Shintaro Takahashi

Kazuki Nakaya

Pastor María Frutos

Antoine Baceiredo

Nathalie Saffon- Merceron

Stéphane Massou

Norio Nakata

Daisuke Hashizume

Vicenç Branchadell

Tsuyoshi Kato

Synthesis of A Stable N-Hetero-Rh I -Metallacyclic Silanone

Keywords: stable silanone, stable silylene, NHSi, metallacycles, rhodium complex

Abstract: A novel N-hetero-Rh I -metallacyclic silanone 2 has been synthesized. The silanone 2, showing an extremely large dimerization energy (G = +86.2 kcal/mol), displays considerable stability and persists in solution up to 60 °C. Above 120 °C, an intramolecular C sp3 -H insertion occurs slowly over a period of two weeks leading to the bicyclic silanol 5. The exceptional stability of 2, related to the unusual electronic and steric effects of Rh I -substituent, should allow for a more profound study and understanding of these new species. Furthermore, the metallacyclic silanone 2 presents two reactive centers (Si=O and Rh), which can be involved depending upon the nature of reagents. Of particular interest, the reaction with H 2 starts with the hydrogenation of Rh I centre leading to the corresponding Rh III -dihydride complex 7 and it undergoes a cis/trans-isomerization via a particular mechanism, demonstrating that addition-elimination processes can also happen for silanones just like their carbon analogues! Silanones are generally highly reactive transient species and are observable only under specific conditions such as in argon matrix at low temperature, [1] in the gas phase [2] or on the solid surface. [3] Particularly, the dimerization and oligomerization of silanones, producing stable Si-O -bonded compounds, are strongly exothermic and barrierless processes. The poor stability of silanones is the obvious bottleneck to advance a more profound study of their reactivity. Recently, various silanones stabilized as donor-or donor/acceptor-complexes, have been synthesized and were demonstrated to be useful stable silanone sources. [4][5][6][7][START_REF] Xiong | For a recent review of silanones and their heavier Group 14 congeners[END_REF] These efficient stabilization methods also allowed to isolate SiO 2 as stable donoracceptor complexes. [9] Nevertheless, not surprisingly, the study of their reactivity is considerably restricted, which hampers profound exploration of their potentials in synthesis. However, the synthesis of stable "base-free" silanones remains a difficult task. Indeed, although the recent studies demonstrated that bulky or/and strongly -or/and -donating substituents efficiently stabilize silanones I-V enough to be isolated in the crystalline form, [10][11][12][13][14] most of them remain unstable in solution at room temperature. To the best of our knowledge, only two silanones, such as the chromium-substituted cationic silanone I [10] and cyclic dialkylsilanone V, [14] are stable in solution at RT. However, even silanone V, protected by extremely bulky substituents, dimerizes above 60 °C. As a consequence, up to date, their reactivity exploration is still largely limited to reactions taking place at room temperature. [15] Here we report the synthesis of a N-hetero-Rh I -metallacyclic silanone 2 which presents a remarkable persistence in solution, and therefore allows reactions requiring thermal activation such as C(sp 3 )-H and H-H bond activations.

We have recently reported the synthesis of an isolable N-hetero-Rh I -metallacyclic silylene (R' = Ph) [16] with a limited stability (t 1/2 = 2 h, at 50 °C). [START_REF] Bruno | reaction involving the phenyl group of DPPE is quite common for binuclear transition metal complexes with a reactive metal centre at the -position of phosphorus atoms of DPPE: a)[END_REF] The silylene 1 with cyclohexyl groups (R' = Cy) on the phosphorus atoms shows an improved stability, and no degradation was observed in toluene at 60 °C for several hours (decomposition at 110 °C, t 1/2 = 8 h).

The silylene 1 readily reacts with N 2 O at corresponding silanone 2 (Scheme 1), which was isolated as highly air sensitive orange crystals from a saturated pentane solution at RT (yield 83 %). Silanone 2 is stable in C 6 D 6 solution degradation was observed after a week at 60 °C spectrum, a signal with a high multiplicity (dddd) appears at significantly low field ( = 81.5 ppm, 1 J SiRh = 83.1 Hz, 44.8 and 170.1 Hz) compared to those of donor (-55 to -91 ppm), [4][5][6][7][START_REF] Xiong | For a recent review of silanones and their heavier Group 14 congeners[END_REF] but in the same region as observed for coordinated silanones II -IV stabilized by -donating substituents (28.8 -71.3 ppm). [11,12,13] Nevertheless, this signal is considerably up-field shifted relative to those observed for other three coordinated silanones without -donor substituent chromium-substituted cationic silanone I (170 ppm) dialkyl silanone V (129 ppm). [14] The 31 P-NMR spectrum displays three signals of an AMM'X pattern with large rhodium coupling constants [P A : dddd,  = 72.0 ppm, 1 J dddd,  = 56.2 ppm, 1 J PRh = 125.6 Hz, P C : dddd, 1 J PRh = 151.0 Hz]. This spectral pattern, showing a large coupling constant ( 2 J PA-PB = 324.4 Hz) and two small constants ( 2 J PA-PC = 26.0 Hz and 2 J PB-PC = 23.7 square planar Rh I complexes, suggesting a classical around the Rh I -center in 2, instead of the tetrahedral geometry observed for silylene 1. [START_REF]The crystal structure clearly demonstrated the distorted tetrahedral geometry around Rh atom of silylene[END_REF] The molecular structure of silanone 2 [1 geometry around the three-coordinated silicon atom ( and a short Si=O bond length [1.540 ( 3) Å] which is longer than those of chromium-substituted silanone I (1.526 Å) dialkylsilanone V (1.518 Å) [14] but is similar to those observed for other -donor substituted silanones II -IV (1.533 The N1-Si1 bond length [1.775 (3) Å] is almost identical to silylene 1 (1.771 -1.781 Å). Although the Si1 Å] is slightly elongated compared to those of 1 is still shorter than classical Si(sp 2 )-Rh I bonds (2.30 which suggests a remaining Rh-Si -interaction. structure of silanone 2 also demonstrates that, a silicon centre, 2 reveals a square planar geometry around centre [ Rh = 30.07° for 2 (Figure 1),  Rh = 63.38 in good agreement with the 31 P-NMR data at -40 °C to give the ), which was isolated as highly air sensitive orange crystals from a saturated pentane solution at RT solution, and no detectable at 60 °C. In the 29 Si-NMR spectrum, a signal with a high multiplicity (dddd) appears at = 83.1 Hz, 2 J SiP = 30.2, donor-stabilized silanones as observed for threedonating substituents this signal is considerably erved for other threedonor substituents such as (170 ppm) [10] and cyclic NMR spectrum displays three signals of an AMM'X pattern with large rhodium-phosphorus J PRh = 110.2 Hz, P B : : dddd,  = 101.5 ppm, his spectral pattern, showing a large trans-Hz) and two small cis-coupling 23.7 Hz), is typical for a classical geometry the unusual distorted which is longer than (1.526 Å) [10] and of but is similar to those observed for (1.533 -1.543 Å). [11,12,13] is almost identical to that of 1 Å). Although the Si1-Rh bond [2.237 (1) (2.141 -2.174 Å), it bonds (2.30 -2.35 Å), [START_REF] Neumann | Si(sp 2 )-Rh bonds: a) E[END_REF] interaction. The X-ray also demonstrates that, after the oxidation of square planar geometry around the metal = 63.38 -76.88° for 1 [START_REF] Mcbee | [END_REF] ], data. The space-filling representation of 2 clearly shows a shielded by substituents (Figure 1 right). geometry, the steric shields of cyclohexyl groups reach to the space around (even beyond) the terminal O atom range steric protection by the particular substituent system explains the exceptional thermal stability To gain more insight into the electronic structure of DFT calculations have been performed a theory. The optimized structure of experimentally observed one (Si-O: 1.556 Å, 2.286 Å,  Rh = 33.4°). on the Si atom of silanone is particularly small even smaller than that calculated for the silanone -and -donating bora-ylide substituent (+1.94). due to an extremely strong -donating effect of Rh I atom. Contrarily, the tetrahedral was predicted to present a strong  well known that -donating substituents trigonal planar geometry of Si atom. the adaptation of square planar geometry of donation and strong -donation) rather than tetrahedral one (strong -donation and strong -acceptation). silanone 2 with a tetrahedral Rh I energy surface. The calculations also indicate dimerization of 2 showing a strong process (G = +86.2 kcal/mol), which experimentally observed high thermal stability of noting that the dimerization of related persistent silanones are thermodynamically favored process kcal/mol respectively). [11,13] In marked contrast smaller substituents (Me groups on the P and N atoms and aryl group instead of Cy, tBu and iPr groups dimerization is strongly exergonic the exceptional stability of 2 is principally due to the efficient kinetic protection by the bulky substituents. much smaller than that calculated for Me clearly indicates that the Si-Rh bond has a 58) relative to the corresponding ), while the Si-N bond order remains almost and 0.45 for 1, Figure 2), suggesting a square planar Rh I substituent toward . Nevertheless, the positive charge is particularly small (+1.69), which is for the silanone IV with a strongly ylide substituent (+1.94). This is probably donating effect of the square planar tetrahedral Rh I substituent in the silylene 1 -accepting character. [16] It is also donating substituents efficiently stabilize a trigonal planar geometry of Si atom. [12,13,22] These probably explain adaptation of square planar geometry of Rh I atom (weak donation) rather than tetrahedral one (strong acceptation). It should be noted that the group is not a minimum on the calculations also indicate the disfavored a strongly endergonic nature of the , which is in good agreement with the high thermal stability of 2. It is worth noting that the dimerization of related persistent silanones II and IV thermodynamically favored processes (G = -30 and -16

