Guillaume Gillot

Nicolas Decourcelle

Gaëlle Dauer

Georges Barbier

Emmanuel Coton

David Delmail

Jérôme Mounier
email: jerome.mounier@univ-brest.fr

-octanol, a self-inhibitor of spore germination in Penicillium camemberti

Keywords: Cheese, quorum sensing, Penicillium camemberti, germination, 1-octanol. Chemical compounds studied 957), ethanol

Penicillium camemberti is a technologically relevant fungus used to manufacture mouldripened cheeses. This fungal species produces many volatile organic compounds (VOCs) including ammonia, methyl-ketones, alcohols and esters. Although it is now well known that VOCs can act as signaling molecules, nothing is known about their involvement in P. camemberti lifecycle. In this study, spore germination was shown to be self-regulated by quorum sensing in P. camemberti. This phenomenon, also called the "crowding effect", is population-dependent (i.e. observed at high population densities). After determining the volatile nature of the compounds involved in this process, 1-octanol was identified as the main compound produced at high-spore density using GC-MS. Its inhibitory effect was confirmed in vitro and 3 mM 1-octanol totally inhibited spore germination while 100 μM only transiently inhibited spore germination. This is the first time that self-inhibition of spore germination is demonstrated in P. camemberti. The obtained results provide interesting perspectives for better control of mould-ripened cheese processes.

Introduction

Fungal spores can be dispersed in different ways, namely through air, dust or water, and are important for fungal dissemination and/or survival. Conidia are dispersed asexual spores and can be released in massive numbers by fungi, in particular by Penicillium genus members [START_REF] Dijksterhuis | Food and crop spoilage on storage[END_REF] including many food-related species. While conidia are easily disseminated, spore germination only occurs when favorable environmental conditions (i.e. temperature, pH, humidity, light and nutrients) are met. Spore germination is also influenced by spore density and is hindered at high spore densities [START_REF] Macko | Germination self-inhibitors of rust uredospores[END_REF]. First evidence for this phenomenon, also called "quorum sensing", was shown for genetic competence induction [START_REF] Felkner | A substance produced by competent Bacillus cereus 569 cells that affects transformability[END_REF][START_REF] Tomasz | Regulation of the transformability of pneumococcal cultures by macromolecular cell products[END_REF] and in light production regulation [START_REF] Nealson | Bacterial bioluminescence: its control and ecological significance[END_REF] in Gram-positive bacteria and marine vibrios, respectively. The ability of microbial cells to chemically sense the density of the surrounding population [START_REF] Fuqua | Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators[END_REF] by extracellular factors has also been found in diverse microorganisms including fungi [START_REF] Hogan | Talking to themselves: autoregulation and quorum sensing in fungi[END_REF]. This phenomenon actually allows for cell density-dependent growth regulation, hence the term "crowding effect", and appears to be prevalent in diverse fungal species [START_REF] Hogan | Talking to themselves: autoregulation and quorum sensing in fungi[END_REF]. Previous studies suggested that intercellular signaling via self-inhibitor compound production prevents premature germination and guarantees that spores only germinate at the suitable time, i.e. in favorable environmental conditions.

Self-inhibitors, which can correspond or not to volatile compounds, have been characterized in numerous fungal genera including Aspergillus, Colletotrichum, Dictyostelium, Fusarium, Glomerella, Penicillium, Puccinia, Syncephalastrum and Uromyces [START_REF] Bacon | Identification of a self-inhibitor from spores of Dictyostelium discoideum[END_REF][START_REF] Barrios-González | Germination of concentrated suspensions of spores from Aspergillus niger[END_REF][START_REF] Chitarra | Germination of Penicillium paneum conidia is regulated by 1-Octen-3-ol, a volatile self-inhibitor[END_REF][START_REF] Garrett | A stable inhibitor of spore germination produced by fungi[END_REF][START_REF] Hobot | The identification of a self-inhibitor from Syncephalastrum racemosum and its effect upon sporangiospore germination[END_REF][START_REF] Lax | Isolation, purification, and biological activity of a self-inhibitor from conidia of Colletotrichum gloeosporioides[END_REF][START_REF] Leite | Mycosporine-alanine: A self-inhibitor of germination from the conidial mucilage of Colletotrichum graminicola[END_REF][START_REF] Lingappa | A self-inhibitor of protein synthesis in the conidia of Glomerella cingulata[END_REF][START_REF] Macko | Germination self-inhibitors of rust uredospores[END_REF]. Regarding the Penicillium genus, [START_REF] Chitarra | Germination of Penicillium paneum conidia is regulated by 1-Octen-3-ol, a volatile self-inhibitor[END_REF][START_REF] Chitarra | 1-Octen-3-ol inhibits conidia germination of Penicillium paneum despite of mild effects on membrane permeability, respiration, intracellular pH, and changes the protein composition[END_REF] Penicillium camemberti, first described by Thom in 1906 [START_REF] Raper | A manual of the Penicillia[END_REF], is mainly used in cheese manufacture and more particularly for mould-ripened soft cheeses such as Camembert, Brie and Coulommiers. P. camemberti growth results in the formation of a characteristic white rind [START_REF] Abbas | Yeasts and Molds: Penicillium camemberti[END_REF]. During spore germination and mycelial growth, lipase and protease activities are involved in lipid and protein degradation in cheese resulting in fatty and amino acid release [START_REF] Beresford | The microbiology of cheese ripening[END_REF]. These latter are then transformed into important taste and aroma compounds such as ammonia, methyl-ketones, primary and secondary alcohols, esters, aldehydes, lactones and sulfur compounds [START_REF] Abbas | Yeasts and Molds: Penicillium camemberti[END_REF] and contribute to overall organoleptic qualities.

Due to the important role of P. camemberti in cheese manufacture, there is a clear interest to understand regulatory mechanisms potentially involved in conidia germination to better control soft mould-ripened cheese production. In this context, the aims of this study were to first investigate the effect of spore density on spore germination in P. camemberti, then to identify the molecules involved and finally estimate the impact of the identified compound and other volatile compounds on spore germination. NJ, USA) by inoculating a spore suspension, conserved at -80°C in 10 % glycerol (v/v), and incubating tubes for 7 days at 25°C. Strains were then sub-cultured on slant PDA and incubated at 25°C for 14 days. After incubation, spores were harvested using 8 mL of sterile distilled water supplemented with 0.1 % Tween 80. Slant agar cultures were scraped using a 1 mL sterile pipette to collect spores. Then, Roux flasks containing 100 mL Malt Extract Agar (MEA, Merck, Darmstadt, Germany) were sowed with 6 mL spore suspension. After incubation for 14 days at 25°C, 25 mL of sterile distilled water supplemented with 0.1 % Tween 80 were added to each Roux flask and the mycelium surface was scraped using a cell scraper (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The resulting spore suspension was transferred into a tube and centrifuged for 5 min at 4000 g at 4°C (CR3i multifunction, Thermo Fisher Scientific Inc., Waltham, MA, USA). After carefully discarding the supernatant, spores were resuspended in 5 mL sterile distilled water supplemented with 0.1 % of Tween 80. Finally, all suspensions of a same strain were pooled together, counted using a Malassez cell and standardized to 2.10 8 and 2.10 6 spores.mL -1 .

Materials and methods

Strains, culture conditions and spore suspension preparations

Effect of spore concentration on germination kinetics in culture broth

Spore germination kinetics were performed as described by [START_REF] Chitarra | Germination of Penicillium paneum conidia is regulated by 1-Octen-3-ol, a volatile self-inhibitor[END_REF][START_REF] Chitarra | 1-Octen-3-ol inhibits conidia germination of Penicillium paneum despite of mild effects on membrane permeability, respiration, intracellular pH, and changes the protein composition[END_REF] with some modifications in Malt Extract Broth (MEB, Merck, Darmstadt, Germany) using fresh spore suspensions. To determine the effect of spore density on germination kinetics, 0.75 mL spore suspensions (either 2.10 8 or 2.10 6 spores.mL -1 ) were mixed with 0.75 mL MEB 2X to obtain a final spore concentration of 10 8 or 10 6 spores.mL -1 . Suspensions were then incubated horizontally in a rotary shaker set to 25°C and 120 rpm. Germination was followed at 0 h, 8 h, then at 2 h intervals for the following 12 h. A minimum of 100 conidia were counted to determine germination kinetics (i.e. percentage of germinated spore as a function of time). A

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spore was considered as germinated when the germinal tube length was superior or equal to the spore diameter itself, as previously described [START_REF] Dantigny | Standardisation of methods for assessing mould M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT germination: a workshop report[END_REF]. In addition, in order to examine whether spore germination capacity was related to spore density, high density spore suspensions obtained after 20 h incubation were diluted to 10 6 spores.mL -1 in fresh MEB 1X and incubated for another 18 h. Moreover, to assess whether inhibitory molecules were produced during incubation at high spore density, filter sterilized cultures obtained after 20 h incubation were inoculated at 10 6 spores.mL -1 and also incubated for another 18 h. For each spore concentration level, three technical replicates and two biological replicates were performed.

Effect of spore density on spore germination and radial growth in agar medium

In these experiments, a Petri dish containing Malt Extract Agar (MEA, Merck, Darmstadt, Germany) layers at the bottom and the top (in the lid) was used as described previously [START_REF] Chitarra | Germination of Penicillium paneum conidia is regulated by 1-Octen-3-ol, a volatile self-inhibitor[END_REF]. The top MEA layer was surface inoculated with 50 μL of a 10 6 or 10 8 spores.mL -1 suspension while 5 μL of a 10 7 spores.mL -1 suspension were deposited into the center of the plate on the bottom layer. Control cultures were also performed without inoculating the top layer. Colony diameters were measured in two perpendicular directions every 2 days for 14 days (plates were incubated at 25°C). Five replicates per condition were performed. Mean values ± 95 % confidence intervals were calculated for each condition.

Extraction of volatile compounds

Volatile compounds produced by P. camemberti in liquid cultures after 20 h incubation were extracted as previously described [START_REF] Husson | Effect of linoleic acid induction on the production of 1-octen-3-ol by the lipoxygenase and hydroperoxide lyase activities of Penicillium camemberti[END_REF]. Briefly, 0.5 g NaCl and 1 mL diethyl ether were added to 1 mL culture suspension followed by centrifugation at 15,000 g for 2 min

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at room temperature. The organic phase (superior phase) was recuperated and Na 2 SO 4 was added to remove any traces of water.

