Anne Marie Farnet Da Silva

Leila Qasemian

Frédérique Peter-Valence

Florence Ruaudel

Jean-Michel Michel Savoie

Elisée Ferré

Anne-Marie Farnet
email: anne-marie.farnet@imbe.fr

Capacity for colonization

Keywords:

Introduction

Mushroom cultivation is a valuable biotechnological process which produces healthy food while converting a wide range of residues from the agro-industries. Agaricus subrufescens Peck is a cultivated mushroom also known as Agaricus blazei Murill. and Agaricus brasiliensis Wasser, Didukh, Amazonas & Stamets (Kerrigan, 2005;[START_REF] Wasser | Current findings, future trends, and unsolved problems in studies of medicinal mushrooms[END_REF]. It was firstly isolated in Brazil in the 1960s and is widely consumed both in Brazil and in oriental countries such as Japan or China for its nutritional properties (edible mushrooms are known for their high concentrations of vitamins or proteins in the biomass). Moreover, this mushroom is reputed to produce bioactive molecules that prevent or combat diseases such as diabetes and cancer but their physiological roles are still not clear [START_REF] Oliveira Lima | Does the Agaricus blazei Murill mushroom have properties that affect the immune system? An integrative review[END_REF]. Despite its economic importance, little information about A. subrufescens cultivation is available in the literature. A recent review [START_REF] Largeteau | The medicinal Agaricus mushroom cultivated in Brazil: biology, cultivation and nonmedicinal valorisation[END_REF] has described the data available about its cultivation process. Yet, there is clearly a need to improve methods of cultivation of A. subrufescens in Brazil (currently involving, for instance, long cultivation cycles of 90-120 days from casing) because production levels are very low compared to A. bisporus [START_REF] Siqueira | Cultivation of Agaricus blazei ss. Heinemann using different soils as source of casing materials[END_REF], the button mushroom commonly cultivated in Europe. Recently, Zied et al. (2011a) estimated the average A. subrufescens production in Brazil at 8-16% after 120 days of cultivation on substrates made from local wastes, such as coffee pulp or sugar cane bagasse.

Strains used for the cultivation of A. subrufescens in Brazil are marketed as varieties collected indigenously, selected through domestication and adaptation to the cultivation conditions of the farms (type and formulation of the compost and local environmental conditions). The consequences are great variability in production yields, long growth cycles and a lack of control over the specific growth characteristics of the strains. Despite the probable tropical origin of the species, several specimens had been isolated in temperate countries [START_REF] Kerrigan | Agaricus subrufescens, a cultivated edible and medicinal mushroom, and its synonyms[END_REF] and could be a source of new cultivars adapted for cultivation in northern countries with interesting yield potentials [START_REF] Llarena Hernández | Phenotypic variability in cultivars and wild strains of Agaricus brasiliensis and Agaricus subrufescens[END_REF]Zied et al., 2011b).The different steps in the process used for A. subrufescens cultivation are usually similar to those involved in A. bisporus cultivation. This means that it should be possible to develop efficient cultivation in Europe, where A. bisporus is cultivated on a large scale. However certain conditions such as the cultivation substrate or temperature used in Brazil vary from those found in Europe: sugarcane bagasse-based substrate is one of the most commonly employed, and relatively high temperatures (27 °C) are selected (Zied et al., 2011a). Testing whether European culture conditions used for A. bisporus (wheat-straw-based substrate, temperature, relative humidity, time allowed for growth) are suitable for an efficient cultivation of A. subrufescens, and how they can be improved, is of prime importance for the development of the diversification in industry of Agaricus mushroom. Because of the risks of contaminations and development of competitors, the first days following the spawning are critical for the success of the culture, the aim of this work was to assess the capacity of A. subrufescens to colonize and transform rapidly horse manure and straw based compost. We thus focused on the preliminary phase of mushroom cultivation i.e. spawn-running during the two first weeks, using analyses of the functional diversity of substrate microbial communities and degradation of lignocelluloses. Mesocosms were used to measure the capacity of different strains of A. subrufescens to colonize a wheatstraw-based compost: wild strains of various origins (Brazil and Europe) and cultivars were selected and an A. bisporus strain was used as control for substrate colonization. The substrate colonizing potential of the fungal strains was tested by monitoring (i) both lignocellulolytic activities produced by the different strains and laccase isoform electrophoretic patterns to assess the potential diversity of these enzymes involved in phenolic compound degradation (the recalcitrant fraction of culture substrate), (ii) H 2 O 2 production, which is known to be correlated to substrate colonization [START_REF] Savoie | Hydrogen peroxide concentration measured in cultivation substrates during growth and fruiting of mushrooms, Agaricus bisporus and Pleurotus spp[END_REF], (iii) the chemical transformation of lignocellulosic material using 13 C solid-state NMR spectroscopy, (iv) the functional diversity of bacterial communities of the substrates using a catabolic level physiological profile (CLPP). The A. bisporus strain Bs527 is a copy of the cultivar 30A (Euromycel) placed in the CGAB in 1997.

Methods

The fungal strains

Mesocosm experimental set-up

Precultures were performed in glass Petri dishes containing 12 g dry weight of rye grains (provided by Somycel, Langeais, France) inoculated with 6 plugs (1 Â 1 cm) of malt agar cultures. These precultures were incubated in a climatic chamber at 25 °C and 90% RH (relative humidity) for 10 days and were used to inoculate the mesocosms. 450 g of fresh mushroom compost (135 g dry weight) were placed in a 2 L glass pot in order to obtain a final volume of compost of 20 Â 16 Â 6 cm. They were inoculated with 3% (w/w) of rye grains (dry weight), which were thoroughly homogenized with the compost under aseptic conditions. The compost was a commercial compost produced by SARL Renaud et Fils at Avy, France, for the cultivation of A. bisporus. The main raw materials used were horse manure and wheat straw, and the phase I of composting was performed indoor. The quality of the compost was assessed by the yields obtained in commercial cultivation facilities that reached standards. Three mesocosms were prepared for each strain and were incubated in a climatic chamber at 25 °C and 90% of RH for 15 days in darkness. An un-inoculated control (with the compost alone), UIC, was also prepared and incubated under the same conditions.

Microbial catabolic profiles via Biolog EcoPlate™

For each mesocosm (three for each strain), one gram of substrate was vigorously shaken in 100 mL of a sterilized desorption-solution (Sodium pyrophosphate 0.08 g/L) for 1 h and then brought to a final OD = 0.001 at 595 nm. Hundred fifty microlitter of the extracted solution were used to inoculate all 96 wells of a BIOLOG EcoPlate (Biolog, California, USA). One EcoPlate™ was used for three mesocosm extracts since each 96-well plate containing three replicates, each with 31 different carbon sources, one per well, and a water blank. The plates were incubated at 25 °C for 48 h and the absorbance was read at 490 nm using a microplate reader (Tecan, France). To measure the functional diversity (FD), the Shannon's diversity index [START_REF] Shannon | A mathematical theory for communication[END_REF] was calculated [START_REF] Zack | Functional diversity of microbial communities: a quantitative approach[END_REF] from:

FD = À P pi(ln pi), where pi is the ratio of color development of well i to the sum of color development of all positive wells. Microbial activity for each microplate was assessed as average well-color development (AWCD) calculated as follows: AWCD = P ODi/31 where ODi is the optical density for each well.

Hydrogen peroxide concentration in compost

To evaluate the mycelial colonization of the substrate, the level of H 2 O 2 extractable from the compost was quantified as described by [START_REF] Savoie | Hydrogen peroxide concentration measured in cultivation substrates during growth and fruiting of mushrooms, Agaricus bisporus and Pleurotus spp[END_REF]. Briefly, 150 mg of lyophilized substrate milled to powder was added to 4 mL of phosphate buffer, 0.1 M pH6 and incubated at 30 °C for 15 min. After centrifugation (10,000g for 4 min), 2 mL of supernatant were placed at 80 °C for 40 min. Samples were centrifuged (10,000g for 4 min) and 250 lL of supernatant was added to 500 lL of 3-dimethylaminobenzoic acid, 7.5 mM, 125 lL of 3-methyl benzothiazolinone hydrazine hydrochloride, 0.6 mM, and 250 lL of peroxidases in phosphate buffer 0.1 M pH 6. Absorption was measured at 590 nm and a standard curve was realized with H 2 O 2 concentrations ranging from 3.1 to 32 lM.

Lignocellulolytic activity assays

For each mesocosm, enzyme extraction was performed as previously described by Velazquez-Cedeno et al. ( 2004) using 10 g of substrate in a 1 L flask containing 200 mL of an extraction solution (Polyvinylpolypyrolidone 5.7 g, CaCl2, 0.2 M, Tween 80, 0.05%). These samples were subjected to axial shaking for 1 h at 120 rpm at room temperature. Solids were eliminated by centrifugation at 10,000g for 15 min and filtration through Whatman GF/D filters (2.7 lm) and through Whatman GF/C filters (1.7 lm). The filtrates Author's personal copy of each extract were concentrated using polyethyleneglycol to a final volume of 10% of initial volume.

