewasthaly | Phase-locked mutants of Mycoplasma agalactiae: defining the molecular switch of high-frequency Vpma antigenic variation[END_REF]. The phase variation, which is important for the host immune evasion and the in-host survival of the pathogen is controlled to a major extent by the Xer1 recombinase, also named DNA invertase Mar, that is encoded by the vpma loci [START_REF] Chopra-Dewasthaly | Comprehensive RNA-Seq profiling to evaluate the sheep mammary gland transcriptome in response to experimental Mycoplasma agalactiae infection[END_REF][START_REF] Czurda | Xer1-mediated site-specific DNA inversions and excisions in Mycoplasma agalactiae[END_REF]. Recently a novel role of Vpmas as major adhesins of M. agalactiae mediating differential adhesion and invasion of host cells has been unravelled by the use of phase locked mutants expressing specific well-characterized Vpma lipoproteins [START_REF] Hegde | Novel role of Vpmas as major adhesins of Mycoplasma agalactiae mediating differential cell adhesion and invasion of Vpma expression variants[END_REF].

In order to better understand the origin of M. agalactiae infections and their mode of propagation within and among herds with the aim of controlling the disease, molecular epidemiological tools including variable-number tandem repeat (VNTR) and multi-locus sequence typing (MLST) have been developed. They have been used successfully to trace M. agalactiae outbreaks and emergence or re-emergence after eradication campaigns [START_REF] Tardy | Emergence of atypical Mycoplasma agalactiae strains harboring a new prophage and associated with an alpine wild ungulate mortality episode[END_REF][START_REF] Nouvel | Molecular typing of Mycoplasma agalactiae: tracing European-wide genetic diversity and an endemic clonal population[END_REF][START_REF] Manso-Silván | Phylogeny and molecular typing of Mycoplasma agalactiae and Mycoplasma bovis by multilocus sequencing[END_REF][START_REF] De La Fe | Unexpected genetic diversity of Mycoplasma agalactiae caprine isolates from an endemic geographically restricted area of Spain[END_REF][START_REF] Poumarat | Diversity and variation in antimicrobial susceptibility patterns over time in Mycoplasma agalactiae isolates collected from sheep and goats in France[END_REF][START_REF] Tardy | Contagious agalactia monitoring in caprine herds through regular bulk tank milk sampling[END_REF]. While certain European countries such as Spain seem to have endemic clones, in particular ST5, others such as Greece have to deal with several different clones including ST2, ST4, ST6, and ST8 [START_REF] Nouvel | Molecular typing of Mycoplasma agalactiae: tracing European-wide genetic diversity and an endemic clonal population[END_REF][START_REF] Tardy | Contagious agalactia monitoring in caprine herds through regular bulk tank milk sampling[END_REF][START_REF] Ariza-Miguel | Molecular characterization of Mycoplasma agalactiae reveals the presence of an endemic clone in Spain[END_REF][START_REF] Kabiri | Homogeneity of Mycoplasma agalctiae vaccine strains in an agalaxy-high-burden environment[END_REF]. It was also shown that M. agalactiae strains circulating in the ibex (Capra ibex) population in the French Alps were highly interrelated and most likely originated from a single parental clone that has also spread to chamois (Rupicapra rupicapra), another wild ungulate species in the same geographical area [START_REF] Tardy | Emergence of atypical Mycoplasma agalactiae strains harboring a new prophage and associated with an alpine wild ungulate mortality episode[END_REF]. The clone contains some genetic loci closely related to Mycoplasma conjunctivae, a pathogen that is frequently found in wild ungulates with infectious kerato-conjunctivitis (IKC) [START_REF] Tardy | Emergence of atypical Mycoplasma agalactiae strains harboring a new prophage and associated with an alpine wild ungulate mortality episode[END_REF][START_REF] Fernández-Aguilar | Postepizootic persistence of asymptomatic Mycoplasma conjunctivae infection in Iberian ibex[END_REF][START_REF] Gelormini | Infectious keratoconjunctivitis in wild Caprinae: merging field observations and molecular analyses sheds light on factors shaping outbreak dynamics[END_REF][START_REF] Zimmermann | Specificity of Mycoplasma conjunctivae strains for alpine chamois Rupicapra r. rupicapra[END_REF]. However, the molecular basis of the potential specificity or predilection of the clone to the wild Caprinae ibex and chamois is still unknown. The current manuscript describes a particular strain of M. agalactiae of a previously undetected sequence type, ST35 that caused an uncommon outbreak of contagious agalactia affecting specifically goats. The outbreak strain had a particularly small genome size and lacked several characteristic genes involved in host adhesion and variable antigenicity.

Materials and methods

Observation farm

The observation farm was located in Aridea (northwestern Greece), a lowland at 120 m above sea level. The farm harboured a mixed herd of 850 Lacaune milking sheep (Ovis aries) and 320 Anglo-nubian milking goats (Capra aegagrus hircus) of the age of 1-12 years. About one third (106) of the animals were aged 15 -17 months and were the first time in the milking season. Sheep and goats were kept in separate barns but shared meadows and were milked at the same milking place. Generally, all adult animals were vaccinated twice a year against contagious agalactia with a commercial vaccine (Agalax Uno; Laboratorios Syva, Leon, Spain) based on inactivated bacteria of Mycoplasma agalactiae. However, the year of the observed outbreak, no vaccination was recorded for both sheep and goats.

Clinical examinations

A veterinarian conducted a general clinical routine examination inspecting eye, ears, mouth, body, skin, hair, rectum vagina, udder, legs, hooves and records of appetence as recommended by Nagy and Pugh [START_REF] Nagy | Handling and examining sheep and goats[END_REF] giving special attention to udders as described for previous studies [START_REF] Giadinis | Milk-drop syndrome of ewes": investigation of the causes in dairy sheep in Greece[END_REF].

Bacteriological diagnosis

Composite milk samples (CMS) were collected from seven randomly selected goats that suffered from clinical mastitis, before any treatment was administered. Briefly, following proper disinfection of the teats and the withdrawal of the initial 3 to 4 squirts of milk, CMS (ca. 30 mL each), were collected in sterile falcon tubes. The samples were refrigerated and submitted in insulated boxes containing coolants to the laboratory of microbiology and infectious diseases at the Faculty of Veterinary Medicine of Aristotle University, Thessaloniki within 12 h of collection. Aliquots of 10 μL milk were cultured on Columbia Agar supplemented with 5% sheep blood (Oxoid Ltd., Cambridge, United Kingdom), MacConkey Agar (Biolife Italiana S.r.l., Milan, Italy) and Sabouraud Dextrose Agar (Oxoid Ltd., Cambridge, United Kingdom) aerobically and in an atmosphere that contained 5% CO 2 at 37 o C.

Detection of mycoplasmas, isolation, culture, extraction of genomic DNA, genome sequencing and annotation

In order to detect the presence of mycoplasmas 0.5 mL of each CMS was inoculated in 5 mL of Heyflicks broth and incubated at 37 °C under an atmosphere of 5% CO 2 . Additionally 50 μL milk volume was cultured on Heyflicks agar and incubated at 37 °C under an atmosphere of 5% CO 2 . Identification of Mycoplasma colonies was performed using quick lysis DNA extraction and polymerase chain reaction (PCR) using primers, specific for M. agalactiae and the Mycoplasma mycoides cluster [START_REF] Grand | Assessment of PCR for routine identification of species of the Mycoplasma mycoides cluster in ruminants[END_REF][START_REF] Subramaniam | Species identification of Mycoplasma bovis and Mycoplasma agalactiae based on the uvrC genes by PCR[END_REF].

A strain (GrTh01) of M. agalactiae was obtained from one of the M. agalactiae isolates by filter cloning through a 0.2 μm filter and sub-culture of a single colony as described [START_REF] Barile | Isolation and characterization of Mycoplasma arginini: spec[END_REF]. For preparing genomic DNA the strain was grown in Heyflicks broth under aerobic atmosphere for 72 h. Mycoplasma DNA was extracted using the Guanidine thiocyanate extraction method [START_REF] Pitcher | Rapid extraction of bacterial genomic DNA with guanidium thiocyanate[END_REF] with two subsequent phenol extractions and two ethanol precipitations.