In marked contrast, for a model 2' with (Me groups on the P and N atoms and aryl Pr groups respectively, Figure 2), the (G = -64.8 kcal/mol). Therefore, is principally due to the efficient kinetic protection by the bulky substituents. However, this value is much smaller than that calculated for Me 2 Si=O (G = -95 kcal/mol), suggesting considerable thermodynamic stabilization provided by the electronic effect of N-and Rh(I)-based substituents.

The highest occupied molecular orbital (HOMO, -4.83 eV) corresponds to the d(z 2 ) orbital of Rh atom. As expected, due to the presence of electron-donating Rh-substituents, the energies of n O (HOMO-4 and -7: -5.99 and -6.20 eV) and  Si=O (HOMO-10: -6.99 eV) bond orbitals are significantly higher than those of Me More interestingly, at 120 °C, an intramolecular C(sp 3 )-H insertion involving a t-butyl group gives the bicyclic silanol 5. Due to the high thermal stability of 2, the reaction is particularly slow and takes two weeks to reach complete conversion. Both reactions are diastereoselective, probably due to the unsymmetrical environment around the Si=O moiety related to the distorted square planar geometry around the Rh atom in 2. In contrast to these reactions at the Si=O function, the reaction of 2 with CO takes place at the Rh centre to form the corresponding carbonyl complex 6. This has been clearly indicated by the 29 Si-NMR signal for the unreacted silanone fragment ( = 102 ppm) as well as the appearance of a new carbonyl carbon signal in a typical region in 13 C-NMR ( = 206 ppm, J RhC = 56.3 Hz). Silanone 2 also reacts with H 2 via an oxidative addition to the Rh center at room temperature, affording the corresponding Rh IIIdihydride-substituted silanone 7 as a mixture of cis/trans-isomers (cis : trans = 1 : 2, Scheme 3). The oxidative addition of H 2 is reversible at room temperature as indicated by the H 2 -pressuredependent ratio between 2 and 7, and the constant ratio of cis/transisomers of 7 at different pressures. The complete conversion to 7 was achieved under 3 bar of H 2 -pressure, and the initial Rh Isubstituted silanone 2 was completely recovered after removal of H 2 -pressure. Although all attempts to isolate 7 failed, its formation as a mixture of two isomers is supported by a multi-nuclear NMR analysis showing particularly large trans-coupling constants of two nuclei on the Rh atom such as J HP (124 Hz) [22] for 7-cis and J HH (12.6 Hz) [23] and J SiP (176 Hz) [START_REF]No detectable 2 JHH coupling was observed for the 7-cis[END_REF] for 7-trans. As expected, for both isomers, the large coupling constants between two apical phosphorous atoms are also observed (J PP(trans) = 372 Hz for 7-cis and 373 Hz for 7-trans). The remaining silanone functions are also confirmed by their 29 Si-NMR signals appearing in the typical region (117.8 ppm for 7-cis and 65.4 ppm for 7-trans). Under the H 2 pressure (3bars), the Rh III -substituted silanones 7 are stable at room temperature, while, at 60 °C, they evolve further via a hydrogenation of silanone moiety to give the corresponding silanol 8. The formation of 8 was confirmed by an X-ray diffraction analysis.

To understand better the reaction of 2 with H 2 , we performed DFT calculations with a simplified model 2' (Figure 3). The calculations indicate slightly exergonic nature of the first hydrogenation reaction to form the Rh III -substituted silanone 7'-cis (G 2'  7'cis = 3.6 kcal/mol) and its low energy barrier (G ǂ 2'  7'cis = 11.1 kcal/mol), which is in good agreement with the experimentally observed reversibility of the reaction at room temperature. The calculations also indicate that the cis/trans isomerization of H 2 Rh III complex 7'-cis starts with the 1,2-migration of hydrogen from the Rh to the Si atom to form a zwitter-ionic intermediate 9'-cis. The successive conformational transformation of 9'-cis into 9'-trans, changing the side of H-Rh bond relative to the H-Si bond, proceeds through the formation of -hydride (O=SiH-Rh) complex as intermediate. [21] Then, the reverse 1,2-hydrogen migration from Si to Rh centre affords the corresponding trans-isomer 7'-trans. [START_REF] Lekich | A similar mechanism of cis-trans isomerization was reported for the a pincer-type carbonyl-Ir complex, proceeding through the reversible hydride migrations between Ir and C=O ligand[END_REF] All transformations in the cis/trans isomerization process cost little energy and the trans-isomer is only slightly more stable than the cisisomer, which explains the formation of 7-trans as the major isomer. Despite the intervention of transition metal moiety, this cis/transisomerization (hydride attack on the silanone to form a silanolate intermediate 9 / elimination-readdition of Rh to change sides of Rh-H bond / reverse hydrogen migration to reform the silanone) [21] demonstrates that the addition-elimination process, which is one of the most important reactions of the carbonyl group, can also happen for silanones. [START_REF] Ishida | elimination of KBr from the corresponding silanolate ion is known[END_REF] Concerning the mechanism of the hydrogenation of silanone 2 into silanol 8, three possibilities can be considered: 1) direct hydrogenation of Rh I -substituted silanone 2, 2) direct hydrogenation of H 2 Rh III -substituted silanone 7 followed by a dehydrogenation at Rh III centre, 3) isomerization of H 2 Rh III -substituted silanone 7 via hydrogen migrations. The first two possibilities were excluded by the excessively high energy barriers calculated for these processes (G ǂ 2'  8' = 31.6 kcal/mol and G ǂ 7'-cis  8' = 34.2 kcal/mol). The hydrogenation process by the isomerization of H 2 Rh III -substituted silanone 7' proceeds in three steps: 1) the Rh → Si hydrogen migration to generate the twitter-ionic intermediate 9-cis, 2) the isomerization of 9'-cis to 9'-trans, 3) Rh→O hydrogen migration to give 7'-trans. All steps cost low energy (G ǂ = 4.6 -20.7 kcal/mol). The most costly step is the last one, the Rh→O hydrogen migration, while the reaction barrier (G ǂ 9trans-8' = 20.7 kcal/mol) is much smaller than those calculated for the direct H 2 addition processes. In addition, the moderately elevated reaction barrier is in good agreement with the experimental conditions requiring a slight thermal activation (60 °C) for the reaction to occur. These results demonstrate that the hydrogenation of silanone 2 proceeds indirectly, the Rh substituent acting as an activator of H 2 . In addition, in contrast to the diasteroselective reactions of 2 with H 2 O and PhSiH 3 , the hydrogenation affords silanol 8 as a mixture of two diastereomers (major : minor = 85 : 15), which also supports the proposed indirect hydrogenation mechanism and the rapid cis/trans isomerization of Rh-hydrogenated intermediate 7.