Laccase activities were measured by monitoring the oxidation of syringaldazine to quinone (e M = 65,000 M À1 cm À1 ) at 525 nm in acetate buffer, 0.1 M, pH 5.4 on a spectrophotometer Biomate 3 (Fischer Bioblock Scientific). One unit (U) of laccase activity is defined as the amount of lmoles of quinone of syringaldazine produced per min and per mL of sample at room temperature. Three assays were performed for each enzyme extract.

CM-cellulase activities were measured with carboxymethylcellulose (CMC) and using a simplified methodology adapted from that of Somogyi-Nelson as described by [START_REF] Farnet | A modified method based on arsenomolybdate complex to quantify cellulase activities: application to litters[END_REF]. Solution A was composed of 50 mL of CuSO 4 , 2%, Na 2 SO 4 , 4 g, complemented with 50 mL of a solution of Na 2 CO 3 , 25 g, NaHCO 3 , 20 g and sodium potassium tartrate, 25 g, q.s.p. 1 L. Solution B was composed of ammonium-molybdate, 25 g, H 2 SO 4 , 21 ml, and Na 2- HAsO 4 , 7H 2 O, 3 g, q.s.p. 450 mL. To measure CM-cellulase activity, 0.1 mL of the extract was incubated at 50 °C for 1 h in 0.9 mL of 50 mM acetate buffer (pH 5.0) with 0.1% CMC. 1 mL of solution A was added to 1 mL of the reaction medium and 1 mL of distilled water; then the mixture was boiled for 20 min and cooled down in a cold water bath for 15 min. One milliliter of solution B was added and the mixture was left for 50 min at room temperature and then centrifuged for 2 min at 10,000g. The absorption was measured at 870 nm. Dilutions were performed when necessary. For the calibration curves, 1 mL of solution A was added to 1 mL, 750 lL, 500 lL, 250 lL of a solution of glucose (80 mg/L) q.s. 1 mL. Three assays were performed for each enzyme extract.

Laccase isoform patterns on PAGE

PAGE were performed using 4% stacking gel and 10% separating gel at 220 V with the Mini-Protean III electrophoresis cell (Biorad) in non-denaturing conditions. The protein extracts obtained from the culture substrate were loaded onto the gels. Gel staining was performed using p-phenylenediamine at a final concentration of 0.1% (v/v) in acetate buffer 0.1 M, pH 5.4 for 30 min. Electrophoresis was performed in triplicate in each case.

2.7. The cross-polarization magic angle spinning 13 C nuclear magnetic resonance (CP/MAS 13 C NMR) procedure CP/MAS 13 C NMR spectra were obtained on a Bruker DSX 400 MHz spectrophotometer operating at 100.7 MHz. Samples (600 mg) were spun at 10 kHz at the magic angle. Contact times of 2 ms were applied with a pulse width of 2.8 ls and a recycle delay of 3 s. Chemical shift values were referenced to glycine signal (carbonyl C at 176.03 ppm). The 13 C NMR spectra were divided into seven chemical-shift regions, according to Ziarelli (2004). Dmfit 2003 software was used to determine the intensity of each chemical-shift-region [START_REF] Massiot | Modelling one-and two-dimensional solid-state NMR spectra[END_REF]. An index of decomposition (HR#1 Alkyl-C/O-Alkyl-C) was calculated as described by [START_REF] Baldock | Assessing the extent of decomposition of natural organic materials using solidstate C-13 NMR spectroscopy[END_REF]. An aromaticity ratio was also calculated to estimate the degree of humification of organic matter and Alkyl-C/COOH-C ratio HR#2 was used to assess organic matter transformation using the alkyl chain length of organic acids.

Statistical analysis

The significance of differences (enzyme activities, AWCD and H 0 from Biolog, RMN ratio) between means of three experiments was tested by nonparametric multiple comparison of Kruskal-Wallis. Each experiment was considered as an independent sample. Pearson correlation was tested between enzyme activities and chemical groups defined by 13 C NMR and ratios calculated from these data.

The optical absorbances of the 30 wells in the ECO plate were subjected to Principal Component Analysis (PCA) after 48 h of incubation, considering each substrate as a variable. The intensities of the 22 peaks obtained via CP/MAS NMR of 13 C analysis of the culture substrate were used for PCA. Statistica Vs 6 (StatSoft, Maison-Alfort, France) was used for statistical analysis and p-value <0.05 is considered as significant.

Results and discussion

3.1. Lignocellulolytic activities, H 2 O 2 concentration and laccase electrophoretic pattern obtained from culture substrate Microbiological and chemical markers of substrate colonization were used to assess the respective potential of six A. subrufescens strains to grow in solid-state fermentation cultures, under conditions used for A. bisporus. Lignocellulolytic activities were determined, since they are involved in the transformation of the main polymers of wheat-straw-based substrates. Previous studies have described factors affecting lignocellulolytic enzyme production in solid-state fermentation, mainly: the nature of nitrogenous sources and of lignocellulosic material, moisture level especially for lignin degradation and, obviously, the fungus under study [START_REF] Colauto | Alternative to peat for Agaricus brasiliensis yield[END_REF][START_REF] Elisashvili | Lentinus edodes and Pleurotus species lignocellulolytic enzymes activity in submerged and solid-state fermentation of lignocellulosic wastes of different composition[END_REF][START_REF] Sharma | Production of lignocellulolytic enzymes and enhancement of in vitro digestibility during solid state fermentation of wheat straw by Phlebia floridensis[END_REF]. Here, variations in lignocellulolytic activities were potentially due to the diversity of the strains of A. subrufescens studied, since all the culture conditions remained constant. The literature suggests that, in mushroom compost before inoculation, no laccase activities are generally observed while cellulases produced by the microflora present at the end of the composting process are often detected. With A. bisporus, it has previously been observed during the incubation phase that laccase activity increased while cellulase activity decreased dramatically with colonization by the mushroom mycelium [START_REF] Savoie | Changes in enzyme activities during early growth of the edible mushroom, Agaricus bisporus, in compost[END_REF]. Here, we observed that laccase and cellulase activity levels differed depending on the strain considered (Table 1). Three strains of A. subrufescens (CA 572, CA 646 and CA 647) produced high laccase activities and weak cellulase activities were observed compared to the other A. subrufescens strains. However, their cellulase activities in the compost were similar to those of A. bisporus. Thus, these three strains more recently cultivated, are apparently those with the highest compost colonizing ability as revealed by lignocellulolytic enzymes.

No strong differences in lignocellulolytic activities between A. bisporus and most of the A. subrufescens strains were observed. However, it is noteworthy that certain A. subrufescens strains (CA 572 and CA 646) produced higher laccase activities than the A. bisporus strain. This is interesting since laccases in spent mushroom substrates of A. subrufescens cultivated in Brazil have been shown to be of interest for soil bioremediation [START_REF] Ribas | Use of spent mushroom substrates from Agaricus subrufescens (syn. A. blazei, A. brasiliensis) and Lentinula edodes productions in the enrichment of a soil-based potting media for lettuce (Lactuca sativa) cultivation: Growth promotion and soil bioremediation[END_REF]. The high potential for laccase production observed here in a European compost is encouraging, having developmental implications for the environmental sciences. The cultivation substrate might indeed be a source of crude laccases for enzymatic oxidation of phenolic compounds or degradation of aromatic pollutants such as PAH (polycyclic aromatic hydrocarbons), as proposed for A. bisporus [START_REF] Trejo-Hernandez | Residual compost of Agaricus bisporus as a source of crude laccase for enzymic oxidation of phenolic compounds[END_REF]. Moreover, in our study, a high diversity of laccase isoforms was observed in compost colonized by A. subrufescens. Laccase electrophoretic patterns of enzyme extracts obtained from the culture substrate showed four bands whatever the A. subrufescens strains considered, whereas only two bands were observed with A. bisporus (data not shown). This result is in accordance with the isoform pattern obtained in liquid culture where the same number of apparent isoforms (four) was observed (data not shown). [START_REF] Ullrich | Laccase from the medicinal mushroom Agaricus blazei: production, purification and characterisation[END_REF] have purified a laccase from an A. blazei strain from Vietnam and found only one iso-form in liquid culture. As stated by [START_REF] Ullrich | Laccase from the medicinal mushroom Agaricus blazei: production, purification and characterisation[END_REF] little is known about the lignocellulolytic system of this mushroom, thus the laccase diversity of the strains under study here seems to be promising and further investigations about their biochemical properties are needed.