The genome sequence of M. agalactiae strain GrTh01 was generated using a combination of Pacific Biosciences RSII and Illumina HiSeq sequencing. After sequencing and circularization using Pacific Biosciences (PacBio) RSII (Swiss Institute of Bioinformatics, Lausanne, Switzerland), the sequence was corrected with Illumina HiSeq data for the substitutions due to sequencing errors and for the frameshifts, using the Celera Assembler (CA) PacBio correction module PBcR (version 7.0) [START_REF] Koren | Hybrid error correction and de novo assembly of single-molecule sequencing reads[END_REF]. Illumina reads were obtained using the Nextera XT DNA protocol for the library preparation and the V3 chemistry to produce 2 × 150 bp paired-end reads that resulted in 9.1 million read pairs (GATC Biotech, Konstanz, Germany). The resulting full genome was annotated using the Prokka pipeline [START_REF] Seemann | Prokka: rapid prokaryotic genome annotation[END_REF] and the NCBI Prokaryotic Genome Annotation Pipeline PGAP https:// www. ncbi. nlm. nih. gov/ genome/ annot ation_ prok/ and deposited at NIH genetic sequence database GenBank. The GenBank/ EMBL accession number of the full genome sequence of M. agalactiae strain GrTh01 is CP039447. Detailed analysis of Vpma loci, potential virulence genes and comparisons of genomes of M. agalactiae strains were performed using the software package Geneious ® software package 9.1.8 (Biomatters Ltd., Auckland 1141, New Zealand), Virulencefinder https:// cge. cbs. dtu. dk/ servi ces/ Virul enceF inder/ [START_REF] Joensen | Real-time whole-genome sequencing for routine typing, surveillance, and outbreak detection of verotoxigenic Escherichia coli[END_REF] and Artemis [START_REF] Carver | Artemis: an integrated platform for visualization and analysis of high-throughput sequence-based experimental data[END_REF]. Multi locus sequence typing (MLST) was performed using the M. agalactiae MLST Databases (https:// pubml st. org/ magal actiae/) extracting the gene-loci of dnaA, gltX, gyrB, metS and tufA from the genome sequence [START_REF] Mcauliffe | Multilocus sequence typing of Mycoplasma agalactiae[END_REF].

Results

Outbreak description

Between April and June 2016, 87 out of 106 young milking goats of the age of 15 to 17 months that were their first milking season showed a general malaise, absence of appetite, and signs of clinical mastitis. In most of the cases, both the right and the left udder were hot, swollen and tender while the milk became discolored and granular. No signs of respiratory complications, arthritis or keratitis were detected. Furthermore, the sheep population of the same farm remained unaffected, showing no clinical signs of contagious agalactia. The study of the medical records of the farm showed that, for an undefined reason, the whole farm (sheep and goats) was not vaccinated against contagious agalactia during the year preceding the outbreak. After the outbreak the goats of the farm were culled.

Bacteriological analyses

Milk samples of 7 of the 87 affected goats were analysed bacteriologically of which all 7 showed M. agalactiae by culture and subsequent species differentiation using PCR. No other bacterial species were diagnosed from the 7 milk samples. One M. agalactiae isolate was twice filter cloned, and the strain isolated named GrTh01 was used for full genome analysis.

Whole genome sequencing and analysis of strain GrTh01

The whole genome sequence (WGS) of M. agalactiae strain GrTh01was obtained by combining PacBio and Illumina sequencing and revealed a circular chromosome of 841′635 bp (GenBank full Genome accesion nr. CP039447). The Illumina HiSeq coverage was of 1895 x. As for the other M. agalactiae strains so far sequenced, no plasmid was identified in GrTh01. Annotation of the genome using the NCBI Prokaryotic Genome Annotation Pipeline PGAP revealed a total of 767 genes of which 623 encode proteins. Strain GrTh01 contains two full sets of rRNAs (5S, 16S, 23S), 34 tRNAs and 3 non coding RNAs (Table 1).

The GrTh01 MLST analysis revealed a new sequence type, namely ST35, having the following profile: dnaA: 21; gltX: 1; gyrB: 1, metS: 2; tufA: 1. ST35 stands apart because of its novel dnaA allele, which sequence was verified by additional Sanger sequencing using primers recommended by Mc Auliffe [START_REF] Mcauliffe | Multilocus sequence typing of Mycoplasma agalactiae[END_REF]. The MLST analyses also indicated that the ST35 GrTh01 strain clustered together with several strains isolated from Greece, with dnaA being the only discriminating allele and from the PG2 type strain isolated in Spain, with two discriminating alleles (Table 2). The dnaA allele in strain GrTh01 leads to a T/S change at a.a. position 91. While strain GrTh01 is currently the only strain among M. agalactiae and the phylogenetically closely related Mycoplasma bovis, the mutation is in a serine-rich area of DnaA and a serine residue is found at the analogous site of DnaA of Mycoplasma simbae (GenBank accession nr. WP 051630113).

Of the currently publicly available M. agalactiae genomes, the GrTh01 genome is the smallest showing ca. 36 and 165 Kbp less than the two representative M. agalactiae strains with circularized genome, namely PG2 and 5632 [START_REF] Sirand-Pugnet | Being pathogenic, plastic, and sexual while living with a nearly minimal bacterial genome[END_REF][START_REF] Nouvel | Comparative genomic and proteomic analyses of two Mycoplasma agalactiae strains: clues to the macro-and micro-events that are shaping mycoplasma diversity[END_REF] (Table 1). More specifically, comparison of GrTh01 with these two strains revealed that GrTh01 lacks the 23-kb Integrative Conjugative Element (ICE) found in 5632 in three copies and the 20-kb ICE vestige of PG2 [START_REF] Nouvel | Comparative genomic and proteomic analyses of two Mycoplasma agalactiae strains: clues to the macro-and micro-events that are shaping mycoplasma diversity[END_REF][START_REF] Frisoni | ICEA of Mycoplasma agalactiae: a new family of self-transmissible integrative elements that confers conjugative properties to the recipient strain[END_REF] (Fig. 1). Interestingly, GrTh01 displayed 2 small ICE vestiges of ca 2.4 and 6.0 kb that are composed of only few CDSs and pseudogenes. The smallest is found in the three strains at the same locus, while the largest is only found in 5632 and GrTh01 (Fig. 1). Of note, PG2 and GrTh01 are deprived of IS elements, a situation that contrasts with 5632 having 15 belonging to the IS30 family [START_REF] Nouvel | Comparative genomic and proteomic analyses of two Mycoplasma agalactiae strains: clues to the macro-and micro-events that are shaping mycoplasma diversity[END_REF].

Other smaller deletions, detected in GrTh01 when compared to PG2 or 5632, mostly affect gene families encoding surface proteins. More specifically, the repertoire of the Bacteroides like surface protein A (BspA) gene family is the smallest with 4 entire and 4 pseudogenes in GrTh01 for 9 and 12 full copies in PG2 and 5632, respectively [START_REF] Nouvel | Comparative genomic and proteomic analyses of two Mycoplasma agalactiae strains: clues to the macro-and micro-events that are shaping mycoplasma diversity[END_REF]. Of note, this family was previously described and annotated as drp genes by Nouvel et al. [START_REF] Nouvel | Comparative genomic and proteomic analyses of two Mycoplasma agalactiae strains: clues to the macro-and micro-events that are shaping mycoplasma diversity[END_REF] or as PARCEL in M. bovis by Wise et al. [START_REF] Wise | Complete genome sequence of Mycoplasma bovis type strain PG45 (ATCC 25523)[END_REF] and were characterized by the presence of the motif DUF285, also present in BspA. In this study, we refer to all this CDS as bspA. The function of the proteins carrying a DUF285 domain is not known but these occurred across a large number of mycoplasma species and bacteria. In strain GrTh01, the spma locus that encodes a family of surface lipoprotein with unknown function is 13,475 bp and similar to PG2. Finally, as shown in Fig. 1, strain GrTh01 displayed two vpma loci as in 5632, a situation that contrast with the single locus of PG2 [START_REF] Nouvel | Comparative genomic and proteomic analyses of two Mycoplasma agalactiae strains: clues to the macro-and micro-events that are shaping mycoplasma diversity[END_REF].