Detection and identification of volatile compound(s) by GC-MS

Detection and identification of volatile compounds were performed by gas chromatography (Agilent Technologies 7820A GC Systems, Agilent Technologies, Santa Clara, CA, USA) coupled with mass spectrometry (Agilent Technologies 5975 Series MSD, Agilent Technologies, Santa Clara, CA, USA) using a HP-5ms (30 m x 250 μm x 0.25 μm) column.

The program used for the oven was as follows: 50°C for 5 min, 4°C.min -1 between 50°C and 200°C then 10°C.min -1 from 200°C to 270°C, followed by a final step at 270°C for 10 min.

The injector and detector temperatures were set to 250°C and 300°C, respectively. The conditions of the mass spectrometer were mode electronic impact (EI); temperature source 250°C; scanning speed 1 scan.s -1 ; mass acquisition 50-300 uma. The vector gas was H 2 at a 1.5 mL.min -1 flow rate. For identification, mass spectra were compared to the NIST mass spectral library and were confirmed using the retention index and mass spectrum of pure compounds.

Effect of pure volatile compounds on spore germination in MEB medium

The impact of the major volatile compound identified by GC-MS on the spore germination process was evaluated in MEB for two strains (P. camemberti A and B) at concentrations ranging between 0 and 4 mM. The effect of other volatile compounds potentially produced by P. camemberti, i.e. ethanol, ammonia, 2-heptanone, 2-nonanone, 1-octen-3-ol, trans-2-octen-1-ol, 2-methyl-1-butanol, 3-methyl-1-butanol and 3-octanone, was also investigated. To perform these assays, after solubilization of the tested compound in propylene glycol and serial dilutions, the percentage of germinated conidia from a 10 6 spore.mL -1 suspension was

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determined in MEB as described above, after 10 and 16h incubation at 25°C with agitation (120 rpm). Control cultures without volatile compound but containing the same amount of propylene glycol were also performed.

Effect of initial spore concentration on the growth of P. camemberti determined by ergosterol analysis in a cheese matrix model

The cheese matrix model was prepared as described previously [START_REF] Le Dréan | Quantification of Penicillium camemberti and P. roqueforti mycelium by real-time PCR to assess their growth dynamics during ripening cheese[END_REF].

Briefly, to prepare 100 g, 23 g milk protein concentrate, 20 g anhydrous milk fat, 50 mL sterile distilled water, 1.5 g NaCl and 1.26 mL lactic acid were added. Prepared model cheeses were inoculated with either P. camemberti A or B spore suspensions to yield a final spore concentration ranging from 10 2 to 10 6 spores.g -1 . Then, 50 g cheese portions were transferred to sterile crystallizing dishes (5.6-cm diameter) and incubated for 9 days at 12°C.

After incubation, a 32-mm diameter and ~5 mm depth layer was removed from the cheese surface with a sterile scalpel. Sufficient sterile 2% (w/v) trisodium citrate was added to yield a 1:10 dilution and the resulting suspension was homogenized with an Ultra-Turrax (IKA, Staufen, Germany) at 24,000 rpm.min -1 for 1 min. Five mL of the trisodium citrate / cheese mixture were then centrifuged in a 12-mL screw cap tube at 10,000 g for 10 min at 4°C. The supernatant was discarded and the mycelium pellets resuspended in 0.5 mL of methanol.

After vortexing, 2.5 mL of 24 % methanolic KOH (24 %) were added and incubated for 2h at 85°C. After cooling in melted ice for 10 min, 2 mL of petroleum ether were added and vortexed 3 times for 20 sec with a cooling step on ice for 2 min after each homogenization treatment. This step was repeated and the extract was centrifuged for 10 min at 3000 rpm at 4°C. The organic phase was collected and evaporated under nitrogen flow for ~15 min and stored at -20°C before use. Prior to HPLC analysis, the dry extract was dissolved in 0.5 mL methanol and filtered through a 0.45-μm acetate filter. Ergosterol was quantified, using

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external ergosterol standards (Sigma, St Louis, MO, USA), with a high-performance liquid chromatograph Agilent 1100 series (Agilent Technologies, Santa Clara, CA, USA) equipped with an Interchrom Lichrospher C18 column and an UV detector set at 282 nm. The mobile phase was methanol with a flow rate set to 1.2 mL.min -1 and injection volume was 50 μl.

Each sample and standard were analyzed in triplicate. Mean values ± 95 % confidence intervals were calculated for each strain and condition.

Results

Effect of spore concentration on germination kinetics in broth medium

Germination kinetics according to initial spore population for the 2 studied P. camemberti strains in MEB are presented in Fig. 1. Independently of the studied strains, cultures containing 10 6 spores.mL -1 presented higher germination percentages compared to 10 8 spores.mL -1 cultures. Indeed, after 20h incubation, >90 % and <3% spores had germinated at 10 6 versus 10 8 spores.mL -1 . Similar results were also obtained on P. camemberti strains UBOCC-A-101392, UBOCC-A-108097 and CBS 299.48 T (data not shown). To determine whether germination inhibition was the consequence of higher spore density, 10 8 spores.mL -1 cultures obtained after 20 h incubation were diluted to 10 6 spores.mL -1 and further incubated for 18 h. Germination percentages for both strains were ~ 90 % indicating that a "crowdingeffect" was responsible for this inhibition.

In order to check for self-inhibitory molecules in the high density spore suspension, 20-h 10 8 spores.mL -1 culture filtrates were inoculated with a new spore suspension (10 6 spores.mL -1 ) and germination kinetics were compared to a control (Fig. 2). While 98 % of spores had germinated in the control, germination rates in the culture filtrates were only 13 % and 8 % for P. camemberti A and B, respectively (Fig. 2), thus, highlighting that one or several selfinhibitors were present in the culture medium previously containing 10 8 spores.mL -1 .

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Effect of spore density on spore germination and radial growth in agar medium

To examine if spore density could impact spore germination and radial growth in solid medium, a Petri dish containing MEA layers at both the bottom and top (in the lid) was used as previously described by [START_REF] Chitarra | Germination of Penicillium paneum conidia is regulated by 1-Octen-3-ol, a volatile self-inhibitor[END_REF][START_REF] Chitarra | 1-Octen-3-ol inhibits conidia germination of Penicillium paneum despite of mild effects on membrane permeability, respiration, intracellular pH, and changes the protein composition[END_REF]. As shown in Fig. 3, growth in the bottom of the Petri dish was significantly delayed as compared to the control (Fig. 3A) when the lid was inoculated with 50 μL of a spore suspension containing 10 6 (Fig. 3B) or 10 8 (Fig. 3C) spores.mL -1 , the latter having the strongest effect. Indeed, mean radial growth rates were 4.14, 0.57 and 0.47 mm.d -1 for the control and the lid agar inoculated with 10 6 and 10 8 spores.mL -1 , respectively. Overall, these results strongly suggested that volatile compounds, able to move from the MEA layer of production to the MEA layer of action, could be responsible for the observed effects.

Detection and identification of volatiles compound(s) by GC-MS

The volatile compounds present in MEB cultures containing 10 8 spores.mL -1 of P. camemberti strain A or B were analyzed by GC/MS and compared to those of the noninoculated MEB medium (Fig. 4). Major differences in volatile compound profiles between the inoculated media (Fig. 4A) and control (Fig. 4B) were observed. Noteworthy, the most abundant volatile compound produced at high spore density exhibited a retention time of 10.997 min. The nature of this compound was assigned to 1-octanol based on its mass spectrum (Fig. 4C) and confirmed after comparison of its mass spectrum with that of pure 1octanol (Fig. 4D). This compound was also verified to have a different retention time and mass spectrum from those of 1-octen-3-one, 3-octanone, 2-octanone, octanal, trans-2-octen-1ol and 1-octen-3-ol (retention times were 7. 237, 8.03, 8.201, 8.488, 11.935 and 12.796 min, respectively) in the tested conditions (data not shown).

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Effect of pure volatile compounds on germination efficiency in MEB

The effects of 1-octanol, as well as other volatile compounds potentially produced by P. camemberti, on germination efficiency were studied in vitro. The germination inhibitory effect of 1-octanol was confirmed in MEB. Indeed, total inhibition of spore germination was achieved in the presence of 3 mM 1-octanol while a concentration as low as 100 μM transiently delayed spore germination after 10h but not after 16h. Indeed, at the latter concentration, the percentage of germinated spores was reduced by 10 % as compared to the control. It is worth mentioning that 1-octen-3-ol, a compound previously identified to act as a self-inhibitor in P. paneum [START_REF] Chitarra | 1-Octen-3-ol inhibits conidia germination of Penicillium paneum despite of mild effects on membrane permeability, respiration, intracellular pH, and changes the protein composition[END_REF][START_REF] Chitarra | Germination of Penicillium paneum conidia is regulated by 1-Octen-3-ol, a volatile self-inhibitor[END_REF], and its isomer trans-2-octen-1-ol had a similar minimum inhibitory concentration (MIC) than 1-octanol. Other compounds, namely, ethanol, ammonia, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-heptanone and 3-octanone did not inhibit spore germination in the tested conditions (MIC>4 mM).

Effect of spore concentration on P. camemberti growth as determined by ergosterol analysis in a cheese matrix model

Fungal biomass obtained after 9 days incubation at 12 °C as a function of the initial spore concentration (10 2 to 10 6 spores.g -1 of cheese) is shown in Fig. 5. Independently of the tested strain, there was no significant difference in fungal biomass whatever the initial spore concentration used.