H 2 O 2 production was also monitored as an indicator of culture substrate colonization, as proposed by [START_REF] Savoie | Hydrogen peroxide concentration measured in cultivation substrates during growth and fruiting of mushrooms, Agaricus bisporus and Pleurotus spp[END_REF] for A. bisporus. This method is used to characterize colonization kinetics of lignocellulosic substrates: H 2 O 2 measured in compost can be generated either directly by enzymes involved in sugar oxidation or from active oxygen species also produced during substrate transformation. However, though the method has been proved to efficiently discriminate the colonization potential of various white-rot fungi, it seems to be of less value for intraspecies comparisons of substrate colonization potential [START_REF] Savoie | Hydrogen peroxide concentration measured in cultivation substrates during growth and fruiting of mushrooms, Agaricus bisporus and Pleurotus spp[END_REF]. Here, we found that all the A. subrufescens strains had the same level of H 2 O 2 production: only strain CA 647 exhibited higher H 2 O 2 production compared to CA 646 and CA 454.

Catabolic level physiological profile of the microbial communities of the culture substrate

The nature of the cultivation substrate strongly influences the equilibrium of the microbial communities, which can enhance the growth of competing or even pathogenic microorganisms [START_REF] Colauto | Alternative to peat for Agaricus brasiliensis yield[END_REF]. This is why the chemical and microbiological composition of the substrate is of high importance in mushroom cultivation. Whereas there are several studies on microbial communities during the composting phase [START_REF] Silva | Microbial diversity in a bagasse-based compost prepared for the production of Agaricus brasiliensis[END_REF][START_REF] Székely | DGGE and T-RFLP analysis of bacterial succession during mushroom compost production and sequence-aided T-RFLP Profile of mature compost[END_REF], little is known about how mushroom growth modifies microbial communities in mushroom compost during colonization. Here, the microbial characterisation of the substrate was investigated via functional diversity. Average Well Color Development (AWCD) and Shannon-Weaver index of biodiversity (H 0 ) were calculated from optical densities obtained and are shown in Table 1. For AWCD, differences were observed between mesophilic bacterial communities of compost inoculated with certain Agaricus spp. strains and those from UIC. Certain microbial populations may have been eliminated from the compost because of the growth of the mushroom mycelium, which would explain a weaker AWCD in mesocosms inoculated with the A. bisporus strain and with strains CA 560 and CA 647 than that of UIC. AWCD was also weaker for substrates inoculated with strains CA 560 and CA 647 compared to those inoculated with strains CA 572 and CA 646. H' varies in the same way as AWCD: the catabolic diversity of the microbial communities of the substrate decreased and differed between (i) substrates inoculated with A. bisporus, strain CA 560, strain CA 647 (0.21 and 0.29 respectively) and UIC (1.36) and (ii) substrates inoculated with CA 560 and 647 (0.21 and 0.29 respectively) and substrate inoculated with CA 572 and CA 646 (1.32 and 1.34, respectively). These results thus show that, when the substrate is inoculated either with A. bisporus or with A. subrufescens strains CA 560 and CA 647, both the catabolic diversity and the global activity (characterized by AWCD) of the microbial communities of the compost are affected. The growth of these Agaricus strains may have altered the equilibrium of the microbial communities of the compost, leading to a shift in biodiversity. [START_REF] Cai | Microbial community structure of casing soil during mushroom growth[END_REF] found a change in the microbial diversity of casing soil when using Phospholipid fatty acid (PLFA) as markers, depending on the stage of A. bisporus development. However, here H' and AWCD did not change for A. subrufescens strains CA 454, CA 487, CA 572 and CA 646 compared to UIC. Thus, our results suggest that for most of the A. subrufescens strains studied (4 out of 6) the diversity of the mesophilic microbial communities of the compost is maintained during the two first weeks of colonization. This is of major importance, since these communities are known to contribute to the electivity of the substrate. Previous findings have indeed demonstrated the major role played by the microbial communities of mushroom substrates in preventing antagonist contamination [START_REF] Velazquez-Cedeno | Role of Bacillus spp. in antagonism between Pleurotus ostreatus and Trichoderma harzianum in heat-treated wheat-straw substrates[END_REF]. Moreover, the microbial communities of substrate used for mushroom cultivation can modify nutrient allocation, and microenvironmental conditions in substrate are known to strongly influence mushroom growth [START_REF] Carabajal | Effect of co-cultivation of two Pleurotus species on lignocellulolytic enzyme production and mushroom fructification[END_REF].

PCA was also performed from Ecoplate data and results are shown in Fig. 1 (F1 and F2 explain 72% of variance). Projections of both the A. bisporus strain and A. subrufescens strains CA 560 and CA 647 are clearly separated from UIC and the other A. subrufescens strains according to F1 (62% of the variance), which corroborates our previous results obtained with H' and AWCD. PCA also shows that strains CA 454, CA 487, CA 572 and CA 646 are differentiated from the control according to F2 (10% of variance). This supplementary result indicates that the catabolic structures of the substrate microbial communities differ from those of the control when substrate is inoculated with these A. subrufescens strains. In this case, though functional diversity did not decrease, as shown by H 0 index (Table 1), it may have been altered as a result of fungal inoculation and growth. This result is in accordance with [START_REF] Wang | Effect of inoculation with Penicillium expansum on the microbial community and maturity of compost[END_REF] who also found modifications in the structure of microbial communities (involved in cellulose degradation or nitrogen cycle) in compost inoculated with Penicillium expansum. The two first weeks of compost colonization by the mycelium of A. subrufescens and A. bisporus modified the equilibrium of the microbial communities of the compost with either a decrease or a shift in Author's personal copy functional diversity. This is in agreement with the proposed explanation of the decrease in cellulase activities due to changes in microbial communities with colonization by the mycelium.

Compost transformation assessed via CP/MAS NMR of 13 C

An aromaticity ratio and two humification ratios HR1 (Alkyl-C/ O-Alkyl-C) and HR2 (Alkyl-C/COOH-C) were calculated to assess degradation of culture substrate (Fig. 2a, b andc). The aromaticity ratio significantly decreased for substrates inoculated with strains CA 454, CA 572 and CA 647 compared to that inoculated with A. bisporus and UIC, showing that the aromatic compounds were more efficiently transformed with these three A. subrufescens strains. Thus, concerning these strains (CA 454,CA 572 and CA 647), this result differs from those obtained by González Matute et al. ( 2010) who found that lignin was actively transformed after fruiting body production only. HR1 significantly increased compared to UIC for composts inoculated with the A. bisporus strain and strains CA 560, CA 646 and CA 647. The same result was found for HR2, except for substrate with strain CA 647. The HR2 increase (Alkyl-C/COOH-C) may be explained by a higher amount of fungal biomass production characterized by lipids [START_REF] Lundberg | 13 C NMR spectroscopy studies of forest soil microbial activity: glucose uptake and fatty acid biosynthesis[END_REF]. Here both HR1 and the aromaticity ratio prove reliable as chemical indicators of organic matter transformation and our results show that the culture substrate was transformed to the same extent both with the A. bisporus strain and with certain A. subrufescens strains. However, for HR1, no correlations were found with either lignocellulolytic activities or H 2 O 2 production, as explained in the next paragraph.

PCA based on the 22 NMR peaks is shown in Fig. 3a and b (F1 and F2 explain more than 60% of the variance). While the chemical characteristics of the substrate inoculated differ from control according to F1 (except for strain CA 454), the chemical characteristics of substrate inoculated with A. bisporus strain is clearly discriminated from those inoculated with the A. subrufescens strains according to F2 (20% of variance). It is also noteworthy that strain CA 454 (expected to be a copy of the original strain from which the cultivation of A. subrufescens was developed) and strain CA 560 (one of the oldest cultivars in Brazil) were separated from the other strains. As shown in Fig. 3b, control and CA 454 were separated from the other strains by a negative correlation with C-Alkyl peaks (P17, P19 and P20) and a positive correlation with C-OAlkyl (P12, 74 ppm). A bisporus was separated from the A. subrufescens strains (except CA 454) by a negative correlation with Aromatic-C (P5, 128 ppm), C-COOH (P1, 172 ppm) and C-OAlkyl (P13, 71 ppm) and a positive correlation with C-Phenol (P3, 151 ppm). These differences in degradation patterns between old and new cultivars might be the result of a selection by mushroom growers based on visual estimations of compost colonization. For these chemical parameters as for the microbial markers, the European wild isolate CA 487 was grouped with the cultivars of Brazilian origin (CA 646, CA 647 and CA 572), indicating its potential as a new cultivated strain. The lignocellulosic material used in the cultivation substrate is of major importance to obtain acceptable yields of mushroom production. As stated by [START_REF] Royse | Effects of spawn, supplement and phase II compost additions ans time of re-casing second break compost on mushroom (Agaricus bisporus) yield and biological efficiency[END_REF], the substrate is the main expense in mushroom production and it is of great importance to identify substrates with high productivity, 2010) examined the biological efficiency (BE) obtained with substrates based on sunflower seed hull and wheat straw and found BE ranging from 30 to 40%. [START_REF] Wang | Yield, dry matter and polysaccharides content of the mushroom Agaricus blazei produced on asparagus straw substrate[END_REF] used asparagus straw complemented with either cottonseed hull or cow manure and found that BE was higher (44%) when both lignocellulosic materials were combined. However, these results, compared to those obtained with A. bisporus -for instance, [START_REF] Sanchez | Scytalidium thermophilum-colonized grain, corncobs and chopped wheat straw substrates for the production of Agaricus bisporus[END_REF] found a BE of 99.3% -indicate that investigations to improve A. subrufescens cultivation are still needed and of crucial importance.