One striking feature of the GrTh01 loci is that they are highly degenerated, with only four genes encoding a signal peptide, a conserved N-terminal and a vpma block of amino acids (Fig. 2). Multiple copies of sequences encoding either a signal peptide and a few amino acids or a Vpma block without a signal sequence were identified on both loci (Fig. 2). These loci also contained multiple "vis" motifs, a highly conserved sequence of 21 nt that is recognized by the Xer recombinase, which one is responsible for DNA rearrangements [START_REF] Glew | Surface diversity in Mycoplasma agalactiae is driven by site-specific DNA inversions within the vpma multigene locus[END_REF] (Fig. 2). As in 5632, each of the two vpma loci contains a highly conserved xer1 gene (100% nucleotide identity in between the two copies). In PG2 and 5632, each vpma locus possesses a single conserved promoter placed upstream of the expressed vpma gene, so that a maximum of 1 and 2 Vpma are expressed in PG2 and 5632, respectively. Here, the conserved promotor sequence was detected, but no entire vpma gene was found directly behind raising the question of whether the sequenced GrTh01 strain expressed a Vpma or whether another promoter, yet unidentified, is used by this strain for Vpma expression at the surface of the cell. Remarkably, GrTh01 locus II is flanked by two short direct repeats also found in 5632 in which they were generated upon IS integration (Fig. 2). Full identical copies including promoter and ribosome binding site were found in both strains PG2 and GrTh01 for the virulence genes nifS -nifU [START_REF] Baranowski | Experimental infections with Mycoplasma agalactiae identify key factors involved in host-colonization[END_REF], the gene encoding the adhesin P40 (Fleury et al., 2002), and the lipoprotein P80 [START_REF] Tola | Sequence, cloning, expression and characterisation of the 81-kDa surface membrane protein (P80) of Mycoplasma agalactiae[END_REF] that is involved in neutrophil extracellular trap (NET) formation [START_REF] Cacciotto | Mycoplasma lipoproteins are major determinants of neutrophil extracellular trap formation[END_REF].

Discussion

A particular strain of M. agalactiae, strain GrTh01, was isolated from an outbreak of caprine contagious agalactia that was restricted to Carpinae and did not affect sheep that were grazing on the same meadows and kept in the same farm as the affected goats. Also rather uncommon was the profile disease symptoms that was restricted to mastitis and did not reveal arthritis, conjunctivitis or respiratory distress. Full genome analysis revealed that the chromosome of GrTh01 was significantly shorter than type strain PG2 and other fully sequenced M. agalactiae strains. Type strain of M. agalactiae PG2 was originally isolated from a goat but shown in several infection experiments to efficiently infect sheep [START_REF] Baranowski | Experimental infections with Mycoplasma agalactiae identify key factors involved in host-colonization[END_REF]. There are no differences between PG2 and GrTh01 in the essential housekeeping genes, the virulence associated genes nifS and nifU that are required for host invasion, protein P40 involved in cell adhesion or the lipoprotein P80 involved in NET formation. However, strain GrTh01 lacks a locus of 20 kbp including conjugal transfer genes such as traE and DDE transposase and genes belonging to a potential type IV secretion system whose function in M. agalactiae is unknown. Furthermore, GrTh01 fully lacks genes encoding transposases such as the IS30 transposase gene that is present in type strain PG2. Most interestingly, strain GrTh01 shows deletions in two groups of genes that both are potentially involved in adhesion: i) genes encoding the Bacteroides-like surface protein A (BspA) and ii) the vpma genes of the variable proteins of M. agalactiae. All currently sequenced strains of M. agalactiae possess several gene copies coding BspA family leucine-rich repeat surface proteins. While strain PG2 contains 9 full copies and 3 truncated bspA genes, and strain 5632 has 12 full, and one truncated copy, strain GrTh01 only contains 4 full copies and 4 truncated versions of bspA. The BspA protein includes a domain that seems to have regulatory function by suppressing the σ54 dependent transcription and was found to have adhesin functions. In Tannerella forsythia the BspA-like protein is essential in pathogenesis [41]. In Entamoeba histolytica the BspA-like protein binds to human TNF receptor 1 (TNFR1) and is essential for invasion of the human colonic mucosa possibly via a fibronectin/fibrinogen integrin binding process The vpma loci of M. agalactiae are considered as pathogenicity island-like loci possessing key features of pathogenicity islands such as the tRNA-lys gene, the invertase-recombinase xer1 and the virulence associated vpma genes [START_REF] Glew | Surface diversity in Mycoplasma agalactiae is driven by site-specific DNA inversions within the vpma multigene locus[END_REF]. Furthermore, the Vpma proteins have been shown to be involved in virulence features such as immune evasion and immunomodulation and colonization of M. agalactiae in particular and Mycoplasmas in general [START_REF] Tardy | Emergence of atypical Mycoplasma agalactiae strains harboring a new prophage and associated with an alpine wild ungulate mortality episode[END_REF][START_REF] Citti | Phase and antigenic variation in mycoplasmas[END_REF][START_REF] Nouvel | Occurrence, plasticity, and evolution of the vpma gene family, a genetic system devoted to high-frequency surface variation in Mycoplasma agalactiae[END_REF]43]. More recently, their involvement in differential cell adhesion and invasion was shown by the analysis of phase locked mutants that steadily express single well-characterized Vpma antigens [START_REF] Chopra-Dewasthaly | Comprehensive RNA-Seq profiling to evaluate the sheep mammary gland transcriptome in response to experimental Mycoplasma agalactiae infection[END_REF][START_REF] Hegde | Novel role of Vpmas as major adhesins of Mycoplasma agalactiae mediating differential cell adhesion and invasion of Vpma expression variants[END_REF]. Coinfections of sheep with phase locked mutants stably expressing VpmaY and VpmaU revealed a dominance in the host of VpmaY expressors in particular during early stages of conjunctival and intra-mammary infections compared to VpmaU expressers, indicating differential roles of the antigenic variants in infection. Their data also revealed the importance of antigenic variation for survival and persistence inside the immunocompetent host as Xer1 independent antigenic variation was detected in the sheep infection model with Xer1 phase locked mutants [START_REF] Chopra-Dewasthaly | Comprehensive RNA-Seq profiling to evaluate the sheep mammary gland transcriptome in response to experimental Mycoplasma agalactiae infection[END_REF]. While the surface antigenic variation caused by the Vpma proteins was mostly shown to be implicated as important mechanisms for better survival and persistence of M. agalactiae inside the host, a recent study using phase-locked mutants expressing the various Vpmas of strain PG2 revealed the role of Vpmas in adhesion [START_REF] Hegde | Novel role of Vpmas as major adhesins of Mycoplasma agalactiae mediating differential cell adhesion and invasion of Vpma expression variants[END_REF]. Using sheep primary mammary epithelial cells and mammary stromal cells Hegde and collaborators showed that VpmaV exhibited the highest adhesion rate of the various Vpmas. Furthermore, they also could show a strong correlation between functions of Vpma proteins in cell adhesion and invasion. This strongly indicates that the variability of these lipoproteins not only causes evasion of the host's immune response, but also changes the adhesion characteristics of the pathogen to allow it to adapt and spread in different host niches. Interestingly strain GrTh01 reveals two vpma loci similar to M. agalactiae strain 5632 [44], one located at the tRNA-lys gene (locus I) and the other at the 16S rRNA gene (locus II) (Fig. 2). Both loci contain a full-length xer1 gene but many strongly truncated vpma genes respectively residues with locus I having full length copies of vpmaW, vpmaX and vpmaZ and locus II showing solely vpmaY in full length. VpmaV, which was shown to have highest adhesion properties to sheep cells, is absent in strain GrTh01. The reduced set of available vpma genes and in particular the lack of vpmaV could explain the reduced host range and a predilection of the outbreak strain GrTh01 for Caprinae and for the mammary gland as revealed from the clinical and epidemiological data of the outbreak. Our clinical and molecular genetic data on strain GrTh01 show certain correlations to the observations by Chopra-Dewasthaly and collaborators [START_REF] Hegde | Novel role of Vpmas as major adhesins of Mycoplasma agalactiae mediating differential cell adhesion and invasion of Vpma expression variants[END_REF] that the various Vpma lipoproteins are involved in host and tissue specificity. However, additional analysis of expressions of Vpma lipoproteins in strain GrTh01 under in vitro and under in vivo conditions would be necessary to confirm these correlations. We assume that the antigenic variation gives M. agalactiae the opportunity to infect and invade various hosts and tissues, hence generally causing multiple symptoms in various small ruminants unless certain adhesins are missing thus reducing host range and tissue tropism.