Discussion

Self-regulation of spore germination as a function of spore density has been previsouly characterized in a wide range of fungal species including Penicillium species [START_REF] Chitarra | Germination of Penicillium paneum conidia is regulated by 1-Octen-3-ol, a volatile self-inhibitor[END_REF][START_REF] Chitarra | 1-Octen-3-ol inhibits conidia germination of Penicillium paneum despite of mild effects on membrane permeability, respiration, intracellular pH, and changes the protein composition[END_REF]. In the present study, we demonstrated for the first time that this phenomenon,

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which was reversible, also occurred in P. camemberti, an industrially relevant fungus used in the dairy industry. We also showed that 1-octanol, a volatile compound, was produced at high spore density and that this compound hindered spore germination in vitro. In nature, Penicillium spp. produce high spore numbers which are directly exposed to air, thus explaining why volatile self-inhibitors may be more widespread than non-volatile ones. Thus, as suggested by [START_REF] Chitarra | Germination of Penicillium paneum conidia is regulated by 1-Octen-3-ol, a volatile self-inhibitor[END_REF] for 1-octen-3-ol, we can hypothesize that 1-octanol is produced by P. camemberti conidia and released into the air in order to inhibit germination until appropriate environmental conditions are met. Likewise, as previously described for 1octen-3-ol in P. paneum and Aspergillus nidulans [START_REF] Chitarra | 1-Octen-3-ol inhibits conidia germination of Penicillium paneum despite of mild effects on membrane permeability, respiration, intracellular pH, and changes the protein composition[END_REF][START_REF] Herrero-Garcia | 8-Carbon oxylipins inhibit germination and growth, and stimulate aerial conidiation in Aspergillus nidulans[END_REF], conidia treated with 3 and 4 mM 1-octanol did not enter into isotropic growth, meaning that 1-octanol prevented the initiation of the spore swelling process which precedes polarized growth and the formation of a germ tube. Thus, this volatile compound may have an effect on fungal membrane permeabilisation which controls water entry into conidia [START_REF] Chitarra | 1-Octen-3-ol inhibits conidia germination of Penicillium paneum despite of mild effects on membrane permeability, respiration, intracellular pH, and changes the protein composition[END_REF].

More generally, eight-carbon (8-C) volatiles are ubiquitous among fungi and characteristic of fungal aromas [START_REF] Combet | Eight-carbon volatiles in mushrooms and fungi: properties, analysis, and biosynthesis[END_REF]. They result from the oxidation and cleavage of fatty acids, in particular linoleic acid. Such products constitute for 44.3-97.6% of the total amount of volatiles produced, depending on the extraction method used [START_REF] Maga | Mushroom flavor[END_REF]Tressl et al., 1982;Venkateshwarlu et al., 1999). The well-known 1-octen-3-ol presenting a mushroomlike aroma is the most abundant VOC while 1-octanol is characterized by a detergent, soap and orange-like aroma [START_REF] Combet | Eight-carbon volatiles in mushrooms and fungi: properties, analysis, and biosynthesis[END_REF]. In the present study, 1-octanol was the only 8-C volatile compound identified in cultures with high conidial density while 1-octen-3-ol was not detected. Likewise, P. commune, which is considered as an ancestral form of the domesticated P. camemberti (Pitt et al., 1986), was not found to produce 1-octen-3-ol at high spore density [START_REF] Chitarra | 1-Octen-3-ol inhibits conidia germination of Penicillium paneum despite of mild effects on membrane permeability, respiration, intracellular pH, and changes the protein composition[END_REF]. Nevertheless, 1-octen-3-ol was found, here, to block the germination

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process in vitro. Given that the role of this compound as a self-inhibitor of germination has also been demonstrated in several fungal genera and species [START_REF] Chitarra | Germination of Penicillium paneum conidia is regulated by 1-Octen-3-ol, a volatile self-inhibitor[END_REF][START_REF] Herrero-Garcia | 8-Carbon oxylipins inhibit germination and growth, and stimulate aerial conidiation in Aspergillus nidulans[END_REF], it could be considered as a cross-talk molecule. In addition, 1-octen-3-ol is produced at high levels by P. camemberti during growth, especially in cheese [START_REF] Abbas | Yeasts and Molds: Penicillium camemberti[END_REF], therefore its role in controlling other steps of the P. camemberti growth cycle cannot be excluded and should be further studied. Finally, 3-octanone, a methyl-ketone derived from linoleic acid which is also produced by P. camemberti [START_REF] Adda | Production de styrène par Penicillium camemberti Thom[END_REF], also deserves further attention since this compound was identified as a conidiogenic compound in A. nidulans [START_REF] Herrero-Garcia | 8-Carbon oxylipins inhibit germination and growth, and stimulate aerial conidiation in Aspergillus nidulans[END_REF].

In the last part of the present study, we investigated whether the initial spore inoculum could impact the P. camemberti colonization process in model cheeses. We found that estimated biomass by ergosterol measurements was similar regardless of initial inoculum, suggesting that the germination and growth coordination phenomena could also occur in cheese. In addition, low inoculum levels containing 10 2 -10 3 spores.g -1 of cheese could be sufficient to maximize P. camemberti growth. In the cheese industry, the inoculation level of P. camemberti is empirically determined as a function of the organoleptic properties desired in final product (surface appearance, texture and flavor). Better knowledge of the phenomena governing the P. camemberti colonization process could be useful to better control growth and metabolic activities during cheese ripening.

Conclusions

In the present study, self-regulation of spore germination by quorum sensing in P. camemberti was shown. The volatile nature of the involved compounds was demonstrated and 1-octanol was found to be the main volatile compound produced at high spore density. Its inhibitory effect was verified in vitro, showing that 3 mM 1-octanol totally inhibited spore germination

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while 100 μM only transiently inhibited this process. This is the first time that spore germination self-inhibition is detected in P. camemberti and such knowledge could be useful to better control the ripening process of mould-ripened cheeses.

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Tressl, R., Bahri, D., Engel, K.H., 1982. Formation of eight-carbon and ten-carbon components in mushrooms (Agaricus campestris). J. Agric. Food Chem. 30, 89-93. doi:10.1021/jf00109a019 Venkateshwarlu, G., Chandravadana, M.V., Tewari, R.P., 1999. Volatile flavour components of some edible mushrooms (Basidiomycetes). Flavour Fragr. J. 14, 191-194. doi:10.

Figure legends

Fig. 1. Germination of Penicillium camemberti strain A (A) and B (B) conidia at 25°C in malt extract broth as a function of the tested spore density (10 6 spores.mL -1 , and 10 8 spores.mL -1 , ). The arrows represent the time (20h) at which the spore suspensions were diluted to 10 6 spores.mL -1 . Two biological replicates and three technical replicates were performed for each strain. Error bars represent standard deviations.

Fig. 2.

Germination kinetics (inoculum level of 10 6 spores.mL -1 ) of Penicillium camemberti strain A ( ) and B ( ) in filter-sterilized 10 8 spores.mL -1 cultures obtained after 20-h incubation (doted lines) as compared to germination of strain A ( ) and B ( ) in fresh malt extract broth (solid lines). Error bars represent standard deviations. • Spore germination in Penicillium camemberti is self-regulated by quorum sensing.

• Quorum sensing is observed when spore densities are high.

• Compounds involved in quorum sensing are volatile.

• 1-octanol was found to be the main volatile compound produced at high-spore density.

  ol influenced different developmental processes during the Penicillium paneum lifecycle, including spore germination inhibition.

  Two commercial P. camemberti strains (coded P. camemberti A and B for confidentiality reasons) isolated from mould-ripened cheeses and P. camemberti UBOCC-A-101392 and UBOCC-A-108097, and CBS 299.48 T respectively obtained from the Université de Bretagne Occidentale Culture Collection (UBOCC, Plouzané, France) and the Central Bureau Voor Schimmelcultures (Baarn, Netherlands) were used in this study. Pre-cultures were prepared M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT on slant Potato Dextrose Agar (Difco PDA, Becton, Dickinson and Company, Franklin Lakes,

  1002/(SICI)1099-1026(199905/06)14:3<191::AID-FFJ810>3.0.CO;2

Fig. 3 .

 3 Fig. 3. Inhibition of spore germination and radial growth of Penicillium camemberti cultivated on malt extract agar in the absence (A) or in the presence of a 50 μL at 10 6 (B) or 10 8 (C) spores.mL -1 inoculated in the top layer (in the lid) of a Petri dish. CI95%: 95%

Fig. 4 .

 4 Fig. 4. Chromatograms showing the volatile compounds found in malt extract broth containing 10 8 spores.mL -1 of Penicillium camemberti strain A after 20 h incubation at 25°C

Fig. 5 .

 5 Fig. 5. Fungal biomass (mean ± 95 % confidence interval) of Penicillium camemberti strain A

Acknowledgements

The authors are thankful to CBB développement and the Région Bretagne for their financial support. We thank Danielle Arzur for technical assistance and Doctor Monika Coton for English revision.

13187), 1-octen-3-ol (PubChem CID: 18827), trans-2-octen-1-ol (PubChem CID: 5318599), 2-methyl-1-butanol (PubChem CID: 8723), 3-methyl-1-butanol (PubChem CID: 31260) and 3-octanone (PubChem CID: 246728).


N El Hassan

M N Kaydouh

H Geagea

H El Zein

K Jabbour

S Casale

H El Zakhem

P Massiani
email: pascale.massiani@upmc.fr

Low temperature dry reforming of methane on rhodium and cobalt based catalysts: active phase stabilization by confinement in the mesoporous SBA-15

Keywords: Rhodium, cobalt, SBA-15, methane dry reforming, confinement effect

Highlights:

 Co nanoparticles are stabilized against sintering in the mesopores of SBA-15 during dry methane reforming.

 The decrease of reduction temperature in presence of Rh leads to more numerous Co species confined in the pores.

 Deactivating carbons are formed in lower amount in presence of both rhodium and cobalt.

Introduction

Nowadays, one of the major problems faced by industrial societies is global warming. Two of the most abundant greenhouse gases are carbon dioxide (CO 2 ), largely produced by human activities, and methane (CH 4 ), which is the major component of natural gas and has a global warming potential 25 times higher than that of CO 2 . Methane can be used to produce energy by combustion in air but one drawback of this energy resource lies in the large amounts of natural gas located in remote areas, so their transportation is very expensive. Therefore, the conversion of methane into heavier -more easily transportable -compounds such as liquid fuels (substitute of petroleum feedstock) represents an attractive and challenging target, offering environmental as well as economical and energetic benefits [1].

The available processes for the conversion of methane into liquid fuels currently consist in two main steps: methane is converted by reforming reaction to a gaseous mixture consisting of mainly hydrogen and carbon monoxide, called synthesis gas (or syngas), then the syngas is transformed to hydrocarbons by appropriate routes [2]. At present, the first step of methane reforming is commonly carried out in industry by steam reforming in the presence of water (CH 4 + H 2 O → 3H 2 + CO). However, dry reforming of methane (DRM) in which carbon dioxide is used as co-reactant (CH 4 + CO 2 → 2H 2 + 2CO) could bring two important advantages [3].

