Ricardo A R Machado
email: ricardo.machado@unine.ch

Arthur Muller

Shimaa M Ghazal

Aunchalee Thanwisai

Sylvie Pages

Helge B Bode

Mona A Hussein

Kamal M Khalil

Louis S Tisa Photorhabdus

Sylvie Pagès

Louis S Tisa

P Namnaonensis Pb45

P Luminescens

Subsp Luminescens

P Cinerea Dsm

T P Thracensis Dsm

P Hainanensis Dsm

T P Akhurstii Dsm

P Temperata Dsm

P Heterorhabditis

Subsp Aluminescens

P Aegyptia Ba1

T P Noenieputensis Dsm

Keywords: aluminescens subsp. nov., Photorhabdus heterorhabditis subsp. heterorhabditissubsp. nov., Photorhabdus australis subsp. thailandensis subsp. nov., Photorhabdus australis subsp. australis subsp. nov., and Photorhabdus aegyptia sp. nov. isolated from Heterorhabditisentomopathogenic nematodes heterorhabditis subsp. heterorhabditis subsp. nov., Photorhabdus australis subsp. thailandensis subsp. nov., Photorhabdus australis subsp. australis subsp. nov., dDDH, Heterorhabditis, Photorhabdus, polyphasic classification, whole genome sequencing. Whole genome

come

To cite this version:

Ricardo A. R. Machado, Arthur Muller, Shimaa M. Ghazal, Aunchalee Thanwisai, Sylvie Pages, et al.. Photorhabdus heterorhabditis subsp. aluminescens subsp. nov., Photorhabdus heterorhabditis subsp. heterorhabditissubsp. nov., Photorhabdus australis subsp. thailandensis subsp. nov., Photorhabdus australis subsp. australis subsp. nov., and Photorhabdus aegyptia sp. nov. isolated from Heterorhabditisentomopathogenic nematodes. International Journal of Systematic and Evolutionary Microbiology, 2021, 71 (1), 10.1099/ijsem.0.004610. hal-03143425 Species of the bacterial genus Photorhabdus live in a close symbiotic relationship with Heterorhabditis entomopathogenic nematodes (EPNs) [START_REF] Clarke | Photorhabdus: a tale of contrasting interactions[END_REF]. EPNs are soil-inhabiting organ-isms that parasitize and reproduce inside small arthropods [START_REF] Poinar | A new strain of entomopathogenic nematode and geographical distribution of Neoaplectana carpocapsae Weiser (Rhabditida, Steinernematidae)[END_REF][START_REF] Khan | Chromonema heliothidis n. gen., n. sp. (Steinernematidae, Nematoda), a parasite of Heliothis zea (Noctuidae, Lepidoptera), and other insects[END_REF]. They colonize their prey by penetrating through the cuticle or natural openings such as the mouth, spiracles or the anus, and crawl towards the haemocoel where they release their Photorhabdus symbiotic bacterial partners [START_REF] Kaya | Entomopathogenic nematodes[END_REF]. Photorhabdus bacteria multiply, produce inmunosuppressors, digestive proteins and secondary metabolites that cause toxaemia, septicemia and eventually kill the infected organism [START_REF] Clarke | Photorhabdus: a tale of contrasting interactions[END_REF][START_REF] Lacey | Entomopathogenic nematodes for control of insect pests above and below ground with comments on commercial production[END_REF][START_REF] Tobias | Genome comparisons provide insights into the role of secondary metabolites in the pathogenic phase of the Photorhabdus life cycle[END_REF][START_REF] Joyce | A hexA homologue from Photorhabdus regulates pathogenicity, symbiosis and phenotypic variation[END_REF][START_REF] Blackburn | A novel insecticidal toxin from Photorhabdus luminescens, toxin complex A (TCA), and its histopathological effects on the midgut of Manduca sexta[END_REF]. The genus Photorhabdus was described by [START_REF] Boemare | DNA relatedness between Xenorhabdus spp. (Enterobacteriaceae), symbiotic bacteria of entomopathogenic nematodes, and a proposal to transfer Xenorhabdus luminescens to a new genus, Photorhabdus gen. nov[END_REF] to include symbiotic bacteria of Heterorhabditis EPNs [START_REF] Boemare | DNA relatedness between Xenorhabdus spp. (Enterobacteriaceae), symbiotic bacteria of entomopathogenic nematodes, and a proposal to transfer Xenorhabdus luminescens to a new genus, Photorhabdus gen. nov[END_REF]. Since then, several species and subspecies have been described [START_REF] Boemare | DNA relatedness between Xenorhabdus spp. (Enterobacteriaceae), symbiotic bacteria of entomopathogenic nematodes, and a proposal to transfer Xenorhabdus luminescens to a new genus, Photorhabdus gen. nov[END_REF][START_REF] Akhurst | Taxonomy of Australian clinical isolates of the genus Photorhabdus and proposal of Photorhabdus asymbiotica subsp. asymbiotica subsp. nov. and P. asymbiotica subsp. australis subsp. nov[END_REF][START_REF] Ferreira | Photorhabdus luminescens subsp. noenieputensis subsp. nov., a symbiotic bacterium associated with a novel Heterorhabditis species related to Heterorhabditis indica[END_REF][START_REF] Ferreira | Photorhabdus heterorhabditis sp. nov., a symbiont of the entomopathogenic nematode Heterorhabditis zealandica[END_REF][START_REF] Fischer-Le Saux | Polyphasic classification of the genus Photorhabdus and proposal of new taxa: P. luminescens subsp. luminescens subsp. nov., P. luminescens subsp. akhurstii subsp. nov., P. luminescens subsp. laumondii subsp. nov., P. temperata sp. nov., P. temperata subsp. temperata subsp. nov. and P. asymbiotica sp. nov[END_REF][START_REF] Glaeser | Photorhabdus luminescens subsp. namnaonensis subsp. nov., isolated from Heterorhabditis baujardi nematodes[END_REF][START_REF] Hazir | Two new subspecies of Photorhabdus luminescens, isolated from Heterorhabditis bacteriophora (Nematoda: Heterorhabditidae): Photorhabdus luminescens subsp. kayaii subsp. nov. and Photorhabdus luminescens subsp. thracensis subsp. nov[END_REF][START_REF] Machado | Photorhabdus khanii subsp. guanajuatensis subsp. nov., isolated from Heterorhabditis atacamensis, and Photorhabdus luminescens subsp. mexicana subsp. nov., isolated from Heterorhabditis mexicana entomopathogenic nematodes[END_REF][START_REF] Machado | Whole-genome-based revisit of Photorhabdus phylogeny: proposal for the elevation of most Photorhabdus subspecies to the species level and description of one novel species Photorhabdus bodei sp. nov., and one novel subspecies Photorhabdus laumondii subsp. clarkei subsp. nov[END_REF][START_REF] Tailliez | Phylogeny of Photorhabdus and Xenorhabdus based on universally conserved protein-coding sequences and implications for the taxonomy of these two genera. proposal of new taxa: X. vietnamensis sp. nov., P. luminescens subsp. caribbeanensis subsp. nov., P. luminescens subsp. hainanensis subsp. nov., P. temperata subsp. khanii subsp. nov., P. temperata subsp. tasmaniensis subsp. nov., and the reclassification of P. luminescens subsp. thracensis as P. temperata subsp. thracensis comb. nov[END_REF][START_REF] Tóth | Photorhabdus temperata subsp. cinerea subsp. nov., isolated from Heterorhabditis nematodes[END_REF][START_REF] Thomas | Xenorhabdus gen. nov., a genus of entomopathogenic, Nematophilic bacteria of the family Enterobacteriaceae[END_REF][START_REF] Szállás | Phylogenetic evidence for the taxonomic heterogeneity of Photorhabdus luminescens[END_REF][START_REF] Orozco | Characterization and phylogenetic relationships of Photorhabdus luminescens subsp. sonorensis (γ-Proteobacteria: Enterobacteriaceae), the bacterial symbiont of the entomopathogenic nematode Heterorhabditis sonorensis (Nematoda: Heterorhabditidae)[END_REF][START_REF] An | Photorhabdus temperata subsp. stackebrandtii subsp. nov. (Enterobacteriales: Enterobacteriaceae)[END_REF][START_REF] An | Photorhabdus luminescens subsp. kleinii subsp. nov. (Enterobacteriales: Enterobacteriaceae)[END_REF]. Currently, the genus Photorhabdus contains the following [START_REF] Tóth | Photorhabdus temperata subsp. cinerea subsp. nov., isolated from Heterorhabditis nematodes[END_REF] [START_REF] Machado | Photorhabdus khanii subsp. guanajuatensis subsp. nov., isolated from Heterorhabditis atacamensis, and Photorhabdus luminescens subsp. mexicana subsp. nov., isolated from Heterorhabditis mexicana entomopathogenic nematodes[END_REF][START_REF] Machado | Whole-genome-based revisit of Photorhabdus phylogeny: proposal for the elevation of most Photorhabdus subspecies to the species level and description of one novel species Photorhabdus bodei sp. nov., and one novel subspecies Photorhabdus laumondii subsp. clarkei subsp. nov[END_REF].