Fig. 1

 1 Fig. 1 Genome comparison of M. agalactiae strains PG2 (Type strain) (upper), GrTh01 (mddle) and 5632 (lower) whose full circularized genomes are available. Orange circles: bpsA. Green, vpma loci. Red blocks show homologous genome stretches. Magneta: spma locus. Purple blocks: ICE or ICE vestiges (smaller blocks). : IS elements

Fig. 2

 2 Fig. 2 Genetic loci of vpma genes in M. agalactiae strain GrTh01. Arrowhead-Boxes represent open reading frames (ORF). Green arrowheads: vpma. Hatched green arrowheads partial or degenerated vpma genes. Black arrowheads: promoter sequences. Red arrowheads: 21 nt 'vis' motif encoding the Xer1 recombinase recognition site. Grey boxes labelled with 'S': signal sequences. HP: hypothetical protein

Table 1

 1 General properties of M. agalactiae circularized genomes and corresponding strains. Note the full genome sequence of M. agalactiae, strain JF4428 is also available but not included in this table as JF4428 is a sample of strain PG2 that was propagated in vitro as internal diagnostic control for several decencies

		PG2	5632	GrTh01
	Date of isolation	1952	< 1991	2017
	Country	Spain	Spain	Greece
	Source	unknown	articulation	milk
	Host	caprine	caprine	caprine
	Genome size (bp)	877,438	1,006,702	841,635
	G + C (%) Total number of CDS	29.70 657	29.60 782	29.80 626
	rRNAs sets	2	2	2
	tRNAs	34	34	34
	GenBank accession number	CU179680	FP671138	CP039447
	ICE number	0 (+ 2 vestigial)	3 (+ 2 vestigial)	0 (+ 2 vestigial)
	Transposases	1 (+ 2 pseudogenes)	15 (+ 2 pseudogenes)	0 (+ 2 pseudogenes)

Table 2

 2 Allelic profiles and sequence types (ST) of M. agalactiae type strain PG2 and field strains isolated from Greece. Data taken from https:// pubml st. org/ magal actiae/

	Strain	Origin	Year of isolation	Allelic profile					ST
				dnaA	gltX	gyrB	metS	tufA
	NCTC 10123 (PG2)	USA	1983	1	1	1	1	1	1
	GrTh01	Greece	2016	21	1	1	2	1	35
	222F03	Greece	2003	1	1	1	2	1	2
	233F03	Greece	2003	1	1	1	2	1	2
	240F03	Greece	2003	1	1	1	2	1	2
	235F03	Greece	2003	1	1	2	2	1	4
	234F03	Greece	2003	1	1	2	4	1	6
	224F03	Greece	2003	1	2	2	3	1	8
	225F03	Greece	2003	1	2	2	3	1	8
	227F03	Greece	2003	1	3	1	2	1	9

Acknowledgments

During the study, our colleague George Filioussis, who wrote the first draft of the manuscript sadly passed away. Corresponding author JF is grateful to all coauthors who helped to accomplish this study in memoriam of George. We are grateful to Richard Ellis, Animal and Plant Health Agency, Weybridge, UK, for the annotation of the sequence type number strain GrTh01.

Funding

The study partially funded by CEVA HELLAS LLC.

Availability of data and materials

Full genome sequence data of this work are publicly available from NCBI Gen-Bank https:// www. ncbi. nlm. nih. gov/ genba nk/, a member of the International Nucleotide Sequence Database Collaborations under the accession number CP039447.

Authors' contributions

GF and JF conceived the study and wrote the main draft. GF carried out the microbiological analyses. JF performed full genome sequencing and submission to GenBank. GB, EP and NDG performed the clinical studies. L-XN and CC performed the bioinformatic analysis and conceived the figures. All authors read and approved the manuscript prior to submission.

Declarations Ethics approval and consent to participate

All milk samples obtained from animals were collected from routine milking for veterinary medical health check of farm animals. The study therefore did not involve controls under EU Directive 2010 (2010/63/EU). For the purpose of this study, consent from the farmers were sought in advance for the use of these samples by the Laboratory of Microbiology and Infectious Diseases, Faculty of Veterinary Medicine, School of Health Science, Aristotle University of Thessaloniki.

Consent for publication

For the purpose of this study, consent from the animal owners were sought in advance for publishing data from these studies.

Competing interests

The authors declare that they have no financial or personal relationship(s), which may have inappropriately influenced them in writing this article.

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Negisa Ebadi Pour

Franck Dumeignil

Benjamin Katryniok

Laurent Delevoye

Bertrand Revel

Sébastien Paul
email: sebastien.paul@centralelille.fr

Investigating the active phase of Ca-based glycerol polymerization catalysts: On the importance of calcium glycerolate

Keywords: Glycerol, Polyglycerol, Ca-glycerolate, Active phase, Mechanism

Ca-based catalysts are of strong interest for glycerol polymerization reaction due to their high catalytic performances, their wide availability and the absence of any toxicity. In the present study, we investigated the active phase of CaO, Ca(OH)2, CaCO3 and calcium diglyceroxide used as catalysts for this reaction. The solids were analyzed by XRD, solid-state 13 C-NMR and TGA-DSC. XRD evidenced the presence of calcium glycerolate in the spent catalysts when CaO, Ca(OH)2 and calcium diglyceroxide were used as starting materials. This was corroborated by the TGA-DSC results. Further, the SS NMR results showed that the as-formed calcium glycerolate is a mixture of linear Ca(C3H7O3) and cyclic-branched Ca(C3H6O3). Moreover, the recycled catalyst issued, e.g., from CaO was more efficient that the initial catalyst with a glycerol conversion of 47% (vs. 22% initially for CaO) and a 70% (vs. 55%) selectivity to polyglycerols higher than tetraglycerol at 245 °C after 4 h in the presence of 3.5 mol.% of catalyst. As the main outcome, we have shown that Ca-glycerolate is the actual solid active phase, which is formed in situ from CaO and Ca(OH)2 playing the role of catalyst precursors. A mechanism involving the dissolution of CaO and Ca(OH)2, the formation of Ca-glycerolate and its precipitation followed by crystallization, supported by characterization studies, is proposed.

Introduction

Polyglycerols (PGs) are water soluble highly functionalized compounds (i.e., containing many hydroxyl groups), biocompatible and with a high thermal stability. These properties make them highly desirable for many applications in the food, cosmetics, plastic or biomedical sectors [START_REF] Martin | Oligomerization of glycerol -a critical review[END_REF][START_REF] Ciriminna | Understanding the glycerol market: Understanding the glycerol market[END_REF]. While polyglycerols are currently produced from biosourced epichlorohydrin, the change in customer demand -more particularly for cosmetics and food -require products derived from nontoxic and preferably natural ingredients. This strongly encourages the manufacturers to produce PGs directly from glycerol [START_REF] Ebadipour | Alkaline-Based Catalysts for Glycerol Polymerization Reaction: A Review[END_REF].