Firstly, the consumption of CO 2 together with CH 4 in DRM allows the simultaneous valorization of these two greenhouse gases into molecules with higher added value. Secondly, the syngas obtained from DRM has an equimolar composition (H 2 :CO ratio of 1) that is the best suited for direct use in Fischer-Tropsch (FT) synthesis to produce hydrocarbons with high yield [2]. By comparison, the H 2 :CO ratio is much higher (in the order of 3) in the case of methane steam reforming and adjustments, such as the addition of a membrane separation unit to the process, are then required before FT synthesis can be performed.

In spite of the above mentioned benefits, dry reforming of methane still faces several difficulties that need to be overcome before any application of the process at an industrial scale can be foreseen. Compared to steam reforming, this reaction is thermodynamically favored at higher temperatures and it is therefore carried out under conditions (usually above 650°C) in which the catalysts can suffer fast deactivation by sintering of the active phase and by coke deposition, the latter also possibly leading to reactor blockage. Amongst the metals (Pt, Pd, Rh, Ir, Ru, Ni, Co) already tested in DRM [4,5], noble metals are recognized as the most stable ones.

Nevertheless, their limited availability and high cost represent a major drawback for industrial use. Alternatively, nickel-based catalysts have been extensively studied in the last decade due to their high activity in DRM, however improvement of their stability is still needed and this represents an active field of research, as recently reviewed [6]. By comparison to Ni, fewer attention has been devoted to Co, less performing in DRM but having however the advantage of less coke deposition [7][8][9][10][11][12][13][14][15][16][17]. From this point of view, studies dedicated to the increase of the activity of Co-based catalysts in DRM while keeping their good stability appear highly relevant.

Interestingly, bi-metallic Ni-Co catalysts were recently investigated in dry reforming of methane and they were found more stable than Ni-based ones [17][18][19][20][21]. Furthermore, we showed in a recent paper that cobalt activity in DRM could be enhanced by dispersion of the metal nanoparticles in a SBA-15 silica support [15]. Such type of mesoporous support was already found to favor high cobalt oxide dispersion [22][23][24]. Furthermore, for Ni-based systems, it was shown to provide a very efficient way to increase catalysts stability in DRM by protecting the metal nanoparticles against sintering and coke deposition through their stabilization inside the SBA mesopores [25][26][27][28][29][30][31].

Another way to increase the performances of transition metal catalysts in DRM is the addition of dopants, often a noble metal in low amounts. This has been already well demonstrated for Ni-based catalysts [32][33][34][35][36] but more rarely studied in the case of Co-based ones. Nevertheless, we observed in a recent paper that doping Co/SBA-15 catalysts by small amounts of ruthenium can lead to increased performances in DRM, even if the catalysts still suffered from some lack of stability due to formation of carbon derivatives [15]. In this respect, rhodium could be a more appropriate doping agent since it was identified as an efficient promoter of nickel over different oxide supports such as boron nitride [37], alumina [38], silica [39] or MCM-41 [40]. A similar effect was also very recently reported on Co/alumina [12]. Even though a promotional effect of rhodium on cobalt was claimed in these previous studies, there is no evidence of a real synergetic effect, rhodium being possibly simply involved as additional active phase for the dry reforming reaction. To clarify this point, it is of interest to compare the activity of bimetallic catalysts containing both cobalt and rhodium phases to those loaded with only either rhodium or cobalt.

Based on these considerations, the aim of this study was to enhance the activity of Co-based catalysts in DRM, as well as their resistance against active phase sintering and coke deposition, by combining two effects: (i) the addition of low amounts of rhodium that could play the role of either a promoter or of a complementary active phase and (ii) the stabilization of the cobalt species by confining them in the channels of mesoporous SBA-15 silica. To this end, Rh/SBA-15 (reference), Co/SBA-15 and Rh-Co/SBA-15 catalysts were prepared, their structural and morphological properties were identified and their performances in dry reforming of methane were compared up to 800°C. A non-porous Rh-free Co-containing sample with commercial silica as support was also prepared for comparison. For stability measurements, we chose to carry out the reaction at 550°C because working at such medium temperature would allow using such systems in membrane reactors without losing the permeation of the membrane [41,42]. It is worth noting that in spite of such potentiality, only few studies describe the performances of methane dry reforming catalysts at such medium temperatures [13,15,[43][44][45][46].

Experimental

Catalysts Preparation

The mesoporous SBA-15 support was prepared according to a well-known method [47]. After drying, the powder was calcined in a muffle furnace (Nabertherm) at 500C for 9 hours (heating rate 2⁰C.min -1 ). The cobalt active phase (12 wt%) was deposited by a two solvents method [48,49] using cobalt (II) nitrate hexahydrate (Sigma Aldrich, 239267) as cobalt precursor: briefly, 1 g of support was suspended in 20 mL of n-hexane for few minutes, then an appropriate amount of solution containing the metal salt precursor was added drop by drop with manual stirring. After drying at room temperature for 24 hours, the sample was calcined in a muffle furnace at 450C for 5 hours (heating rate 0.5⁰C.min -1 ) to give sample Co 12 /SBA-15. Two different rhodium loadings (0.2 and 0.5 wt. %) were then added to this material by the two solvents method, using rhodium (III) nitrate hydrate (Sigma Aldrich, 83750) as Rh precursor salt.

The samples obtained after calcination (same conditions as for Co 12 /SBA-15) are labeled Rh x Co 12 /SBA-15 where x is the Rh content (in wt%). A Co-free reference sample with 0.5 wt% Rh (labeled Rh 0.5 /SBA-15) was similarly prepared from the parent SBA-15 support. Moreover, a non-porous silica based sample (denoted Co 12 /SiO 2 ) was prepared using fumed silica (Sigma Aldrich 381276), following the same procedure as for the Co 12 /SBA-15 sample.

Characterization techniques

N 2 sorption experiments were performed on an ASAP 2020 Micrometrics. Prior to analysis, each sample was outgassed under vacuum (10 -5 bar) for 2 hours at 250C. The external surface areas were calculated from the BET equation applied at relative pressures between 0.05 and 0.25.

The microporous volumes were estimated using the t-plot method; the obtained values were subtracted from the total porous volumes measured at P/P 0 = 0.998 to evaluate the mesoporous volumes. The average pore size diameters were estimated by the BJH methodology applied to the desorption branch of the isotherms. X-Ray Diffraction (XRD) experiments were performed on a Panalytical XPert 3 diffractometer using CuKα radiation (λ=1.54 nm) and operating at 30 kV and 10 mA. All diffractograms were registered in the angular range 5° < 2Θ < 90°. The crystallite sizes were calculated from the full width at half maximum (FWHM) of the peak at 65° (440 reticular plan), using the Scherrer's equation and assuming spherical morphologies.

High Resolution Transmission Electron Microscopy (HRTEM) experiments were conducted on a JEOL JEM-200 electron microscope operated at 200 keV (LaB 6 gun) and equipped with an energy dispersive X-ray spectroscopy (EDXS). The preparation method of the ultra-thin sections was done as described in a previous work [22]. In order to determine the average particle size of cobalt oxides (on fresh calcined catalysts) or metallic cobalt nanoparticles (on spent catalysts), the software "Comptage des Particules" was used.

Temperature Programmed Reduction (TPR) experiments were conducted on the fresh catalysts using an Autochem 2920 Micrometrics. During these experiments, the use of a cooling bath (ice and NaCl salt at -10 °C) allowed to trap all the water (desorbed or produced during reduction) and thus ensure that all TCD signal is related to H 2 consumption. The flow rate of the 5 vol% H 2 /Ar mixture was 20 mL.min -1 and the temperature was increased from room temperature to 900C at 10°C.min -1 .

Combined Temperature Programmed Hydrogenation and Mass Spectrometry (TPH-MS) studies were carried out on spent catalysts up to a maximum temperature of 900C (heating rate of 10C.min -1 , 5 vol% H 2 /Ar flow of 30 ml.min -1 ). The same apparatus and same cooling trap as for TPR was used and the exit gas lines were connected to an online quadrupole mass spectrometer (MKS, Cirrus 2) for exhaust gas analysis. The deposited carbon was classified according to its temperature of gasification: Cα up to 350°C, Cβ between 350 and 500°C and Cγ above 500°C [31]. The origins of these distinct carbon deposits (as reported in the literature) will be detailed in section 3.4.

Catalytic tests

Prior to reaction, the calcined sample (80 mg) was placed in the reactor and a pretreatment under 5 vol% H 2 /Ar flowing at 20 mL.min -1 was performed. The temperature was increased from room temperature up to 650C (heating rate 10°C.min -1 ) and kept at 650 C for two hours. Then the temperature was decreased to 200C before an equimolar mixture of CH 4 and CO 2 diluted in argon was introduced into the reactor. The temperature was next increased up to 800C (heating rate 5°C.min -1 ) under a total GHSV of 67 L.g -1 .h -1 . The composition of the effluent feed (CH 4 and CO 2 as reactants and CO and H 2 as main products) was measured using a Inficon Micro-GC equipped with two columns (Molecular Sieve and Plot-U) and a TCD detector. An ice-salt trap was also used at the exit of the reactor to trap any water that can be formed during the reaction.

Finally, the reactor was cooled down to 550C in order to run the stability test for 550 minutes.

The reactants conversions were calculated as follows: CH 4 conversion = (CH 4in -CH 4out )/CH 4in and CO 2 conversion = (CO 2in -CO 2out )/CO 2in . Note that these values take into account all consumed reactants (no longer present in the flow at the exit of the reactor), which includes not only those converted by main DRM reaction but also those involved in side reactions (particularly C deposits and reverse water gas shift).