Correlation between microbial activities and chemical characteristics of the substrate in mesocosms

Pearson's correlations between chemical characteristics ( 13 C NMR signals) and microbial activities (cellulases, laccases and H 2 O 2 production) are shown in Table 2. A very significant negative correlation was found between laccase activities and aromaticity ratio (p < 0.01). As a corollary, laccase activities were negatively correlated with aromatic-C signal (p < 0.05). This negative correlation can be explained by the fact that these enzyme activities are involved in the degradation of recalcitrant fractions of organic matter from plants. Laccases are thus actively involved in these changes in aromaticity ratio. Both the aromaticity ratio and the aromatic-C signal are also negatively correlated with H 2 O 2 concentration (p < 0.05 and p < 0.01, respectively), showing that H 2 O 2 concentration used as an indicator of substrate colonization can also be considered as an indicator of substrate degradation and transformation of aromatic compounds, as hypothesized by [START_REF] Savoie | Hydrogen peroxide concentration measured in cultivation substrates during growth and fruiting of mushrooms, Agaricus bisporus and Pleurotus spp[END_REF]. On the other hand, no significant correlations were observed with cellulase activities confirming that H 2 O 2 concentration does not vary in the same way as these activities, as previously stated by [START_REF] Savoie | Hydrogen peroxide concentration measured in cultivation substrates during growth and fruiting of mushrooms, Agaricus bisporus and Pleurotus spp[END_REF].

Conclusions

This study showed that the capacity for colonization and degradation of horse manure and wheat-straw-based compost by different strains of A. subrufescens during the first two weeks of culture was strain dependent, but globally as efficient as that of the button mushroom. Strains produced higher laccase activities with four isoforms and this biodiversity could be used for biotechnological purposes. Significant changes in 13 C NMR aromaticity signal ratios correlated with laccase activities showing the efficient transformation of compost. Our results also suggest that a European wild isolate and the cultivars currently used appeared to be more efficient than the older strains. They will be used to improve the culture of this medicinal mushroom.

Fig. 1 .

 1 Fig. 1. PCA (based on Pearson corelation) performed using OD 48h of each Ecoplate Carbone source (n = 31) as variables. The two first factors (F1 + F2) counted for 72% of total variance. Catabolic profile of compost microbial communities was obtained for UIS: Un-Inoculated Substrate and compost inoculated with Agaricus bisporus (strain Bs 527) or six strains of A. subrufescens (CA 454, CA 487, CA 560, CA 572, CA 646, CA 647).

Fig. 2 .

 2 Fig. 2. Aromaticity ratio and two humification ratios, HR#1, Alkyl-C/O-Alkyl-C and HR#2, Alkyl-C/COOH-C, calculated from the relative intensities of chemical functions from 13 C NMR data from Un-Inoculated Substrate (UIS) and horse manure and straw-based compost inoculated with six different Agaricus subrufescens strains (CA 454, CA 487, CA 560, CA 572, CA 646, CA 647) and A. bisporus, after incubation at 25 °C for 14 days. The different letters indicate when values differ significantly according to Kruskal-Wallis test and Dunn procedure for multiple comparisons of means (p < 0.05).

Fig. 3 .

 3 Fig. 3. PCA (based on Pearson correlation) performed using 13 C NMR peaks (n = 22, P1-P2, COOH-C, P3-P6, Aromatic-C, P7-P16, O-Alkyl-C, P17-P22, Alkyl-C, ranging from 0 to 200 ppm) measured in horse manure and straw based compost samples as variables. The two first factors (F1 + F2) counted for 60% of total variance. The compost samples are: Un-inoculated substrate (UIS), compost inoculated with Agaricus bisporus (strain Bs 527) or with A. subrufescens CA 454, CA 487, CA 560, CA 572, CA 646, CA 647 and incubated at 25 °C for 14 days.

  Six strains of A. subrufescens and one strain of A. bisporus from the Collection of Germplasms of Agaricus in Bordeaux (CGAB), INRA, France, were used. CA 487 is a wild strain of A. subrufescens isolated in France in 2006. CA 454 is a subculture of the original strain cultivated for the first time in Brazil in the 1960s and registered as A. blazei ATCC 76739. CA 646 and CA 647 are two cultivars sold in Europe by Mycelia under the reference numbers 7700 and 7703, respectively. CA 560 was isolated in Botucatu, Brazil (1999) from a commercial farm and CA 572 is a Brazilian cultivar purchased by D.C. Zied in 2007. Their original reference numbers are ABL-99/28 and ABL-07/58 respectively, in the Culture Collection of the Edible and Medicinal Mushroom Module of São Paulo State University (UNESP).

Table 1

 1 Laccase activities, Cellulase activities and H 2 O 2 concentration (n = 3) measured in the horse manure-and straw-based compost inoculated with Agaricus bisporus (Bs 527) or six strains of A. subrufescens after incubation at 25 °C for 14 days. AWCD and Shannon Weaver index (H 0 ), based on Ecoplate data (n = 3) after 48 h of incubation of the microbial extract obtained from the substrate. The letters define the mean values which are statistically different from each other according to multiple comparison of Kruskal-Wallis (Dunn Procedure).

		Agaricus strains
		Bs 527	CA 560	CA 454	CA 572	CA 646	CA 647	CA 487
	Laccase	15.79 ± 2.49 ab	26.83 ± 2.90 abc	24.80 ± 3.71 abc	77.99 ± 7.61 d	48.65 ± 10.41 cd	40.96 ± 4.87 bcd	13.04 ± 0.26 a
	Cellulase	0.46 ± 0.10 ab	3.39 ± 0.83 bc	5.91 ± 1.00 c	0.265 ± 0.11 a	0.10 ± 0.26 a	0.68 ± 0.36 abc	2.50 ± 0.22 bc
	H 2 O 2 concentration	19.13 ± 0.81 a	64.17 ± 1.92 abcd	55.28 ± 1.08 abc	81.39 ± 2.74 cd	27.23 ± 1.68 ab	104.21 ± 5.65 d	78.76 ± 5.41 bcd
	AWCD	0.075 ± 0.002 ab	0.025 ± 0.03 a	0.669 ± 0.056 abc	0.887 ± 0.089 bc	0.930 ± 0.282 bc	0.019 ± 0.016 a	0.594 ± 0.112 abc
	H'	0.87 ± 0.10 ab	0.21 ± 0.21 a	1.25 ± 0.14 bc	1.32 ± 0.04 bc	1.34 ± 0.07 c	0.29 ± 0.32 a	1.27 ± 0.07 abc

Table 2

 2 Correlation coefficients (r values) between 13 C NMR data, presented here as chemical groups and ratios, and H 2 O 2 concentration, laccase and cellulase activities. Significant correlations are marked bold with ⁄ p < 0.05, ⁄⁄ p < 0.01 and ⁄⁄⁄ p < 0.001. needs an effective colonization by the mycelium. Various materials have been tested as substrate for A.subrufescens: González Matute et al. (

		Laccase activities	Cellulase activities	H 2 O 2 concentration
	Aromaticity ratio	À0.52 ⁄⁄	0.07	À0.48 ⁄
	Humification ratio #1 (Alkyl C/O-Alkyl C)	0.25	À0.27	0.12
	Humification ratio #2 (Alkyl C/COOH C)	0.32	À0.34	0.05
	COOH C	0.26	À0.16	0.25
	Aromatic C	À0.48 ⁄	0.04	À0.60 ⁄⁄
	O-Alkyl C	À0.06	0.17	À0.19
	Alkyl C	0.37	À0.34	0.14

which

A.-M. Farnet et al. / Bioresource Technology 131 (2013) 266-273

Acknowledgements

We thank Diego Cunha Zied for purchasing Brazilian A. subrufescens strains and Mrs Marjorie Sweetko for English language revision. This work was supported by the French ANR, project ANR-09-BLAN-0391.