In the last two decades, attention was paid to glycerol polymerization over alkaline earth oxides-based catalysts such as CaO, BaO and SrO due to their higher catalytic activity [START_REF] Ruppert | Glycerol Etherification over Highly Active CaO-Based Materials: New Mechanistic Aspects and Related Colloidal Particle Formation[END_REF][START_REF] Nieuwelink | CaO/CNF for the Oligomerization of Glycerol[END_REF][START_REF] Gholami | Selective Etherification of Glycerol over Heterogeneous Mixed Oxide Catalyst: Optimization of Reaction Parameters[END_REF] compared to other catalysts such as alkali-modified zeolites [START_REF] Krisnandi | Glycerol Upgrading over Zeolites by Batch-Reactor Liquid-Phase Oligomerization: Heterogeneous versus Homogeneous Reaction[END_REF] or MgAl mixed oxides derived from layered double hydroxides [START_REF] García-Sancho | Etherification of glycerol to polyglycerols over MgAl mixed oxides[END_REF][START_REF] Pérez-Barrado | Influence of acid-base properties of calcined MgAl and CaAl layered double hydroxides on the catalytic glycerol etherification to short-chain polyglycerols[END_REF]. For instance, CaO, SrO and BaO have shown a conversion of nearly 80% with 90% selectivity to di-and triglycerol at 220 °C [START_REF] Ruppert | Glycerol Etherification over Highly Active CaO-Based Materials: New Mechanistic Aspects and Related Colloidal Particle Formation[END_REF], while 96 % glycerol conversion with a 88% selectivity to acrolein was obtained with MgAl-CaAl layered double hydroxides as catalysts at 235 °C [START_REF] Pérez-Barrado | Influence of acid-base properties of calcined MgAl and CaAl layered double hydroxides on the catalytic glycerol etherification to short-chain polyglycerols[END_REF].

Among alkaline earth oxides catalysts, Ca-based catalysts such as bulk CaO, CaO supported on carbon nanofibers [START_REF] Kirby | CaO as Drop-In Colloidal Catalysts for the Synthesis of Higher Polyglycerols[END_REF], dolomite (CaO-MgO mixed oxides) [START_REF] Barros | Glycerol Oligomerization Using Low Cost Dolomite Catalyst[END_REF] exhibited a higher catalytic activity towards oligomers production. Such higher performances were explained by the possible in situ formation of Ca-glyceroxides [START_REF] Ruppert | Glycerol Etherification over Highly Active CaO-Based Materials: New Mechanistic Aspects and Related Colloidal Particle Formation[END_REF][START_REF] Sangkhum | Ca-Mg-Al ternary mixed oxides derived from layered double hydroxide for selective etherification of glycerol to short-chain polyglycerols[END_REF][START_REF] Barros | Glycerol Oligomerization Using Low Cost Dolomite Catalyst[END_REF] during the glycerol polymerization reaction but without any direct evidence.

Ca glyceroxides may present various structures. Potential structures of calcium monoglyceroxide, described by Ca(C3H6O3) and Ca(C3H7O3) formulas, and calcium diglyceroxides (CaDG) with the formula Ca(C3H7O3)2, are represented in Scheme 1. However, in the literature CaDG is referred generally as Ca glyceroxides (plural form) [START_REF] Granados | Leaching and homogeneous contribution in liquid phase reaction catalysed by solids: The case of triglycerides methanolysis using CaO[END_REF] or for sake of simplicity as Ca glyceroxide (singular form) [START_REF] Kouzu | Solid base catalysis of calcium glyceroxide for a reaction to convert vegetable oil into its methyl esters[END_REF][START_REF] Reyero | Heterogenization of the biodiesel synthesis catalysis: CaO and novel calcium compounds as transesterification catalysts[END_REF][START_REF] Esipovich | The structure, properties and transesterification catalytic activities of the calcium glyceroxide[END_REF]; calcium monoglyceroxide being similarly referred as Ca glycerolate [START_REF] Taylor | Preparation and Properties of a Glycerolatocalcium Complex[END_REF].

Thus, to avoid any confusion, in the present work, we will use CaG for Ca glycerolate (i.e., monoglyceroxide) and CaDG for Ca diglyceroxide. Synthesis of Ca-glyceroxides was first reported using a CaO-glycerol system at temperatures from 10 to 150 °C by Grün and Bockish [START_REF] Ad | Glycerinate der Erdalkalien[END_REF] and Fujii and Kondo [START_REF] Fujii | Calcium glyceroxides formed in the System of calcium oxide-glycerol. With 4 figures[END_REF]. Fujii and Kondo reported the formation of 4 types of crystalline glyceroxides depending on the temperature range, namely CaG (CaC3H6O3) (Scheme 1(1-3), further noted CaG(1), CaG(2) and CaG(3)); CaDG (Ca(C3H7O3)2) (Scheme 1 (4-6) , further noted CaDG(4), CaDG(5) and CaDG( 6)); tricalcium octa-glyceroxide (Ca3(C3H7O3)6.(C3H8O3)2) and calcium hexa-glyceroxide (Ca(C3H7O3)2.(C3H8O3)4) using a mass ratio calcium oxide:glycerol of 3:100 at > 100 °C, 40-100 °C, 20-60 °C, < 20 °C, respectively. They reported that different Ca crystalline glyceroxides structures can be formed and interconverted depending on the temperature. For instance, calcium hexa-glyceroxide could be transformed into tricalcium octa-glyceroxide at 18-30 °C, tricalcium octa-glyceroxide to CaDG at 44 °C, while the decomposition of CaDG into CaG was observed in the 130-160 °C temperature range [START_REF] Fujii | Calcium glyceroxides formed in the System of calcium oxide-glycerol. With 4 figures[END_REF].

Similarly, Taylor et al. [START_REF] Taylor | Preparation and Properties of a Glycerolatocalcium Complex[END_REF] synthetized CaG (CaC3H6O3) as a pharmaceutical product, by heating calcium hydroxide and glycerol above 180 °C in a microwave oven. The exact structure of the asobtained CaG obtained was not discussed in this paper.

Later, Kouzu's group demonstrated that CaDG was formed in situ during the transesterification reaction to produce biodiesel starting from CaO as the catalyst precursor [START_REF] Kouzu | Heterogeneous catalysis of calcium oxide used for transesterification of soybean oil with refluxing methanol[END_REF][START_REF] Kouzu | Active phase of calcium oxide used as solid base catalyst for transesterification of soybean oil with refluxing methanol[END_REF]. Although CaDG was reported to be an active catalyst itself for the synthesis of biodiesel by several groups, CaO showed slightly higher catalytic activity compared to that of in situ formed CaDG [START_REF] Kouzu | Active phase of calcium oxide used as solid base catalyst for transesterification of soybean oil with refluxing methanol[END_REF] or to that of CaDG synthesized prior to the reaction [START_REF] Reyero | Heterogenization of the biodiesel synthesis catalysis: CaO and novel calcium compounds as transesterification catalysts[END_REF][START_REF] Catarino | Calcium diglyceroxide as a catalyst for biodiesel production[END_REF]. To the best of our knowledge, except its catalytic role clearly identified in the transesterification reaction, no study has been conducted so far to elucidate the formation of CaG during the glycerol polymerization reaction.

In the present work, we investigate the nature of the active phase in the glycerol polymerization reaction when the catalyst precursors were CaO, Ca(OH)2 , CaCO3 or CaDG. Besides, to explain the insitu CaG formation, a mechanism based on a characterization study using X-ray diffraction (XRD), solid-state 13 C NMR, thermal analysis and scanning electron microscopy is proposed. Furthermore, a mechanism for PGs formation is also proposed based on the mechanism of CaG formation.

Method and Materials

Materials

Calcium hydroxide was purchased from Alfa Aesar. Glycerol (99% purity), calcium nitrate tetrahydrate, calcium carbonate, and HPLC grade methanol (99.9% purity) and ethanol (99.5% purity) were purchased from Sigma-Aldrich. All the chemicals were used as such without any pre-treatment or purification.