Results and discussion

Textural and structural properties

For the SBA-15 based samples, the N 2 sorption isotherms are of type IV (Fig. 1), typical of mesoporous SBA-15 silica materials. This indicates that the hexagonal pore system of the parent SBA-15 support was systematically preserved after impregnation(s) and calcination(s) treatments, confirming the good stability of the synthesized support, already highlighted in previous papers [14,22,23]. Both microporous and mesoporous volumes decreased after cobalt addition, and the effect was accentuated after rhodium addition (Table 1 and Fig. 1). Such decrease is attributable to partial filling of the pores by metal species and/or to some collapse of the silica walls during successive impregnation and calcination treatments. Similarly, amongst metal loaded SBA-15 samples, the total surface area is the highest for Rh 0.5 /SBA-15 (low metal loading, one impregnation step), it is intermediate for Co 12 /SBA-15 (high metal loading, one impregnation step) and it is the lowest for Rh 0.5 Co 12 /SBA-15 (high metal loading, two impregnation steps). Note also, the significant decrease of the micropores volume in all samples, showing their sensitivity to shrinking during impregnation treatments in solution then calcination. This, however, did not modify the average pore diameter that remains comparable in all materials, between 3.6 and 3.8 nm (Table 1). As expected, the surface area of the Co 12 /SiO 2 was the lowest (171 m 2 .g -1 ). 1). The size value is much higher in Co 12 /SiO 2 , close to 16 nm, in agreement with the non-porous character of the support.

Figure 1 and Table 1

Figure 2

The cobalt dispersion in the calcined samples and the location of the nanoparticles on the support were also analyzed by transmission electron microscopy. The observations were carried out on ultra-thin sections (microtomic slices) in order to facilitate the identification of the internal porosity and thus avoid any ambiguity between internal and external locations, as can occur on SBA-15 grains due to their thickness. Figure 3 shows typical micrographs obtained for Rh-free Co 12 /SBA-15 (images a to c) and for Rh containing Rh x Co 12 /SBA-15 materials (images a' to c'). On these images, the mesoporous SBA-15 support appears in grey while the metal nanoparticles appear darker due to their higher electronic density. When the silica grain is parallel to the electronic beam, one can easily observe the channels arrangement (Fig. 3a,a'); the other orientation (grain perpendicular to the electronic beam) permits the observation of the very regular hexagonal pore apertures (Fig. 3b,b'), especially at high magnification (Fig. 3c,c').

Figure 3

In both orientations, partial filling of the pores by occluded cobalt oxide nanoparticles is clearly seen. The nanoparticles are in majority located inside the pores, with however a significant fraction outside in the case of Rh-free Co 12 /SBA-15, appearing as big particles located at the silica surface (Fig. 3a-c). Such external particles, particularly well visible on highresolution images of grains orientated perpendicular to the beam (Fig. 3c), are on the contrary absent -or at least much less numerous -when Rh is present, in addition to Co, in the sample (Fig. 3a'-c'). Statistical particle diameter counting was done for all samples, on images taken perpendicular to the channels, by considering at least 500 nanoparticles inserted in the pores (see typical image, Fig. 4a), which led to rather narrow size distributions in all samples. The average size of about 8.1 nm thus found for Co 12 /SBA-15 is slightly smaller than the size estimated from XRD (Table 1), in line with the presence in this sample of bigger external particles that are not considered in the TEM evaluation. On the contrary, both HRTEM and XRD size estimations are quite consistent for both Rh 0.2 Co 12 /SBA-15 and Rh 0.5 Co 12 /SBA-15 (Table 1), confirming that cobalt in these Rh-enriched samples is predominantly located inside the SBA-15 mesopores. It can be also noted that the average size values thus estimated are higher (around 7-8 Å) than the pore diameters evaluated above (below 4 Å). This can be explained in part by the presence of external nanoparticles on the surface of the silica grains and by the aggregated ones appearing in the form of elongated chains on the HRTEM images recorded in parallel direction (Fig. 4b,c).

Moreover, it can be recalled that such occurrence of confined nanoparticles slightly bigger than the average pore diameter is often observed in the case of zeolites, suggesting that some local framework destruction takes place during nanoparticle growth, leading to a small increase of the channel diameter around the particle. In fact, careful observation of the HRTEM micrographs in Figures 3 c andc' confirms the bigger diameter of the occluded nanoparticles compared to the channels. On the images of the calcined Rh-richest Rh 0.5 Co 12 /SBA-15 sample, individual rhodium-based nanoparticles smaller than 1 nm can be also detected inside the pores (chemical nature confirmed by EDS), appearing as very small black spots well distributed on the silica supports (see arrows in Fig. 4d), independent of the elongated cobalt oxide aggregates.

Interestingly, these nanoparticles are distinct from Co ones, suggesting that Rh and Co active sites do not form common bimetallic phases and should therefore behave as individual active phases during the catalytic act.

Reducibility of the supported rhodium and cobalt oxide species

The TPR profiles obtained upon reduction of the calcined samples are compared in Figure 5.

The non-porous Co 12 /SiO 2 catalyst contains a majority of cobalt oxide reducible below 450°C.

Mesoporous Co 12 /SBA-15 shows a peak centered at 350°C and a weaker one around 400°C, both attributable to the reduction of Co 3 O 4 particles but a broad intense signal above 600°C is also present, indicating reduction of cobalt species in stronger interaction with the support [50,51]. This difference between the two Rh-free samples shows that the interaction between cobalt oxides nanoparticles and their silica support depends on the textural and surface properties of the latter, SBA-15 favoring stronger interaction compared to non-porous fumed silica. After rhodium addition, the shape of the reduction profile drastically changed, due to hydrogen activation by the noble metal (and therefore easier reduction of all species, including cobalt). The overlapped peaks with maxima at around 110°C and 190°C are typical of the reduction of rhodium oxide Rh 2 O 3 [52] and isolated RhO x [53], respectively. The broader overlapped peaks between 200 and 440C correspond to the reduction of cobalt oxide species, which occurs at lower temperature than on Co 12 /SBA-15 due to the above mentioned hydrogen activation and hydrogen spillover process in presence of rhodium [15,53] and also possibly to some Rh-Co interaction. On these samples, full reduction is achieved at 650°C (temperature of in situ reduction before catalytic tests). The total hydrogen consumptions reported in table 1 for all the samples remain within the limits of the expected values, revealing the good accessibility of hydrogen to the active sites.

Finally, cobalt free Rh 0.5 /SBA-15 shows a single peak around 100°C, a temperature that fits well with Rh 2 O 3 reduction [52].

Figure 5

Activity and stability in DRM

After reduction under 5 vol% H 2 /Ar at 650°C for 2h, the performances of the catalysts in dry reforming of methane were evaluated. For both Rh-free Co 12 /SBA-15 and Co 12 /SiO 2 catalysts, conversions of both CH 4 (Fig. 6A) and CO 2 (Fig. 6B) start to be detected around 500°C. The process is facilitated in presence of Rh, since such conversions start to be already observed about 100°C below, including in the absence of cobalt, which means that rhodium sites are more active at lower temperature than cobalt ones. At this low temperature, Co free Rh 0.5 /SBA-15 is as active as Rh 0.5 Co 12 /SBA-15, showing that rhodium is the only active phase participating to the reaction in these conditions, in spite of its low content compared to Co. Nevertheless, the situation is no longer the same when the temperature increases above 500°C, since cobalt sites becomes also active. A significant amelioration of activity is then seen when combining Co and Rh in a same catalyst and, for the metal-richest Rh 0.5 Co 12 /SBA-15 catalyst, the conversions are close to the maximum thermodynamic limit values allowed for the applied test conditions [25]. On this catalyst, full conversion of methane is reached below 650°C while the highest CO 2 conversion is attained at around 700°C. This temperature is lower than that previously observed for Ni/SBA-15 catalysts studied in comparable conditions on which reactant conversions were below 90% at 650°C [25]. This attests of the very good performances of the Co and Rh enriched SBA-15 materials, in which both metals participate to the reaction, most probably in a separate way, in view of both TEM and catalytic data.

Figure 6

From stability experiments (Fig. 7) carried out at 550°C, it can be seen that all catalysts are fully stable under stream for 500 min. This figure highlights again the limited conversion level obtained at this temperature with cobalt alone (Co 12 /SBA-15 and Co 12 /SiO 2 catalysts), much below than with Rh 0.5 /SBA-15 in spite of the lower metal content (and therefore smaller number of active sites) in the latter. The good performances of Rh 0.5 Co 12 /SBA-15 compared to Rh 0.5 /SBA-15 nevertheless demonstrate the positive effect of the presence of Co. From figure 7, it also appears that CO 2 conversions are always slightly higher than methane ones on both Co 12 /SBA-15 and Co 12 /SiO 2 catalysts. This indicates the occurrence of side reactions, probably reverse water gas shift at medium temperatures (CO 2 + H 2 → CO + H 2 O) [24,26]. The conversions of both reactants are on the contrary very close on the Rh-containing samples. It was previously reported for Ni containing MCM-41 catalysts that Rh incorporation decreases the relative significance of the reverse water gas shift reaction with respect to dry reforming of methane [41]. Therefore, not only the activity of the Co/SBA-15 catalysts but also the side reactions occurrence decreased by the addition of rhodium, in line with a previous paper on Rhdoped Co/Al 2 O 3 catalysts tested in DRM [12]. This result can also be seen on figure 8 that reports the H 2 :CO ratios measured during stability tests. Thus, the ratio is near one in presence of rhodium, as expected from the DRM reaction alone, but it is significantly lower, below 0.6, for both cobalt monometallic catalysts, confirming strong occurrence of side reactions. In the case of the spent Co 12 /SBA-15 catalyst, the HRTEM images reveal significant cobalt migration outside the pores associated with a consequent sintering of the nanoparticles, both leading to the formation of external particles with diameters as big as 10-30 nm (Fig. 9a-c). For the reduced Co 0 nanoparticles still present inside the pores, their average size is also increased since it reaches 11.6 nm whereas it was 8.6 nm for the cobalt oxide species present before reduction and catalytic run. It is worth recalling here that, in absence of sintering, the average size should be lower in the reduced catalyst due to the replacement of bulkier Co 3 O 4 by reduced Co 0 . This further demonstrates the high sensitivity of the Rh-free Co 12 /SBA-15 sample to sintering. Contrarily, the shape and location of the metal nanoparticles were found much more stable in Rh 0.2 Co 12 /SBA-15 in which the active metal phase was predominantly maintained inside the pores after the catalytic run (Fig. 9 a'-c'). Furthermore, the average size of the reduced cobalt particles in spent Rh 0.2 Co 12 /SBA-15 (8.3 nm) was only slightly higher than on the fresh sample (around 6.6 nm), thus further demonstrating that sintering is more limited in presence of rhodium, similarly to the case of Ni/Al 2 O 3 DRM catalysts doped with noble metals [54].