Through the years, the taxonomy and nomenclature of this bacterial group have undergone profound changes as more strains have been isolated and new technological capabilities to characterize them have emerged [START_REF] Boemare | DNA relatedness between Xenorhabdus spp. (Enterobacteriaceae), symbiotic bacteria of entomopathogenic nematodes, and a proposal to transfer Xenorhabdus luminescens to a new genus, Photorhabdus gen. nov[END_REF][START_REF] Fischer-Le Saux | Polyphasic classification of the genus Photorhabdus and proposal of new taxa: P. luminescens subsp. luminescens subsp. nov., P. luminescens subsp. akhurstii subsp. nov., P. luminescens subsp. laumondii subsp. nov., P. temperata sp. nov., P. temperata subsp. temperata subsp. nov. and P. asymbiotica sp. nov[END_REF][START_REF] Machado | Whole-genome-based revisit of Photorhabdus phylogeny: proposal for the elevation of most Photorhabdus subspecies to the species level and description of one novel species Photorhabdus bodei sp. nov., and one novel subspecies Photorhabdus laumondii subsp. clarkei subsp. nov[END_REF][START_REF] Tailliez | Phylogeny of Photorhabdus and Xenorhabdus based on universally conserved protein-coding sequences and implications for the taxonomy of these two genera. proposal of new taxa: X. vietnamensis sp. nov., P. luminescens subsp. caribbeanensis subsp. nov., P. luminescens subsp. hainanensis subsp. nov., P. temperata subsp. khanii subsp. nov., P. temperata subsp. tasmaniensis subsp. nov., and the reclassification of P. luminescens subsp. thracensis as P. temperata subsp. thracensis comb. nov[END_REF][START_REF] Szállás | Phylogenetic evidence for the taxonomic heterogeneity of Photorhabdus luminescens[END_REF]. As a result, the taxonomy of Photorhabdus is apparently clearer and whether a bacterial strain represents a new taxon or not is more straightforward to determine than in the past. However, there are still several taxonomic uncertainties, especially regarding bacterial strains that are highly similar to the type strain of the species that they were circumscribed or that were not formally characterized at the taxonomic level. This is the case of at least three bacterial strains: BA1, PB68.1 and Q614 [START_REF] Ferreira | Photorhabdus heterorhabditis sp. nov., a symbiont of the entomopathogenic nematode Heterorhabditis zealandica[END_REF][START_REF] Ghazal | Draft genome sequence of Photorhabdus luminescens strain BA1, an entomopathogenic bacterium isolated from nematodes found in Egypt[END_REF][START_REF] Thanwisai | Diversity of Xenorhabdus and Photorhabdus spp. and their symbiotic entomopathogenic nematodes from Thailand[END_REF][START_REF] Akhurst | A non-luminescent strain of Xenorhabdus luminescens (Enterobacteriaceae)[END_REF][START_REF] Hussein | Isolation and characterization of two heterorhabditid and one steinernematid nematodes from Egypt[END_REF]. They were isolated from the intestines of Heterorhabditis EPNs that were recovered from soil samples in Egypt, Thailand and Australia, respectively. These strains were initially classified as P. luminescens BA1, P. australis subsp. australis PB68.1 and Xenorhabdus luminescens Q614. Based on the most recent taxonomy and nomenclature of this bacterial group, these strains are currently classified as P. luminescens BA1, P. australis subsp. australis PB68.1 and P. heterorhabditis Q614. This classification relies to a great extent on 16S rRNA and/or housekeeping gene sequences. As it recently became evident that housekeeping gene and 16S rRNA gene sequences provide insufficient information to resolve the phylogenetic relationships of this bacterial group, particularly of closely related taxa, we revisited the phylogenetic relationships of these strains based on whole genome sequences [START_REF] Machado | Photorhabdus khanii subsp. guanajuatensis subsp. nov., isolated from Heterorhabditis atacamensis, and Photorhabdus luminescens subsp. mexicana subsp. nov., isolated from Heterorhabditis mexicana entomopathogenic nematodes[END_REF][START_REF] Machado | Whole-genome-based revisit of Photorhabdus phylogeny: proposal for the elevation of most Photorhabdus subspecies to the species level and description of one novel species Photorhabdus bodei sp. nov., and one novel subspecies Photorhabdus laumondii subsp. clarkei subsp. nov[END_REF]. To this end, we conducted whole genome-based phylogenetic analyses and sequence comparative studies and found out that these three strains actually represent new taxa within the genus Photorhabdus. Here we characterize these strains to describe these new taxa and present our taxonomic conclusions.

To physiologically, biochemically and morphologically characterize strains BA1 T , PB68.1 T and Q614 T , we used bacterial cultures from a single primary form colony of each strain. Bacteria primary forms were determined by examining colony characteristics on NBTA plates [Luria-Bertani (LB) agar plates supplemented with 25 mg ml -1 bromothymol blue and 4 mg ml -1 triphenyl-2,3,5-tetrazolium chloride]. The selected primary form colony was further sub-cultured and maintained on LB agar plates at 28 °C. Cell morphology was observed under a Zeiss light microscope at a magnification of ×1000, with cells grown for 5 days at 28 °C on LB agar plates. Motility was tested on soft agar as described [START_REF] Tremblay | Improving the reproducibility of Pseudomonas aeruginosa swarming motility assays[END_REF]. Cytochrome oxidase production was tested on discs containing N,N-dimethyl-p-phenylenediamine oxalate and α-naphthol according to manufacturer's conditions (Sigma-Aldrich). Catalase activity was determined by adding a drop of 10 % (v/v) H 2 O 2 into 50 µl of a liquid LB-grown, 24-h-old bacterial culture. The ability of bacterial strains to absorb dye was tested by growing the cells on NBTA agar containing bromothymol blue and triphenyl-2,3,5-tetrazolium chloride (Sigma-Aldrich) [START_REF] Akhurst | Phenotypic and DNA relatedness between nematode symbionts and clinical strains of the genus Photorhabdus (Enterobacteriaceae)[END_REF]. Bioluminescence was determined from liquid cultures using a TriStar LB 942 Multimode Microplate Reader (Berthold Technologies). API 20E strips were used according to manufacturer's instructions (BioMérieux). In this case, strains BA1 T , Q614 T and PB68.1 T were tested in parallel and the results obtained were compared to those published previously and obtained using all Photorhabdus type strains [START_REF] Machado | Photorhabdus khanii subsp. guanajuatensis subsp. nov., isolated from Heterorhabditis atacamensis, and Photorhabdus luminescens subsp. mexicana subsp. nov., isolated from Heterorhabditis mexicana entomopathogenic nematodes[END_REF].

To molecularly characterize strains BA1 T , Q614 T and PB68.1 T , we reconstructed phylogenetic relationships based on 16S rRNA gene sequences and whole genome sequences, and calculated sequence similarity scores. As the full genome sequences of certain type strains are not publicly available, the set of strains used to reconstruct phylogenetic relationships based on 16S rRNA gene sequences, whole genome sequences and biochemical tests are slightly different. Genome sequences of BA1 T , Q614 T and PB68.1 T were obtained as described previously [START_REF] Tobias | Genome comparisons provide insights into the role of secondary metabolites in the pathogenic phase of the Photorhabdus life cycle[END_REF][START_REF] Machado | Photorhabdus khanii subsp. guanajuatensis subsp. nov., isolated from Heterorhabditis atacamensis, and Photorhabdus luminescens subsp. mexicana subsp. nov., isolated from Heterorhabditis mexicana entomopathogenic nematodes[END_REF][START_REF] Machado | Whole-genome-based revisit of Photorhabdus phylogeny: proposal for the elevation of most Photorhabdus subspecies to the species level and description of one novel species Photorhabdus bodei sp. nov., and one novel subspecies Photorhabdus laumondii subsp. clarkei subsp. nov[END_REF][START_REF] Ghazal | Draft genome sequence of Photorhabdus luminescens strain BA1, an entomopathogenic bacterium isolated from nematodes found in Egypt[END_REF]. Whole genome sequence similarities were calculated by the digital DNA-DNA hybridization (dDDH) method using formula 2 of the Genome-to-Genome Distance Calculator (GGDC) web service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) [START_REF] Meier-Kolthoff | Genome sequence-based species delimitation with confidence intervals and improved distance functions[END_REF][START_REF] Meier-Kolthoff | Complete genome sequence of DSM 30083(T), the type strain (U5/41(T)) of Escherichia coli, and a proposal for delineating subspecies in microbial taxonomy[END_REF][START_REF] Auch | Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison[END_REF][START_REF] Auch | Standard operating procedure for calculating genome-to-genome distances based on high-scoring segment pairs[END_REF]. Whole genome-based phylogenetic relationships were reconstructed using the Reference sequence Alignment based Phylogeny builder realphy 1.12 and Fast-Tree 2. 1.10 [35-39]. 16S rRNA genes were amplified by PCR and sequenced by Sanger sequencing [START_REF] Marchesi | Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA[END_REF][START_REF] Hill | Identification of Photorhabdus symbionts by MALDI-TOF MS[END_REF]. The 16S rRNA gene-based phylogenetic relationships were reconstructed using the maximum-likelihood method based on the Kimura two-parameter model in mega7 [START_REF] Kimura | A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences[END_REF][START_REF] Kumar | mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets[END_REF]. Sequences were aligned with muscle (version 3.8.31) [START_REF] Edgar | Muscle: multiple sequence alignment with high accuracy and high throughput[END_REF]. The tree with the highest log likelihood (-3554.61) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying neighbour-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories (+G, parameter=0.7354)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 87.62 % sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. There were a total of 1348 positions in the final dataset. Graphical representation and editing of the phylogenetic tree were performed with the Interactive Tree of Life (version 3.5.1) [START_REF] Chevenet | TreeDyn: towards dynamic graphics and annotations for analyses of trees[END_REF][START_REF] Letunic | Interactive tree of life (iTOL) V3: an online tool for the display and annotation of phylogenetic and other trees[END_REF]. Phylogenetic reconstruction based on 16S rRNA gene sequences and 16S rRNA gene sequence similarity calculations indicate that the closest relatives of strain BA1 T are P. akhurstii DSM 15138 T (99.1 %) and P. hainanensis DSM 22397 T (99.1 %), the closest relative of strain Q614 T is P. heterorhabditis SF41 T (99.3 %) and the closest relative of strain PB68.1 T is P. australis DSM 17609 T (99.7 %) (Figs 1 and2). Lower sequence similarities were found to all validly described species of the genus Photorhabdus.

Given the high 16S rRNA gene sequence similarity scores observed and that 16S rRNA and housekeeping gene sequences provide insufficient information to resolve the phylogenetic relationships of this bacterial group, particularly of very closely related species [START_REF] Machado | Photorhabdus khanii subsp. guanajuatensis subsp. nov., isolated from Heterorhabditis atacamensis, and Photorhabdus luminescens subsp. mexicana subsp. nov., isolated from Heterorhabditis mexicana entomopathogenic nematodes[END_REF][START_REF] Machado | Whole-genome-based revisit of Photorhabdus phylogeny: proposal for the elevation of most Photorhabdus subspecies to the species level and description of one novel species Photorhabdus bodei sp. nov., and one novel subspecies Photorhabdus laumondii subsp. clarkei subsp. nov[END_REF], and to fully meet the guidelines of the ad hoc Committee on Reconciliation of Approaches to Bacterial Systematics [START_REF] Wayne | Report of the ad hoc committee on reconciliation of approaches to bacterial systematics[END_REF] that recommends the dDDH method as the gold standard for bacterial species circumscription, we reconstructed phylogenetic trees based on core genomes and calculated dDDH scores to determine the taxonomic position of these strains. Strain BA1 T clusters together with P. akhurstii DSM 15138 T and P. hainanensis DSM 22397 T , Q614 T clusters together with P. heterorhabditis SF41 T , and PB68.1 T clusters together with P. australis DSM 17609 T (Fig. 3). The dDDH scores between BA1 T and all Photorhabdus species were lower than 70 % (Fig. 4). The dDDH scores between Q614 T and all Photorhabdus species, except P. heterorhabditis SF41 T , were lower than 70 %. The dDDH scores between PB68.1 T and all Photorhabdus species, except P. australis DSM 17609 T , were lower than 70 %. Strains Q614 T and P. heterorhabditis SF41 T share 75.4 % dDDH (CI: 72.4-78.1 %), and strains PB68.1 T and P. australis DSM 17609 T share 76.6 % isDDH (CI: 73.6-79.4 %). Given that the thresholds for species and subspecies delimitation are 70 and 79 % dDDH, respectively, we propose to classify BA1 T as a new Photorhabdus species, Q614 T as a new subspecies within the species P. heterorhabditis and PB68.1 T as a new subspecies within the species P. australis [START_REF] Machado | Photorhabdus khanii subsp. guanajuatensis subsp. nov., isolated from Heterorhabditis atacamensis, and Photorhabdus luminescens subsp. mexicana subsp. nov., isolated from Heterorhabditis mexicana entomopathogenic nematodes[END_REF][START_REF] Meier-Kolthoff | Complete genome sequence of DSM 30083(T), the type strain (U5/41(T)) of Escherichia coli, and a proposal for delineating subspecies in microbial taxonomy[END_REF][START_REF] Wayne | Report of the ad hoc committee on reconciliation of approaches to bacterial systematics[END_REF]. Biochemical characterization supports the status of BA1 T , Q614 T and PB68.1 T as new taxa since they exhibit unique biochemical profiles, which differ from the profiles of other strains from other taxa (Table 1). Citrate utilization, acetoin and indole production, and urease activity are particularly suitable biochemical tests to differentiate the different species of the genus Photorhabdus (Table 1).