A novel stable N-hetero-Rh I -metallacyclic silanone 2, has been successfully synthesized. The silanone 2, showing an extremely large dimerization energy (G = +86.2 kcal/mol), displays considerable stability and persists in solution up to 60 °C, while it slowly isomerizes at 120 °C via an intramolecular insertion into C(sp 3 )-H bond. The exceptional stability of silanone 2, related to the unusual electronic and steric effects of the Rh I -substituent (strong and -donations and long-range steric protection), should allow for a more profound study and understanding of silanone derivatives. Furthermore, the Rh-metallacyclic silanone 2 presents two reactive centers (Si=O and Rh), which can be involved depending upon the nature of reagents. Notably, the reaction with H 2 , resulting in the hydrogenation of silanone moiety, proceeds via cooperative involvement of both sites, and it starts with the reaction at Rh I centre. Of particular interest, the resulting silanone 7 presenting a hexacoordinate Rh III -dihydride moiety, undergoes a cis/transisomerization via a particular mechanism, demonstrating that the addition-eliminations processes, which are classical carbonyl reactivities, can also be observed with silanones. More detailed investigations on the reactivity of this new stable silanone 2 are under active investigation.

Entry for the Table of

Figure 1 .

 1 Figure 1. Molecular structure (left) and space filling representations (rignt) of silanone 2. Thermal ellipsoids represent 30 % probability. H and disordered atoms are omitted for clarity. Selected bond lengths [Å] and angles [°]: Si1-O 1.540(3), N1-Si1 1.775(3), Rh Rh-P1 2.310(1), Rh-P2 2.315(1), Rh-P3 2.362(1), P1 C1-C2 1.359(5), C2-N1 1.376(4), O-Si1-N1 106.8(2), O 129.0(1), N1-Si1-Rh 122.7(2), Si1-Rh-P1 92.1(1), P2 Rh-P1-C1 116.9(1), P1-C1-C2 130.9(3), C1-C2-Si1 124.8(2), Rh = 30.0°, Si = 358.39°. Rh : dihedral torsion angle between two planes, Rh-Si1-P1 and Rh-P2-P3. ( planar, Rh = 90°; tetrahedral)

(

  left) and space filling representations Thermal ellipsoids represent 30 % probability. H and disordered atoms are omitted for clarity. Selected bond lengths Si1 1.775(3), Rh-Si1 2.237(1), P3 2.362(1), P1-C1 1.776(4), N1 106.8(2), O-Si1-Rh P1 92.1(1), P2-Rh-P3 84.4(1), -N1 130.1(3), C2-N1-: dihedral torsion angle P3. (Rh = 0°: square [19] reveals a planar coordinated silicon atom ( si ° = 358.39°)

Figure 2 .

 2 Figure 2. Calculated NBO charges of acetone, dimethylsilanone, ylidesubstituted silanone IV and 2 as well as dimerization energies 2 and its simpler model 2-simple. W

2 clearly

 2 shows a Si=O moiety sterically well-(Figure1right). Thanks to the square planar cyclohexyl groups reach to the space the terminal O atom of silanone. This longby the particular substituent system probably stability of 2 (Figure1, right). ht into the electronic structure of silanone 2, have been performed at the M06/6-31G(d) level of theory. The optimized structure of 2 agrees quite well with the O: 1.556 Å, Si-N: 1.791 Å, Si-Rh: and bond orders (in parenthesis) -substituted silanone II, bora-ylide as well as dimerization energies (G) of WBI: Wiberg bond index.

  2 Si=O [HOMO(n O ): -9.56 eV, HOMO-1( Si=O ): -10.27 eV]. In contrast, the lowest unoccupied orbital (LUMO), corresponding to the * Si=O orbital mixed with the rhodium d-orbital (-0.78 eV) is similar to that of Me 2 Si=O [LUMO(* Si=O ): -0.89 eV]. These results are in good agreement with the high reactivity of silanone 2.

Scheme 2 .

 2 Scheme 2. Reactions of (amino)(rhodium) silanone 2

Scheme 3 .

 3 Scheme 3. Hydrogenation of silanone 2

Figure 3 .

 3 Figure 3. Calculated reaction pathways for the hydrogenation of 2' and calculated Gibbs energies G (kcal/mol) for each step of the reaction. In parenthesis are calculated reaction barriers G ǂ (kcal/mol).

  Contents (Please choose one layout) has been synthesized. This silanone, showing an extremely large dimerization energy (G = +86.2 kcal/mol), is stable in solution at 60 °C and only slowly isomerizes at 120 °C, via an intramolecular C(sp 3 )-H bond insertion. This Rh I -substituted silanone can react either on both sites, the Si=O function or the Rh center, depending on the type of reagents. Of particular interest, the reaction with H2, resulting in the hydrogenation of silanone moiety, proceeds via cooperative involvement of both sites.

We are grateful to the CNRS (IEA-SiM), the Spanish MINECO (grant CTQ2016-77978-R), the CNRS-JSPS Bilateral Open Partnership Joint Research Projects (SANDTEC-PICS, no. I2017650) and Marelli corporation (Marelli next-generation scholarship) for financial support of this work. We also thank Dr. K. Sugamata (College of Science, Rikkyo Universityon the WWW under http://www.angewandte.org or from the author.


Highlights

• Lactococcus lactis from environmental niches show high esterolytic activity • Higher metabolic diversity is seen in environmental versus dairy L. lactis strains • SGNH hydrolase family of esterases may be linked to high esterolytic activity

Abstract: Lactococcus lactis strains are widely used in the dairy industry in fermentation processes for production of cheese and fermented milks. However, the esterolytic activity of L. lactis is not generally considered high. For this reason, purified microbial lipases and esterases are often added in certain dairy processes to generate specific flavors in the final food product. This work demonstrates the superior esterolytic activity of a collection of L. lactis strains isolated from different environmental sources compared with that of dairyderived strains. It provides further evidence of the more diverse metabolic capabilities displayed by L. lactis strains from environmental sources compared to their domesticated dairy counterparts. Furthermore, the presence of a 1,287-bp gene encoding a 428-amino acid SGNH hydrolase in the high-esterolytic environmental strains suggests a possible link between superior esterolytic activity and the presence of the esterase from the SGNH hydrolase family.

L ipolysis is an important biochemical event for flavor diversifi- cation in dairy manufacture. The pathway generates free fatty acids, di-and monoglycerides, and glycerol, which contribute to the flavor profile of fermented dairy products and act as substrates for other highly flavored components [START_REF] Thierry | Lipolysis and Metabolism of Fatty Acids in Cheese[END_REF]. Many mold-ripened cheeses, such as blue cheese, undergo significant lipolytic activity through the actions of Penicillium roqueforti, whereby volatile and nonvolatile aroma compounds (mainly methyl ketones) are generated to provide unique flavors [START_REF] Collins | Lipolysis and free fatty acid catabolism in cheese: A review of current knowledge[END_REF][START_REF] Martín | Blue Cheese[END_REF]. The key enzymes involved in this lipolytic process are lipases and esterases, which catalyze hydrolysis and synthesis of esters and triglycerides, contributing to flavor development [START_REF] Broadbent | Cheese flavor and the genomics of lactic acid bacteria[END_REF]. The free fatty acids released by these enzymes act as precursors for flavor compounds such as esters, methyl ketones, lactones, and secondary alcohols [START_REF] Thierry | Lipolysis and Metabolism of Fatty Acids in Cheese[END_REF][START_REF] Mcauliffe | Symposium review: Genomic investigations of flavor formation by dairy microbiota[END_REF].