Catalyst preparation

Calcined calcium oxide (CaO) was freshly prepared from calcium nitrate tetrahydrate (Ca(NO3)2.4H2O) treated under static air in a calcination oven from room temperature to 700 °C during 2 h with a ramp of 10 °C.min -1 .

CaDG was prepared by a modified procedure previously described in the literature [START_REF] Kouzu | Solid base catalysis of calcium glyceroxide for a reaction to convert vegetable oil into its methyl esters[END_REF][START_REF] Esipovich | The structure, properties and transesterification catalytic activities of the calcium glyceroxide[END_REF] . Namely, a mixture of methanol, glycerol and commercial CaO (molar ratio 38 : 4.2 : 1) was prepared and heated at 60 °C for 4 h. Then, the obtained yellowish precipitate was washed with methanol to remove unreacted glycerol before the remaining solvent was evaporated under vacuum at 90 °C. A yield of 91% to CaDG was obtained.

Catalytic test

Catalytic tests were performed with 218 mmol (20 g) of pure glycerol in a 100 mL Schlenk tube maintained at 245 °C and stirred at 800 rpm with a magnetic bar under N2 atmosphere in the presence of 3.5 mol.% of catalyst (based on moles of glycerol) including Ca(OH)2, CaCO3 and CaDG (loadings of catalysts in wt.% are given in Table 1). After 4 h of reaction, the catalyst was separated by centrifugation and washed with ethanol. The collected catalysts in pasty form were characterized by XRD, before drying at 80 °C in an oven for 4 h prior to further uses or analyses.

Glycerol and polyglycerol analyses

The glycerol analyses were carried out by high performance liquid phase chromatography (HPLC) using a Phenomenex column (organic acid H+) 300 x 7.8 mm and equipped with a refractive index detector, where the mobile phase was 5 mM H2SO4 fed at 0.6 mL/min. The glycerol conversion was calculated by Equation ( 1):

𝐺𝑙𝑦𝑐𝑒𝑟𝑜𝑙 𝑐𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛(%) = 100 * 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑚𝑜𝑙 𝑜𝑓 𝑔𝑙𝑦𝑐𝑒𝑟𝑜𝑙-𝑚𝑜𝑙 𝑜𝑓 𝑢𝑛𝑟𝑒𝑎𝑐𝑡𝑒𝑑 𝑔𝑙𝑦𝑐𝑒𝑟𝑜𝑙 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑚𝑜𝑙 𝑜𝑓 𝑔𝑙𝑦𝑐𝑒𝑟𝑜𝑙 (1)
Polyglycerols analyses were conducted by direct infusion Electrospray Ionization Mass Spectrometry (ESI-MS) in a Waters ESI-MS/MS SYNAPT G2-Si HDMS (High Definition Mass Spectrometer) set in the positive ion mode. ESI source conditions were as follows: heated capillary temperature 300 °C, spray voltage 3 kV and capillary voltage of 60 V. By this technique, PG2 to PG6 were detected.

As a quantitative analysis would have required a pure PG standard for each product, whereas the available standards were mixtures of PGs, a relative PG selectivity has been chosen to evaluate and compare the catalysts performances.

Hence, as indicated in Equations ( 2) and (3) a relative polyglycerol selectivity was defined as being the ratio of the desired polyglycerol (PGx) peak intensity divided by the sum of the peaks intensities of all the PGs observed by direct ESI-MS:

𝑃𝐺 2-3 𝑠𝑒𝑙𝑒𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 100 * 𝑆𝑢𝑚 𝑜𝑓 𝑃𝐺2 𝑎𝑛𝑑 𝑃𝐺3 𝑝𝑒𝑎𝑘 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑖𝑒𝑠 𝑇𝑜𝑡𝑎𝑙 𝑃𝐺𝑠 𝑝𝑒𝑎𝑘 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑖𝑒𝑠 (2)
Where PG2 and PG3 are di-and tri-glycerols respectively.

𝑃𝐺 4+ 𝑠𝑒𝑙𝑒𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 100 * ∑ 𝑃𝐺 𝑖 6 𝑖=4 𝑝𝑒𝑎𝑘 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑖𝑒𝑠 𝑇𝑜𝑡𝑎𝑙 𝑃𝐺𝑠 𝑝𝑒𝑎𝑘 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑖𝑒𝑠 (3)
Where PG4+ represent the polyglycerols including tetra-, penta-and hexaglycerols.

Characterization of the catalysts

XRD

X-ray diffraction patterns of the catalysts were recorded on a D8 Discover X-Ray Diffractometer from Bruker using an X-Ray tube in Cu (Kα) radiation (λ = 1.54060 Å). The diffraction angle 2θ was taken in the 10-60° range with steps of 0.01° per second.

Thermal Analyses

Thermo Gravimetric and Differential Scanning Calorimetry (TGA and DSC) were performed using a Setaram Labsys instrument. A continuous heating was applied from room temperature to 900 °C with a heating rate of 10 °C min -1 under a flow of nitrogen of 30 mL/min.

Solid state-NMR

13 C solid-state NMR spectra were recorded using a 400 MHz Bruker BioSpin spectrometer equipped with AVANCE-III consoles at a spinning rate of 10 kHz.

SEM

To study the morphology of the catalysts, scanning electron microscopy (SEM) pictures were recorded on a Hitachi S3600N electron microscope equipped with a Thermo Ultradry EDS detector using an acceleration voltage of 15 kV.

Results and discussion

Catalytic performances

The catalytic performances were evaluated in presence of 3. The eye-catching similar catalytic activity observed for CaO, Ca(OH)2 and CaDG suggests that the polymerization reaction was most probably catalyzed by the same in-situ formed active phase. Thus, to investigate the actual nature of this active phase, a characterization study was performed on all the used catalysts.

Performance of recycled catalysts

The catalytic performance of used catalysts obtained from CaO (CaG-O) was assessed by its reutilization in the reaction. The results revealed that, in the same reaction conditions, 47% of glycerol conversion and 70% of selectivity to PG4+ could be reached when recycled CaG-O was used as catalyst, while conversion and selectivity were only 22% and 55%, respectively, when starting initially from CaO (Figure 1). This increase can be explained by the dissolution of CaG in the new reaction medium where the reaction is then catalyzed homogenously. This dissolution of CaG in the 2 nd run, which is not observed in the 1 st test can be explained by the mechanism of CaG formation proposed in Section 3.4.

Catalyst characterization

XRD

First, XRD analyses were carried out over the collected spent materials recovered after the reaction when CaO, Ca(OH)2, CaCO3 and CaDG were used initially as catalysts. To verify the structural transformation of the active phase without affecting the latter, the collected catalysts were analyzed in the pasty form without any intermediate washing and drying steps.

The XRD diffractograms evidenced structural modifications of calcium oxide, calcium hydroxide and CaDG under test conditions. Figure 2(e-g) exhibits XRD patterns of the used catalysts. A sharp peak at 2θ = 10.4° (200) (main peak) was observed for spent CaO, Ca(OH)2 and CaDG after 4 h of reaction. This diffraction pattern was similar to those previously reported in the literature [START_REF] Taylor | Preparation and Properties of a Glycerolatocalcium Complex[END_REF][START_REF] Fujii | Calcium glyceroxides formed in the System of calcium oxide-glycerol. With 4 figures[END_REF] for CaG with a cubic lattice (001) and also for other crystallized glycerolate metal complexes such as Zn (Zn(C3H6O3)) with a cubic lattice (100) [START_REF] Reinoso | Zinc glycerolate as a novel heterogeneous catalyst for the synthesis of fatty acid methyl esters[END_REF]. Moreover, no peaks attributed either to CaO (Figure 1-c) with 2θ at 32.2, 37.4 and 53.9 (PDF-99-0070) nor to Ca hydroxide (Figure 1-b) with 2θ at 18.14°, 28.7°, 34.2°, and 50.8° (PDF-00-087-0673) were observed for the used catalysts, suggesting that the latter was transformed quantitively to CaG. On the other hand, the XRD pattern of the used catalyst CaCO3 clearly showed that the latter remained in its initial calcite form (PDF-01-071-3699). Thus, these results show that the higher catalytic performances of CaO, Ca(OH)2 and CaDG compared to that of CaCO3 could be attributed to the in-situ formation of CaG as an active phase. XRD analysis was also carried out on the used sample issued from CaO after washing with ethanol and drying at 80 °C. The diffractograms (not shown here) revealed only one peak at 2θ = 10.4° corresponding to the CaG structure. This showed that -after all -the washing and drying steps did not affect the crystalline structure of CaG. Thus, other characterization techniques were performed on the washed and dried collected samples.