Based on the results of this polyphasic approach, we propose the creation of Photorhabdus aegyptia sp. nov. with the type strain BA1 T (=DSM 111180 T =CCOS 1943 T =LMG 31957 T ), Photorhabdus heterorhabditis subsp. aluminescens subsp. nov. with the type strain Q614 T (=DSM 111144 T =CCOS 1944 T =LMG 31959 T ) and Photorhabdus australis subsp. thailandensis subsp. nov. with the type strain PB68.1 T (=DMS 111145 T =CCOS 1942 T ). These propositions automatically create Photorhabdus heterorhabditis subsp. heterorhabditis subsp. nov. with SF41 T as the type strain (currently classified as P. heterorhabditis) and Photorhabdus australis subsp. australis subsp. nov. with DSM 17609 T as the type strain (currently classified as P. australis).

EMENDED DESCRIPTION OF PHOTORHABDUS AUSTRALIS (AKHURST ET AL. 2004) MACHADO ET AL. 2018

Photorhabdus australis ( aus. tra′lis. L. fem. adj. australis: southern; the type strain of this species was detected in the southern hemisphere).

Maximum temperature for growth is 40 °C. Yellow or no pigment; weakly pigmented. Most isolates are positive for DNase and most are negative for aesculin hydrolysis. Negative for urease and indole production. Most isolates produce acid from gluconate, variable for acid production from aesculin and negative for trehalose. Proteinaceous inclusions are rare. β-Galactosidase weak or positive. Annular haemolysis is variable on sheep blood and horse blood agars. Most isolates are negative for Tween 60 and Tween 80 esterases and most grow on myo-inositol. Natural habitat is Heterorhabditis EPNs; all isolates were obtained from human clinical specimens in Australia. The type strain of the species is 9802892 T (=CIP 108025 T =ACM 5210 T ). S1.

DESCRIPTION OF PHOTORHABDUS AUSTRALIS SUBSP. AUSTRALIS SUBSP. NOV.

Photorhabdus australis subsp. australis ( aus. tra′lis. L. fem. adj. australis: southern; the type strain of the species was detected in the Southern Hemisphere).

Maximum temperature for growth is 40 °C. Yellow or no pigment; weakly pigmented. Most isolates are positive for DNase and most are negative for aesculin hydrolysis. Negative for urease and indole production. Most isolates produce acid from gluconate; variable for acid production from aesculin and negative for trehalose. Proteinaceous inclusions are rare. Weak β-galactosidase activity. Annular haemolysis is variable on sheep blood and horse blood agars. Most isolates are negative for Tween 60 and Tween 80 esterases and most grow on myo-inositol. Natural habitat is Heterorhabditis EPNs; all isolates were obtained from human clinical specimens in Australia. The type strain of the subspecies is 9802892 T (=CIP 108025 T =ACM 5210 T ).

DESCRIPTION OF PHOTORHABDUS AUSTRALIS SUBSP. THAILANDENSIS SUBSP. NOV.

Photorhabdus australis subsp. thailandensis ( thai. land. en′sis N.L. fem. adj. thailandensis, from Thailand, the country where the entomopathogenic nematodes that host the type strain were originally collected).

Cells are large rods and motile. Maximum temperature for growth is 40 °C. Yellow or orange. Mucoid. Negative for urease, indole, acetoin and H 2 S production. Proteinaceous inclusions are rare. Negative for arginine dihydrolase, lysine and ornithine decarboxylase. Positive for β-galactosidase activity. Annular haemolysis is variable on sheep blood and horse blood agars. The type strain is symbiotically associated with Heterorhabditis indica EPNs. Their natural habitat is the intestines of these nematodes and the insects infected by them. The type strain of the subspecies is PB68.1 T (=DMS 111145 T =CCOS 1942 T ). Whole genome sequences of this strain are available in the NCBI data bank under accession number LOMY01.

EMENDED DESCRIPTION OF PHOTORHABDUS HETERORHABDITIS FERREIRA ET AL. 2014

Photorhabdus heterorhabditis ( he. te. ro. rhab′ di. tis. N.L. gen. n. heterorhabditis of the nematode Heterorhabditis).

Cells are Gram-stain-negative, catalase-positive rods. Bioluminescence variable. Aerobic growth is preferred, with growth temperatures ranging from 24 to 42 °C in nutrient broth (NB) and from 24 to 35 °C in tryptic soy broth (TSB). Optimal growth in NB and TSB occurs at 30 °C. Colonies on NBTA are blue or blue-green. Indole production negative. Citrate utilization variable. Urease variable. Tryptophan deaminase variable. Acid is produced from N-acetylglucosamine, d-fructose, d-glucose, glycerol, d-mannose, maltose and d-xylose. Able to ferment glucose, hydrolyse arginine, aesculin and gelatin, and produce urease. Assimilates glucose, d-mannose, N-acetylglucosamine, maltose and potassium gluconate (weakly). Nitrate is not reduced. This strain was isolated from Heterorhabditis S1.

zealandica EPNs collected in South Africa. The type strain of the species is SF41 T (=ATCC BAA-2479 T =DSM 25263 T ).

DESCRIPTION OF PHOTORHABDUS HETERORHABDITIS SUBSP. HETERORHABDITIS SUBSP. NOV.

Photorhabdus heterorhabditis subsp. heterorhabditis ( he. te. ro. rhab′ di. tis. N.L. gen. n. heterorhabditis of the nematode Heterorhabditis).

Cells are Gram-stain-negative, catalase-positive rods. Bioluminescent. Aerobic growth is preferred, with growth temperatures ranging from 24 to 42 °C in NB and from 24 to 35 °C in TSB. Optimal growth in NB and TSB occurs at 30 °C. Colonies on NBTA are blue or blue-green. Acid is produced from N-acetylglucosamine, d-fructose, d-glucose, glycerol, d-mannose, maltose and d-xylose. Able to ferment glucose, hydrolyse arginine, aesculin and gelatin, and produce urease. Assimilates glucose, d-mannose, N-acetylglucosamine, maltose and potassium gluconate (weakly). Indole production negative. Citrate utilization negative. Urease negative. Tryptophan deaminase positive. Nitrate is not reduced. This strain was isolated from Heterorhabditis zealandica EPNs collected in South Africa.

The type strain of the subspecies is SF41 T (=ATCC BAA-2479 T =DSM 25263 T ).

DESCRIPTION OF PHOTORHABDUS HETERORHABDITIS SUBSP. ALUMINESCENS SUBSP. NOV.

Photorhabdus heterorhabditis subsp. aluminescens ( a. lu. mi. nes'cens. N.L. part. adj. aluminescens non-luminescing; for its incapability to produce bioluminescence).

Cells are large motile rods (4.5×1.0-10.0×2.0 µm). Colonies are mucoid, circular, slightly irregular margins, yellow or orange in colour with a diameter of approximately 2 mm after 48 h growth on LB agar. Maximum temperature for growth is 33-34 °C. Negative for β-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and for H 2 S, indole and acetoin production. Positive for citrate utilization, for urease and gelatinase activity, and for glucose oxidation. Annular haemolysis is observed on sheep blood agar. Natural habitat is Heterorhabditis EPNs. The type strain of the subspecies is Q614 T (=DSM 111144 T =CCOS 1944 T =LMG 31959 T ). Whole genome sequences of this strain are available in the NCBI data bank under accession number JABBCS01. S1. heterorhabditis subsp. aluminescens subsp. nov.Q614 T were tested in parallel. The experiment was conducted twice. The obtained results were compared to those published previously [START_REF] Machado | Photorhabdus khanii subsp. guanajuatensis subsp. nov., isolated from Heterorhabditis atacamensis, and Photorhabdus luminescens subsp. mexicana subsp. nov., isolated from Heterorhabditis mexicana entomopathogenic nematodes[END_REF]. For further information refer to the original studies [START_REF] Boemare | DNA relatedness between Xenorhabdus spp. (Enterobacteriaceae), symbiotic bacteria of entomopathogenic nematodes, and a proposal to transfer Xenorhabdus luminescens to a new genus, Photorhabdus gen. nov[END_REF][START_REF] Akhurst | Taxonomy of Australian clinical isolates of the genus Photorhabdus and proposal of Photorhabdus asymbiotica subsp. asymbiotica subsp. nov. and P. asymbiotica subsp. australis subsp. nov[END_REF][START_REF] Ferreira | Photorhabdus luminescens subsp. noenieputensis subsp. nov., a symbiotic bacterium associated with a novel Heterorhabditis species related to Heterorhabditis indica[END_REF][START_REF] Ferreira | Photorhabdus heterorhabditis sp. nov., a symbiont of the entomopathogenic nematode Heterorhabditis zealandica[END_REF][START_REF] Fischer-Le Saux | Polyphasic classification of the genus Photorhabdus and proposal of new taxa: P. luminescens subsp. luminescens subsp. nov., P. luminescens subsp. akhurstii subsp. nov., P. luminescens subsp. laumondii subsp. nov., P. temperata sp. nov., P. temperata subsp. temperata subsp. nov. and P. asymbiotica sp. nov[END_REF][START_REF] Glaeser | Photorhabdus luminescens subsp. namnaonensis subsp. nov., isolated from Heterorhabditis baujardi nematodes[END_REF][START_REF] Hazir | Two new subspecies of Photorhabdus luminescens, isolated from Heterorhabditis bacteriophora (Nematoda: Heterorhabditidae): Photorhabdus luminescens subsp. kayaii subsp. nov. and Photorhabdus luminescens subsp. thracensis subsp. nov[END_REF][START_REF] Machado | Photorhabdus khanii subsp. guanajuatensis subsp. nov., isolated from Heterorhabditis atacamensis, and Photorhabdus luminescens subsp. mexicana subsp. nov., isolated from Heterorhabditis mexicana entomopathogenic nematodes[END_REF][START_REF] Machado | Whole-genome-based revisit of Photorhabdus phylogeny: proposal for the elevation of most Photorhabdus subspecies to the species level and description of one novel species Photorhabdus bodei sp. nov., and one novel subspecies Photorhabdus laumondii subsp. clarkei subsp. nov[END_REF][START_REF] Tailliez | Phylogeny of Photorhabdus and Xenorhabdus based on universally conserved protein-coding sequences and implications for the taxonomy of these two genera. proposal of new taxa: X. vietnamensis sp. nov., P. luminescens subsp. caribbeanensis subsp. nov., P. luminescens subsp. hainanensis subsp. nov., P. temperata subsp. khanii subsp. nov., P. temperata subsp. tasmaniensis subsp. nov., and the reclassification of P. luminescens subsp. thracensis as P. temperata subsp. thracensis comb. nov[END_REF][START_REF] Tóth | Photorhabdus temperata subsp. cinerea subsp. nov., isolated from Heterorhabditis nematodes[END_REF][START_REF] Szállás | Phylogenetic evidence for the taxonomic heterogeneity of Photorhabdus luminescens[END_REF][START_REF] An | Photorhabdus temperata subsp. stackebrandtii subsp. nov. (Enterobacteriales: Enterobacteriaceae)[END_REF][START_REF] An | Photorhabdus luminescens subsp. kleinii subsp. nov. (Enterobacteriales: Enterobacteriaceae)[END_REF][START_REF] Akhurst | A non-luminescent strain of Xenorhabdus luminescens (Enterobacteriaceae)[END_REF] : more than 80% of strains and/or conducted tests showed a positive reaction, "-": more than 80% of strains and/or conducted tests showed a negative reaction, "v": variable, about 50% of strains/conducted tests showed a positive reaction, "w": weak, more than 80% of strains and/or conducted tests showed a weak, positive reaction. Photorhabdus aegyptia ( ae. gyp′ti.a. L. fem. adj. aegyptia pertaining to Egypt, the country where the entomopathogenic nematodes that host the type strain were originally collected).