Lactic acid bacteria, including Lactococcus lactis, are usually considered to have weak esterolytic activity compared with other bacterial species such as Flavobacterium, Acinetobacter, Propionibacterium, and Pseudomonas [START_REF] Collins | Lipolysis and free fatty acid catabolism in cheese: A review of current knowledge[END_REF][START_REF] Thierry | Lipolysis and Metabolism of Fatty Acids in Cheese[END_REF]. However, L. lactis strains isolated from environmental (or nondairy) niches exhibit much greater diversity in their metabolic capabilities than their dairy counterparts [START_REF] Alemayehu | Characterization of plant-derived lactococci on the basis of their volatile compounds profile when grown in milk[END_REF][START_REF] Cavanagh | Assessment of wild non-dairy lactococcal strains for flavour diversification in a mini-Gouda type cheese model[END_REF]. Environmental lactococcal strains exhibit certain adaptation capabilities such as higher tolerance to salt and alkaline conditions, high glutamate dehydrogenase (GDH) activity, and diverse metabolization of carbohydrates, including sugars usually found in plant environments such as arabinose and xylose, which has been demonstrated to affect the production of flavor compounds in certain dairy processes [START_REF] Alemayehu | Characterization of plant-derived lactococci on the basis of their volatile compounds profile when grown in milk[END_REF][START_REF] Cavanagh | Assessment of wild non-dairy lactococcal strains for flavour diversification in a mini-Gouda type cheese model[END_REF][START_REF] Cavanagh | Evaluation of Lactococcus lactis isolates from nondairy sources with potential dairy applications reveals extensive phenotypegenotype disparity and implications for a revised species[END_REF]. Although no significant difference was found in lipase production by dairy and nondairy L. lactis in a previous study [START_REF] Nomura | Phenotypic and molecular characterization of Lactococcus lactis from milk and plants[END_REF], [START_REF] Kalbaza | Identification and major technological characteristics of Lactococcus and Lactobacillus strains isolated from "hamoum", an Algerian fermented wheat[END_REF] demonstrated higher lipolytic activity in nondairy L. lactis than in Lactobacillus strains.

In this study, we investigated the esterolytic activity of a group of 16 dairy and environmental L. lactis strains (Table 1). The dairy isolates were of the subspecies lactis, whereas all nondairy isolates, with the exception of DPC6853, were of the subspecies cremoris. However, we have shown in previous studies that environmental L. lactis strains that are genotypically subspecies cremoris behave phenotypically like dairy subspecies lactis [START_REF] Cavanagh | Evaluation of Lactococcus lactis isolates from nondairy sources with potential dairy applications reveals extensive phenotypegenotype disparity and implications for a revised species[END_REF]; therefore, we phenotypically compared the dairy subspecies lactis strains to the environmental subspecies cremoris strains. Cell extracts were prepared according to a method previously described [START_REF] Stefanovic | Genetic, enzymatic and metabolite profiling of the Lactobacillus casei group reveals strain biodiversity and potential applications for flavour diversification[END_REF] from overnight cultures grown in M17 (Oxoid, Basingstoke, UK) supplemented with 5 g/L lactose monohydrate (L-M17; VWR, Leuven, Belgium) for dairy strains or M17 supplemented with 5 g/L d(+)-glucose monohydrate (G-M17; VWR) for environmental strains. A quantitative esterase assay, relying on the principle of hydrolysis of p-nitrophenyl dodecanoate (Sigma-Aldrich, Arklow, Ireland) to dodecanoic acid and p-nitrophenyl (PNP) was used [START_REF] Bertuzzi | Modification of cheese flavour through the use of surface microbiota[END_REF]. Although both dairy and environmental strains showed esterase activity, there were clear differences between the 2 groups of strains in relation to the levels of esterase activity. The dairy strains showed activities in the range of 10 to 22 μmol of PNP/mg, with a mean of 18.5 μmol PNP/mg (Figure 1). However, environmental strains showed the greatest activity (range of 58-100 μmol of PNP/mg; mean of 78.5 μmol of PNP/mg), except for strain DPC6853, the activity of which was 17.5 μmol of PNP/mg. The environmental strain used as a reference in our study, KF147, shared high esterase activity with the environment-derived group of strains. The means of the 2 groups analyzed (dairy and environmental) differed significantly (P = 0.00003). Our findings further confirm the more variable metabolic activities of environmental L. lactis strains (Alemayehu

Superior esterolytic activity in environmental

Lactococcus lactis strains is linked to the presence of the SGNH hydrolase family of esterases Desirée Román Naranjo, 1,2 Michael Callanan, 2,3 Anne Thierry, 4 and Olivia McAuliffe 1,3 * et al., 2014; Cavanagh et al., 2014, 2015). Interestingly, one environmental strain, DPC6853, did not show this high esterolytic activity. This strain was the only one in our collection isolated from corn, and further investigation is required to determine whether there is a link between the observed activity and specific environmental conditions.

To determine a possible genetic link to the high esterolytic activity observed in the environmental strains, comparative genome analysis was performed on available genome sequences of the environmental strain set (Table 1). The genome data were analyzed using the Artemis 16.0.0 genome browser [START_REF] Carver | ACT: the Artemis comparison tool[END_REF] and the BLASTP web server (Madden et al., 1996), using default parameters. Analysis of the draft genome of strain DPC6855, isolated from grass, revealed the presence of one open reading frame (FIB60_02895) related to esterolytic activity, which encodes the predicted product SGNH/GDSL hydrolase family protein (Figure 2A). Subsequent analysis of the genomes available for 3 other environmental strains in our collection also revealed the presence of this 1,287-bp gene encoding the 428-AA SGNH/GDSL hydrolase in DPC6856 and DPC6860. Examination of the literature revealed that the SGNH-hydrolase family is a recently classified subgroup of the GDSL group of esterase and lipase enzymes that possess multifunctional properties such as regiospecificity and broad substrate specificity [START_REF] Akoh | GDSL family of serine esterases/lipases[END_REF]. A conserved XynE-like domain is associated with the SGNH hydrolase subfamily and has the consensus AA sequence of Ser-Gly-Asn-His (SGNH) found in the active site. This motif provides a catalytic mechanism different from the classical GxSxG motif-containing hydrolases, such as the lack of nucleophile elbow and the presence of a flexible active site [START_REF] Akoh | GDSL family of serine esterases/lipases[END_REF][START_REF] Reina | Isolation, characterization, and localization of AgaSGNH cDNA: A new SGNH-motif plant hydrolase specific to Agave americana L. leaf epidermis[END_REF][START_REF] Oh | Carboxylic ester hydrolases in bacteria: Active site, structure, function and application[END_REF]. The SGNH hydrolase encoded by FIB60_02895 is related to the putative arylesterase/acylhydrolase encoded by the xynE gene located in a xylanase gene cluster in the rumen microbe Prevotella bryantii [START_REF] Miyazaki | Involvement of the multidomain regulatory protein XynR in positive control of xylanase gene expression in the ruminal anaerobe Prevotella bryantii B(1)4[END_REF].

Interestingly, this gene was not found in any of the publicly available genomes of L. lactis strains from dairy sources, or indeed, the genome of strain DPC6853 from corn, which displayed lower levels of esterase activity than the other environmental strains. The presence of the gene was confirmed in other environmental strains for which genome sequence information is publicly available, such as L. lactis ssp. lactis NCDO 2118 (isolated from frozen peas; [START_REF] Oliveira | Genome sequence of Lactococcus lactis ssp. lactis NCDO 2118, a GABA-producing strain[END_REF] and L. lactis ssp. cremoris KW2 (isolated from fermented corn; [START_REF] Kelly | Interaction between the genomes of Lactococcus lactis and phages of the P335 species[END_REF]. Indeed, 2 loci encoding SGNH/GDSL hydrolase proteins were identified in strain KF147. The first is LLKF_RS02565, which encodes a GDSL family lipase. This 858-bp gene encodes a 286-AA protein and contains an Ypmr_like conserved domain. The second is a 1,286-bp gene (LLKF_0950) that encodes a 428-AA predicted protein product with 98% similarity to that found in DPC6855, DPC6856, and DPC6860 and described as a SGNH hydrolase superfamily protein, also related to esterolytic activity.

To determine the presence of the gene encoding SGNH hydrolase in the strains used in this study where whole-genome sequences are not yet available, we designed a set of primers, SGNH-F (5′-TGAGTGGTACGGCCTTTCGC-3′) and SGNH-R (5′-GAAAATAATCAATCAAGCACATACAT-3′), to amplify the partial gene sequence. No amplification product was detected in any of the dairy strains tested, whereas the 994-bp product was detected in all environmental strains except for the corn-derived strain with low esterase activity (DPC6853; Figure 2B). Subsequent sequencing of the amplified products revealed 100% identity to the gene found in DPC6855. Thus, except for the corn-derived DPC6853, all 7 environmental isolates in this study possessed the SGNH hydrolase gene, whereas it was not detected in any of the tested dairy strains using these PCR conditions. This correlates with our phenotypic analysis because the dairy strains showed low esterase activity compared with the environmental strains (<22 vs. ~80 μmol of PNP/mg, respectively), confirming a link between the presence of the FIB60_02895 gene and higher esterase activity.