Figure 9

In addition to metal sintering effects, coke formed during reaction may also participate in deactivation. Due to the medium temperature (550°C) applied during stability tests, the coke content was very low (hardly identified by HRTEM) but high enough (1-2 wt%) to be detected on the spent catalysts by TPH/MS, a technique that can provide useful information about carbon nature [55,56]. In this technique, the coke reacts with hydrogen to form methane that is analyzed by following its mass (16). One advantage compared to other techniques currently used for coke identification (e.g. temperature programmed oxidation or thermal gravimetric analysis) is that it is carried out in the absence of oxygen, which ensures the absence of interferences with other possible phenomena such as metal active phase reoxidation. Moreover, it permits to discriminate the three different types of coke (Cα, Cβ and Cγ) commonly identified depending on the temperature at which they react with hydrogen (see details in section 2.2). We can recall that Cα is known as a reaction intermediate that does not affect the stability, whereas Cβ (evolved at medium temperature) corresponds to nanotubes precursor species and graphitic Cγ (evolved at high temperatures) is known for its strong deactivation effect [31,56,57,58]. The origins of these different carbons are threefold: (i) active C* (C adsorbed on an active metal site) reacting with adsorbed oxygen species to give CO, (ii) C* leading to inactive C deposit on metal° due to the lack of adjacent oxygen species or (iii) C deposit after carbon migration from metal sites to the support. It is worth noting that the first type of carbon is an intermediate in the reaction, which does not lead to deactivation of the catalyst [59,60].

Figure 10

The percentages of different types of coke C, C and C were deduced from the TPH-MS profiles, following the same methodology as described in our recent paper [56], for the spent Co 12 /SBA-15, Rh 0.2 Co 12 /SBA-15, Rh 0.5 Co 12 /SBA-15 and Rh 0.5 /SBA-15 catalysts and are presented in Figure 10. All the catalysts contain the three types of coke but in different percentages. In the Co 12 /SBA-15 catalyst, 7, 54 and 39% of the deposited carbon are in the forms of C, C and C, respectively. The Rh 0.2 Co 12 /SBA-15 catalyst presents a similar proportion of C, but it contains less C (10% instead of 39%) that is known as the heaviest and most detrimental type of coke for catalysts stability [56]. Finally, it is clear that Rh 0.5 Co 12 /SBA-15 contains the lowest C amount and the highest amount of C that is known to be an intermediate in the DRM reaction, with no deleterious impact on the reaction [56]. In light of these results, we can say that the DRM mechanism is not similar on cobalt and rhodium active sites. For the highest rhodium content, an equivalent percentage of carbon reaction intermediate was obtained which means that adsorbed oxygen species are present in higher percentage in presence of rhodium. This indicates that oxygen activated species are more easily adsorbed on rhodium than on cobalt sites and this is the main reason behind the higher conversion of reactants on the rhodium containing catalysts. Finally, it is worth mentioning that the presence of the two metals, rhodium and cobalt, leads to a minimization of the most deactivating carbon type (Cγ) that originates from the deposition of carbon on metal rather than onto the support. This latter observation shows the importance of using catalysts loaded with the two metals and their advantages compared to monometallic ones.

Conclusion

SBA-15 improves the textural and morphological properties of the catalysts by preserving the high active phase dispersion inside the mesoporosity of the support when used instead of nonporous silica. The use of mesoporous silica SBA-15 as support of cobalt active nanoparticles ( 12wt%) together with doping by small amounts of rhodium (up to 0.5 wt%) leads to highly active and stable catalysts in DRM as good as those Ni/SBA-15 catalysts described in the literature.

The occlusion of the cobalt nanospecies inside the mesopores of the silica grains strongly enhances their stability against sintering. Rhodium promotes their reducibility under hydrogen and it enhances the catalytic performances by participating to the reaction. Furthermore when present with cobalt, rhodium affects the nature of the coke formed on the spent catalysts by minimizing the formation of the most destructive type of coke (Cγ).

Figure 2 shows

 2 Figure2shows XRD patterns of calcined samples at wide angles (2θ range 15-85°). The wide

Figure 7 and Figure 8 3. 4

 84 Figure 7 and Figure 8

Figures CaptionsFigure 1 :

 1 Figures CaptionsFigure1: N 2 sorption isotherms on the SBA-15 support and on calcined impregnated samples.

Figure 2 :

 2 Figure 2: XRD patterns of calcined materials (* and ○ show the expected peak positions for crystalline Co 3 O 4 and for Rh oxide, respectively).

Figure 3 :

 3 Figure 3: Typical TEM images of grains of (a-c) Co 12 /SBA-15 and (a'-c') Rh 0.5 Co 12 /SBA-15 calcined materials with porous network oriented (a,a') along or (b,b' and c,c') perpendicular to the electron beam.

Figure 4 :

 4 Figure 4: Typical HRTEM images showing (a) Co 3 O 4 nanoparticles occluded in the pores of Co 12 /SBA-15, (b) their aggregation along the channels in Rh 0.2 Co 12 /SBA-15 and (c) the presence of very small isolated Rh oxide nanoparticles (indicated by the arrows) in Rh 0.5 Co 12 /SBA-15.

Figure 5 :

 5 Figure 5: TPR profiles of calcined samples.

Figure 6 :

 6 Figure 6: Evolution of (A) CH 4 and (B) CO 2 conversions with temperature on the reduced catalysts (P = 1 bar, CH 4 :CO 2 = 1 and GHSV of 67 L.g -1 .h -1 ).

Figure 7 :

 7 Figure 7: Evolution of (A) CH 4 and (B) CO 2 conversions as a function of time-on-stream during stability tests at 550°C (P = 1 bar, CH 4 :CO 2 = 1 and GHSV of 67 L.g -1 .h -1 ).

Figure 8 :

 8 Figure 8: Evolution of H 2 :CO ratio products ratio as a function of time-on-stream during stability tests at 550°C (P = 1 bar, CH 4 :CO 2 = 1 and GHSV of 67 L.g -1 .h -1 ).

Figure 9 :

 9 Figure 9: Typical TEM images of the spent (a-c) Co 12 /SBA-15 and (a'-c') Rh 0.5 Co 12 /SBA-15 catalysts.

Figure 10 :

 10 Figure 10: Distribution of the different types of coke C α , C β and C γ on the spent catalysts.

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10

 12345678910 Figure 1

Table 1 :

 1 Textural properties and mean diameter of the Co-based nanoparticles in the calcined and spent samples

SBA-15 Co 12 /SBA-15 Rh 0.5 /SBA-15 Rh 0.2 Co 12 /SBA- 15 Rh 0.5 Co 12 /SBA- 15 Co 12 /SiO 2

	Total Surface Area (m 2 .g -1 )	803	639	668	448	446	171
	Microporous volume (cm 3 .g -1 )	0.32	0.20	0.19	0.13	0.13	0.01
	Mesoporous volume (cm 3 .g -1 )	0.48	0.43	0.53	0.33	0.32	-
	Average pore diameter (nm)	3.7	3.7	3.8	3.7	3.6	-
	Total H 2
	consumption	-	2356	171	2807	2843	2324
	(µmol.g -1 )
	dCo 3 O 4 (a)	-	8.6	-	7.1	7.1	16.4
	dCo 3 O 4 (b)	-	8.1	-	7	6.8	-
	dCo 0 (b)	-	11.6	-	8.3	n.d. (c)	-
	(a) Average particle diameter (nm) estimated from XRD (440 reticular plan)
	(b) Average diameter (nm) of spherical particles determined from TEM (± 0.2 nm)
	(c) Not determined

Acknowledgments

Authors would like to thank the UOB research council for financial support through the BIRG 14/2012 and the CEDRE France-Lebanon Hubert-Curien (PHC) program for project N° 30962ZH (2014)(2015). Thanks also to Miss Hala Fallah for the help in the experimental part (N 2 sorption, X-Ray diffraction and particles counting) and Miss Ranim Hajal for particles counting.


M.D Cécile Bouchet-Doumenq

Jérémie H Lefevre

M.D Malika Bennis

M.D Najim Chafai

M.D Emmanuel Tiret

M.D Yann Parc

M.D Jeremie H Lefevre
email: jeremie.lefevre@sat.aphp.fr

Jeremie Pr

Lefevre

Management of postoperative bladder emptying after proctectomy in men for rectal cancer. A retrospective study of 190 consecutive patients

Keywords: rectal cancer, colorectal anastomosis, urinary dysfunction, suprapubic catheterization, transurethral catheterization

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INTRODUCTION

Modern rectal cancer treatment is based on radical surgery with nerve preservation and total mesorectal excision [START_REF] Heald | Total mesorectal excision is optimal surgery for rectal cancer: a Scandinavian consensus[END_REF]21]. Mesorectal excision improves oncological outcome but it may result in decreased sexual [START_REF] Maas | Radical and nerve-preserving surgery for rectal cancer in The Netherlands: a prospective study on morbidity and functional outcome[END_REF], urogenital and anorectal function [START_REF] Doeksen | Sexual and urinary functioning after rectal surgery: a prospective comparative study with a median follow-up of 8.5 years[END_REF][START_REF] Gerstenberg | Bladder function after abdominoperineal resection of the rectum for anorectal cancer. Urodynamic investigation before and after operative in a consecutive series[END_REF][START_REF] Regenbogen | Urinary tract infection after colon and rectal resections: more common than predicted by risk-adjustment models[END_REF], unless careful attention is given to nerve preservation.

Urinary dysfunction remains a clinical problem for up to two thirds of patients after rectal resection, especially for those with low rectal cancer [START_REF] Benoist | Optimal duration of urinary drainage after rectal resection: a randomized controlled trial[END_REF]. The percentage of urinary dysfunction after rectal cancer surgery varies in the literature and depends on surgical expertise and patients' characteristics. There are several possible reasons for urinary dysfunction. Intraoperative nerve damage seems to be the main cause. Impaired preoperative urinary function may also contribute to the development of postoperative dysfunction [START_REF] Benoist | Optimal duration of urinary drainage after rectal resection: a randomized controlled trial[END_REF][START_REF] Burgos | Risk factors for developing voiding dysfunction after abdominoperineal resection for adenocarcinoma of the rectum[END_REF][START_REF] Gerstenberg | Bladder function after abdominoperineal resection of the rectum for anorectal cancer. Urodynamic investigation before and after operative in a consecutive series[END_REF]. Overall, several risk factors have been suggested: male gender, advanced age, tumour location, rectal cancer rather than colon cancer, longer operative time, additional pelvic procedure, surgical drain, neoadjuvant chemoradiotherapy and pelvic infection [START_REF] Changchien | Postoperative urinary retention after primary colorectal cancer resection via laparotomy: a prospective study of 2,355 consecutive patients[END_REF].