Cells are motile, non-spore-forming rods (approx. 1.0 µm wide and 1.5-2.0 µm long), Gram-stain-negative, oxidasenegative and catalase positive. Colonies are mucoid, circular, slightly irregular margins, pale yellow in colour with a diameter of approximately 2 mm after 48 h growth on LB agar and produce light. Good growth occurs on LB at 28-30 °C. Negative for β-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and for H 2 S production. Positive for citrate utilization, for urease and gelatinase activity, for glucose oxidation and for indole and acetoin production. The type strain is symbiotically associated with Heterorhabditis indica EPNs. Their natural habitat is the intestines of these nematodes and the insects infected by them. The type strain is BA1 T (=DMS 111180 T =CCOS 1943 T =LMG 31957 T ). Whole genome sequences of this strain are available in the NCBI data bank under accession number JFGV01

Fig. 1 .

 1 Fig. 1. Maximum-likelihood phylogenetic tree of Photorhabdus bacterial strains reconstructed from 1348 nucleotide positions of 16S rRNA gene sequences. Numbers at nodes represent bootstrap values based on 100 replications. Bar represents 0.01 nucleotide substitutions per sequence position. Accession numbers of gene sequences used for the reconstruction are shown in TableS1(available in the online version of this article).

Fig. 2 .

 2 Fig. 2. Pairwise nucleotide similarities (%) of 16S rRNA gene sequences of Photorhabdus strains. 1348 nucleotide positions were analysed. Accession numbers of gene sequences used are shown in TableS1.

Fig. 3 .

 3 Fig. 3. Phylogenetic reconstruction based on core genome sequences of Photorhabdus bacterial strains. Numbers at the nodes represent SH-like branch supports. Bar represents 0.01 nucleotide substitutions per sequence position. Accession numbers of the genome sequences used for the reconstruction are shown in TableS1.

Fig. 4 .

 4 Fig. 4. Pairwise comparison of digital DNA-DNA Hybridization (dDDH) scores (%) of Photorhabdus strains. Accession numbers of gene sequences used are shown in TableS1.

  Photorhabdus temperata and Photorhabdus thracensis. Photorhabdus laumondii is divided into two subspecies: P. laumondii subsp. laumondii and P. laumondii subsp. clarkei; Photorhabdus khanii is divided into two subspecies: P. khanii subsp. khanii and P. khanii subsp. guanajuatensis; and Photorhabdus luminescens is divided into two subspecies: P. luminescens subsp. luminescens and P. luminescens subsp. mexicana

	species with validly published names: Pho-
	torhabdus akhurstii, Photorhabdus asymbiotica, Photorhabdus
	australis, Photorhabdus bodei, Photorhabdus caribbeanensis,
	Photorhabdus cinerea, Photorhabdus hainanensis, Photorhab-
	dus heterorhabditis, Photorhabdus kayaii, Photorhabdus khanii,
	Photorhabdus kleinii, Photorhabdus laumondii, Photorhabdus
	luminescens, Photorhabdus namnaonensis, Photorhabdus
	noenieputensis, Photorhabdus stackebrandtii, Photorhabdus
	tasmaniensis,

Table 1 .

 1 API20E-based phenotypic characters to differentiate all Photorhabdus species/subspecies. Strains P. australis subsp. thailandensis subsp. nov. PB68.1 T , P. aegyptia sp. nov. BA1 T , and P.

  . 1: P. akhurstii, 2: P. asymbiotica, 3: P. australis subsp. australis subsp. nov., 4: P. australis subsp. thailandensis subsp. nov, 5: P. bodei, 6: P. caribbeanensis, 7: P. cinerea, 8. P. aegyptia sp. nov., 9: P. hainanensis, 10: P. heterorhabditis subsp. heterorhabditis subsp. nov.,11: P. heterorhabditis subsp. aluminescens subsp. nov.,12: P. kayaii, 13: P. khanii subsp. khanii subsp. nov., 14: P. khanii subsp. guanajuatensis subsp. nov., 15: P. kleinii, 16: P. laumondii subsp. laumondii, 17: P. laumondii subsp. clarkei, 18: P. luminescens subsp. luminescens subsp. nov., 19: P. luminescens subsp. mexicana subsp. nov., 20: P. namnaonensis, 21: P. noenieputensis, 22: P. stackebrandtii, 23: P. tasmaniensis, 24: P. temperata, 25: P. thracensis. "+"

Acknowledgements

We thank the Swiss National Science foundation for financial support, the next Generation Sequencing Platform and the Interfaculty Bioinformatics Unit of the University of Bern for performing whole genome sequencing and providing high performance computing infrastructure, the Institute of Biology of the University of Neuchâtel (Switzerland) and the National Research Centre (Cairo, Egypt) for their support.

P. laumondii subsp. clarkei BOJ-47 T P. cinerea DSM 19724 T P. australis subsp. thailandensis subsp. nov. PB68.1 T P. heterorhabditis subsp. heterorhabditis subsp. novSF41 T P. australis subsp. australis subsp. nov. DSM 17609 T P. asymbiotica DSM 15149 T P. namnaonensis PB45.5 T P. laumondii subsp. laumondii TT01 T P. kayaii HUG-39 P. khanii subsp. guanajuatensis MEX20-17 T P. heterorhabditis subsp. aluminescens subsp. nov.Q614 T P. tasmaniensis DSM 22387 T P. thracensis DSM 15199 T P. temperata Meg1 P. stackebrandtii DSM 23271 T P. khanii subsp. khanii DSM 3369 T P. luminescens subsp. mexicana MEX47-22 T P. noenieputensis DSM 25462 T P. caribbeanensis HG29 T P. aegyptia sp. novBA1 T P. akhurstii DSM 15138 T P. hainanensis DSM 22397 T Funding information The work of R.A.R.M. and A.M. is supported by the Swiss National Science Foundation (grant 186 094 to R.A.R.M.). The work of M.A.H. is supported by the National Research Centre (grant 12050137).

Conflicts of interest

The authors declare that there are no conflicts of interest.


Camille Bihanic

Andrii Stanovych

Franck Pelissier

Claude Grison

Putting Waste to Work: The Demonstrative Example of Pyrite Quarry Effluents Turned into Green Oxidative Catalysts

Keywords: Pyrite quarrying, Turning wastes into catalysts, Sustainable oxidative catalysts, Green oxidative cleavage, Green epoxidation

In the context of exponentially growing problems of pollution and waste, the design of ecofriendly processes for turning the current treatment of effluents into sustainable catalysts represents a great opportunity. Wastewater arising from pyrite quarrying is selected as a model example. The acidity of pyrite effluents leads to solubilization of useful cations: Mn II and Fe II /Fe III . Their chemical properties are an opportunity to promote innovative studies of oxidation in organic synthesis. The controlled addition of NaOH and a co-oxidant (H 2 O 2 ) onto the effluents allows generation of new, oxidative catalysts. MP-AES analysis, XPS, XRPD, BET, TEM, HRTEM, and EDX studies were performed to characterize the new and efficient catalysts. Oxidation of alcohols into carbonyl compounds, oxidative cleavage of 1,2diols, and epoxidation of alkenes are described.

INTRODUCTION

Intensive mining, quarrying, and industrial activities are causing serious dissemination of "Trace metallic Elements" (or "TEs") throughout the environment. This is an alarming problem because TEs are among the most harmful pollutants, as they are not biodegradable. 1 Beyond the environmental consequences, the health risks are real: damage to the nervous and renal systems, as well as to lung and bone tissues, has been proven. Metals are transported either as dissolved species in water or as suspended sediments through rivers and streams. They accumulate in river bed sediments or seep into the groundwater, thereby contaminating underground sources. Problems related to protecting drinking water and groundwater flux are becoming a major priority.

For example, pyrite quarrying exposes sulfide and disulfide minerals to air and water, facilitating a natural chemical reaction producing sulfuric acid. This reaction increases acidity in water (often to pH as low as 2 or 3), thus rendering water quality unacceptable. The oxidation of pyrite (iron disulfide) produces sulfuric acid, but also FeSO 4 and MnSO 4 . Under low pH conditions, ferrous sulfate may be oxidized into ferric sulfate (Figure 1).

Currently, the quality of effluents before discharge must meet European Performance Standards. The solutions implemented are often unsatisfactory. Managers of quarries and mines have to treat more than 200 000 m 3 of effluents per quarry and per year with soda or lime in order to comply with discharge standards. This costly, inadequate treatment produces new waste in the form of an alkaline sludge rich in metal hydroxides. In the end, the problem remains unresolved. A similar situation is observed with effluents from chemical plants. Today, a major challenge is to innovate and find ecoresponsible and sustainable solutions.

In a context of exponentially growing problems of pollution and waste, the design of ecofriendly processes for transforming contaminated wastes into new catalysts and reagents is a powerful opportunity. Herein, we propose ways to turn the current treatment of effluents into bioinspired promoters of oxidation (Figure 2).

The acidity of effluents from pyrite quarries allows solubilization of catalytically efficient cations: Mn II and Fe II / Fe III . Their natural properties are an opportunity to promote innovative oxidation processes according to three strategies using the specific chemical composition of quarrying effluents:

(1) studying the conditions of alcohol oxidation into carbonyl compounds by manganese-based catalysts; (2) testing these catalysts as promoters of oxidative cleavages; and (3) carrying out experimental studies on the use of effluents for the epoxidation of alkenes. The polymetallic composition and the activity, selectivity, and recyclability of novel catalysts will be showcased in selected reactions, such as reactions traditionally employing oxidants prohibited by REACH regulations 2 and the synthesis of monomers from terpenes. The key benefits of the proposed system are the recovery of problematic wastes, namely, the effluents from pyrite quarries, the possibility of recycling and reuse of these wastes, the efficiency of the system in terms of yield, the wide range of its applications, the ease of control of the reaction outcome, and the mild and ecofriendly reaction conditions.

EXPERIMENTAL SECTION

Chemicals. Reagents and solvents were purchased from Sigma-Aldrich and were used without further purification.