In conclusion, this work provides further evidence of more diverse genotypic and phenotypic traits in L. lactis strains from environmental sources compared with their dairy counterparts. An SGNH hydrolase protein was identified that is potentially related to the higher esterase activity observed in these strains, and work is currently ongoing to associate the role of this gene with the functionality observed. Alternative knockout methods are being tested because the traditional knockout by double recombination has proven ineffective in this case. In addition, the metabolite profiles and the ability of these environmental strains to hydrolyze milk glycerides compared with the less-active dairy strains are being examined. These strains represent potential options for in situ production of lipolytic enzymes in dairy processing.

1

  Teagasc Food Research Centre, Moorepark, Fermoy, Cork, Ireland, 2 Cork Institute of Technology, Cork, Ireland, 3 VistaMilk SFI Research Centre, Moorepark, Fermoy, Cork, Ireland, 4 STLO, INRAE, Institut Agro, Rennes, France. *Corresponding author: Olivia.mcauliffe@ teagasc .ie. © 2020, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http: / / creativecommons .org/ licenses/ by/ 4 .0/ ). Received July 06, 2020. Accepted October 02, 2020.

Figure 1 .

 1 Figure 1. Esterase activity [expressed in μmol of p-nitrophenyl (PNP)/mg of cell-free extracts] of dairy and environmental Lactococcus lactis strains on the substrate p-nitrophenyl dodecanoate. The concentration of p-nitrophenyl released was determined from a standard curve obtained for a set of standards of p-nitrophenyl phosphate (0-500 nmol; Sigma-Aldrich, St. Louis, MO). The concentration of protein in each sample was calculated using the Qubit Protein assay kit (ThermoFisher Scientific, Waltham, MA). Experimental results from the different groups (dairy and environmental) were statistically examined by running a t-test (2 samples assuming equal variances) in Excel (Microsoft Corp., Redmond, WA) with an α value of 0.05. Error bars represent SD of 3 independent experiments. SeeTable 1 for sources of strains.

27

  Figure 2. (A) Graphical representation of FIB60_02895 gene locus encoding SGNH hydrolase protein and surrounding genes [FIB60_02890 (glycosyl hydrolase) and glf (UDP-galactopyranose mutase)] from the draft genome sequence of Lactococcus lactis DPC6855 isolated from grass. Also shown are the locations of the SGNH_Forward and SGNH_Reverse primers used to generate the 994-bp product in the PCR-based assay. The predicted SGNH hydrolase protein isshown, revealing the location of the conserved domain "Xyn_E-like" as well as the catalytic triad site and oxyanion hole found within the enzyme. Graphic was generated using SnapGene software (Insightful Science; snapgene.com). (B) PCR-based detection of the SGNH gene in the dairy and environmental strain sets. A 1% agarose gel was used and HyperLadder 1 kb (Bioline, London, UK) was used as the molecular weight marker. The dairy strains are represented in lanes 1 to 6: DPC141 (1), DPC155 (2), DPC176 (3), DPC220 (4), DPC266 (5), DPC420 (6), and the environmental strains are represented in lanes 7 to 15: KF147 (7), DPC6853 (8), DPC6854 (9), DPC6855 (10), DPC6856 (11), DPC6857 (12), DPC6858 (13), DPC6859 (14), DPC6860 (15). See Table1for sources of strains.

Table 1 .

 1 Lactococcus lactis strains used in this study

	DPC 1 code	Species/subspecies (ssp.)	Isolation source	Source or reference 1	Accession no.
	Dairy
	141	L. lactis ssp. lactis	Mixed-strain starter culture	DPC CC
	155	L. lactis ssp. lactis	Mixed-strain starter culture	DPC CC
	176	L. lactis ssp. lactis biovar diacetylactis	Mixed-strain starter culture	DPC CC
	220	L. lactis ssp. lactis biovar diacetylactis	Mixed-strain starter culture	DPC CC
	260	L. lactis ssp. lactis	Mixed-strain starter culture	DPC CC
	266	L. lactis ssp. lactis	Mixed-strain starter culture	DPC CC
	420	L. lactis ssp. lactis	Mixed-strain starter culture	DPC CC
	Nondairy
	6853	L. lactis ssp. lactis	Corn	Cavanagh et al. (2015); DPC CC	LAVD00000000.1
	6854	L. lactis ssp. cremoris	Grass	DPC CC
	6855	L. lactis ssp. cremoris	Grass	Roman Naranjo et al. (2019); DPC	VERW00000000.1
				CC
	6856	L. lactis ssp. cremoris	Bovine rumen	Cavanagh et al. (2015); DPC CC	LAVW00000000.1
	6857	L. lactis ssp. cremoris	Grass	DPC CC
	6858	L. lactis ssp. cremoris	Grass	DPC CC
	6859	L. lactis ssp. cremoris	Grass	DPC CC
	6860	L. lactis ssp. cremoris	Grass	Cavanagh et al. (2015); DPC CC	LAVX00000000.1
	KF147	L. lactis ssp. cremoris	Mung bean sprouts	Siezen et al. (2010)	CP001834

1 DPC CC = Teagasc DPC Culture Collection housed at the Teagasc Food Research Centre, Moorepark, Fermoy, Cork, Ireland.

  Table 1 for sources of strains.

Román Naranjo et al. | Esterolytic activity in environmental lactococci

Román Naranjo et al. | Esterolytic activity in environmental lactococci

Notes

The authors thank Jennifer Mahony (University College Cork, Ireland) for provision of strain KF147.

The authors have not stated any conflicts of interest.


Clément Lynde
email: clement.lynde@cea.fr

Eva Montbarbon

Matthieu Hamel

Amélie Grabowski

Camille Frangville

Guillaume H V Bertrand

Giacomo Galli

Frédérick Carrel

Vincent Schoepff

Ziad El Bitar

P Tns

Optimization of the Charge Comparison Method for Multiradiation Field Using Various Measurement Systems

Keywords: Particles classification, pulse shape discrimination, charge comparison method, scintillator, fission chamber

come   L'archive ouverte pluridisciplinaire

I. INTRODUCTION

ULSE-SHAPE discrimination (PSD) is a widely used technique to identify particles by analyzing the electrical signal induced in a detector [START_REF] Knoll | Radiation Detection and Measurement[END_REF] and in particular with plastic scintillator [START_REF] Bertrand | Pulse shape discrimination between ( fast or thermal ) neutrons and gamma rays with plastic scintillators : State of the art[END_REF]. Usually, such an electrical signal consists of a succession of pulses, each of them representing the detection of a neutron, X-or γ-rays, β or α particles, or the noise occurrence within the detector. Due to its simplicity and performance, one of the most frequently used PSD method is the charge comparison method (CCM) [START_REF] Flaska | Identification of shielded neutron sources with the liquid scintillator BC-501A using a digital pulse shape discrimination method[END_REF]- [START_REF] Zaitseva | Scintillation properties of solution-grown transstilbene single crystals[END_REF]. The CCM generally allows the identification of the phenomenon at the origin of each pulse solely based on the value of the tail-to-total integral. This latter involves the integration of each pulse of the detection signal over two different time periods. Since the optimal values are not known in all cases, several solutions have been considered in the literature [START_REF] Flaska | Optimization of an offline pulse-shape discrimination technique for the liquid scintillator BC-501A[END_REF]- [START_REF] Sardet | Spectres en énergie des neutrons prompts de fission : optimisation du dispositif expérimental et application à l' 238 U[END_REF] to solve this problem. As detailed in Section III.A, these methods were not entirely satisfactory for many applications; therefore, we propose a novel procedure capable of carefully choosing the integration durations.

This paper presents the discrimination principle of the associated measurement system used in it. Then, the pulse shape discrimination processed on signals is detailed. In Section IV, the optimization procedure is explained. Finally, this article presents the improvement due the optimization and focuses on three case studies.