Although voiding difficulties are transitory in most patients, temporary bladder drainage is justified [START_REF] Chaudhri | Successful voiding after trial without catheter is not synonymous with recovery of bladder function after colorectal surgery[END_REF].

In the literature the optimal duration of postoperative urinary drainage is unclear [START_REF] Zmora | Urinary bladder catheter drainage following pelvic surgery--is it necessary for that long?[END_REF]. The authors keep urinary catheters in situ between one and five days. Lee and al. showed that urinary catheter removal before the fifth postoperative day was an independent risk factor for acute urinary retention after rectal cancer surgery [START_REF] Lee | Risk factors and preventive measures for acute urinary retention after rectal cancer surgery[END_REF]. The type of drainage is not clear either. Suprapubic catheterization seems to give rise to fewer bladder and urethral symptoms but, to our knowledge, has not yet been proven to be superior to transurethral catheterization [START_REF] Benoist | Optimal duration of urinary drainage after rectal resection: a randomized controlled trial[END_REF][START_REF] Jahn | Types of indwelling urinary catheters for long-term bladder drainage in adults[END_REF][START_REF] Ratnaval | Suprapubic versus transurethral catheterisation of males undergoing pelvic colorectal surgery[END_REF]. Current French Guidelines recommend suprapubic catheterization (SPC) if the anticipated duration of drainage exceeds four days [START_REF] Alfonsi | French guidelines for enhanced recovery after elective colorectal surgery[END_REF] but do not specify a standard delay of bladder drainage after colorectal surgery.

In our department of digestive surgery, SPC is used in the majority of male patients undergoing rectal cancer surgery. Some studies suggest that the rates of urinary tract infection [START_REF] Sethia | Prospective randomized controlled trial of urethral versus suprapubic catheterization[END_REF][START_REF] Vandoni | Bacteriuria during urinary tract catheterization: suprapubic versus urethral route: a prospective randomized trial[END_REF], second catheterization and urinary symptoms (pain, bladder irritation) are lower with SPC than with transurethral catheterization (TUC) [15,[START_REF] Ratnaval | Suprapubic versus transurethral catheterisation of males undergoing pelvic colorectal surgery[END_REF]. The benefits of SPC include both easier management and better tolerance. Moreover, it allows for testing bladder voiding without drainage removal. On the other hand, it is a more invasive system.

The aim of this study was to evaluate our practice of urinary drainage after rectal resection and to identify factors predictive of postoperative urinary dysfunction.

METHODS

Patients

We included retrospectively 200 male patients from our department undergoing rectal resection. The sample consisted of 50 patients with abdomino-perineal resection (APR), 50 patients with mechanic coloanal anastomosis (MechCAA), 50 patients with manual coloanal anastomosis (ManCAA) and 50 patients with colorectal anastomosis (CRA). Inclusions were performed using a retrospective database of all rectal cancer patients operated in our department. Inclusions were performed back to front (??) starting on 1/12/2012 until 50 patients were included in each group. This method allowed us to have a sample of successive, recent cases and the same number of patients in each group.

Database

The following baseline characteristics were documented: comorbidity, ASA (American Society of Anaesthesiologists) score, tumour characteristics (including origin (?) and location), operative details, histopathological examination of the resected specimen, postoperative analgesia, early and late complications.

All data were added to an anonymous database. In addition, we recorded type and duration of bladder drainage as well as any associated complications and their management.

Surgical Technique

The standard surgical technique for all patients included full mobilization of the splenic flexure, high ligation of the inferior mesenteric artery and vein, and total or partial mesorectum excision [START_REF] Pocard | A prospective study of sexual and urinary function before and after total mesorectal excision with autonomic nerve preservation for rectal cancer[END_REF]. The choice of intervention was defined by tumor localization, previous surgery and past medical history. The type of reconstruction and anastomotic technique were left to the surgeons' discretion. A straight coloanal anastomosis was avoided whenever possible. The pelvis was routinely drained with a suction drain in the presacral space, which was removed 48 hours after surgery [START_REF] Bennis | Morbidity risk factors after low anterior resection with total mesorectal excision and coloanal anastomosis: a retrospective series of 483 patients[END_REF]. Rectal resections with a coloanal anastomosis were routinely covered with a temporary loop ileostomy. Rectal resections with a colorectal anastomosis were protected if the anastomosis was low. An omentoplasty was realized in all abdominoperineal resections without shred (??).

Postoperative Urinary Management

If a suprapubic catheter (SPC) was used, it was inserted at the end of the procedure. It was clamped on the fifth postoperative day and removed if the urinary residual was less than 50cc. If the urinary residual was not satisfactory, the patient received an alpha-blocker (Alfuzonide LP 10) and the clamping test was repeated until the urinary residual was acceptable. Patients with a persistently negative test were discharged with the SPC and referred to an urologist.

If the choice was transurethral catheterization (TUC), the catheter was taken out once morphine analgesia had been discontinued. It was removed on day 5 in patients without complications or bladder resection.

Analgesic protocol

Analgesic protocols were morphine patient controlled analgesia (PCA) with or without a wound catheter delivering local analgesia or epidural morphine pump alone. Pumps were routinely removed on the third postoperative day prior to clamping of the urinary catheter.

Evaluation of Urinary Function

We recorded preoperative dysfunction, type of catheter, interval between the first clamping of SPC and its removal, complications after catheter removal and their management. Urinary dysfunction was defined as catheter removal after the sixth postoperative day and/or any episode of acute urinary retention.

Statistical analysis

Descriptive analyses are presented as median (range) or mean +/-standard deviation for quantitative data and as number of patients (percentage of patients) for categorical data. The correlation between the urinary dysfunction and the variables of interest was studied by univariate analysis (Chi-square). All tests were two-sided, and a pvalue <0.05 was considered to be significant. The multivariate analysis was performed using a backward stepwise logistic regression model that included all variables with a p-value <0.2 in univariate analysis. Results of this multivariate analysis are shown as odds ratio (OR); [95 percent confidence interval]. All analyses were performed using JMP9 (SAS Institute Inc, Cary, NC, USA).

RESULTS

Patients

In order to obtain 50 men in each group, patients operated between December 2012 and January 2006 were included. Median age was 63 years and 173 patients (86.5%) had an ASA score of 1 or 2. Patients' characteristics did not differ between the four groups (Table 1).

Due to the location of the tumor, the rate of neoadjuvant radiotherapy was significantly different in the 4 groups: APR (84%), MechCAA (76%), ManCAA (70%), CRA (18%), p<0.001.

Intervention

A laparoscopic approach was performed in 84 patients (42%). The rate of laparoscopy ranged from 68% for CRA to 22% for APR (P<0.0001). However, the conversion rate was not significantly different between the four groups. The bladder was drained with a suprapubic catheter (SPC) in 142 patients (71%) and a transurethral catheter (TUC) in 58 patients (29%). A significant difference in the rate of SPC use was observed between patients who underwent total mesorectal excision (TME) (APR, ManCAA or MechCAA) (82%-90%) versus partial mesorectal excision (PME) with CRA (28%), p<0.001.

Urinary Management

Associated urological procedures

During surgery, there were 3 bladder resections, 4 prostatic resections, 2 JJ stents (for bladder and ureteric lesions) and 1 ureteric resection. All patients with bladder lesions were drained with TUC for 10 to 17 days. All patients with prostatic lesions were drained with SPC for 7 to 57 days. As urinary drainage was longer in patients with associated urological procedures , we excluded these patients from the study (n=190).

Suprapubic catheterization without associated urological surgery (n=136)

SPC was used in the majority of patients undergoing TME (90%). The complication rate of SPC was 15% and mainly due to leakage (4.3%) or accidental catheter removal (4.3%), (table 2). The clamping test was positive for 69 patients (51%) and their SPC were removed before or on POD6. There were no episodes of acute urinary retention in this group, giving the clamping test a positive predictive value of 100%. The rate of removal before POD6 ranged from 44% after MechCAA to 62% after ManCAA (table 2).

For the remaining patients with a negative test, removal of SPC was possible at POD10 or at POD21 for 82.7% and 93.5% of patients, respectively. The SPC could not be taken out in 9 patients (6.5%) and they left the hospital with the catheter in situ.

Urinary catheter without associated urological surgery (n=54)

Transurethral catheterization was mainly used after PME and CRA (67%), table 2. Its complication rate was 6% (infection: n=8, 5.8%). The TUC was removed in all patients before POD6. After TUC removal, 2 patients (4%) developed AUR and were discharged with an indwelling TUC. (Table 2)

Urinary dysfunction

Overall, 72 patients (38%) had urinary dysfunction: 11 (6%) were discharged with an indwelling urinary catheter and in 61 (32%) patients the bladder catheter was removed after POD6.

Ablation Failures

Details of patients with failed catheter removal are given in table 3. All patients except one, had at least one of the three classical predisposing factors (diabetes mellitus, previous urological history or neoadjuvant radiotherapy). They were all treated with alpha-blockers and recovered a normal urinary function after a median period of drainage ranging from 22 to 259 days. The patient with the longest drainage period (259 day) had a local recurrence needing a prolonged drainage, (table 3).

Risk factors of urinary dysfunction

The rates of urinary dysfunction were 51% for APR, 50% for MechCAA, 36.2% for ManCAA and 14.3% for CRA. There was no significant difference between APR, MechCAA and ManCAA (p=0.269) but urinary dysfunction was significantly less frequent after PME and CRA (p< 0.0001).

In univariate analysis, five risk factors were significantly associated with urinary dysfunction: diabetes mellitus (p=0.036), previous urological history (p=0.003), neoadjuvant radiotherapy (p=0.016), laparoscopic surgical procedure (p=0.002) and total mesorectal excision (p<0.0001), table [START_REF] Burgos | Risk factors for developing voiding dysfunction after abdominoperineal resection for adenocarcinoma of the rectum[END_REF].

In multivariate analysis, only three risk factors were independent: diabetes mellitus (OR=2.9 (1.2-7.7)), previous urological history (OR=2.9 (1.2-7.6)) and total mesorectal excision (OR=5.2 (2.3-13.7)), table 4.