Preparation of Pyr-Mn. Hydrogen peroxide solution (30% w/w in H 2 O) was added to effluents from the Pleyber-Christ mining site (France, Table 1), and the solution was stirred at room temperature. After 30 min, NaOH (2 M) was added drop-by-drop until the desired pH was reached. A precipitate formed. The solution was let under stirring at room temperature for 12 h. The solid was then recovered by filtration and washed with deionized water (3 times) and then with absolute ethanol (3 times). The resulting solid was dried in an oven at 140 °C for 24 h.

Preparation of Synthetic Mn Catalyst. Manganese sulfate monohydrate (972 μmol) was put in ultrapure water (1 L). Hydrogen peroxide solution (30% w/w in H 2 O) was added, and the solution was stirred at room temperature. After 30 min, NaOH (2 M) was added drop-by-drop until the pH reached 11. A precipitate formed. The solution was let under stirring at room temperature for 12 h. The solid was then recovered by filtration and washed with deionized water (3 times) and then with absolute ethanol (3 times). The resulting solid was dried in an oven at 140 °C for 24 h.

Preparation of Synthetic Mn-Ca Catalyst. Manganese sulfate monohydrate (243 μmol) and calcium sulfate (211 μmol) were put in ultrapure water (1 L). Hydrogen peroxide solution (30% w/w in H 2 O) was added, and the solution was stirred at room temperature. After 30 min, NaOH (2 M) was added drop-by-drop until the pH reached 11. A precipitate formed. The solution was let under stirring at room temperature for 12 h. The solid was then recovered by filtration and washed with deionized water (3 times) then with absolute ethanol (3 times). The resulting solid was dried in an oven at 140 °C for 24 h.

Preparation of Synthetic Mn-Mg Catalyst. Manganese sulfate monohydrate (243 μmol) and magnesium sulfate (270 μmol) were put in ultra pure water (1 L). Hydrogen peroxide solution (30% w/w in H 2 O) was added and the solution was stirred at room temperature. After 30 min, NaOH (2 M) was added drop-by-drop until pH reached 11. A precipitate formed. The solution was let under stirring at room temperature for 12 h. The solid was then recovered by filtration and washed with deionized water (3 times) then with absolute ethanol (3 times). The resulting solid was dried in an oven at 140 °C for 24 h.

Preparation of Synthetic Mn-Ca-Mg Catalyst. Manganese sulfate monohydrate (243 μmol), calcium sulfate (211 μmol) and magnesium sulfate (270 μmol) were put in ultra pure water (1 L). Hydrogen peroxide solution (30% w/w in H 2 O) was added and the solution was stirred at room temperature. After 30 min, NaOH (2 M) was added drop-by-drop until pH reached 11. A precipitate formed. The solution was let under stirring at room temperature for 12 h. The solid was then recovered by filtration and washed with deionized water (3 times) and then with absolute ethanol (3 times). The resulting solid was dried in an oven at 140 °C for 24 h.

Characterization of the Catalysts. Mineral composition of the catalysts was determined using an Agilent 4200 microwave plasmaatomic emission spectrometer (MP-AES) coupled with a SPS4 autosampler. The samples were digested in 6 mL of reversed aqua regia (1:2 hydrochloric acid (37%)/nitric acid (65%)) under an Anton Paar Multiwave Go microwave-assisted digestion, with the following program: 20-164 °C in 20 min then 10 min isothermal at 164 °C. Samples were filtered and then diluted to 0.2 g L -1 in nitric acid (1%). Three blanks were recorded for each step of the dilution procedure. Three analyses were carried out for each sample in order to determine the standard deviation of the measurement.

The specific surface area and pore size distribution of the catalysts were inferred from the measurements of adsorption-desorption of gaseous nitrogen at 77 K with the use of a Micromeritics Tristar unit. Prior to the experiment, each sample was degassed down to 2 Pa at 120 °C for 12 h. X-ray powder diffraction (XRPD) data measurements on the samples dried at 100 °C for 2 h were provided by using a BRUKER diffractometer (D8 Advance, with Cu Kα radiation at 1.54086 Å) equipped with a Lynxeyes detector. X-ray photoelectron spectroscopy (XPS) analyses were carried out with an ESCALAB 250 X-ray photoelectron spectrometer from Thermo Electron. The spectra were generated by Al Kα X-ray at 1486.6 eV. The surface of the sample that was analyzed had a diameter of 400 μm. All the binding energies were referenced to the C 1s core-level energy at 284.8 eV.

Transmission electron microscopy (TEM) images were taken using a JEOL 1200EX2 transmission electron microscope at an accelerating voltage of 100 kV, equipped with a CCD EMSIS Olympus 11 Mpx Quemesa CCD at the MEA platform (University of Montpellier, France). In each case, ecocatalysts were grounded into a thin powder and put in suspension in ethanol. Each suspension of catalyst was dropped on a carbon coated copper grid (300 mesh).

High resolution transmission electron microscopy (HRTEM) analyses were performed at 200 kV on a JEOL 2200 FS equipped with a CCD Gatan Ultrascan 4000 CCD (4092 × 4092 px 2 ) at the MEA platform (University of Montpellier, France). Scanning transmission electron microscopy-energy dispersive x-ray spectroscopy (STEM-EDX) was used to obtain chemical mapping, with a probe size of 0.7 nm. The EDS spectra were recorded on a Silicon Drift X-MAX n 100TLE detector from Oxford Instrument. Sample were embedded in LR White resin and cut (thin section of 70 nm) with a Leica UC7 ultramicrotome equipped with a Diatome diamond. Sections were deposited on carbon coated copper grid (300 mesh).

Benzyl Alcohol Oxidation. In a typical procedure, benzyl alcohol (1 mmol), catalyst (0.3 equiv Mn), and anhydrous toluene (10 mL) were added to a three-neck flask with a magnetic stirring bar. The reaction mixture was stirred under reflux at 110 °C with air flow for 5 h. The catalyst was then filtered and washed with ethyl acetate (3 times) and ethanol (3 times) before being dried in an oven at 140 °C for 24 h.

Oxidative Cleavage of 1,2-Diols. In a typical procedure, 1,2-diol (0.5 mmol), catalyst (0.3 equiv Mn), and anhydrous toluene (5 mL) were added to a three-neck flask with a magnetic stirring bar. The reaction mixture was stirred under reflux at 110 °C with air flow for 5 h. The catalyst was then filtered and washed with ethyl acetate (3 times) and ethanol (3 times) before being dried in an oven at 140 °C for 24 h.

Epoxidation of Alkenes. In a typical procedure, the effluent (0.00006 equiv Mn), NaHCO 3 (0.88 mmol), and alkene (0.18 mmol) were stirred in t-BuOH or acetone (2.6 mL) at room temperature for 10 min. H 2 O 2 (30% w/w in H 2 O, 1.75 mmol) was added and the mixture was stirred during 2 h at 30 °C.

Analyses of Reaction Products. Gas chromatography-mass spectroscopy (GC-MS) analyses were performed on a Thermo Scientific Trace 1300 GC coupled with an ISQ QD quadrupole and a Thermo TG-5SILMS column (0.18 μm × 0.18 μm × 20 mm) with hydrogen as carrier gas (1 mL min -1 ). The program followed was: 30 s isothermal at 80 °C, 80 °C-280 °C at 40 °C min -1 then 30 s isothermal at 280 °C. Conversion and selectivity were established by GC-MS, using dodecane or biphenyl as intern standard. Mass spectra were recorded in impact electronic mode at 70 V, and identification was made by using the NIST 14 database.

Fourier transform infrared spectroscopy (FTIR) spectra were recorded using a PerkinElmer Spectrum100 FT-IR spectrometer, between 1400 and 1600 cm -1 .

RESULTS AND DISCUSSION

A feature of effluents from pyrite quarrying was the variation in their chemical composition, which depends on climatic conditions and the natural composition of rock. We studied the composition of a model quarrying site, Pleyber-Christ in France, during different seasons (autumn and summer). The MP-AES was used to determine the exact chemical composition of effluents (Table 1). As expected, autumnal effluents were more concentrated than summer ones, as the result of seasonal precipitation leaching. However, whichever season, the mineral composition of effluents followed the same trend: the analysis clearly revealed that Mn was the major transition metal (9-13%), accompanied by an amount of 1-7% Fe. An amount of 3-5% Al was also noted. Whereas the content of cations (Al, Na, K) was stable in the samples, significant differences were observed for Mn, Fe, Mg, and Ca. Transition metals present in effluents are an intended source of oxidation activity, but a difference in Mg and Ca content could modulate the catalytic activity by some synergetic effects. Indeed, a cooperative effect between Mn and Ca for oxidation is possible. The cofactor (Mn 4 CaO 5 ) in photosystem II, which is able to oxidize H 2 O, is a demonstrative natural example. [START_REF] Bricker | Introduction to Oxygen Evolution and the Oxygen-Evolving Complex[END_REF] On the contrary, Mg is known to show an antagonist effect on the Mn oxidizing property. [START_REF] Pascal | Nouveau traitéde chimie mineŕale[END_REF] Therefore, it was interesting to study the synthetic potential of this polymetallic composition as sustainable catalytic alternatives in oxidation reactions.

Studying the Conditions of Alcohol Oxidation into Carbonyl Compounds. The selective oxidation of alcohols into carbonyl compounds needs new green catalytic alternatives. The catalytic oxidation with H 2 O 2 is widely used, because it presents numerous advantages: it is considered to be clean and cheap, is an excellent oxygen donor, and does not generate waste or coproduct. [START_REF] Anastas | Green Chemistry: Theory and Practice[END_REF] Among the transition elements known to catalyze oxygen transfer process, Mn is clearly preferred because of its low toxicity and its abundance in the Earth's crust. [START_REF] Gerber | Carcinogenicity, Mutagenicity and Teratogenicity of Manganese Compounds[END_REF]7 Oxidations by Mn IV are usually soft and selective. Their chemoselectivity is interesting; for example, there is no oxidation of phenol and no overoxidation of benzylic aldehydes. [START_REF] Cahiez | Encyclopedia of Reagents for Organic Synthesis[END_REF] MnO 2 is considered as a potential green oxidant. It is not soluble in either organic solvents or water, and thus, it is recyclable via filtration, with very low dispersion into the environment and low toxicity. However, there are drawbacks to the standard system: MnO 2 needs to be activated (chemical treatments, use of KMnO 4 ). Additionally, high excess (10-15 equiv) and long reaction times are necessary. The development of catalytic methodology for soft oxidation by Mn IV was motivating. Our recent reports using catalysts prepared from Mn-rich plant extracts, [START_REF] Escande | Phytoextracted Mining Wastes for Ecocatalysis: Eco-Mn®, an Efficient and Eco-Friendly Plant-Based Catalyst for Reductive Amination of Ketones[END_REF][START_REF] Escande | Acid Catalysis and Green Oxidations: Sequential Tandem Oxidation Processes Induced by Mn-Hyperaccumulating Plants[END_REF][START_REF] Escande | Switchable Alkene Epoxidation/Oxidative Cleavage with H 2 O 2 /NaHCO 3 : Efficient Heterogeneous Catalysis Derived from Biosourced Eco-Mn[END_REF][12] called Eco-Mn and Eco-MnOx, revealed an unique polymetallic composition and displayed superior catalytic performances than conventional Mn catalysts in oxidative transformations. These results prompted us to investigate the preparation of novel Mn-rich polymetallic systems by the controlled addition of NaOH and co-oxidant To ensure an optimal preparation of efficient catalysts, the best conditions for precipitation of useful species as a function of pH had to be taken into account. The catalysts synthesis followed the same steps as for the synthesis of already reported Eco-MnOx 12 but using effluents as manganese source. Pyr-Mn were prepared by oxidation of the elements present in effluents by H 2 O 2 and subsequently precipitated by addition of NaOH. The precipitate was collected by filtration. Analysis of the mineral composition of the catalysts synthesized at different pH was provided by MP-AES (Table 2).