II. DISCRIMINATION PRINCIPLE AND ASSOCIATED MEASUREMENT SYSTEMS

A. Between fast neutrons and gamma rays with organic scintillator

The scintillation mechanism in organic scintillators has been described by Birks [START_REF] Birks | Scintillations from Organic Crystals: Specific Fluorescence and Relative Response to Different Radiations[END_REF]. In the case of a charged particle (e.g. alpha, beta, proton) or a secondary ionizing particle (mainly proton for neutron and electron for gamma ray), its motion in the material will excite the scintillator whose de-excitation is accompanied by the emission of visible photons. The discrimination between neutrons and gamma rays in scintillators has been theorized by Voltz and Laustriat [START_REF] Voltz | Radioluminescence des milieux organiques -I. Etude cinétique[END_REF]. The difference between the types of particles, identifiable on the electrical signals, is due to the density of the excited states generated by the ionizing particle. The ionization power of a recoil proton being greater to a recoil electron, the density of excitation of the scintillator is therefore more important. The energy deposition of a proton thus generates more delayed deexcitation than that of an electron. As a result, for the same amount of deposited energy, the pulse generated by an interaction with neutrons will be more spread over time than a pulse generated by an interaction with gamma rays (see an example in Fig. 1). In the work presented in this article, two homemade organic scintillators were used:

• A liquid cell (left photograph in Fig. 2), filled with BC-501A liquid scintillator marketed by Saint-Gobain Crystals and Detectors. The surface of the cell was 1.5×1.5 cm 2 and the height 4 cm.

•

A plastic scintillator (right photograph in Fig. 2), composed of a high concentration of 2,5-diphenyloxazole and a small quantity of 9,10-diphenylanthracene in polystyrene. This formulation should be closed to that of EJ-276 (formerly EJ-299-33) from Eljen Technology. The diameter of the scintillator was 1.5 cm and the height 1.5 cm. We used two different photosensors to collect the light emitted from those two scintillators:

• A Hamamatsu R7724-100 photomultiplier tube (PMT), left photograph in Fig. 3.

•

A SensL ArrayC-30035-16P silicon photomultiplier (SiPM), right photograph in Fig. 3. In this article, the plastic scintillator was tested with both photosensors whereas the liquid scintillator only with the PMT.

B. Between beta and gamma rays with phoswich scintillator

For mixed radiation field, a phoswich (phosphor sandwich) detector can also be used [START_REF] Knoll | Radiation Detection and Measurement[END_REF], [START_REF] Wilkinson | The phoswich -a multiple phosphor[END_REF]- [START_REF] Huang | A gamma and neutron phoswich read out with SiPM for SPRD[END_REF]. This kind of detector consists of a combination of two or more layers of dissimilar scintillators optically coupled to only one photodetector. Scintillators must have decay times such that the pulse at the output of the photodetector is dependent on the emitted light. The differences accessible within the pulse will then identify the scintillator(s) in which the interaction occurs. In our case, a two layers phoswich plastic scintillator was used (see Fig. 4). The first 0.15 mm layer was composed of the "fast" scintillator EJ-212 (decay time of 2.4 ns) and the second 5 mm layer of the "slow" one EJ-240 (decay time of 285 ns). In order to maximize the light collection and minimize the energy deposition by beta in the reflector material, the surface of the phoswich was recovered by polytetrafluoroethylene (PTFE) with an entry window. The phoswich detector was optically coupled to the PMT described in the Section A. In this configuration, low-energy betas (< 100 keV) only interact in the first scintillator and induce very short pulses. High-energy betas (> 100 keV) interact in both scintillators and induce pulses with a fast and slow component. Due to the large volume difference (~30×), gamma rays interact mainly and only in the second scintillator and induce long pulses. The representative pulse for each of the three pulse shapes is shown in Fig. 5.

C. Between neutrons and partial discharges with fission chamber

Sodium-cooled fast neutron reactors are among the advanced reactors selected by the Generation IV International Forum. The fission chamber (see Fig. 6) has been identified as the most appropriate neutron detector for use in the vessel of this type of reactor [START_REF] Blanc | EFR98 -Synthesis on core instrumentation[END_REF]. This detector -namely High Temperature Fission Chamber (HTFC), must be able to operate under high irradiation (up to 10 10 neutrons.cm -2 .s -1 ) and high temperature (up to 650 °C). One of the effect of this hostile environment is the occurrence of partial discharges (PD). Under given temperature and bias voltage conditions, PD can induce a pulse in fission chambers. The physics underlying creation of PD is still under investigation. Therefore, the rejection of signals induced by this phenomenon has been studied [START_REF] Hamrita | Rejection of partial-discharge-induced pulses in fission chambers designed for sodium-cooled fast reactors[END_REF]. It has been experimentally proven that the neutron-induced and the partial-dischargeinduced signals have temporal differences from which it is possible to discriminate them. The representative pulse for each of the two pulse shapes is shown in Fig. 7. Fig. 7. Representatives pulses obtained with a fission chamber taken from [START_REF] Hamrita | Rejection of partial-discharge-induced pulses in fission chambers designed for sodium-cooled fast reactors[END_REF]. Left: 252 Cf neutron-induced pulses at room temperature. Right: PD-induced pulses at 600 °C.

III. PULSE SHAPE DISCRIMINATION

The required format for input data for the implementation of PSD and our optimization procedure is the temporal evolution of pulse recorded using a digitizer or a digital oscilloscope. Pulse measurements involving scintillators were performed with a CAEN "DT5743" digitizer (500 MHz of bandwidth, 12 bits of resolution) at 800 mega-samples/s with PMT and 400 mega-samples/s with SiPM, and involving fission chambers with a digital LeCroy "wavePro 725Zi" oscilloscope (2.5 GHz of bandwidth, 10 bits of resolution) at 20 gigasamples/s.

A. Charge comparison method

The Charge Comparison Method (CCM) is one of the signal processing methods used to perform PSD to discriminate signals according to their origin. The CCM can be applied when observable signals have temporal differences characteristic of their origin. It consists in integrating the pulse, , over two different time periods, a so-called slow period, , and a socalled total period, according to ( 1) and ( 2). For each pulse, its start,

, is evaluated and it is defined as the first time step from which the pulse is greater than 5% of the amplitude. Fig. 8 illustrates the placement of the other two integration gates, and . = .

(1) The tail-to-total integral used to differentiate the origin of pulses is the ratio, " ##$ , of the slow integral to the total integral, defined by following equation:

= . ! (2)
" ##$ = % (3)
The main disadvantage of the CCM is that the detector's discrimination performance is highly dependent on the choice of the integration periods. However, it is known [START_REF] Rawat | Pulse shape discrimination properties of Gd3Ga3Al2O12:Ce,B single crystal in comparison with CsI:Tl[END_REF] that each part of the acquisition chain and each parameter setting influences the integral duration values corresponding to optimal discrimination. The usual experimental setup in the laboratory where the CCM is used involves plastic scintillators (with most of the time about the same decay times) and the PMT presented in Section II.A. Therefore, the integration periods have only been optimized for this acquisition chain and are worth 800 ns for the total period and 56 ns for the fast period.

In many applications, multi-radiation fields and numerous measurement systems are encountered, so optimization methods presented in Section I were not entirely satisfactory for several reasons:

• the need for prior calibration during which the nature of the phenomena at the origin of the detection signal is known by another discrimination method; • limitations to two types of particles; • lack of solutions to exclude results that are inconsistent with the physical phenomena involved.

As an example of inconsistent results to be excluded, we can cite the case where, although only one type of particles is involved, an approximation by a function of two Gaussians may be obtained (Fig. 9). With such limitations, an optimization of the integration gates when the detector is exposed to an unknown flux of two or more types of particles is difficult to achieve.

B. Figure of Merit

When there are only two origins of pulses, the classical neutron-γ Figure of Merit (FoM), determined from an analysis on the distribution of tail-to-total integrals, is used to assess discrimination capabilities of a scintillator in a defined energy domain [START_REF] Winyard | Pulse shape discrimination in inorganic and organic scintillators. I[END_REF]. An example, obtained with a mixed neutron-gamma emitting 252 Cf source measured through the plastic scintillator and the PMT, of this analysis is shown in Fig. 10. This distribution was fit with a two-component Gaussian function (solid line in Fig. 10). The FoM is then evaluated from the two means (& '' and & ( ) ) and the two full widths at half maximum (*+,-'' and *+,- Where the coefficients α k,l are defined according to the measurement objective. An example is given in the Section V.B.