We evaluated the dysfunction urinary rate according to the number of independent risk factors. Out of 35 patients without any risk factors, only four patients (11%) developed urinary dysfunction. Among patients with one, two or three risk factors, the rates of urinary dysfunction were: 36% (40/110), 61% (25/40) and 75% (3/4), respectively.

DISCUSSION

We studied retrospectively the urinary function in 200 men after proctectomy for rectal cancer. In the majority of patients (71%) the bladder was drained with a SPC. We observed few complications (15%) and the clamping test had an accuracy of 100%. The urinary function was a clinical problem for 72 men (38%) including 11 patients (5.8%) who were discharged with an indwelling urinary catheter. During follow-up all SPC could eventually be removed. We found three independent risk factors: diabetes, urologic history and TME.

One of the major interests of this study is its homogeneous population consisting exclusively of male patients undergoing surgery for rectal cancer. To our knowledge, all previous publications included either colorectal resections and/or women or evaluated fewer patients. Moreover, we studied four different procedures for rectal cancer resection according to the tumour location, which allows a precise comparison. SPC were used in the majority of patients (71.6%), as this is the policy of our unit. The lower the anastomosis was, the more frequently a SPC was used. The complication rate associated with SPC was 15% and all complications were minor. Infection rates were similar for SPC and TUC. However, there was no episode of acute urinary retention (AUR) after SPC removal unlike TUC removal (4% of AUR). This rate is much lower than the one reported by Benoist et al. in their randomized study comparing one day versus five days of urinary drainage after proctectomy [START_REF] Benoist | Optimal duration of urinary drainage after rectal resection: a randomized controlled trial[END_REF]. They observed a 31% rate of voiding problems in the 1-day group and 10% in the 5-days group even though half of the included patients were women. We believe that this difference may be explained by both the wide use of SPC and the routine clamping test in our unit. Indeed, in their comparative study, the rate of acute retention was 72% for patients operated for a low rectal cancer with 1-day urinary drainage. This observation confirms the interest of a more prolonged drainage with SPC.

Gerstenberg et al. studied bladder function by urodynamic investigation before and after APR for cancer. The urinary dysfunction rate was 7.7% for both genders [START_REF] Gerstenberg | Bladder function after abdominoperineal resection of the rectum for anorectal cancer. Urodynamic investigation before and after operative in a consecutive series[END_REF] but no significant difference was found in any parameters of cystometric and urodynamic investigations in women after surgery (p>0.10 for all comparisons) confirming that male patients are more at risk. Moreover, this study showed that patients with negligible symptoms of bladder outlet obstruction before surgery were likely to develop clinically relevant urinary symptoms after APR.

Previous urological history was an independent risk factor in both our and Benoist's studies [START_REF] Benoist | Optimal duration of urinary drainage after rectal resection: a randomized controlled trial[END_REF].

There are conflicting opinions regarding the superiority of open versus laparoscopy surgery for preservation of bladder function after rectal cancer surgery. Lim et al.'s recently published meta analysis did not show any difference between the two surgical approaches [START_REF] Lim | Postoperative bladder and sexual function in patients undergoing surgery for rectal cancer: a systematic review and meta-analysis of laparoscopic versus open resection of rectal cancer[END_REF]. Similarly, we did not observe any influence of the approach. In the present work, there was however a significant positive influence of laparoscopy (??). However, laparotomy was chosen for more complicated patients. Also, the rate of laparoscopy was lower for APR while this group was most at risk of urinary dysfunction.

We observed that risk factors for urinary dysfunction were not significantly different between APR, MechCAA and ManCAA as did Benoist et al. [START_REF] Benoist | Optimal duration of urinary drainage after rectal resection: a randomized controlled trial[END_REF] We also found that partial mesorectal excision was a protective factor of urinary function. We believe that SPC can be avoided after PME and CRA. In our department the rate of SPC after PME and CRA was only 33%. Currently, the majority of male patients undergoing a PME are drained with TUC.

Our score of three independent risk factors allowed to select low risk patients and to carry out a clamping test before POD5. Further studies are required to assess if a 3-day drainage would be feasible. If so, TUC may be preferable to SPC, which is recommended by the current guidelines [START_REF] Alfonsi | French guidelines for enhanced recovery after elective colorectal surgery[END_REF]. Moreover, validation of this score on another population is mandatory.

Finally, the best type of drainage is still unknown. In their meta-analysis Jahn et al. were unable to draw practical conclusions from the comparison of SPC with TUC. SPC was not associated with a higher morbidity than TUC [START_REF] Jahn | Types of indwelling urinary catheters for long-term bladder drainage in adults[END_REF]. Ratnaval et al. compared SPC versus TUC in fifty consecutive male patients after rectal surgery in a prospective study [START_REF] Ratnaval | Suprapubic versus transurethral catheterisation of males undergoing pelvic colorectal surgery[END_REF]. Acute urinary retention rate was 20.8% (5 patients) for SPC and 23.1% (6 patients) for TUC. Recatheterization was necessary for 2 patients in SPC group and all of the TUC patients (7 AUR).

Frequent voiding was recorded in 2 SPC versus 11 TUC (p<0.05). They concluded that SPC allowed a controlled return of normal voiding with fewer bladder and urethral symptoms. Studies on long-term urinary drainage do not add any useful information. Katsumi et al. studied the bladder management of male patients with spinal cord injury [START_REF] Katsumi | Urethral versus suprapubic catheter: choosing the best bladder management for male spinal cord injury patients with indwelling catheters[END_REF]. In this study of 179 patients with a long-term catheter there was no significant difference between the complication rates for the two catheter groups. However, the current trend in managing patients requiring an indwelling urinary catheter is to use a SPC in order to avoid the morbidity specifically associated with TUC.

Our study has several limits. Its retrospective design is associated with a risk of lost of data. However, due to the high volume of proctectomy in our department the inclusion period was short and the majority of data were collected exhaustively by our paramedical staff. Finally, the number of patients included should reduce this bias.

Conclusion

In this study the urinary dysfunction rate was 38% after surgery for rectal cancer. Drainage by SPC is not associated with a higher morbidity. SPC management is easier and more confortable for the patients. Three risk factors of urinary dysfunction (preoperative urological history, total mesorectal excision and diabetes mellitus) may serve to select those patients who are good candidates for early (POD2 or POD3) catheter removal.

Overall

Table 1 .

 1 Characteristics of the cohort of 200 male patients operated for rectal cancer.

	APR	MechCAA	ManCAA	CRA	p

Table 2 .

 2 Management and complications of urinary drainage

	N° Age	Diabe tes	Urolog ical history	Radio therapy	Surgery	Morbidit y Clavien Score	pT	Type of bladder drainage	Total time of drainage	AUR	Urinar y infecti on
	1	71 YES		YES	APR	LT	2	4	SPC	44
	2	71		YES		APR	LT	0	2	SPC	57
	3	77		YES	YES	APR	LT	0	0	SPC	41	YES
	4	48			YES	ManCA A	LC	1	2	SPC	60	YES
	5	56			YES	ManCA A	LT	1	2	SPC	45	YES
	6	58			YES	MechC AA	LC	3	2	SPC	50	YES
	7	77 YES	YES	YES	MechC AA	LC	3	2	SPC	64
	8	68				MechC AA	LC	1	1	SPC	40
	19 69		YES	YES	MechC AA	LT	3	3	TUC	58	YES	YES
	10 41 YES		YES	CRA	LT	3	4	SPC	259
	11 62				CRA	LC	3	3	TUC	22	YES

APR: abdomino-perineal amputation, MechCAA: mechanic colo-anal anastomosis, ManCAA: manual coloanal anastomosis, CRA: colorectal anastomosis, SPC: suprapubic catherization, TUC: transurethral catheterization, AUR: acute urinary retention, LC: laparoscopy, LT: laparotomy.

Table 3 .

 3 Characteristics of patients with failed catheter removal

			Univariate analysis		Multivariate analysis
		N	Urinary	No urinary		Odd Ratio	P value
			Dysfunction	dysfunction	P value	(IC95%)
			N=72	N=118
	Patients' characteristics
	ASA score 1-2	168	62 (36.9%)	106 (63.1%)
					0.4370
	ASA score 3-4	22	10 (45.5%)	12 (54.6%)
	Age>63 years	85	37 (56.5%)	48 (43.5%)			0.337
					0.1497
	Age<63 years	105	35 (33.3%)	70 (66.7%)
	BMI>25	92	38 (41.3%)	54 (58.7%)
					0.3843
	BMI<25	94	33 (35.1%)	61 (64.9%)
	Abdominal surgery history	74	33 (44.6%)	41 (55.4%)			0.153
					0.1284
	No previous surgery	116	39 (33.6%)	77 (66.4%)
	Diabetes	38	20 (53%)	18 (47.4%)		2.9(1.2-7.7)	0.0494
	No diabetes	152	52 (34.2%)	100 (65.8%)	0.0363
	Urological history	24	14 (58.3%)	10 (41.7%)		2.9 (1.2-7.6)	0.0233
	No Urological history	166	58 (34.9%)	108 (65.1%)	0.00272
	Neoadjuvant Radiotherapy	116	52 (44.8%)	64 (55.2%)			0.399
					0.0163
	No neoadjuvant radiotherapy	73	20 (27.4%)	53 (72.6%)
	Surgical procedure
	Laparoscopic approach	82	21 (25.6%)	61 (74.4%)			0.05
					0.0024
	Open approach	108	51 (47.2%)	57 (52.8%)
		142				5.2 (2.3-13.5) <0.0001
	TME		65 (45.8%)	77 (54.2%)	<
	PME	48	7 (14.6%)	41 (85.4%)	0.0001
	Operative time >275 min	85	33 (38.8%)	52 (61.2%)
					0.6269
	Operative time <275 min	99	35 (35.4%)	64 (64.7%)
	Pathological exam
	pT0-T1-T2 tumors	76	30 (39.5%)	46 (60.5%)	0.714
	pT3-T4 tumors	114	42 (36.8%)	72 (63.2%)
	Analgesic protocol
	Morphine PCA	92	31 (33.7%)	61 (66.3%)
					0.2471
	No Morphine PCA	98	41 (41.8%)	57 (58.2%)
	Epidural analgesia	84	34 (40.5%)	50 (59.5%)	0.5138

Table 4 .

 4 Univariate and multivariate analysis of risk factors associated with post-operative urinary dysfunction.