As expected, by using solubility product constants of the different metallic hydroxides, [START_REF] Charlot | Les reáctions chimiques en solution; l'analyse qualitative mineŕale[END_REF] the maximal percentage of Fe and Mn was obtained at pH 9.5 (Table 2, Figure S1). It should be noted that the ratio Mn/Ca was close to the one of Mn 4 CaO 5, the cofactor of photosystem II. At pH 11, Mg became the major element, whereas at pH 14 Mg and Ca represented the most abundant species. In the two latter cases, Mn was dispersed in a mineral matrix of alkaline earth metal cations. The substantial amount of Al can be of special importance as mixed Mn/Al catalysts have shown oxidizing properties. [START_REF] Neatu | Oxidation of 5-Hydroxymethyl Furfural to 2,5-Diformylfuran in Aqueous Media over Heterogeneous Manganese Based Catalysts[END_REF] High chemodiversity of metal elements, such as Fe, Mn, Ca, and Al, is an opportunity to create synergistic effects for green oxidations. Studying the morphology, microstructure, and properties of these novel catalysts Pyr-Mn was required to understand and control interactions between metals and reagents for innovation in oxidative synthesis. We focused on Pyr-Mn 1 and Pyr-Mn 2 , as Pyr-Mn 3 showed very poor reactivity in benzylic alcohol oxidation, taken as our first model reaction (Table 4, entries 5 and 6).

Catalysts Characterization. The specific surface area S BET of Pyr-Mn was determined by the Brunauer-Emmett-Teller method (BET). The specific surface area of Pyr-Mn catalysts obtained at pH 9.5 and 11 were compared to commercial activated MnO 2 and other MnO 2 catalysts with different morphologies. The data are summarized in Table 3. Pyr-Mn 1 and Pyr-Mn 2 exhibited high specific surface areas, with 209 and 119 m 2 g -1 , respectively. Furthermore, these values mostly exceeded those of classic MnO 2 materials.

XRPD was performed to deliver insights regarding the structure of Pyr-Mn 1 and Pyr-Mn 2 . Only one component was detected: calcium sulfate, originally present in effluents. Manganese oxide was not present in a crystalline form, which is consistent with the structure of active manganese dioxide. [START_REF] Cahiez | Encyclopedia of Reagents for Organic Synthesis[END_REF] In order to check the valence state of Mn ions in near surface areas, XPS was performed. Pyr-Mn 1 and Pyr-Mn 2 exhibited similar XPS spectra. In the Mn 2p spectra, peaks at 642.4 and 654.4 eV corresponded to the spin orbit doublet of Mn 2p 3/2 and Mn 2p 1/2 respectively (Figure S2). Mn 2p 3/2 binding energy seemed to correspond to Mn IV present in MnO 2 . [START_REF] Carver | Use of X-Ray Photoelectron Spectroscopy to Study Bonding in Cr, Mn, Fe, and Co Compounds[END_REF][START_REF] Aoki | X-Ray Photoelectron Spectroscopic Studies on ZnS: MnF2 Phosphors[END_REF] Nevertheless, a clear distinction between Mn III and Mn IV oxidation states is difficult by using Mn 2p 3/2 photopeak position, especially in the case of a combination of different oxidation state species, which would result in overlapping Mn 2p peaks. Hence, in the present case, a great number of simple and mixed stoichiometric manganese oxides was not to exclude. A deconvolution of Mn 2p spectra into several subbands, using LabSpec5 and fitting parameters from Biesinger et al., [START_REF] Biesinger | Resolving Surface Chemical States in XPS Analysis of First Row Transition Metals, Oxides and Hydroxides: Cr, Mn, Fe, Co and Ni[END_REF] was carried out. However, the poor resolution of the spectra made it difficult to draw any reliable conclusions. The absence of a satellite peak at 647.4 eV, characteristic for Mn II , confirmed that MnO was not present in the catalysts. [START_REF] Nesbitt | Interpretation of XPS Mn(2p) Spectra of Mn Oxyhydroxides and Constraints on the Mechanism of MnO 2 Precipitation[END_REF] To overcome this problem, multiplet splitting of Mn 3s photopeak is widely used as a more accurate approach to distinguish the two oxidation states or to estimate their ratios in mixed oxides. Unfortunately, Mn 3s spectra could not give more information because of the interference with Mg peak.

Significant differences concerning Pyr-Mn catalysts and commercial activated MnO 2 , used as the reference system, led us to study the morphology of these materials by using TEM, HRTEM, and EDX performed by using STEM.

TEM images revealed thin lamellar particles aggregated into crumpled-tissue-paper structures of about 100-200 nm diameter, for both Pyr-Mn 1 and Pyr-Mn 2 (Figure 3). These particles looked more uniformly aggregated for Pyr-Mn 2 than for Pyr-Mn 1 , even if no clear organization was visible in both cases, confirming the results of XRPD. This type of arrangement is in accordance with different types of conventional MnO 2 structures. [START_REF] Portehault | A Core-Corona Hierarchical Manganese Oxide and Its Formation by an Aqueous Soft Chemistry Mechanism[END_REF][START_REF] Ching | Self-Assembly of Manganese Oxide Nanoparticles and Hollow Spheres. Catalytic Activity in Carbon Monoxide Oxidation[END_REF][START_REF] Luo | Synthesis of Petal-like δ-MnO2 and Its Catalytic Ozonation Performance[END_REF] Commercial activated MnO 2 exhibited a totally different structure with rodlike particles of about 10-30 nm width over 50-100 nm length (Figure S3). HRTEM images did not only confirm these structures but also support the results obtained by N 2 adsorption-desorption measurements. Indeed, Pyr-Mn 1 and Pyr-Mn 2 showed type IV isotherms with a H3 hysteresis, confirming a nonrigid aggregates of plate-like particles with slit-shaped pores (Figure S4a andb). Pore diameters were estimated using the Barrett-Joyner-Halenda (BJH) method from the desorption branch of the nitrogen isotherm. Following similar results obtained by Augustin et al. [START_REF] Augustin | Manganese Oxide Phases and Morphologies: A Study on Calcination Temperature and Atmospheric Dependence[END_REF] for the isotherms, the "pores" detected could correspond to the space between particles in the network. This assumption is in good agreement with the broad pore size distributions obtained for Pyr-Mn 1 and Pyr-Mn 2 , ranging from 3 nm to more than 500 nm (Figure S4a andb). For commercial activated MnO 2 , the pore size distribution from 3 to 20 nm and from 29 to 37 nm could be explained in the same way (Figure S4c). It should be noticed that the spike in the N 2 desorption pore size distribution of Pyr-Mn 2 , not appearing in the adsorption pore size distribution, reflected an artifact (Figure S4b). This artifact is caused by a spontaneous evaporation of the capillary condensate, phenomena which can occur, for instance, for ink-bottleneck type cavities. STEM-EDX mapping was used to establish chemical composition mapping of our catalysts. The different elemental distributions gave some useful information on the overall composition. The presence of manganese, iron, calcium, magnesium and aluminum was in accordance with MP-AES analysis. Sulfide certainly stemmed from sulfate from pyrite effluents and supported XRPD analysis. In each case, the manganese and oxygen maps were overlaid, confirming the formation of some manganese oxides (Figures 4 andS5). Otherwise, STEM-EDX mapping results showed an aggregation of all elements in the particles, where manganese and oxygen were concentrated. This information is of the utmost importance, as it supports the polymetallic structure of the catalysts.

Catalyst Performance Evaluation through the Aerobic Oxidation of Alcohols. The aerobic oxidation of benzyl alcohol into benzaldehyde was chosen as model reaction to design the best reaction conditions. Oxygen from air was used as co-oxidant to regenerate the catalysts. In stoichiometric quantities, high yields were obtained with Pyr-Mn 1 (Table 4, entry 1). No retention of the reagents and products onto the catalyst mineral matrix was observed unlike for Pyr-Mn 2 and Pyr-Mn 3 (Table 4, entries 3 and 5). However, when Pyr-Mn 1 and Pyr-Mn 3 were involved in catalytic quantity, the conversion was poor, even under air flow (Table 4, entries 2 and 6). On the contrary, at pH 11, 79% conversion was obtained with only 0.3 equiv Mn (Table 4, entry 4). In all cases, complete selectivity toward benzaldehyde was obtained, without any overoxidation to benzoic acid.

To better understand the reactivity of the catalysts, and the synergetic/antagonist effects of other elements present in the polymetallic structures (mostly calcium and magnesium), synthetic catalysts were prepared from manganese, calcium and/or magnesium salts. The oxidation reaction of benzyl alcohol was also tested with those synthetic catalysts.

Total conversion and selectivity were reached for manganese alone and manganese-calcium catalysts (Table 5, entries 2 and 3). These results testified the synergetic or neutral effect of calcium over the Mn oxidizing properties. As these results could not give clear evidence of calcium role, the two synthetic catalysts were engaged in the oxidation of 5-hydroxymethyl-2furfural (HMF). In this case, only 20% conversion was obtained with Mn synthetic catalyst against 36% conversion with Mn-Ca synthetic catalyst. This last example confirmed the synergetic role of calcium over manganese oxidizing activity. On the other hand, magnesium antagonist role was clear, as GC-MS yield dropped to 66% and 89% for Mn-Mg and Mn-Ca-Mg synthetic catalysts, respectively (Table 5, entries 4 and 5). The activity of Pyr-Mn 2 was also compared with other manganese catalysts and well-known oxidative reagents. In the same reaction conditions, only 36% yield was obtained with commercial activated MnO 2 , supporting the performance of Pyr-Mn 2 (Table 5, entries 1 and 6). To afford 89% yield, 12 equiv of MnO 2 and 23 h of reaction time were needed for Highet and Wildman 24 (Table 5, entry 7). Son et al. used octahedral molecular sieves (K-OMS-2) in the same reactional conditions and also obtained 90% yield in benzaldehyde (Table 5, entry 8). Different amorphous MnO x catalysts were engaged in benzyl alcohol oxidation by Hu et al., and yields between 17% and 72% were reached in 1.5 h 25 (Table 5, entry 9). On the contrary, crystalline γ-MnO 2 showed very low activity for aerobic oxidation of benzyl alcohol (Table 5, entry 10). Shurz et al. supported the highest catalytic activity of amorphous manganese oxides compared to crystalline oxides, as crystalline MnO, Mn 2 O 3 , and Mn 3 O 4 did not show any activity. [START_REF] Schurz | Octahedral Molecular Sieves of the Type K-OMS-2 with Different Particle Sizes and Morphologies: Impact on the Catalytic Properties in the Aerobic Partial Oxidation of Benzyl Alcohol[END_REF] Chromium-based reagents, [START_REF] Warrener | A Comparative Study of Jones's, Collins's and Corey's Reagents in the Chromium(VI) Oxidation of an Epimeric Pair of Allylic Alcohols in the Benzo-Fused Norbornen-5-Ol Series[END_REF] hypervalent iodine reagents such as Dess-Martin Periodinane, [START_REF] Dess | Readily Accessible 12-I-5 Oxidant for the Conversion of Primary and Secondary Alcohols to Aldehydes and Ketones[END_REF] or DMSO-activated reagents [START_REF] Mancuso | Activated Dimethyl Sulfoxide: Useful Reagents for Synthesis[END_REF] were figured among the most widely used oxidative reagents (Table 5, entries 11-13). Unlike those reagents, Pyr-Mn 2 can be used in catalytic quantities, while conserving competitive conversions and yields, within a reasonable reaction time and in an acceptable solvent, contrary to CMR solvent like chloroform (Table 5, entry 7), dichloromethane (Table 5, entries 11 and 13) or acetonitrile (Table 5, entry 12). Its nontoxic and ecofriendly use contributed to make it an efficient and green system, as much in the catalyst it-self-as in the reaction conditions.