C. Rejection Ratio

The relationship between FoM and the probability of pulse classification error, also called rejection ratio (RR), has been identified by Winyard [START_REF] Winyard | Pulse shape discrimination in inorganic and organic scintillators. I[END_REF]. In the classical neutron/gamma discrimination case, MacDonald [START_REF] Mcdonald | A wearable sensor based on CLYC scintillators[END_REF] described it by ( 6) and used the intersection of the two Gaussian components as a selection criterion. This rejection ratio uses the complementary error function, denoted FGHI. Therefore, this implies that the rejection rate evaluated for all measurement systems in this work is based on an idealized Gaussian. As McDonald notes, for high values of the FoM, a rejection ratio computed using (6) will be affected by any non-Gaussian long tails or other artifacts in the peaks. It corresponds to the probability of classifying a gamma interaction as a neutron when its tail-to-total integral is greater than the intersection value. The advantage of using the intersection as a criterion is that the rejection ratio is also equivalent to the probability of classifying a neutron event as a gamma when its tail-to-total integral is lower than the intersection value. This second parameter complements the FoM because it allows a more concrete representation of particles discrimination performance.

"" = J K • FGHI 2 • √ln 2 • *.- (6)
For applications where more than two components are expected, the rejection ratio between each kind of particle can be calculated from the distributions evaluated by the Gaussian fit:

"" P, = Q P G G R S T = Q G G J R S (7)
With:

• " U , intersection point of D k and D l ;

• D k , D l , respectively the distribution of the k-th and the lth components of the Gaussian function.

D. Energy calibration

The pulse distribution as a function of the total integral is exploited for energy calibration of the acquisition chains (here 137 Cs and 22 Na examples in Fig. 11). It was done for the following cases: liquid scintillator with PMT, plastic scintillator with PMT and plastic scintillator with SiPM. The results, presented in Section V, for these three measurement systems were performed for energy above 250 keVee. It is not usual to perform an energy calibration for the other two systems: phoswich with PMT and fission chambers. The calibration was carried out according to the procedure proposed in [START_REF] Swiderski | Measurement of Compton edge position in low-Z scintillators[END_REF], [START_REF] Li | Energy calibration for plastic scintillation detectors based on Compton scatterings of gamma rays[END_REF]. When there were identifiable, we use the Compton edge energies of 137 Cs (477 keV) and 22 Na (341 keV and 1062 keV) and the photopeaks of 241 Am (60 keV), 133 Ba (81 keV) and 57 Co (122 keV). A linear energy response function was then applied for the calibration. Thanks to this energy calibration, the calculation of the FoM value can be performed in a given energy domain in order to assess the impact of the energy on discrimination performance. The bi-parametric histogram presented in Fig. 12 corresponds to the two-dimensional graphical representation of the distribution of tail-to-total integral as a function of the total integral. To highlight densely populated areas, the maximum value of the color level was chosen for a number of five occurrences. Two areas are visible, the top area corresponds to neutron interactions and the bottom area contains the contribution of gamma rays. One can also notice that there are pulses down to about 1 a.u., then none between 0.5 and 1 a.u. and then some between 0.25 and 0.5 a.u. This singular distribution is due to the presence of thermionic emissions of PMT.

IV. OUR PROCEDURE FOR OPTIMIZING CCM

The goal behind our algorithm development is to identify optimal integration periods by analyzing unclassified detection signals produced by two or more types of particles in order to allow the best use of an acquisition chain without characterizing each part of it. In addition, to solve the problems described in Section III.A, we developed an unsupervised method that:

1. reduces the pre-processed (baseline-subtracted) digitized pulses to two features using two adjustable reduction parameters, 2. combines the two features into one score using CCM, 3. builds a distribution of the score, 4. obtains FoM values by fitting the distribution of scores with Gaussians, 5. optimizes the adjustable reduction parameters with respect to FoM, 6. and reports performance based on RR.

A. Principle

The principle of the proposed procedure is divided into three interlocked parts:

• The main program corresponds to the first sequence (top in Fig. 13) and evaluates the FoM for all possible integration periods. From this evaluation, the maximum value of the FoM is identified, the optimal integration periods that led to this value are extracted and the RR is calculated.

•

The second sequence (center of Fig. 13) corresponds to the subprogram evaluating the FoM called by the first sequence for each combination of integration periods. Then a set of Gaussian fits whose number of components varies from one to the expected number of components is made on the distribution of the tail-to-total integral. The compliance with an acceptance criterion, the value of which is defined by the user, on the correlation coefficients of all fits is checked and, where applicable, the FoM and rejection ratio calculated.

•

The third sequence (bottom in Fig. 13) corresponds to the subprogram of the second sequence verifying compliance with the acceptance criterion in terms of the correlation coefficients of all fits and calculating the FoM. The FoM is only calculated when the correlation coefficient of the Gaussian fit whose number of components is equal to the expected number of components meets the acceptance criterion, otherwise its value is zero.

B. Discussion

In the application cases where the expected number of components is two (such as the classical neutron/gamma discrimination), the FoM and the rejection ratio are calculated according to ( 4) and [START_REF] Blanc | Neutron/gamma pulse shape discrimination in plastic scintillators: Preparation and characterization of various compositions[END_REF]. When the expected number of components is greater than two, one or more new FoMs are to be defined specifically according to the needs of the experiment. In this case, it is possible to use the FoM and the rejection ratio with ( 5) and [START_REF] Zaitseva | Scintillation properties of solution-grown transstilbene single crystals[END_REF] proposed in Section III.

By jointly implementing the conditions on the expected number of components and the correlation coefficient, the procedure is able to discard results that would correspond to a satisfactory approximation with regard to the correlation coefficient, but which would not reflect the physical reality of the involved phenomena.

V. EXPERIMENTAL RESULT AND DISCUSSION

Firstly, we summarized in Table 1, a comparison of the results between the integral periods usually used in the laboratory (56 ns -800 ns) and the optimal ones found with our procedure. Then, the results of our optimization procedure applied to three measurement systems are detailed:

• Neutron/gamma discrimination with the plastic scintillator and the SiPM; • Discrimination between beta and gamma rays with the phoswich organic scintillator and the PMT; • Discrimination between signal collected from neutron or partial discharge within a fission chamber.

A. Neutron/gamma discrimination with plastic scintillator and SiPM

The result of the optimization of the acquisition chain composed of the plastic scintillator coupled to the SiPM and exposed to a mixed neutron/gamma 252 Cf source is shown in Fig. 14. Fig. 15 shows the two-dimensional graphical representation of the distribution of tail-to-total integral as a function of the total integral. The distribution of the tail-to-total integrals for energy deposits greater than 250 keVee (red bar on the left in Fig. 15) and the Gaussian fit obtained for the optimal integration periods are shown in Fig. 16 (left). As shown in Fig. 14, the maximum FoM value of 0.88 is obtained for a total integration gate of 1400 ns and a fast integration gate of 165 ns. With such an optimization, the pulses detected with energy above 250 keVee have a rejection ratio of 1.92%. To illustrate the contribution of this optimization procedure to neutron/gamma discrimination performance, on the right in Fig. 16 is the result obtained by using the integration periods usually used in the laboratory. The FoM obtained is 0.58, 34% lower than the optimized one. The rejection ratio would then have been 8.6%.

B. Beta/gamma rays discrimination with phoswich organic scintillator

The application of the CCM optimization procedure applied for the discrimination of beta and gamma rays with a phoswich organic scintillator and a PMT exposed to a mixed beta/gamma rays field of 137 Cs (only the 662 keV γ-ray was accessible) and 90 Sr/ 90 Y sources is shown below in Fig. 17. In our case, it was difficult to differentiate between high-energy beta and gamma rays because of the similar shape of the pulses. Therefore, the coefficients α k,l in [START_REF] Zaitseva | Scintillation properties of solution-grown transstilbene single crystals[END_REF] were chosen so that the FoM is maximum when the difference between the approximation function corresponding to high-energy betas and the one corresponding to gamma rays is maximum. This means that the coefficient α k,l between high-energy beta and gamma rays is taken equal to 1 and all the other coefficients equal zero. As shown in Fig. 17, the maximum FoM value of 0.85 is obtained for a total integration gate of 25 ns and a fast integration gate of 11 ns. Fig. 18 shows the two-dimensional graphical representation of the distribution of tail-to-total integral as a function of the total integral. The distribution of the number of pulses as a function of the tail-to-total integral for this combination of optimal integration gates and the Gaussian fit are shown in Fig. 19. With such an optimization, pulses for which the tail-to-total integral is less than about 0.55 have a 98.86% probability of having a beta as their origin. In addition, pulses for which the tail-to-total integral is greater than about 0.55 have a 99.73% probability of having a gamma as their origin.