Author's personal copy Ziarelli, F., 2004


Laura Selliez
email: laura.selliez@cnrs-orleans.fr

C Briois

N Carrasco

L Thirkell

R Thissen

M Ito

F.-R Orthous- Daunay

G Chalumeau

F Colin

H Cottin

C Engrand

L Flandinet

N Fray

B Gaubicher

N Grand

J.-P Lebreton

A Makarov

S Ruocco

C Szopa

V Vuitton

P Zapf

F.-R Orthous-Daunay

Identification of organic molecules with a laboratory prototype based on the Laser Ablation-CosmOrbitrap

Keywords: Astrobiology, Space mass spectrometry, High mass resolution, Chemical identification, Orbitrap, CosmOrbitrap

, based on the Orbitrap™ technology. This work challenges the use of LAb-CosmOrbitrap, a space instrument prototype combining Laser Ablation ionisation and the CosmOrbitrap mass analyser, to identify solid organic molecules of relevance to the future space exploration. For this purpose a blind test was jointly organised by the JAXA-HRMS team (Japan Aerospace Exploration Agency-High Resolution Mass Spectrometry) and the CosmOrbitrap consortium. The JAXA team provided two organic samples, whereas the CosmOrbitrap consortium analysed them without prior information. Thanks to the high analytical performances of the prototype and our HRMS data post-processing, we successfully identified the two molecules as HOBt, hydroxybenzotriazole (C 6 H 5 N 3 O) and BBOT, 2,5-Bis(5-tert-butyl-benzoxazol-2-yl)thiophene (C 26 H 26 N 2 O 2 S), with a mass resolving power of, respectively, 123 540 and 69 219. The success of this blind test on complex organic molecules shows the strong potential of LAb-CosmOrbitrap for future space applications.

The capability to study organic molecules and organic-rich environments in the Solar System is important for astrobiology [START_REF] Horneck | Exobiology, the study of the origin, evolution and distribution of life within the context of cosmic evolution: a review[END_REF]. This allows a better understanding of the chemical evolution that leads to the emergence of Life on Earth and provides constraints on the possible habitability of other planets or moons. Detection of organic molecules and traces of extinct or extant Life is therefore driving space exploration concepts for objects like Europa, Titan and Enceladus.

Many space missions to these objects are either in preparation or proposed: the JUICE (JUpiter ICy moons Explorer) mission [START_REF] Grasset | JUpiter ICy moons Explorer (JUICE): An ESA mission to orbit Ganymede and to characterise the Jupiter system[END_REF], the Europa Clipper mission [START_REF] Phillips | Europa Clipper Mission Concept: Exploring Jupiter's Ocean Moon[END_REF] or, among others, the Dragonfly mission (Principal Investigator E. Turtle, see the JHU/APL website for further details) selected in 2017 as finalist of the NASA New Frontiers program.

One of the best analytical tools used in space missions for chemical analysis is mass spectrometry. This technique allows to assess the molecular composition of the environments studied. The detection and the identification of compounds lead to a better understanding of the chemistry occurring on diverse objects of the Solar System. A high diversity in terms of targets studied (planets, moons, small bodies etc.) and sample types (solid rocks, gaseous compounds in atmosphere, aerosols, ions etc.) is possible. In part, this is due to the multiple combinations of ion sources and mass analysers existing. Mass spectrometers have been boarded since the beginning of the space exploration and are still an instrument family essential to the future space missions. On the Cassini-Huygens mission, the instruments INMS (Ion and Neutral Mass Spectrometer) [START_REF] Waite | The Cassini Ion and Neutral Mass Spectrometer (INMS) Investigation[END_REF] or CAPS (CAssini Plasma Spectrometer) [START_REF] Young | Cassini Plasma Spectrometer Investigation[END_REF] enabled the collection of a large amount of data and the improvement of our knowledge about Titan upper atmosphere [START_REF] Waite | The Process of Tholin Formation in Titan's Upper Atmosphere[END_REF] and Enceladus plumes [START_REF] Waite | Cassini finds molecular hydrogen in the Enceladus plume: Evidence for hydrothermal processes[END_REF]. Thanks to modelling, attributions of a number of peaks in INMS data were successfully proposed, up to m/z < 100. However, the mass resolution of these instruments (including INMS) did not enable to directly decipher the composition of the complex organic matter. That explains why the moons of Saturn are still highly requested in the discovery

M A N U S C R I P T A C C E P T E D

ACCEPTED MANUSCRIPT 6 programs of space agencies. The best mass resolving power (m/Δm or MRP at Full-Width Half Maximum (FWHM)) of a space mass spectrometer, was provided by the DFMS (Double Focusing Magnetic Mass Spectrometer) instrument of the ROSINA (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis) experiment on board the Rosetta mission with m/Δm 9,000 FWHM at m/z 28 [START_REF] Balsiger | Rosina -Rosetta Orbiter Spectrometer for Ion and Neutral Analysis[END_REF]. With these performances, the detection of species like N 2 and O 2 on a comet was made possible for the first time [START_REF] Bieler | Abundant molecular oxygen in the coma of comet 67P/Churyumov-Gerasimenko[END_REF][START_REF] Rubin | Molecular nitrogen in comet 67P/Churyumov-Gerasimenko indicates a low formation temperature[END_REF]. In addition, a prebiotic compound, the amino acid glycine, at m/z 75, has been detected [START_REF] Altwegg | Prebiotic chemicals-amino acid and phosphorus-in the coma of comet 67P/Churyumov-Gerasimenko[END_REF]. Despite its high MRP, ROSINA/DFMS covered a mass range from 12 to 150 mass units (u) excluding the analysis of heavy organic molecules about hundreds of mass units.

It became a critical need to develop a new generation of mass spectrometers to go further in these studies, with high analytical performances required. In term of mass accuracy, a range of less than 1 to 5 ppm is needed in order to provide relevant molecular formula attributions. About the MRP, few tens of thousands (from 50,000 to 100,000) allow the separation of isobaric interferences at high m/z (up to m/z 500) of organic species with, for instance, an exobiological interest. Detection and identification of complex organic molecules involved in chemical mechanisms occurring on Solar System bodies, such as the organic chemistry observed on Titan [START_REF] Hörst | Titan's atmosphere and climate[END_REF] are thus possible. In the laboratory, the Orbitrap TM technology is now currently applied to the analysis of complex organic material of interest to space exploration, such as analogues of Titan's aerosols [START_REF] Pernot | Tholinomics-Chemical Analysis of Nitrogen-Rich Polymers[END_REF] and soluble organic matter of meteorites [START_REF] Bonnet | Compositional and structural investigation of HCN polymer through high resolution mass spectrometry[END_REF][START_REF] Gautier | Development of HPLC-Orbitrap method for identification of N-bearing molecules in complex organic material relevant to planetary environments[END_REF][START_REF] Orthous-Daunay | Midinfrared study of the molecular structure variability of insoluble organic matter from primitive chondrites[END_REF]. It enables to cover a dynamic mass range, up to several thousands in mass units [START_REF] Makarov | Electrostatic Axially Harmonic Orbital Trapping: A High-Performance Technique of Mass Analysis[END_REF], with a data acquisition time of about 1 second.

A mass analyser designed for space and based on the Orbitrap technology is currently being developed under the name of CosmOrbitrap [START_REF] Briois | Orbitrap mass analyser for in situ characterisation of planetary environments: Performance evaluation of a laboratory prototype[END_REF], which would bring a technical breakthrough for direct in situ analysis. Mainly based on the analysis of metals with a LAb-CosmOrbitrap prototype, [START_REF] Briois | Orbitrap mass analyser for in situ characterisation of planetary environments: Performance evaluation of a laboratory prototype[END_REF] have shown the high analytical performances of this mass spectrometry technique, expecting this mass analyser to be part of a future space mass

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 7 spectrometer.
In this previous work, high performances have been achieved with a simple instrumental configuration of the prototype using direct laser ablation ionisation coupled with a CosmOrbitrap mass analyser through an Einzel lens. They obtained mass resolutions at FWHM of 474,000 on beryllium (m/z 9) and of 90,000 on lead (m/z 208), close to mass resolution of 60,000 at m/z 400 obtained with a commercial LTQ-Orbitrap XL instrument [START_REF] Perry | Orbitrap mass spectrometry: Instrumentation, ion motion and applications[END_REF]. [START_REF] Briois | Orbitrap mass analyser for in situ characterisation of planetary environments: Performance evaluation of a laboratory prototype[END_REF] have demonstrated a mass accuracy within less than 15 ppm over the 12 to 115 m/z range with the LAb-CosmOrbitrap prototype, rather larger than the 1-5 ppm given by commercial Orbitrap TM based instruments.