As Pyr-Mn 2 showed the best reactivity for alcohol oxidation, we studied the versatility of Pyr-Mn 2 on the aerobic oxidation of other alcohols. Results were reported in Table 6. The activity of Pyr-Mn 2 remained quite similar regarding primary (Table 6, entries 1-3), secondary (Table 6, entry 4) and allylic alcohol (Table 6, entry 5). These results prove the generality of Reaction conditions: benzyl alcohol (1 mmol), catalyst (0.3 eq. Mn), anhydrous toluene (10 mL), under air flow, temperature 110 °C, 5 h.

b Conversion, yield and selectivity were established by GC-MS, using dodecane as intern standard. c The selectivity was calculated as the ratio of yield over conversion. Table 6. Extension of the Process to the Oxidation of Other Alcohols by Pyr-Mn 2 a a Reaction conditions: alcohol (0.5 mmol), catalyst (0.3 eq. Mn), anhydrous toluene (5 mL), under air flow, temperature 110 °C, 5 h. b Conversion, yield and selectivity were established by GC-MS, using dodecane as intern standard. c The selectivity was calculated as the ratio of yield over conversion.

the proposed method. It should also be noticed the total selectivity obtained for each case.

Pyr-Mn 2 was also tested in the selective oxidation of 5hydroxymethyl-2-furfural (HMF) in 2,5-diformylfuran (DFF). As it derives from the dehydration of carbohydrates such as fructose, glucose, cellulose, and insulin, [START_REF] Lewkowski | Synthesis, Chemistry and Applications of 5-Hydroxymethyl-Furfural and Its Derivatives[END_REF][START_REF] Kuster | A Review Focussing on its Manufacture[END_REF][START_REF] Chheda | Production of 5-Hydroxymethylfurfural and Furfural by Dehydration of Biomass-Derived Mono-and Poly-Saccharides[END_REF] HMF represents a renewable alternative from fossil-fuel resources. HMF is considered a building block platform for the synthesis of many top-value-added chemicals. [START_REF] Rosatella | 5-Hydroxymethylfurfural (HMF) as a Building Block Platform: Biological Properties, Synthesis and Synthetic Applications[END_REF] Selective oxidation of the hydroxyl group in aldehyde is of particular interest as DFF finds various applications. [START_REF] Gandini | Furans in Polymer Chemistry[END_REF][START_REF] Hopkins | Extended Aromatic Furan Amidino Derivatives as Anti-Pneumocystis Carinii Agents[END_REF][START_REF] Ma | Efficient Aerobic Oxidation of 5-Hydroxymethylfurfural to 2,5-Diformylfuran, and Synthesis of a Fluorescent Material[END_REF] 60% conversion was reached by using Pyr-Mn 2 , with 77% selectivity in DFF (Table 6, entry 6). An IR analysis and a GC-MS analysis of the products obtained after silylation by N,O-bis(trimethylsilyl)trifluoroacetamide revealed that no carboxylic acids were formed. This result proved one more time that no overoxidation occurred using Pyr-Mn catalysts. Reaction conditions: diol (0.5 mmol), catalyst (0.3 eq. Mn), anhydrous toluene (5 mL), under air flow, 110 °C, 5 h. b Conversion and yields were established by GC-MS using biphenyl as interne standard.

Table 8. Oxidative Cleavage of 1,2-Diols Catalyzed by Pyr-Mn or Other Mn Polymetallic Catalysts a Reaction conditions: diol (0.5 mmol), catalyst (0.3 eq. Mn), anhydrous toluene (5 mL), under air flow, 110 °C, 5 h. b Conversion and yields were established by GC-MS using biphenyl as intern standard. Reaction conditions: alkene (0.18 mmol), effluent (0.00006 eq. Mn), NaHCO 3 (0.88 mmol), H 2 O 2 (1.75 mmol), t-BuOH/acetone (2.6 mL), under air, 30 °C, 2 h. b Conversion, yield and selectivity were established by GC-MS using dodecane as intern standard. c The selectivity was calculated as the ratio of yield over conversion.

Our approach presents several advantages compared to the current processes. First of all, the reaction does not need high pressure as it is performed under atmospheric conditions. Moreover, an air flow succeeds in regenerating the catalysts so pure oxygen flow or oxygen atmosphere are avoided. The quantity of Mn involved into the oxidation reaction is far below the necessary quantities reported into the literature. Last but not least, our synthetic process meets European Performance Standards as the remaining manganese concentration into the effluents after treatment is below 0.1 ppm (Table S6). The effluent can be discharged into the environment, without any health risks for nearby population nor pollution of ecosystems in surrounding areas.

Experimental Studies of Effluents as a Promoter of the Oxidative Cleavages. Oxidative cleavage of 1,2-diols is one of the paramount oxidation reactions. Whereas the stoichiometric oxidants such as periodate, tetra-acetate lead and ozonolysis lead to toxicity problems and lack of selectivity, catalytic systems are often based on expensive, precious, nonrecyclable or toxic transition metals (Re, Mo, W, Os, and Co). [START_REF] Kuḧn | Oxidative Cleavage of Olefins[END_REF] In this context, Mn IV catalysts are a real opportunity. They allow the development of innocuous, selective and costeffective systems.

Recently, our group has developed two original methods for the aerobic oxidative cleavage of 1,2-diols with polymetallic catalysts prepared from Mn hyperaccumulating plants. 12,[START_REF] Escande | Heterogeneous Sodium-Manganese Oxide Catalyzed Aerobic Oxidative Cleavage of 1,2-Diols[END_REF] We wished to apply our new Pyr-Mn 2 catalyst derived from pyrite quarrying in oxidative cleavage reactions, to show the compatibility of the catalyst with different conditions and demonstrate its values as powerful synthetic tools. meso-Hydrobenzoin was chosen as a model for the oxidative cleavage of 1,2-diols by Pyr-Mn 2 . As for alcohol oxidation, the oxidative cleavage can be performed under mild conditions. Air atmosphere was sufficient to promote the reaction: no pressure or no atmosphere of O 2 was required. Conversion of 100% was obtained with 71% yield in benzaldehyde after 5 h reaction (Table 7, entry 1). The remaining 29% were assigned to diphenylethanedione. Benzoin was not observed. IR spectra of the crude product also attested that no carboxylic acids were formed during the reaction, contrary to the mixture obtained by Mecozzi et al. [START_REF] Mecozzi | Oxidation of Vicinal Diols to α-Hydroxy Ketones with H2O2 and a Simple Manganese Catalyst[END_REF] What is more, when the diphenylethanedione was engaged in the presence of the catalyst in the same conditions, no cleavage occurred, even after 24 h. This experiment proves that the diphenylethanedione was not an intermediate product of the cleavage to benzaldehyde. Thus, two types of oxidation proceeded simultaneously: the mild oxidation of meso-hydrobenzoin into diketone as well as the oxidative cleavage into benzaldehyde.

In order to study the recycling of Pyr-Mn 2 , the catalyst was reused for three runs. After reaction, Pyr-Mn 2 was separated by filtration and washed three times with ethyl acetate and three times with ethanol before being dried in an oven at 140 °C for 24 h. The recovered catalyst was then reapplied in the oxidative cleavage of meso-hydrobenzoin. As summarized in Table 7, the mineral composition remained virtually the same even after three runs. The conversion and yields were unchanged in the second run, even by letting the reaction proceeding for 24 h (Table 7, entry 2). However, the reactivity of Pyr-Mn 2 decreased when engaged for the third time in the cleavage of meso-hydrobenzoin (Table 7, entries 3 and 4). The conversion increased from only 20% after 5 h to 73% in 24 h.

These results prompted us to look for a new catalyst able to promote preferentially the reaction toward the oxidative cleavage of 1,2-diols. We turned toward Pyr-Mn 3 , whose formation at higher pH was expected to facilitate the oxidation of Mn II into Mn IV .

Benzaldehyde was formed with higher yields with Pyr-Mn 3 for the cleavage of meso-hydroxybenzoin (Table 8, entry 2). The cleavage of 1-phenylethane-1,2-diol is of particular interest. Yield of 100% was reached with Pyr-Mn 3 (Table 8, entry 3) whereas many Mn-catalysts described in the literature do not work in these conditions. For example, Na-Mn LMO needed the presence of NaHCO 3 to reach 53% conversion (Table 8, entry 6). With EcoMnOx, the cleavage of benzylicaliphatic 1,2-diol required 24 h to obtain 57% of conversion (Table 8, entry 5). It should be mentioned that no benzaldehyde was formed when Pyr-Mn 2 was engaged with 1-phenylethane-1,2-diol. Finally, the methodology can be extended to the cleavage of α-hydroxyacid, as illustrated with the example of mandelic acid (Table 8, entry 4), for which more than 95% yield was obtained.

Studying the Catalytic Behavior of Manganese-Based Catalysts for the Epoxidation of Alkenes. The functionalization of alkenes to furnish epoxides in green and sustainable conditions is a motivating challenge. Indeed classical processes, involving organic peroxides or percarboxylic acids such as mchloroperoxybenzoic acid (m-CPBA), [START_REF] Sienel | Ullmann's Encyclopedia of Industrial Chemistry[END_REF] are questionable as regards safety considerations and the large production of wastes. Ideally, ecofriendly oxidants such as H 2 O 2 in combination with Mn or Fe constitute effective catalysts for epoxidation. Recently, our group has reported the first Mnbiosourced catalysts (Eco-Mn) for the epoxidation of relatively reactive olefins. [START_REF] Escande | Switchable Alkene Epoxidation/Oxidative Cleavage with H 2 O 2 /NaHCO 3 : Efficient Heterogeneous Catalysis Derived from Biosourced Eco-Mn[END_REF] Starting from the manganese-hyperaccumulating plant Grevillea exul subsp. rubiginosa, we prepared a heterogeneous Eco-Mn catalyst, by incorporation of biosourced polymetallic complexes into montmorillonite K10. The supported Eco-Mn catalyst gave much higher yields of epoxides than previously described Mn-derived heterogeneous catalysts. [START_REF] Escande | Switchable Alkene Epoxidation/Oxidative Cleavage with H 2 O 2 /NaHCO 3 : Efficient Heterogeneous Catalysis Derived from Biosourced Eco-Mn[END_REF] However, the process required the preparation of intermediary polymetallic chloride catalysts. Active Eco-Mn were prepared via the sequence thermic treatment of natural resource (leaves)/activation by HCl/addition of sodium carbonate and oxidation.