C. Neutron-partial discharge discrimination with fission chamber

This CCM optimization procedure was also applied to High Temperature Fission Chambers. In the experiments performed [START_REF] Hamrita | Rejection of partial-discharge-induced pulses in fission chambers designed for sodium-cooled fast reactors[END_REF], these chambers were first polarized at 400 V and subjected to a 252 Cf neutron source at room temperature, in order to collect the pulses from the neutrons without the interference of the partial discharges. Then, the neutron source was removed and the fission chambers were heated to temperatures between 400 °C and 650 °C, so that the partial discharge pulses could be collected at different temperatures without neutron interference. In this article, to apply the optimization procedure, the two samples were mixed. The result in Fig. 20 shows all the FoMs calculated according to the integration periods, as well as the identification of the optimal periods. As shown in Fig. 20, the maximum FoM value of 1.45 is obtained for a total integration gate of 150 ns and a fast integration gate of 85 ns. Fig. 21 shows the two-dimensional graphical representation of the distribution of tail-to-total integral as a function of the total integral. The distribution of the number of pulses as a function of the tail-to-total integral for this combination of optimal integration gates and the Gaussian fit are shown in Fig. 22. With such an optimization, pulses detected have a rejection ratio of less than 0.01%. To illustrate the contribution of this optimization procedure and the CCM to neutron-partial discharge discrimination, the FWHM was previously [START_REF] Hamrita | Rejection of partial-discharge-induced pulses in fission chambers designed for sodium-cooled fast reactors[END_REF] used as the feature to discriminate pulses and the rejection ratio was around 1%. The comparison is only made between rejection errors because it is not a usual practice to evaluate the FoM for this kind of acquisition chain.

VI. CONCLUSION AND FUTURE WORK

This paper presents a very simple procedure for optimizing the CCM. Thanks to this procedure, the optimal values of the integration gates are determined automatically and without requiring a prior classification of each pulse of the detection signal. This procedure is independent of the acquisition chain and can be used without full knowledge of how it works.

Thanks to the use of this procedure, we report improvement in FoM for various measurement systems. Besides, a deeper understanding can be investigated by studying the quantity of FoM as provided in Equation ( 4) from which the improvement is due. Another advantage of the procedure is that it can be adapted to experimental cases where signals of multiple and very different origins can be encountered. This advantage will be particularly interesting in the development of a detector based on a scintillator capable of also discriminating thermal neutrons, fast neutrons and gamma rays. Future work will also include implementing the proposed algorithm on an FPGA to perform real-time optimization and process data at a high acquisition rate.

Fig. 1 .

 1 Fig. 1. Baseline-subtracted and energy-normalized average pulses from neutron pulses (red line) and gamma ray pulses (blue line) obtained from a plastic scintillator, a SiPM and a 252 Cf source.

Fig. 2 .

 2 Fig. 2. Overview of the organic scintillators used for these tests and manufactured in the laboratory. Left: BC-501A liquid scintillator. Right: EJ-276 plastic scintillator.

Fig. 3 .

 3 Fig. 3. Overview of the photosensors used. Left: Hamamatsu R7724-100 PMT. Right: SensL ArrayC-30035-16P SiPM.

Fig. 4 .

 4 Fig. 4. Overview of the phoswich scintillator used. Left: Photograph. Right: Vertical cross-sectional schematic (not to scale).

Fig. 5 .

 5 Fig. 5. Baseline-subtracted and energy-normalized average pulses from low-energy beta pulses (red line), high-energy pulses (black line) and gamma ray pulses (blue line) obtained from a phoswich plastic scintillator, a PMT, 137 Cs and 90 Sr/ 90 Y sources.

Fig. 6 .

 6 Fig. 6. Simplified schematic of a fission chamber.

Fig. 8 .

 8 Fig. 8. Example of pulse processing.

Fig. 9 .

 9 Fig. 9. Example of a fit with a function of two Gaussians when only one particle is involved.

Fig. 10 .

 10 Fig. 10. Example of analysis of a histogram of tail-to-total integrals obtained from a plastic scintillator, a PMT and a 252 Cf source.For applications where more than two components are expected, we propose to use a figure of merit that could include

Fig. 11 .

 11 Fig. 11. Examples of spectrum obtained from 137 Cs and 22 Na sources with a plastic scintillator and a PMT.

Fig. 12 .

 12 Fig. 12. Example of a bi-parametric histogram of tail-to-total integral as a function of the total integral obtained from a plastic organic scintillator, a PMT and a 252 Cf source. The red bars correspond to the limits of the energy range [250 keVee; 4.5 MeVee].

Fig. 13

 13 Fig. 13 represents the procedure in the form of a flowchart, the notations are explained below: • = [XYZ; \ F]; X^_], the domain to be tested; • = [XYZ; \ F]; X^_], the domain to be tested; • " ##$ , tail-to-total integral; • a , expected number of components; • *bc, the fit acceptance criterion; • " K [_], correlation coefficient of the Gaussian fit with [_] component(s).

Fig. 13 .

 13 Fig. 13. Procedure for optimizing the CCM.

Fig. 14 .

 14 Fig. 14. Example of optimization for neutron/gamma discrimination: FoM value as a function of integration periods obtained from a plastic scintillator, a SiPM and a 252 Cf source.

Fig. 15 .

 15 Fig. 15. Bi-parametric histogram of tail-to-total integrals as a function of the total integral obtained from a plastic scintillator, a SiPM matrix and a 252 Cf source with the optimized integral durations.

Fig. 16 .

 16 Fig. 16. Histogram of the tail-to-total integrals obtained from a plastic scintillator, a SiPM matrix and a 252 Cf source. Left: with optimized integral durations. Right: with the ones usually used in the laboratory.

Fig. 17 .

 17 Fig. 17. Example of optimization for high-energy beta and gamma rays discrimination: FoM value as a function of integration periods obtained from a phoswich organic scintillator, a PMT, 137 Cs and 90 Sr/ 90 Y sources.

Fig. 18 .

 18 Fig.18. Bi-parametric histogram of tail-to-total integrals as a function of the total integral obtained from a phoswich organic scintillator, a PMT,137 Cs and 90 Sr/ 90 Y sources with integral durations optimized for high-energy beta and gamma rays discrimination.

Fig. 20 .

 20 Fig. 20. Example of optimization for neutron-partial discharge discrimination: FoM value as a function of integration periods obtained from a fission chamber at high temperature (650 °C) and a 252 Cf source.

Fig. 21 .Fig. 22 .

 2122 Fig. 21. Bi-parametric histogram of tail-to-total integrals as a function of the total integral obtained from a fission chamber at high temperature (650 °C) and 252 Cf source with optimized integral durations.

TABLE 1 :

 1 Comparison of the results between the integral periods usually used in the laboratory and the optimal ones found with our procedure

	Measurement system	Discrimination	Number of components	d efgd (ns)	Usual integral periods d ghij (ns) FoM Rejection ratio	d efgd (ns)	Optimal integral periods d ghij (ns) Rejection FoM ratio
	Plastic scintillator + PMT					1.39	0.05%	75	1050	1.40	0.05%
	Liquid scintillator + PMT	Neutron/gamma	2			0.68	5.47%	22.5	275	1.50	0.02%
	Plastic scintillator + SiPM Phoswich + PMT	Beta/gamma	3	56	800	0.58 0.33	8.60% 22.02%	165 11	1400 25	0.88 0.85	1.92% 2.28%
	Fission chamber	Neutron/partial discharge	2			1,14	0.36%	170	500	1.85	< 0.01%

French National Agency "ANR" for financial support, within the frame of the Nessyned project (ANR-15-CE39-0006).