The present work focuses on the capability of the LAb-CosmOrbitrap to provide molecular identification, another important question in the development of a space instrument, by performing a blind test on two different molecules. Under a Japanese/French collaboration framework, two organic samples have been chosen by the JAXA HRMS team and sent to the CosmOrbitrap team without any information on their chemical composition. This work reports the blind analysis performed on these two "unknown" samples by the CosmOrbitrap consortium with the LAb-CosmOrbitrap prototype in order to identify them. Updates on the analytical performances of the LAb-CosmOrbitrap, on organics, are also given.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 8 II) Methods a)
The laser ablation -CosmOrbitrap prototype

The blind test experiments have been conducted using a slightly modified version of the laboratory prototype previously described in [START_REF] Briois | Orbitrap mass analyser for in situ characterisation of planetary environments: Performance evaluation of a laboratory prototype[END_REF] To provide a better absorption of organic molecules in the UV resulting into a more efficient ionisation, the UV nitrogen laser at 337 nm was exchanged for a Nd-YAG laser at 266 nm [START_REF] Goesmann | The Mars Organic Molecule Analyzer (MOMA) Instrument: Characterization of Organic Material in Martian Sediments[END_REF]. The latter is the "Brilliant" model provided by Quantel, with 4 ns pulse duration and about 100 µJ energy per pulse. The angle of incidence on the surface of the sample-holder is 50°, resulting in an elliptical shape of the footprint of the laser beam with a minor axis of typically 30 µm and a major axis of 40 µm (measured on silicon wafer) and energy density of 15 J.cm -2 .

The Orbitrap mass analyser cell commercialised by the Thermo Fisher Scientific (Bremen, Germany) is an ion trap in which ions are oscillating in a confining quadro-logarithmic electric field produced by barrel-shaped electrodes. The geometry of the cell and the injection of ions by electrodynamic squeezing was developed by A. Makarov [START_REF] Makarov | Electrostatic Axially Harmonic Orbital Trapping: A High-Performance Technique of Mass Analysis[END_REF]. The Orbitrap cell consists of two external electrodes, one central electrode and a deflector electrode. External electrodes are kept at the ground potential. Ions are injected in the cell after 1.1 kV acceleration, while a transition of the high voltage (from nominal voltages -2500 V to -3500 V) is applied to the central electrode and (from 0 to 350 V) to the deflector electrode. All experiments have been carried out in positive ion mode. We note that negative ions can also be studied by reverting the high voltages on the source, the central and the deflector electrodes. Due to the quadratic potential in the longitudinal direction, trapped ions oscillate at a frequency proportional to their mass to charge (m/z) ratio. The pulsation ω (in rad/s) and the m/z ratio are linked by the formula: ω= (k*(z/m)) ½ , where k is a parameter depending on the shape and the voltage applied on electrodes [START_REF] Makarov | Performance evaluation of a high-field orbitrap mass analyzer[END_REF][START_REF] Perry | Orbitrap mass spectrometry: Instrumentation, ion motion and applications[END_REF]. This Figure 1 represents the laboratory test bench used in this study. The Nd-YAG laser at 266 nm is used for ablation and ionisation. The prototype is composed of two vacuum chambers. The first one contains the sample at 10 -8 mbar. Solid samples can be analysed, as they are usually pressed or dropped on the metallic surface of a small sample-holder (8 mm height, 7 mm diameter) itself screwed at the extremity of a rod. The second vacuum chamber contains the Orbitrap cell and is maintained at a pressure of 10 -9 mbar. Both vacuum chamber are linked by a differential pumping system. The aperture between both vacuum chambers is smaller than 2 mm. Two antennas, one on each external electrodes of the Orbitrap cell, are directed to the pre-amplifier. The whole configuration is named LAb-CosmOrbitrap, standing for Laser Ablation CosmOrbitrap. As defined in [START_REF] Briois | Orbitrap mass analyser for in situ characterisation of planetary environments: Performance evaluation of a laboratory prototype[END_REF], the CosmOrbitrap space mass analyser includes the following elements (in red in Figure 1): the Orbitrap cell itself and its retaining ring, the ultra-stable high voltage power supply, the pre-amplifier and the data acquisition / command control board cards. These elements, composing the CosmOrbitrap, are involved in a TRL (Technology Readiness Level) development. TRL is a scale used by space agencies, such as NASA, with 9 levels. The first one (TRL 1) is the basic principle, an idea of a concept and the last one (TRL 9) a real system, on-board and operational. CosmOrbitrap This simple sample preparation provides a useful internal reference for mass calibration with the positive ion of indium major isotope peak at m/z 114.903, and possible cluster ions between the sample (M) and the indium ion as [In+M] + , [In+2M] + , [2In+M] + , which help for the identification of the parent mass M. These clusters are also observed with other mass spectrometry techniques [START_REF] Bhardwaj | Ion sources for mass spectrometric identification and imaging of molecular species[END_REF][START_REF] Carrasco | Laboratory analogues simulating Titan's atmospheric aerosols: Compared chemical compositions of grains and thin films[END_REF][START_REF] Roy | Inventory of the volatiles on comet 67P/Churyumov-Gerasimenko from Rosetta/ROSINA[END_REF] and with the LAb-CosmOrbitrap on another organic molecule, adenine (C 5 H 5 N 5 ) observed at m/z 135.0545

(unpublished data from the study of [START_REF] Briois | Orbitrap mass analyser for in situ characterisation of planetary environments: Performance evaluation of a laboratory prototype[END_REF]. Adenine is studied in pure solid form (thin white powder) and Figure 3 shows a detail of a mass spectrum obtained from the analysis of adenine powder pressed onto an indium surface (as it was done for samples A and B studied in this work).

Both indium isotopes ( 113 In + and 115 In + ) are visible, as well as the adenine protonated peak [M+H] + .

Cluster of the molecular ion of adenine associated with indium [In+M] + is observed at the nominal m/z 250.

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12 115.3, 135.5, and 136.6

are artifacts due to the FFT treatment

The presence of the indium peak is also an indicator of the laser beam position, as it disappears from the spectrum when the laser beam irradiates the selected organic samples which absorb less energy from the 266 nm UV laser in this setup. At the edge of the sample and the sample-holder, both are visible on the spectrum (Figure 3).

c) Validation of the ionisation method for the blind test

Tests are performed on the sample with a progressive increase of the laser power from 30 µJ up to 750 µJ, until a complex specific pattern is observed in the spectrum. A systematic study of the mass spectra as a function of the laser power was not possible, due to the too small amount of sample available. One of the main features of laser ablation ionisation (LAb) with organic molecules is the fragmentation resulting from the dissociative ionisation of the sample. This can induce a high

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13 variability and diversity of mechanisms generating the mass spectrometric signature for organic molecules, which is not observed for metal, essentially resulting in singly charged atomic ions and clusters. Clusters are positively charged entities from the addition of one metallic ion coming from the surface of the sample-holder to a neutral organic molecule thermo-desorbed from the sample, as described in the previous section.

To ensure that this ionisation method is relevant for this blind test study and appropriate in the ion is expected at m/z 108.0431. We observed it at m/z 108.0427 (-3.7 ppm). Laser ablation and electron ionisation (EI) are no soft ionisation processes and produce molecular ions and subsequent fragmentations. The fragmentation results from molecular ions presenting an unstable structure due to (1) the loss of an electron from the electronic cloud and (2) the internal energy left in the ionic species, imparted either by the incident electron or the plasma generated by the laser. For a given molecular structure, the fragmentation routes that results from this process should be similar. This explains why the main fragment ions observed are similar with both ionisation methods. However differences of peak intensities are observed between the two mass spectra. This is the signature that the internal energy imparted in the ablation plasma is different from the one imparted by the 70 eV electrons. This is understandable as the laser generates micron size plasma, with a lifetime of The clear similarities observed between the two spectra of adenine allow us to conclude that adenine can be identified with the fragmentation patterns obtained in our experiment by comparison with its electron impact fingerprint provided in the NIST database. Note that obvious discrepancies are also observed, for instance in the m/z range 30 to 40 and 90 to 100. In our case, the applied laser power is only high enough to induce a fragmentation pattern. With a higher laser power, more fragment ions would be formed and detected. The electron impact ionization used to obtain mass spectra referenced in the NIST database involves a similar dissociating ionization process, breaking the molecular ion. For one given molecule, some fragments are produced with a higher probability which highly depends on the spatial configuration of the molecule and its chemical bonds. Electron impact ionization and laser ablation are preferentially producing the same fragments and it is why we observe the main fragment ions at the closest masses from the molecular ion in the mass spectra. However, the NIST mass spectrum shows more fragmentation peaks at lower masses

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT
16 than in our mass spectrum. This suggests that the electron ionization at 70 eV is more energetic than the photo-ionisation process induced by the 266 nm laser used (corresponding to 4.6 eV for onephoton transitions). The goal of this comparison (and of those which will be presented in the results section) is to confirm the attribution of a molecular formula and the identification of a compound by finding similar fragment ions between both techniques. We are not looking for two identical mass spectra but only a few and consistent similarities between both in order to confirm our identification.