It is clear that the possibility of a direct preparation of active Mn catalysts would be much more efficient and ecofriendlier than the synthesis of active Eco-Mn. In order to explore this opportunity, we investigated the direct transformation of effluents into active oxidant catalyst. Unlike Burgess et al., the epoxidation reaction by mixing hydrogen peroxide and aqueous sodium hydrogen carbonate in DMF can be catalyzed by Mn II salts in a green solvent instead of DMF. [START_REF] Lane | Manganese-Catalyzed Epoxidations of Alkenes in Bicarbonate Solutions[END_REF] The formation of a manganese peroxycarbonate complex, which mimics the Mn-catalase, is one of the supposed formulated mechanism. [START_REF] Saisaha | Mechanisms in Manganese Catalysed Oxidation of Alkenes with H2O2[END_REF] In that purpose, effluents, as a source of Mn II salt (0.00006 eq. Mn), was stirred with NaHCO 3 (5 equiv) and alkene (1 equiv) in t-BuOH. After 10 min, H 2 O 2 (10 equiv) was added and then the mixture was stirred during 2 h at 30 °C. After extraction, the expected epoxide was analyzed by GC-MS. Results are reported in Table 9.

High conversions were obtained with a large variety of alkenes as well as terminal or cyclic substrates with only 0.00006 equiv of Mn. 100% selectivity was reached with styrene, cyclooctene, and cyclohexene (Table 9, entries 1-3) in t-BuOH, an ecofriendly solvent. For the less reactive substrates, high conversions were obtained in t-BuOH with 5 eq. acetone or pure acetone. Total conversion was reached for tri-or disubstituted alkenes. The double epoxidation of limonene totally occurred (Table 9, entries 5 and 6), while it was predominant for cyclooctadiene with 83% of selectivity for the diepoxides (Table 9, entry 8). As a comparison, only monoepoxides of limonene was obtained with Eco-Mn. [START_REF] Escande | Switchable Alkene Epoxidation/Oxidative Cleavage with H 2 O 2 /NaHCO 3 : Efficient Heterogeneous Catalysis Derived from Biosourced Eco-Mn[END_REF] Interestingly, the epoxide of α-pinene (Table 9, entry 7), which is unstable, was obtained in a quantitative yield. This yield is remarkable and can be advantageously compared with the known systems: 59% yield obtained by Burgess et al. [START_REF] Lane | Manganese-Catalyzed Epoxidations of Alkenes in Bicarbonate Solutions[END_REF] and 75% with Eco-Mn in DMF. [START_REF] Escande | Switchable Alkene Epoxidation/Oxidative Cleavage with H 2 O 2 /NaHCO 3 : Efficient Heterogeneous Catalysis Derived from Biosourced Eco-Mn[END_REF] The epoxides from terpenes are attractive for industrial use as biorenewable substrates. For example, Thomas et al. have reported the alternating copolymerization of α-pinenoxide and glutaric anhydride to build fully biodegradable copolymer. [START_REF] Robert | Tandem Synthesis of Alternating Polyesters from Renewable Resources[END_REF] Limonene oxide has become an excellent comonomer for the preparation of polyester, whereas limonene dioxide can replace the epichlorhydrin. The epoxide of linalool rearranged into cyclized products. The six membered ring was preferentially obtained, with 88% of selectivity (Table 9, entry 9). 76% selectivity was finally reached for nopol (Table 9, entry 10).

Epoxidation is one of the most important functionalization reactions of the C = C bond in order to improve the oxidative stability of unsaturated fatty acids. Epoxidized vegetable oils are used as biolubricants and intermediate key molecules.

Oleic acid is a conventional model compound for epoxidation. The epoxidation of oleic acid (Table 9, entry 11) afforded the desired product with high yield, indicating the efficiency of the process. A ratio of 75/25 of epoxides was probably due to the isomerization of the CC double bond before oxidation. A small amount of ketal derived from dihydroxy acid was observed (18%).

Finally, we applied this system into the epoxidation of one monosubstituted alkene, which is challenging substrate (Table 9, entry 12). While the cleavage of allylic moiety was observed (14%), the most product obtained was the expected epoxide (38%).

CONCLUSION

In conclusion, we have exploited the effluent waste from pyrite quarries and inorganic chemicals by turning these wastes into valuable resources. The characterization of obtained materials exhibited distinctive mineral composition, morphology and structure for promoting oxidation transformations. They are ecofriendly and innocuous catalysts, which proved to be highly efficient in alcohol oxidations, oxidative cleavages, and epoxidations. Good to excellent selectivities and yields were obtained under mild reaction conditions, simple operations, very low catalyst loading, and good substrate scope. The catalysts are compatible with a wide range of functional groups, making them highly competitive with conventional oxidative catalysts and reagents. Finally, this study encourages the development of recycling technologies, which aims at exploring breakthrough solutions for the development of new ecomaterials and processes within the context of sustainable chemistry.

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of chargeat DOI: 10.1021/acssusche-meng.8b06582.

Evolution of catalyst mineral composition as a function of the pH of precipitation; Mn 2p XPS spectra of Pyr-Mn; HRTEM image of commercial activated MnO 2 ; N 2 adsorption-desorption isotherm of Pyr-Mn 1 , Pyr-Mn 2 , and commercial activated MnO 2 and pore size distribution of the corresponding samples; STEM-EDX mapping of Pyr-Mn 1 ; study of the mineral composition of effluents after filtration (PDF)

Figure 1 .

 1 Figure 1. Formation of pyrite quarry effluents.

Figure 2 .

 2 Figure 2. Transformation of pyrite quarry effluents into new oxidative catalysts as an alternative of current processes.

Figure 3 .

 3 Figure 3. TEM images of (a) Pyr-Mn 1 and (b) Pyr-Mn 2 .

Figure 4 .

 4 Figure 4. STEM-EDX mapping of Pyr-Mn 2 .

Table 1 .

 1 Study of Mineral Composition of Different Effluents Derived from Pyrite Quarrying According to the Seasons

	composition (ppm)

Table 2 .

 2 Mineral Composition of the Catalysts Synthesized at Different pH of Precipitation

	composition (wt % ± SD)

Table 3 .

 3 Specific Surface Area of Pyr-Mn Catalysts, Commercial Activated MnO 2 , and Other MnO 2 Catalysts

	catalyst	refs	S surface area (m 2 g -1 )
	Pyr-Mn 1	pH 9.5 [this article]	209
	Pyr-Mn 2	pH 11 [this article]	119
	commercial activated MnO 2	[this article]	88
	Mn 0.74 Cu 0.01 Al 0.25	14	75-115
	amorphous MnO 2	15	189
	OL-1) birnessite-type MnO 2 (Na-	15	57
	γ-MnO 2	15	38
	OMS-1	15	47
	OMS-2	15	81
	OMS-6	15	53
	OMS-7	15	17

Table 4 .

 4 Comparison of the Activity of Different Catalysts in the Aerobic Oxidation of Benzyl Alcohol a

	entry catalyst	equiv Mn	conversion [%] b	yield [%] b	selectivity [%] b,c
	1	Pyr-Mn 1	1	70	69	>99
	2 d	Pyr-Mn 1	0.3	22	21	>99
	3	Pyr-Mn 2	1	≤86 e	≥44 e
	4 d	Pyr-Mn 2	0.3	79	78	>99
	5	Pyr-Mn 3	1	≤45 e	≥16 e
	6 d	Pyr-Mn 3	0.3	10	9	>99

a Reaction conditions: benzyl alcohol (1 mmol), catalyst, anhydrous toluene (10 mL), temperature 110 °C, 5 h. b Conversion, yield and selectivity were established by GC-MS, using dodecane as internal standard. c The selectivity was calculated as the ratio of yield over conversion. d Reaction under air flow. e Loose of reagents and products onto catalyst matrix.

Table 5 .

 5 Comparison of the Activity of Pyr-Mn 2 with that of Synthetic and Commercial Catalysts in Benzyl Alcohol Oxidation

	entry		oxidative agent	equiv	t [h]	solvent	conversion [%] b	yield [%] b
	1 a	Pyr-Mn 2		0.3	5	toluene	79	78
	2 a	synthetic Mn catalyst	0.3	5	toluene	>99	99
	3 a	synthetic Mn-Ca catalyst	0.3	5	toluene	>99	99
	4 a	synthetic Mn-Mg catalyst	0.3	5	toluene	67	66
	5 a	synthetic Mn-Ca-Mg catalyst	0.3	5	toluene	90	89
	6 a	Activated MnO 2		0.3	5	toluene	37	36
	7	MnO 2	24		12	23	chloroform	89
	8	K-OMS-2 30		0.5	4	toluene	90	89
	9	Amorphous MnO x	25		1.5	toluene	18-73	17-72
	10	γ-MnO 2 25		0.5	1.5	toluene	39	38
	11	dipyridine Cr(IV) oxide 31	6	0.4	dichloromethane	95
	12	Dess-Martin Periodinane 32	1	2	acetonitrile	>99	99
	13	DMSO-(COCl) 2 33	2.2	0.3	dichloromethane	>99	99
	a

Table 7 .

 7 Reusability of Pyr-Mn 2 Catalyst in the Oxidative Cleavage of meso-Hydrobenzoin a

					composition (wt % ± SD)
	entry	run	Mn	Na	K	Mg	Ca	Fe	Al	temp [h]	conv [%] b	2 [%] b 4 [%] b
	1	no. 1	3.0 (±0.2)	0.03 (±0.02)	0.05 (±0.02)	12.7 (±0.8)	4.53 (±0.7)	2.7 (±0.2)	1.2 (±0.1)	5	100	71	29
	2	no. 2	2.9 (±0.4)	0.1 (±0.1)	0.1 (±0.1)	12.0 (±1.8)	4.2 (±0.6)	2.6 (±0.4)	1.2 (±0.2)	24	100	72	28
	3 4	no. 3	2.7 (±0.1)	0.01 (±0.01)	0.1 (±0.1)	11.3 (±0.1)	2.4 (±1.7)	2.5 (±0.1)	1.1 (±0.1)	5 24	2 0 73	1 4 31	6 42
	a

Table 9 .

 9 Epoxidation of Alkenes with Effluents, in the Presence of NaHCO 3 and H 2 O 2

	a

ACKNOWLEDGMENTS

The authors would like to thank CNRS and ENS for financial support. Colas Centre Ouest and GBN are acknowledged for the effluents. Plateforme Microscopie Electronique et Analytique of University of Montpellier has our thanks for TEM and HRTEM analyses.

AUTHOR INFORMATION

Corresponding Author *E-mail: claude.grison@cnrs.fr.

ORCID

Claude Grison: 0000-0002-2687-1520

Notes

The authors declare no competing financial interest.