Thermal analyses

TGA was performed on the used samples to determine the thermal stability of the CaG obtained after reaction using initially CaO and Ca(OH)2 as catalysts. The samples were labeled "CaG-O" and "CaG-H", respectively.

The TGA curve for CaG-O showed multistep weight loss behavior with a first weight loss of 5 wt.% between 130 and 343 °C, followed by 2 steps of mass loss of 14 wt.% and 3.5 wt.%, respectively, up to 550 °C. According to the DSC curve, shown in Figure 3-a, each of these mass losses correspond to an exothermic peak, which could be attributed to phase transitions of the glyceroxides crystallites. A final weight loss of 34 wt.% was recorded between 550 and 835 °C. The total weight loss of 56.5 wt.% for CaG-O is in good agreement with the theoretical value of 57 % for CaG, i.e. when it is totally decomposed to form CaO and one molecule of glycerol.

Similarly, the TGA for CaG-H curve (Figure 3-b) also showed a multistep weight loss with a first weight loss of about 5.8 wt.% between 80 and 140 °C followed by 3 steps of mass losses up to 525 °C (12 wt.%). Three main exothermic peaks in DSC curve between 300 and 500 °C also correspond to these weight losses. A final weight loss of 39 wt.% was recorded between 525 and 830 °C. The total weight loss of 56.8 wt.% for the CaG-H is also very close to the theoretical value of 57% for total decomposition of CaG to form CaO and glycerol.

The TGA-DSC profiles of CaDG are shown in Figure 3-c. Four weight loss steps can be observed. The first step was observed between 100 and 260 °C with a mass loss of 31 wt.%. For this step, the DSC curve showed an endothermic peak, whereby the weight loss was attributed to the partial decomposition of CaDG to CaG where the theoretical weight loss for total decomposition is 41.5% (to form CaG and one molecule of glycerol). However, Fujii and Kondo [START_REF] Fujii | Calcium glyceroxides formed in the System of calcium oxide-glycerol. With 4 figures[END_REF] reported a decomposition of CaDG to CaG in the range of 130-160 °C and similarly León-Reina et al. [START_REF] León-Reina | Structural and surface study of calcium glyceroxide, an active phase for biodiesel production under heterogeneous catalysis[END_REF] observed a peak corresponding to CaDG decomposition between 160 and 200 °C. This variation in decomposition temperatures, in the literature and our sample, can be attributed to the presence of different CaDG structures, linear and branched (Scheme 1), which can consequently affect the global thermal stability [START_REF] Wall | Effect of structure on the thermal decomposition of polymers[END_REF].

Further losses were observed in the 260-534 °C temperature range with a total loss of 21 wt.%. The final weight loss of 22.8 wt.%, which was recorded between 534 and 830 °C most likely corresponds to the formation of CaO. The total weight loss of 74.4% is very close to the theoretical one (74.8%) expected when CaDG is totally decomposed to CaO plus two molecules of glycerol.

Solid state-NMR

The structures of CaG-O and CaG-H used samples were further studied by solid-state 13 C NMR. For these compounds, three peaks were observed at 67.9 ppm, 70.2 ppm and 74.9 ppm (Figure 4 a-b).

Based on the various possible CaG structures (Scheme 1), for cyclic CaG(1) of formula Ca(C3H6O3), 2 peaks corresponding to -CH2OCa (2 equivalent carbons) and -CH-OH are expected on the 13 C NMR spectrum. For cyclic-branched form CaG(2), 3 peaks corresponding to -CH2OH, -CH2-OCa and -CH-OCa are expected and for the linear CaG(3) of formula Ca(C3H7O3) also 3 peaks corresponding to the carbons in -CH2OH, -CH2-OCa and -CH-OH are expected.

Thus, the three observed peaks in CaGs spectra at 67.9 ppm, 70.2 ppm and 74.9 ppm (Figure 4 a-b) could be assigned either to the -CH2OH, -CH2-OCa and -CH-OH carbon chains of glycerolate, respectively, in linear form (CaG(3)); or to the -CH2OH, -CH2-OCa and -CH-OCa carbon chains in CaG [START_REF] Ciriminna | Understanding the glycerol market: Understanding the glycerol market[END_REF]. Besides, from these spectra one can concluded that the CaGs are not in a fully cyclic conformation CaG(1) on Scheme 1.

It also should be noted that 13 C CPMAS SS NMR is not a quantitative technique, thus, it is difficult to conclude if the CaGs are in a unique structure or a mixture of CaG(2) and CaG(3). Furthermore, for CaDG six peaks at 63.3 ppm, 64.3 ppm, 65.2 ppm, 65.8 ppm, 71.9 ppm and 75.1 ppm were observed in the 13 C NMR spectrum (Figure 4-c). These peaks can be attributed to the carbon chains of glycerol in the different CaDG structures (Scheme 1 (4-6)). However, 6, 2 and 3 peaks are expected to be observed for CaDG(4), CaDG( 5) and CaDG(6), respectively. Thus, once again due to the fact that 13 C CPMAS SS NMR is not a quantitative technique, it cannot be concluded if CaDG is pure CaDG(4) or a mixture of CaDGs. Nevertheless, similar spectra for CaDG were previously reported by Kouzou et al. [START_REF] Kouzu | Solid base catalysis of calcium glyceroxide for a reaction to convert vegetable oil into its methyl esters[END_REF]. It could thus be concluded that CaDG is not present in the in-situ formed CaG.

SEM

The morphologies of the used catalysts CaG-O, CaDG and CaO were analysed by SEM (Figure 5). Fig. 5a shows that CaG-O presents large "potato-shaped" particles with irregularities on the surface (Fig. 5a-left). For CaDG (Figure 5b), large particles with an angular rock shape were observed. This is in good agreement with what was previously reported in the literature for CaDG [START_REF] Esipovich | The structure, properties and transesterification catalytic activities of the calcium glyceroxide[END_REF][START_REF] Kouzu | Active phase of calcium oxide used as solid base catalyst for transesterification of soybean oil with refluxing methanol[END_REF]. From Fig. 4c, it can be seen that calcium oxide is in the form of large agglomerates with outgrowths.

Main conclusions on the characterization study

From the XRD results, it can be concluded that CaO and Ca(OH)2 are transformed to calcium glycerolate during glycerol polymerization reaction. These catalysts also showed a similar thermal behavior with a total weight loss of 56.

Mechanism of CaGs formation

In order to explain the in situ formation of CaGs, a mechanism based on dissolution-precipitationcrystallization for CaGs formation starting either from CaO and Ca(OH)2 is proposed (Scheme 2). Note that CaG can also be formed from thermal decomposition CaDG (Figure 2-c). A similar mechanism was previously suggested [START_REF] Sánchez-Cantú | Direct synthesis of calcium diglyceroxide from hydrated lime and glycerol and its evaluation in the transesterification reaction[END_REF] for the formation of CaDG from a mixture of glycerol and Ca(OH)2 at 70 °C under transesterification reaction conditions.

Following this mechanism CaG is formed from CaO (or Ca(OH)2) by a dissolution of the precursor; its conversion in the liquid phase is followed by a precipitation step, which allows CaG to eventually crystallize.