The fragmentation becomes a key asset and the final step in the identification process of a molecule and consequently the choice of the laser ablation as ionisation system has been found relevant for the blind test study. and/or elements composing the molecule (as described in the results section and shown in Figure 6 & Figure 9). Artifact peaks (mostly some vertical alignments of points due to ringing phenomenon) are easily detected with this analytical representation, which allows to discard them.

Our identification steps, based on the study of the two data representations enounced and described above, are: (1) to find the parent peak, this means the m/z of the peak to identify; (2) to determine the molecular formula: for this, we make some assumptions on elements possibly composing the molecule and their occurrence (hydrogen and carbon are directly supposed to be present as we are looking for organic compounds but nitrogen, sulphur, oxygen or phosphorous can also be part of the molecular formula); (3) to confirm our identification based on the comparison of mass spectra from the NIST database (electron ionisation at 70 eV) and LAb-CosmOrbitrap mass spectra. This comparison is performed, for the selected species, by looking for a match of the same main fragment ions in both mass spectra (as described in the previous section).

The determination of the molecular formula (step 2) is done with the Attributor software which calculates combinations of molecular formula at one m/z given. Each combination calculated is given with its mass accuracy (based on the difference between the theoretical m/z and the observed one) in ppm. Within this list of candidates, the most interesting ones (depending on their mass accuracy) are chosen and then the step 3 starts (comparison of the fragmentation pattern using the NIST database mass spectra as reference).

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18 III) Results

a) Identification of "sample A"

The surface of "sample A" is scanned with successive laser shots at different locations on the sampleholder. A direct visualisation of mass spectra during the data acquisition allows to locate peaks in the m/z range 50 to 250, with some isolated peaks above m/z 350. Within this quite extended pattern, the identification of a parent molecule is not obvious. Figure 5 presents a representative mass spectrum. Among the most intense peaks detected, we observe the main indium isotope coming from the metallic surface of the sample holder at nominal m/z 115. Among the other intense peaks detected, nominal m/z 136, 250 and 364 are expected to be organic compound(s) or cluster(s) between indium and organics. In addition, in view of the large number of peaks detected, we assume a high fragmentation. This fragmentation is consistent with our laser ablation ionisation process which, as explained in the method section, induces it. Fragmentation comes from the molecular ion, then fragments ions produced can be themselves fragmented.

Between some clusters of peaks we are thus able to identify losses of only one element such as carbon, nitrogen, oxygen etc. These losses corresponding to only one element are good indicators for identifying elements composing the molecular formula. It is what we call "mass spacing": the mass difference between two peaks. The high MRP of the CosmOrbitrap (up to 90,000 at m/z 208 (lead) as referenced in the [START_REF] Briois | Orbitrap mass analyser for in situ characterisation of planetary environments: Performance evaluation of a laboratory prototype[END_REF] allows to calculate them with a precision of four digits in the m/z range 50 to 250. The CosmOrbitrap MRP (as for the conventional laboratory Orbitrap) is decreasing as a function of the square root of m/z [START_REF] Briois | Orbitrap mass analyser for in situ characterisation of planetary environments: Performance evaluation of a laboratory prototype[END_REF][START_REF] Makarov | Electrostatic Axially Harmonic Orbital Trapping: A High-Performance Technique of Mass Analysis[END_REF][START_REF] Perry | Orbitrap mass spectrometry: Instrumentation, ion motion and applications[END_REF]. The power law fitting this evolution of the MRP with the m/z ratio is given by: m/Δm= k*(m/z) - 1/2

. Several nominal mass spacing of 12 u are detected (m/z 11.9999 for instance, attributed to carbon), but also 14 u (m/z 14.0028, attributed to nitrogen Two different trends are evidenced. The first one (top blue line on Figure 6), with a positive MD, is attributed to organic species. Indeed, hydrogen and nitrogen elements have a positive MD (respectively +0.0078 and +0.0031), as explained in the method section carbon has a MD equal to 0 by convention and within these organic elements only the oxygen has a negative MD (-0.0051). From these cluster ions and as calculated previously in this section, we derive a hypothetical m/z of the parent peak at 135.0417, consistent with the observed mass of a peak, located at m/z 135.0426.

With this laser ablation ionisation process and as demonstrated on adenine in the previous section we are observing both molecular and protonated ions. Thus, we assume that the peak at m/z 135.0426 corresponds to the molecular ion and the peak at m/z 136.0503 (more intense) to the protonated ion, with a mass gap between both of 1.0082 u, matching the mass of one hydrogen atom (1.0078 u). Then, we focus on the peaks observed at the nominal m/z 137, to derive more information about the elements present in the molecule. We thus observe two adjacent peaks at this same nominal m/z: 137.0469 and 137.0537. The mass gap between both is 0.0068 u. Calculations based on their distance (in mass) from the protonated ion allow us to consider them both as isotopologues with the replacement in the molecular formula of the protonated ion of, respectively, one 14 N by a 15 N atom (first peak, with a mass gap of 0.9966 u) and one 12 C by a 13 C atom (second peak, with a mass gap of 1.0034 u). According to the nitrogen rule, the even m/z of the protonated ion implies an odd number of nitrogen atoms. At the nominal m/z 138 we do not observe a clear signal, confirming the absence of sulphur and a low abundance of oxygen. Based on all these assumptions (a CHNO molecule, a protonated ion at a m/z close to 136.0503 u, the presence of an odd number of nitrogen atoms and oxygen in low abundance), 3 candidates are found compatible with a precision lower than 15 ppm on the exact mass attribution : have to be consistent together to confirm which molecular group is lost or added between these two peaks. In this case, the molecular group should be CO. This is indeed corresponding (1) in term of m/z (the experimental mass difference of 27.9945 u is very close to the theoretical one of 27.9949 u) and

(2) in term of mass defect calculation where only the MD of oxygen is involved (as the MD of carbon is 0) with an experimental value of -0.0055 close the theoretical value of -0.0051. The first candidate, presenting the best mass accuracy, is also the one that explains the high intensity observed at m/z 432 (matching with a high number of carbon atoms). We decide to focus on this first molecule and to look at its fragmentation mass spectrum on the NIST database. This study shows the capability of the LAb-CosmOrbitrap instrument to identify unknown organic molecules. We also demonstrate that only a small amount of sample is needed to provide a good at m/z 135. Mass accuracies better than 3 ppm (-0.9 ppm for HOBt and -2.2 ppm for BBOT) have been demonstrated. The LAb-CosmOrbitrap is thus a key instrument for future space missions and particularly for organic worlds, with analytical performances never reached in space to this date.

However the study also points out some difficulties to be tackled in the future such as the variability resulting from the ionisation process chosen (laser ablation) but also from the intrinsic properties of the sample (UV absorption, ionisation potential). As a wide energy range can be applied on the sample, a specific Laser Ablation (and Desorption) CosmOrbitrap calibration mass spectra database would be relevant in order to get reference mass spectra at different energies of organic species presenting a potential exobiological interest. This database would allow a post data treatment of the spectra received from a spacecraft. In addition, a laser system with variable output energy should be set for a space configuration, in agreement with present laboratory configurations. On-board, data collection and mass spectra selection will highly depend on space mission parameters such as the mission duration, the scenario of the mission (flyby(s), encounter, escort etc.) and the targeted body.

•

  Efficient ionisation of solid sample by nano-pulsed single laser shot • Powerful analytical performances of CosmOrbitrap mass analyser • Successful blind test identification of organics ionised by Laser-CosmOrbitrap • Laser-CosmOrbitrap relevant technique for space exploration of organic rich worlds Introduction

  perturbation of the potential difference between the external electrodes, providing the signal analysed by the instrument electronics.

Figure 1 :

 1 Figure 1: LAb-CosmOrbitrap laboratory prototype at LPC2E (Orléans). The Nd-YAG laser is used as ablation/ionisation process. The first vacuum chamber holds the sample for ablation and ions extraction. The second chamber contains the Orbitrap cell for the mass analysis. Both chambers are connected by a 2 mm feedthrough. In red are reported the CosmOrbitrap elements involved in a TRL development for space applications. In the present work, CosmOrbitrap elements are at TRL 3.

  of each sample are collected and put on a different indium sample-holder. Sticks are pressed using an agate pestle to be embedded onto the indium target (Goodfellow, high purity: 99,999%, rolled) previously cleaned under consecutive ultrasonic baths of acetone and n-Hexane.The indium targets and the sample deposit are fed into the first vacuum chamber (Figure1).

Figure 2 :

 2 Figure 2: Pictures obtained with an optical microscope of Sample "A" as it was received from the JAXA HRMS team (left) and pressed on the sample holder (right, in the red circle).

Figure 3 :

 3 Figure 3: Detail of a mass spectrum of adenine (M) powder pressed on an indium surface showing the formation of clusters between the sample-holder (indium) and adenine. The left mass window shows the indium main isotope peak 115 In + and its isotope at m/z 113. The mass window in the middle details the molecular ion peak of adenine (M + ) and its protonated ion [M+H] + . One mass unit higher, at m/z 137, we observe an isotopologue of the adenine protonated ion. The right mass window shows the cluster formed between the indium ion 115 In + and the neutral adenine M. This [In+M] + peak is surrounded by two other peaks at nominal m/z 249 and 251, meaning at one hydrogen mass lower and higher. Peaks at m/z 114.5,

  context of a space configuration, we compare the mass spectrometric signature of adenine obtained on the one hand by the well-documented electron ionisation process at 70 eV (NIST database) and on the other hand by the LAb technique of our prototype (Figure 4). Similarities in the fragmentation patterns are observed between both mass spectra. The top panel, in red, shows a LAb-CosmOrbitrap mass spectrum. The lower panel, in black and with a reverse y-scale, shows the electron ionisation mass spectrum, recovered from the NIST database. Intense peaks and parent molecules are identified on the mass spectra. Intensities of ions detected are different but main fragment ions are the same for both ionisation processes. Two of the most intense fragment ions are at m/z 28 and 108. The fragment ion at m/z 108 is interpreted as a loss of an HCN molecule from the adenine molecular ion observed at m/z 135.0527. Based on exact masses calculations, the m/z 108 fragment

Figure 4 :

 4 Figure 4: Mass spectra of adenine obtained with LAb-CosmOrbitrap (red, upper panel) and from the NIST (black, lower panel, inverted scale) showing a comparison of the fragmentation patterns of adenine (M) between both ionisation processes: laser ablation and electron impact. On CosmOrbitrap spectrum, we see the molecular ion of adenine (M + ) and the protonated ion ([M+H] + ) both in positive mode. On the NIST spectrum, we observe only the molecular ion.

  d) Data processing procedures for the blind test Data processing of the spectra is made with the in-house Attributor software of HRMS analysis (described at https://frodsite.wordpress.com/reasearch/attributor/) developed in the Igor Pro environment. A Hann apodisation window and 3 zero padding (signal of 4 million points associated to 12 million points set to 0) are applied before a [6-838] ms FFT. Mass calibration is performed on the 115 In + peak.The study is based on two data representations: (1) the formal mass spectrum and (2) the "Mass Defect versus Exact Mass" diagram (MDvEM diagram). The latter represents the mass defect of each ion as a function of its exact mass. The mass defect (MD) of an element is the mass difference between its exact mass and its closest integer mass (Murray[START_REF] Kermit | Definitions of terms relating to mass spectrometry (IUPAC Recommendations 2013)[END_REF]. By convention, the mass defect of Carbon (MD Carbon ) is set to 0. Indeed, its exact mass is 12 u, which already is the integer mass. If we consider the nitrogen element: its exact mass is 14.0031 u and the closest integer mass is 14 u. The difference between both gives a positive mass defect of +0.0031 u. On the contrary, indium shows a negative mass defect: the exact mass is 114.9038 u and the closest integer mass 115 u, yielding a negative mass defect of -0.0962 u. A valuable asset of this kind of diagram is the observation of specific trend lines. They are representative of repetitive molecular groups thus they signatures. They allow us to make assumptions on the possible molecular groups

  ) and 16 u (m/z 15.9946, attributed to oxygen). Mass spacing of 31.9831 u are observed, which best correspond to losses (or addition) of two oxygen (mass = 31.9898 u for O 2 ). The high precision of this value enables to discard the M A N U S C R I P T A C C E P T E D presence of sulphur in "sample A" (m/z 31.7921). At this time, we are thus looking for a "CHNO" molecule.The kind of mass spectrum presented on Figure5is usually observed at the edge of the sample deposit or after a series of shots, when the metal target starts to emerge. As mentioned in the method section, the ionisation of a solid sample deposited on a metal target can produce cluster ions such as [In+M] + . Consequently, removing the mass of115 In to these cluster ions would lead to the neutral parent mass of sample "A" (molecule M). We obtain a nominal mass of 245 u when subtracting the mass of one115 In from the m/z 364 and a nominal mass of 135 u when subtracting the mass of one115 In from the m/z 250.

Figure 5 :

 5 Figure 5: Overall mass spectrum of sample "A". The blue square indicates the indium peak. Dynamic range is about 25 (based on 115 In + and 113 In + ).

Figure 6 :

 6 Figure 6: Mass Defect versus Exact Mass (MDvEM) diagram of the « Sample A ». Each dot on the diagram corresponds to an ion detected in the mass spectrum (Figure 5). The mass defect is represented as a function of the m/z of each ion. In addition, colour and size of the dots are proportional to the logarithm of the intensity of each ion peak. Lighter colours (such as yellow then orange) corresponds to the most intense peaks and darker ones (dark red then black) to the less intense peaks. Blue lines highlight specific trend lines and cluster ions.

  present mass spacing calculated between peaks on the m/z range 20 to 250 pointing toward the presence of a "CHNO" molecule with mainly C, H and N elements. We therefore expect a parent compound with a positive mass defect.The second trend (bottom blue line with negative MD) is linked to the presence of indium, which has a MD of -0.0962. The yellow point corresponds to the large MD of indium alone, then the other points on this line corresponds to heteroatoms at higher masses: cluster ions of indium and the organic compound are suspected at m/z 249.9455 ([In+M] + ) and at the nominal m/z 365 [2In+M] + .

  C 6 H 6 N 3 O + (-0.9960 ppm), C 8 H 8 O 2 + (8.8018 ppm) and C 4 H 4 N 6+ (-10.7940 ppm). The first possibility shows the lowest error. In addition, an oxygen loss with mass gap calculations excludes the third attribution. To confirm our choice, we look at the C 6 H 5 N 3 O molecule fragmentation pattern in the NIST database. We find that it is consistent with our spectrum (see Figure7), mainly by the similar fragment ions at m/z 107 and 119. As for the comparison NIST/CosmOrbitrap made for adenine, all fragment ions are not comparable. More of them are detected on the NIST mass spectrum, obtained with a more energetic ionisation process. In this case, the m/z 119 fragment ion is relevant enough to confirm our molecule selection.

Figure 7 :Figure 8 :Figure 9 :

 789 Figure 7: Mass spectrum of sample "A" (assumed to be HOBt) obtained with LAb-CosmOrbitrap (top panel) and mass spectrum of HOBt from NIST database (lower panel). Comparison between both fragmentation patterns.

Figure 10 :

 10 Figure 10: Mass spectrum of sample "B" (assumed to be BBOT) obtained with LAb-CosmOrbitrap (top panel, in red) and mass spectrum of BBOT from NIST database (low panel in reverse scale, in black). Comparison of fragmentation patterns observed with these two techniques. Similarities are mainly observed in the m/z range 350 to 431, with some intense fragment ions at nominal m/z 105 and 267. In addition, the m/z range 230 to 300 allows the observation of C x H y O, C x H y O 2 NS and C x H y ON 2 S fragment ions, as described in the MDvEM diagram on the Figure 9.

  analysis. The instrument has an important potential for in situ chemical analysis of organic molecules during space exploration missions, as molecular identification capability is absolutely required for new instrumental developments. Precious clues are given here to anticipate what is needed for a space configuration. We show the crucial importance of the sample preparation and the nature of the sample-holder. Sample deposition on the surface of a metallic sample-holder offers the benefit of clusters formation. Moreover, the metallic sample-holder gives us a useful calibration mass point for the whole mass spectrum. Finally, following the work by the[START_REF] Briois | Orbitrap mass analyser for in situ characterisation of planetary environments: Performance evaluation of a laboratory prototype[END_REF] and to update analytical performances of the LAb-CosmOrbitrap on organics, we show the capability of our reach a mass resolving power (MRP) of 69,219 at m/z 430 and 123,540

Acknowledgements L. S. thanks the CNES (Centre National d'Etudes Spatiales) and the Région Centre-Val de Loire for the funding of her PhD. N.C. thanks the European Research Council for funding via the ERC PrimChem project (grant agreement No. 636829.) We gratefully acknowledged CNES for funding the instrumental development of the CosmOrbitrap, the Labex Exploration Spatiale des Environnements Planétaires (ESEP), and the Région Centre-Val de Loire, Dr Titaina Gibert for lending us the Nd-Yag laser 266 nm used to perform all tests and the JAXA HRMS team for initiating this study.