For the dissolution of CaO (Scheme 2 -I and II) and Ca(OH)2 (Scheme 2 -III), the Ca cation interacts with the oxygen lone pair from the terminal -OH group in glycerol. In the following, the hydroxyl group of calcium hydroxide (or of CaO) abstracts the proton of the as-activated -OH group of glycerol. Consequently, a glycerolate anion and a Ca 2+ cation are formed, which coordinate together with OH - in a Ca-glycerolate-OH -complex. When the medium is saturated, crystallization takes place, whereby Ca coordinates with more glycerolate anions (Scheme 2-IV). Besides, , as reported previously [START_REF] Ruppert | Glycerol Etherification over Highly Active CaO-Based Materials: New Mechanistic Aspects and Related Colloidal Particle Formation[END_REF], colloidal Ca(OH)2 is formed due to the presence of the water concomitantly formed during the reaction.

Scheme 2. Proposed mechanism for CaG formation in the PGs synthesis reaction from glycerol.

However, when CaO and Ca(OH)2 are converted to solid CaG, colloidal Ca(OH)2, Ca 2+ and hydroxide ions in the liquid phase (Eq. 1-3), an equilibrium takes place between CaG and colloidal Ca(OH)2 (Eq(4)).

𝐶𝑎𝑂 + 𝐻 2 𝑂 → 𝐶𝑎(𝑂𝐻) Hence, a simultaneous dissolution of CaG to Ca ion and intermediate glyceroxide species and its reformation takes place due to the presence of colloidal Ca(OH)2, as previously described.

It is clear that after recovering the CaG from the first reaction medium, when CaO was the precursor, and during the washing step, the colloidal forms of Ca(OH)2 were washed out. Thus, CaG in the fresh medium (glycerol) began to solubilize while it could not reform in situ due to the absence of protonated oxygen. This mechanism also confirms that CaG can only be formed from CaO and Ca(OH)2 because of the presence of protonated oxygen in these compounds, and thus cannot form from CaCO3.

Polyglycerol formation mechanism

By understanding the CaG formation mechanism, the polyglycerol formation mechanism can also be explained. Based on our recent review [START_REF] Ebadipour | Alkaline-Based Catalysts for Glycerol Polymerization Reaction: A Review[END_REF], PGs could form in both homogeneously and heterogeneously catalyzed reactions even when the catalyst precursor is initially solid due to its partial solubility under the reaction conditions. As aforementioned in the CaG formation mechanism, Ca and hydroxyl ions are simultaneously present in the reaction media. Thus, at this stage the homogenous reaction can take place as described previously in the literature [START_REF] Martin | Oligomerization of glycerol -a critical review[END_REF][START_REF] Salehpour | Towards the Sustainable Production of Higher-Molecular-Weight Polyglycerol[END_REF]. On the other hand, the heterogeneously catalyzed reaction could also take place by CaG which is formed in situ. The mechanism of polymerization of glycerol by CaG is presented in Scheme 3.

In this proposed mechanism, CaG activates the carbon-oxygen bond of the terminal hydroxyl group of glycerol. By decreasing its electron density through the coordination with Ca, the glycerol can undergo a nucleophilic attack by a glycerolate anion thus removing the hydroxyl group.

It should be noted that we chose here to represent linear CaG structure to simplify the mechanism. Scheme 3. Proposed mechanism for diglycerol formation.

Conclusion

In the present paper, the nature of the active phase in glycerol polymerization reaction was investigated by characterizing the spent catalysts issued from CaO, Ca(OH)2 and CaDG used as catalyst precursors. The XRD diffractograms revealed a good agreement with the glycerolate (CaG) patterns reported in the literature. Besides, while the obtained calcium glycerolate, CaG-O and CaG-H, respectively issued from CaO and Ca(OH)2 showed a similar thermal behavior, namely a total weight loss close to 57 wt.%, which is in very good agreement with the theoretical value for CaG decomposition, the phase transitions observed between 350 °C and 550 °C suggested that Ca glycerolates are simultaneously present under different structures. Furthermore, the SS 13 C NMR results confirmed the XRD and thermal analyses and proved that CaG samples most probably consist in mixtures of cyclic-branched CaG(2) and linear CaG(3) and do contain any cyclic CaG or CaDG. We also showed that the similarity between catalytic performances of CaO, Ca(OH)2 and CaDG is due to the in situ formation of CaG actually acting as the catalyst. Furthermore, 47% of glycerol conversion was obtained on CaG, vs. 22% on CaO used as the catalyst precursor. This increase in catalytic performances can be explained by a mechanism of CaG in situ formation, which comprises the simultaneous dissolution of the catalyst precursors and the precipitation of CaG after recombination of the intermediate with glycerol. A mechanism of glycerol polymerization reaction itself could be subsequently proposed, in which the in situ formed CaG and the glyceroxide ion participate in the formation of diglycerol.

Scheme 1 .

 1 Scheme 1. Potential structures of Ca glyceroxides, 1) cyclic CaG with Ca(C3H6O3) formula; 2) cyclic-branched CaG with Ca(C3H6O3) formula; 3) linear CaG with Ca(C3H7O3) formula; and Ca diglyceroxides with Ca(C3H7O3)2 formula, 4,5) branched CaDG and 6) linear CaDG.

Figure 1 .

 1 Figure 1. Catalytic performances of CaO and CaG-O in glycerol polymerization reaction

Figure 2 .

 2 Figure 2. XRD patterns of a) fresh CaCO3, b) fresh Ca(OH)2, c) fresh CaO and d) freshly prepared CaDG and spent catalyst issued from experiments in presence of e) CaCO3, f) Ca(OH)2, g) CaO and h) CaDG.

Figure 3 .

 3 Figure 3. TGA-DSC profiles for a) CaG-O, b) CaG-H and c) CaDG

Figure 4 .

 4 Figure 4. 13 C SS-NMR spectra of a) CaG-O, b) CaG-H and c) CaDG.

Figure 5 .

 5 Figure 5. SEM analysis of: a) CaG-O, b) CaDG and c) CaO samples

  5 and 56.8 wt.% respectively for CaG-O and CaG-H, which agrees with the theoretical value of 57% when CaG is totally decomposed to form CaO. These results strongly suggest that CaG-O and CaG-H only contain CaGs, i.e., do not contain any CaDG. However, the phase transitions observed between 350 °C and 550 °C for CaG-O and CaG-H suggest that various structures of CaGs are probably present. Solid state13 C NMR results were also consistent with XRD and TGA-DSC analyses, confirming that CaG-O and CaG-H contain no CaDG or other Ca glyceroxides suggesting that these samples are mixtures of the CaG(2) and CaG(3) species.Thus, based on the characterization studies a mechanism is hereafter proposed to explain the in-situ CaGs formation.

Table 1 .

 1 5 mol% of CaO, Ca(OH)2, CaCO3 and of freshly prepared CaDG catalysts. The results are presented in Table1. CaO, Ca(OH)2 and CaDG all led to ~ 22 % glycerol conversion at 245 °C after 4 h of reaction with a selectivity to diglycerols (linear, branched and cyclic) of 12.5, 8 and 19 %, respectively; whereas the glycerol conversion was lower in the presence of CaCO3 compared to other catalysts (10%) and the selectivity to PG2 was very high (83%). Higher polyglycerols (PG4+) including tetra-, penta-and hexaglycerol were obtained in the presence of CaO, Ca(OH)2 and CaDG. Catalytic performance of glycerol in the presence of 3.5 mol% of CaO, Ca(OH)2 , CaCO3 and CaDG.

	Catalyst	Catalyst	Glycerol	PG2	PG3	PG4+
		amount	conversion	Selectivity	Selectivity	Selectivity
		(wt.%)	(%)	(%)	(%)	(%)
	CaO	2.13	22	12.5	32	55.4
	Ca(OH)2	2.8	22	8	37	55
	CaCO3	3.8	10	83	17	-
	CaDG	8.4	23	19	43	38

Acknowledgements

Chevreul Institute (FR 2638), Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation, Région Hauts-de-France and FEDER and Centrale Lille are acknowledged for supporting and funding partially this work. The authors would like to warmly thank Dr. Joël Barrault for providing us PGs samples for the analytic methods calibration.

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: