Christian Hernández

Anne Marie Farnet Da Silva

Fabio Ziarelli

Isabelle Perraud-Gaime

Beatriz Gutierrez-Rivera

José Antonio García-Pérez

Enrique Alarcón
email: enalarcon@uv.mx

Anne-Marie Farnet

Da Silva

Beatriz Gutiérrez-Rivera

Gonzalo Aguirre

Beltrán Sn

Col Emiliano Zapata

Laccase induction by synthetic dyes in Pycnoporus sanguineus and their possible use for sugar cane bagasse delignification

Keywords: Alizarin yellow, Carminic acid, Endoglucanase, 13 C CPMAS solid-state NMR, Cellulose crystallinity

Introduction

Laccases (benzenediol/oxygen oxidoreductase, EC 1.10.3.2) are enzymes with the capacity to degrade lignin and other phenolic compounds, like mono-, di-, and polyphenols, aminophenols, methoxyphenols, aromatic amines, and ascorbic acid [START_REF] Ding | Production and characterization of laccase from Pleurotus ferulae in submerged fermentation[END_REF]. Recently, laccases have been proposed as biocatalyst for many industrial and bioremediation processes, including biobleaching [START_REF] Zheng | Biobleaching of wheat straw pulp with recombinant laccase from the hyperthermophilic Thermus thermophilus[END_REF][START_REF] Monteiro | Pulp bleaching using laccase from Trametes versicolor under high temperature and alkaline conditions[END_REF], azo dyes degradation for wastewater treatment [START_REF] Chhabra | Immobilized laccase mediated dye decolorization and transformation pathway of azo dye acid red 27[END_REF][START_REF] Kanagaraj | Degradation of azo-dyes by laccase: biological method to reduce pollution load in dye wastewater[END_REF][START_REF] Teerapatsakul | Improvement of laccase production from Ganoderma sp. KU-Alk4 by medium engineering[END_REF][START_REF] Teerapatsakul | Dye decolourisation by laccase entrapped in copper alginate[END_REF][START_REF] Junghanns | Towards higher laccase activities produced by aquatic ascomycetous fungi through combination of elicitors and an alternative substrate[END_REF][START_REF] Vanhulle | Effect of mannan oligosaccharide elicitor and ferulic acid on enhancement of laccases production in liquid cultures of bacidiomycetes[END_REF]), herbicide degradation (Pizzul et al. Electronic supplementary material The online version of this article (doi:10.1007/s00253-016-7890-0) contains supplementary material, which is available to authorized users.

2009; [START_REF] Jolivalt | Transformation of N′, N′-dimethyl-N-(hydroxyphenyl)ureas by laccase from the white rot fungus Trametes versicolor[END_REF], petroleum-derivate phenols degradation [START_REF] Vargas | Phenol oxidation of petrol refinery wastewater catalyzed by laccase[END_REF], and biomass delignification [START_REF] Rico | In-depth 2D NMR study of liginin modification during pretreatment of Eucalyptus wood with laccase and mediators[END_REF][START_REF] Ryu | Enhanced lignin biodegradation by a laccase-overexpressed white-rot fungus Polyporus brumalis in the pretreatment of wood chips[END_REF].

For industrial applications, vast amounts of protein are extremely important [START_REF] Songulashvili | Production of a high level of laccase by submerged fermentation at 120-L scale of Cerrena unicolor C-139 grown on wheat bran[END_REF]; thus, many studies have focused in enhancing the production of this enzyme with different organisms. Laccases are produced by bacteria [START_REF] Dhiman | Bioprospecting and molecular characterization of laccase producing bacteria from paper mills of Himachal Pradesh[END_REF][START_REF] Galai | A new Stenotrophomonas maltophilia strain producing laccase. Use in decolorization of synthetic dyes[END_REF], filamentous ascomycetes [START_REF] Cázares-García | Structural and phylogenetic analysis of laccase from Trichoderma: a bioinformatics approach[END_REF], and brownrotting basidiomycetes [START_REF] An | Transcriptional profiles of laccase genes in the brown rot fungus Postia plancenta MAD-R-698[END_REF]; however, white-rot fungi are the desired producers because of the high yields obtained, and the high catalytic laccases produced [START_REF] Viswanath | Fungal laccases and their applications in bioremediation[END_REF][START_REF] Ramírez-Cavazos | Purification and characterization of two thermostable laccases from Pycnoporus sanguineus and potential role in degradation of endocrine disrupting chemicals[END_REF]. The production of laccase by culturing white-rot fungi can be done in liquid media [START_REF] Songulashvili | Basidiomycetes laccase and manganese peroxidase activity in submerged fermentation of food industry wastes[END_REF][START_REF] Songulashvili | Production of a high level of laccase by submerged fermentation at 120-L scale of Cerrena unicolor C-139 grown on wheat bran[END_REF] and in solidstate fermentation [START_REF] Karp | Characterization of laccase isoforms produced by Pleurotus ostreatus in solid state fermentation of sugarcane baggase[END_REF]; in both systems, the use of inducers can enhance the production of laccase [START_REF] Hernández | Ethanol induction of laccase depends on nitrogen conditions of Pycnoporus sanguineus[END_REF][START_REF] Khammuang | Cooper induction of laccase by Lentinus polychrous under liquidstate fermentation[END_REF][START_REF] Meza | Laccase production by Pycnoporus cinnabarinus grown on sugar-cane bagasse: influence of ethanol vapors as inducer[END_REF].

Inducer compounds must be abundant and not expensive to be used at large scale; industrial wastes meet these assumptions. Many metals (e.g., cupper, manganese, and cadmium) phenolic compounds [START_REF] Mann | Use of olive mill wastewater as a suitable substrate for the production of laccase by Cerrena consors[END_REF] and synthetic dyes can induce laccase production in fungi [START_REF] Kuhar | Optimization of laccase production by two strains of Ganoderma lucidum using phenolic and metallic inducers[END_REF], and much of them are discarded in wastewaters. Phenolic dyes are of special interest due to textile industries that generate a large amount of wastewater (120 m 3 /Ton fiber produced) enriched with synthetic dyes (1100-1300 Hazen units; [START_REF] Anjaneyulu | Decolourization of industrial effuents-available methods and emerging technologies: a review[END_REF], which generate environmental problems, e.g., when the azo dyes are degraded in anaerobic conditions, they can generate carcinogenic aromatic amines [START_REF] Arslan | Removal of carcinogenic azo-dyes from water by new cyclodextrin-immobilized iron oxide magnetic nanoparticles[END_REF].

Besides the degradation of many phenolic dyes that has been proven via laccase catalysis [START_REF] Kanagaraj | Degradation of azo-dyes by laccase: biological method to reduce pollution load in dye wastewater[END_REF][START_REF] Younes | Investigation of dyes degradation intermediates with Scytalidium thermophilum laccase[END_REF][START_REF] Bayramoglu | Immobilization of laccase on itaconic acid grafted and Cu(II) ion chelated chitosan membrane for bioremediation of hazardous materials[END_REF], there are few studies that evaluate in vivo induction of laccase by synthetic dyes (Table 1). The induction of high laccase production in vivo by synthetic dyes can be very useful for lignocellulose pretreatment, in order to enhance lignin decay and, indirectly, decrease the cellulose crystallinity index (CI). Achieving the reduction of lignin and CI, cellulose and hemicellulose are more easily hydrolyzed by enzymes [START_REF] Yu | Effect of lignin chemistry on the enzymatic hydrolysis of woody biomass[END_REF][START_REF] Yoshida | Effects of cellulose crystallinity, hemicellulose, and lignin on the enzymatic hydrolysis of Miscanthus sinensis to monosaccharides[END_REF], which are very important for bioethanol production.

This work aimed to test the induction of laccase with four synthetic/phenolic dyes: carminic acid, alizarin yellow, aniline blue, and violet crystal at different concentrations in liquid media and, subsequently, utilize the best inducer to enhance the production of laccase in solid-state fermentation (SSF). Carminic acid is an anthraquinone dye which is naturally present in the cochineal insect Dactylopius coccus [START_REF] Eisner | Red cochineal dye (carminic acid): its role in nature[END_REF]) and has been used since pre-Hispanic time as a textile and ceramic dye in Mexico. On the other hand, alizarin yellow is a common azo dye used in textile industry, such as dye cotton, nylon, and silk [START_REF] Bowes | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF], and as other azo dyes, which are potentially hazardous for the environment because of their toxicity [START_REF] Bae | Influences of new azo dyes to the aquatic ecosystem[END_REF], mutagenic [START_REF] Freeman | Genotoxicity of azo dyes: bases and implications[END_REF] and carcinogenic [START_REF] Hildenbrand | Azo dyes and carcinogenic aromatic amines in cell cultures[END_REF]. The other two tested dyes were aniline blue, a common dye used for wool and cotton staining, and crystal violet, which has uses in paper and ink industries.

The substrate (sugar cane bagasse) modifications were evaluated by 13 C cross-polarization magic angle spinning (CPMAS) nuclear magnetic resonance (NMR), in order to determine if the addition of dyes to lignocellulose promotes a decay in lignin amount and CI values.

Materials and methods

Strain and inoculum

Pycnoporus sanguineus strain was isolated from the sugarcane fields of Jalcomulco, Veracruz, México (19°20′ 00″ N, 96°46′ 00″ W), cultured on a potato-dextrose-agar medium ) as nutrient source; pH was adjusted to 6. The cultures were performed in quadruplicate, in plastic containers of 500 ml. Each experimental replicate was inoculated with two plugs of agar-containing mycelium (6 days old) and were incubated in darkness at 30 °C in an environmental chamber (Binder, GmbH; Tuttlingen, Germany). Samplings were performed each 3 days for 12 days of incubation to test laccase activity (U L -1 ); meanwhile, biomass production (mg of dry mass) and dye degradation (%) were determined at the end of the incubation time.

Analytical methods for liquid cultures

Laccase activity in liquid cultures was estimated according to [START_REF] Criquet | Laccase activity of forest litter[END_REF]. Twenty microliters of a solution of 5 mM syringaldazine was added to 980 μL of the culture sample. The oxidation kinetics from syringaldazine to quinone was followed at 525 nm (ε = 65,000 M -1 cm -1 ) at 37 °C for 90 s. The activity was expressed as μmoles of quinone formed from syringaldazine per minute (U) per one liter of culture (U L -1 ).

Fungal biomass from liquid cultures was calculated at the end of the experiment: cultures were vacuum-filtered and recovered biomass was dried at 60 °C by 48 h. Biomass weight was estimated by the gravimetric method and was reported as milligrams of dry mass (mg.DM -1 ). Aromatic dye degradation was estimated according to linear models obtained from known concentrations (0 to 0.03 mM) and the absorbance measured at the wavelength of the main peaks (nm) in the visible light spectrum (aniline blue: 595, 610 nm; violet crystal: 588, 592 nm; carminic acid 494 nm; alizarine yellow: 358, 363 nm). Absorbance was quantified with a UV spectrophotometer and transformed into mM according to linear models (Table 2).

Experimental design and culture conditions for solid-state fermentation

The conditions that induced the highest laccase activity were used for the second experimental phase. Because the lowest CA concentration in liquid cultures reach the highest laccase activity, different concentrations of carminic acid were tested in solid-state fermentation (SSF): 0.00, 0.003, 0.006, and 0.01 mM; a volume of basal medium enriched with yeast extract and carminic acid was added to 50 g of pre-dried sugar cane bagasse (SCB) until a final humidity of 80 % v/w. The wet SCB was deposited into poly-paper plastic bags and sterilized by autoclaving (120 °C:1.5 atm) for 20 min. After cooling, three-agar squares 0.25 cm 2 in size with reactivated mycelium (7 days of culture) were used as inoculum. The mesocosms were incubated in darkness at 30 °C in an environmental chamber (Binder, GmbH; Tuttlingen, Germany). Each treatment was replicated four times, and samplings to determine laccase and endoglucanase activities were done each for 4 days during 16 days from expendable replicates; consequently, a total of 80 mesocosms (4 replicates × 4 carminic acid concentrations × 5 sampling dates) were set up. SCB samples were analyzed by 13 C CPMAS NMR methodology, in order to determine the substrate modification by P. sanguineus.

Enzyme activity was normalized with the protein and biomass values. Protein was determined according to Bradford (1976).

Analytical methods for SSF phase

Enzymatic activities were calculated from concentrated protein extracts, obtained according to [START_REF] Criquet | Laccase activity of forest litter[END_REF]: 5 g of SSB (fresh weight) were added to 100 ml of 0.2 M CaCl 2 buffer added with Tween 80 0.05 % ,and 6 g of polyvinylpyrrolidone (PVP) stirred per 1 h at ∼220 rpm and then filtered with 0.2 μm cellulose filters. Fifty milliliters of the crude extract was dialyzed within cellulose tubular membranes (Sigma-Aldrich; St. Louis, USA) and polyethylene glycol (PEG-8000) overnight at 4 °C; concentrated proteins were recovery in one volume of 0.2 M sodium phosphate buffer with pH 6 and were used for enzymatic determinations.

The reaction mixture of laccase determination was as follows: 100 μl of protein extract, 880 μl of 0.2 M sodium phosphate buffer pH 6, and 20 μl of syringaldazine 5 mM. Endoglucanase activity was estimated according to [START_REF] Ghose | Measurement of cellulase activities[END_REF], measuring simple sugars released from carboxymethyl cellulose (CMC, 2 %), by the method of dinitrosalicilic acid (DNS). The reaction mixture for endoglucanase estimation was as follows: 250 μl of protein extract was mixed with 250 μl of CMC 2 %; the reaction was incubated in a water bath at 50 °C for 1 h. The reaction was stopped with 500 μl of DNS reagent, boiled for 5 min, and immediately cooled in icy water for 10 min. The reaction mixture was diluted with 5 ml of dH 2 O prior spectrophotometer quantification (540 nm). Absorbance was transformed into grams per liter of glucose by comparison with a standard glucose curve (g L -1 ). In the case of SSF, both enzymatic activities were reported as μmoles of quinone formed from syringaldazine per minute (U) per g of dry mass (U g DM -1 ) of SCB.

Substrate modifications

Changes in the chemical composition of SCB were followed via 13 C CPMAS NMR in a Bruker Advance DSX 400 MHz spectrometer (Bruker, Madison, WI, USA). One hundred milligrams of dried and ground SCB was placed in a zirconium rotor and spun at the magic angle (54.44°) at 10 KHz. The 13 C CPMAS NMR technique was performed with a ramped 1 H pulse during a contact time of 3 ms and with 1 H decoupling during the acquisition time to improve the resolution. Recording 4 K transients with a recycling delay of 2 s represented standard conditions to obtain a good signal-to-noise ratio.

Deconvolution of NMR spectra was performed using the DmFit software [START_REF] Massiot | Modelling one-and twodimensional solid-state NMR spectra[END_REF]. Specifically, regions of NMR spectra were utilized as indicators of chemical shifts in SCB: alkyl C (0-45 ppm) and carboxyl C (160-190 ppm) were related to fungal biomass production since they can be assigned to membrane phospholipids and since only very weak signals were observed in the sugar cane bagasse. Polysaccharides content were estimated measuring the O-alkyl C signal (45-110 ppm) and the content in aromatic C compounds measuring the signal between 110 and 160 ppm. Cellulose crystallinity index was calculated according to [START_REF] Park | Cellulose crystallinity index: measurement techniques and their impact on interpreting cellulase performance[END_REF], by separating the C 4 region of the spectrum into crystalline and amorphous peaks, and calculated by dividing the total area of the crystalline peak (87 to 93 ppm) by the total area assigned to the C 4 peak (80-93 ppm). In addition, changes in aromatic C region were evaluated according to [START_REF] Hallac | Biomass characterization of Buddleja davidii: a potential feedstock for biofuel production[END_REF], measuring the decrease of phydroxyphenyl (162-157 ppm), aromatic quaternary C (160-123 ppm), and aromatic tertiary C (123-103 ppm) signals.

Statistical analysis

Data of laccase activity and biomass production was analyzed by two-way ANOVA (α = 0.05), in order to identify if the different dyes added to the medium or their concentrations had significant effects on these variables; when ANOVA was significant, a post hoc Fisher LSD analysis was made. In addition, Pearson product-moment correlation coefficient (r) between laccase activity, biomass production, and percentage of dyes degradation was done. All the analyses were done in the software STATISTICA 7 (StatSoft Inc. 2004).

Results

The effects of aromatic dyes on biomass and laccase production in liquid cultures Two-way ANOVA showed that the type of dye added to liquid media affects the amount of biomass produced (F 2 = 6.10; P = 0.006); meanwhile, dye concentration had no significant effects (F 2 = 1.29; P = 0.29), but the interaction between dye type and dye concentration was significant (F 4 = 4.31; P = 0.007). Treatments with aniline blue (whatever the concentration), alizarin yellow (0.01 mM), and carminic acid (0.03 mM) were those that produced more fungal biomass (Fig. 1). More precisely, the production of biomass increased with carminic acid concentrations while an inhibiting effect was found for alzarine yellow concentrations of 0.02 and 0.03 mM. Moreover, fungal biomass production was positively correlated with laccase production only in cultures with carminic acid (r = 0.66; P = 0.001). According to our ANOVA results, the addition of different aromatic dyes to culture medium of P. sanguineus affects laccase production (F = 79.07; P < 0.001); meanwhile, the concentration was not significant (F = 2.28; P = 0.11) as well as the interaction between the type of dye and dye concentration (F = 1.31; P = 0.27). Cultures with carminic acid showed the highest (Fig. 2) total laccase activities (i.e., 0.01 mM = 273.65 ± 90.72 U L -1 ), followed by alizarin yellow (0.01 mM = 120.85 ± 81.51 U L -1 ) and control (37.8 ± 12.25 U L -1 ). Cultures with aniline blue showed lower values (0.01 mM = 34.46 ± 10.16 U L -1 ). No laccase activity nor fungal development was observed in cultures with violet crystal (Fig. 2).

Because the effect of the dyes on laccase production was compromised by the effect of the dyes on the biomass development (Fig. 1), total laccase activity was normalized with the amount of total protein (specific activity), and with the amount of total biomass. The higher the concentration of CA, the fewer the activity of laccase (U g protein -1 g biomass -1; Table 3), with the exception of the control. With this basis, we found that carminic acid at the concentration of 0.01 mM was the best inducer of laccase (Table 3), and we determine to use it in the following tests (SSF).

Dye degradation

Carminic acid and alizarin yellow were degraded in P. sanguineus liquid cultures (Table 4). Carminic acid was degraded above of 92 % in all concentrations tested, and alizarin yellow by 97 % in cultures with an initial concentration of 0.01 mM, 48 % in cultures with 0.02 mM, and 60 % in cultures with 0.03 mM. This is consistent with the fact that laccase activity was weaker for alizarin yellow concentrations of 0.02 and 0.03 mM. Dye degradation was positively correlated with laccase activity (carminic acid r = 0.91 P < 0.0001; alizarin yellow r = 0.64, P = 0.02).

Enzyme production in SSF

Enzyme activities in SSF showed maximum values after 12 days for endoglucanase (Fig. 3a) and after 8 days for laccase (Fig. 3b). ANOVA results indicate that the amount of carminic acid added to SCB affects the endoglucanase activity (F 3 = 10.96; P = 0.004). According to Fisher LSD post hoc test, cultures without carminic acid had the highest endoglucanase activity (1116.45 ± 127.12 U g DM -1 ; Fig. 3a); meanwhile, the higher the carminic acid, the lower the endoglucanase activity was observed (Fig. 3a). On the other hand, laccase activity was detected at 4 and 8 days of culture, and laccase increased when carminic acid was added. Treatments with carminic acid showed higher laccase activity, with an average of 1.09 ± 0.40 U g DM -1 , whatever the concentration of CA than control (0.75 ± 0.31 U g DM -1 ). Normalized laccase activity (U biomass -1 ; Table 5) corroborate that CA induces laccase production in SSF, in a range of 21.05 to 42 % more than control, after 8 days of culture. C CPMAS NMR showed variations in the chemical composition of sugarcane bagasse under biological pretreatment with P. sanguineus and at different concentrations of carminic acid over time (after 16 days under SSF; Fig. 4). Cellulose crystallinity index (CI) increased in all the treatments; SCB had an initial CI ratio of 0.34 and increased 27.34 % in the control. When carminic acid was added, the increment in CI was lower (Table 6).

Changes in the chemical composition of SCB were mainly observed for the aromatic C region, which indicates changes in SCB lignin fraction. However, the reduction of these values was higher in control and when 0.003 mM of carminic acid was added, reaching a reduction in aromatic C signals of 32.67 and 30.65 %, respectively. Treatments with 0.006 mM of carminic acid showed a decrease of 16.33 %; meanwhile, when 0.01 mM were added, the aromatic region only decreased by 1.95 %. However, aromatic quaternary C diminish its signal by 17.60 %. 13 C CPMAS NMR analysis also showed that SCB's lignin had very little p-hydroxyphenyl (162-157 ppm) radicals, and that the peaks obtained for aromatic C region corresponded mainly to aromatic quaternary C (160-123 ppm) and aromatic tertiary C (123-103 ppm; Table 6).

Carboxylic C and alkyl C regions of the spectra were related to fungal biomass development (membrane phospholipid production); the increase in those regions was lower when Table 3 Laccase total activity (U L -1 ), specific activity (U g protein -1 ), and normalized activity (U g protein -1 g biomass -1 ). Obtained with the addition of carminic acid and alizarin yellow in liquid cultures. Values are mean and SD; n = 4 U L -1

Protein, g L -1 U g -1 Biomass, g DM -1 U g protein -1 g biomass carminic acid was added to SCB compared to the control (Table 6), which indicated negative effects of carminic acid in biomass production, in SSF. On the other hand, the polysaccharide content (O-alkyl C) was almost unchanged in all treatments (decreased less than 2 %) and no differential effects were observed in treatments with and without carminic acid.

Discussion

Liquid cultures

We observed that carminic acid and alizarin yellow can enhance total laccase by 722 and 317 % more than the control, respectively, in liquid cultures with P. sanguineus. In an early report, Korniłłowicz-Kowalska and Rybczyńska (2014) indicated that carminic acid can induce the production of other phenoloxidases in Bjerkandera adusta CCBAS 930 like Mn peroxidase and horseradish peroxidase, and that the production of these enzymes its accompanied with a degradation of carminic acid and Poly R-478. Low concentrations of alizarin yellow also increase the laccase activity but in less proportion that CA does. This study contributes to the knowledge on this field, proving that alizarin yellow can induce laccase production and that azo dyes and anthraquinonic dyes can be degraded in liquid cultures of P. sanguineus.

Biomass production was also modified by the addition of dyes into culture medium: aniline blue (all concentrations), carminic acid (0.03 mM), and alizarin yellow (0.01 mM) enhanced the production of biomass in liquid cultures of P. sanguineus. The increased biomass production in cultures with these dyes could indicate that P. sanguineus can use them as nutrient sources, i.e., alizarin yellow and aniline blue can function as nitrogen and sulfur sources [START_REF] Dave | Microbial degradation of synthetic dyes in wastewaters[END_REF]; however, the nutrimental role of carminic acid is still unclear. Biomass production was positive correlated with alizarin yellow and carminic acid degradation, and with laccase production.

P. sanguineus does not produce either Mn peroxidase (MnP) or lignin peroxidase (LiP) (Pointing and Vrijmoed 2000;[START_REF] Eggert | Laccase is essential for lignin degradation by the white-rot fungus Pycnoporus cinnabarinus[END_REF]), but it is considered as a good laccase producer (Pointing et al. 2000); thus, the decoloration capacity is probably related to the laccase production observed. Laccase-mediated dye degradation has been previously reported for carminic acid in agar plates and with purified enzymes from Pleutorus ostreatus, Kuehneromyces mutabilis, Trametes versicolor, and Cerrena unicolor [START_REF] Cho | The effects of wood rotting fungi and laccase on destaining of dyes and KP bleaching effluent[END_REF][START_REF] Cho | Effect of fungal laccase and new mediators, acetovanillone and acetosyringone, on decolourization of dyes[END_REF].

In addition, alizarin yellow degradation in cultures of P. sanguineus reached values up to 95 % (with an initial concentration of 0.01 mM), similar to other technological methods, e.g., laser-induced photocatalytic degradation [START_REF] Hayat | Kinetic study of laser-induced photocatalytic degradation of dye (alizarin yellow) from wastewater using nanostructured ZnO[END_REF] or hybrid processes like iron-carbon microelectrolysis combined to aerobic bio-contact oxidation [START_REF] Liang | Enhanced degradation of azo dye alizarin yellow R in a combined process of iron-carbon microelectrolysis and aerobic bio-contact oxidation[END_REF]). This azo dye has been degraded in vitro by chloroperoxidases of Caldariomyces fumago [START_REF] Li | Highly efficient biodecolorization/degradation of Congo red and alizarin yellow R by chloroperoxidase from Caldariomyces fumago: catalytic mechanism and degradation pathway[END_REF]), but we did not find previous reports of laccase-mediated decolorization of alizarin yellow. However, in this study, we found a high correlation with alizarin yellow decolorization and laccase production in vivo. Laccase has been utilized in many studies for azo dyes degradation in vitro [START_REF] Abadulla | Decolorization and detoxification of textile dyes with a laccase of Trametes hirsuta[END_REF]Pointing and Vrijmoed 2000;[START_REF] Rodriguez | Industrial dye decolorization by laccases from ligninolytic fungi[END_REF].

The other tested dyes (aniline blue and crystal violet) were not degraded in liquid cultures, and no laccase production was registered. Thus, carminic acid was the stronger laccase inducer tested here in liquid cultures of P. sanguineus: It promoted higher laccase and biomass production than the other dyes and was degraded more than 95 % at the end of incubation time (12 days); even if the induction mechanism is not clear, some anthraquinone-derived compounds could be involved in the induction of phenol-oxidases. For these reasons, this anthraquinone dye was selected for being utilized in solid-state fermentation, in an attempt to induce laccase activity and consequently depolymerize the lignin faction of sugar cane bagasse (SCB).

Solid-state fermentation

Many studies reported the induction of laccase in SSF using white-rot fungi, and most of them tested the effect of different carbon sources, nitrogen sources, the use of surfactants (e.g., Tween) and metal or organic inducers, like cooper and xylidine [START_REF] El-Batal | Laccase production by Pleurotus ostreatus and its application in synthesis of gold nanoparticles[END_REF][START_REF] Karp | Statistical optimization of laccase production and delignification of sugarcane bagasse by Pleurotus ostreatus in solid-state fermentation[END_REF][START_REF] Boran | Enhanced production of laccase by fungi under solid substrate fermentation condition[END_REF]. However, only few have evaluated the effect of dye for ligninolytic enzymes induction. We found that, as well as in liquid cultures, carminic acid was able to enhance laccase production in SSF, compared to control treatments, even though induction was lower. In spite of that, laccases showed to have none or negative effect on the delignification with respect to control. [START_REF] Usha | Enhanced production of ligninolytic enzymes by a mushroom Stereum ostrea[END_REF] reported that bioabsorption of synthetic dyes such as remazol brilliant blue and remazol brilliant violet 5R, onto lignocellulose (wheat bran and rice bran), and its further use for laccase and lignin peroxidase production was possible, using Stereum ostrea. Other studies reported that malachite green absorbed onto wheat bran could be used as solid substrate to produce lignin peroxidase from Fomes sclerodermeus [START_REF] Papinutti | Removal and degradation of the fungicide dye malachite green from aqueous solution using the system wheat bran-Fomes screlodermeus[END_REF]. Remazol brilliant blue and remazol brilliant violet 5R are anthraquinone dyes, as well as carminic acid; thus, this work along with these previous reports showed evidence that anthraquinone dyes can be added onto lignocellulosic material, and this substrate can enhance phenol-oxidases production.

When carminic acid was added to sugarcane bagasse, cellulase activity decreased, which is positive as an attempt to reach selective delignification, and it aimed to reduce the lignin fraction and the cellulose crystallinity index, while preserving the carbohydrate fraction [START_REF] Ramos | The chemistry involved in the steam treatment of lignocellulosic matherials[END_REF]. Generally, when fungi are used, these characteristics can be achieved with high phenol-oxidase activities (e.g., laccase) and low cellulase activities, in order to reach a selective delignification [START_REF] Dorado | Nitrogenremoval with protease as a method to improve the selective delignification of hemp steamwood by the white-rot fungus Bjerkandera sp. strain B0S55[END_REF]) useful to biomass pretreatments for bioethanol production. Some authors considered that P. sanguineus is capable of selective delignification [START_REF] Gupta | Fungal delignification of lignocellulosic biomass improves the saccharification of cellulosics[END_REF][START_REF] Lomascolo | Pecualiarities of Pycnoporus species for applications in biotechnology[END_REF], and it has been reported that this fungus can produce its own redox mediator which enhances lignin decay in the presence of laccase, i.e., 3hydroxyanthranilic acid (3-HAA; [START_REF] Eggert | A fungal metabolite mediates degradation of non-phenolic lignin structures and synthetic lignin by laccase[END_REF]. This fungal metabolite can mediate the degradation of nonphenolic lignin structures by laccase [START_REF] Eggert | A fungal metabolite mediates degradation of non-phenolic lignin structures and synthetic lignin by laccase[END_REF].

The enzymatic responses in SSF with or without carminic acid had to be considered together with chemical shifts in SCB composition measured by 13 C CPMAS NMR. We found that the main structural shifts in SSF were taking place in the aromatic C region. Other studies that used 13 C CPMAS NMR to evaluate lignocellulosic transformation (sorghum Fig. 4 Specters obtained from 13 C CPMAS NMR analysis. a Cultures without carminic acid. c Cultures with 0.003 mM of carminic acid. b Cultures with 0.006 mM of carminic acid. d Cultures with 0.01 mM of carminic acid. Signals of different carbon functional groups are indicated in the basis of the specters, and the square maximizes the aromatic C region. Note that C4 has two signal peaks, which corresponds to crystalline and amorphous forms and wheat wastes), in solid-state cultures of white-rot fungi, concluded that the growth of such type of fungi mainly led to a decrease in aromatic C amount [START_REF] Akin | Biological delignification of plant components by the white-rot fungi Ceriporiosis subvermispora and Cyathus stercoreus[END_REF], which is similar to our observations. The delignification yields obtained here are interesting when comparing with other studies, which used white-rot fungus in SSF. Delignification yields of 33 % have been reported in SSF using Pleurotus ostreatus and a combination of ferulic acid and cooper sulfate as laccase inducers [START_REF] Karp | Statistical optimization of laccase production and delignification of sugarcane bagasse by Pleurotus ostreatus in solid-state fermentation[END_REF]. Also, our results are similar to those obtained by [START_REF] Masayuki | Evaluation of pretreatment with Pleurotus ostreatus for enzymatic hydrolysis of rice straw[END_REF] using rice straw inoculated with either P. ostreatus (41 %), Phanerochaete chrysosporium (21 %), or Trametes versicolor (37 %). However, it should be noted that the incubation time in these experiments was longer (about 60 days) than that used here (16 days). Even if carminic acid induced more laccase production in SSF, we did not find higher delignification rates; indeed, surprisingly, when carminic acid was added, less of the aromatic C region was degraded. Dyes could be adsorbed by the solid substrate [START_REF] Sen | Fungal decolouration and degradation of azo dyes: a review[END_REF], reducing their availability to the fungi. This could reduce the laccase induction effect that we observed in liquid cultures, and thus the effect on lignin degradation was not evident in SSF.

We observed that the O-alkyl C region, which corresponds to polysaccharides, was slightly affected over time and decreased less than 2 % in all treatments. This result is in accordance to [START_REF] Gupta | Fungal delignification of lignocellulosic biomass improves the saccharification of cellulosics[END_REF] and [START_REF] Lomascolo | Pecualiarities of Pycnoporus species for applications in biotechnology[END_REF], which suggested that P. sanguineus is a fungus with a selective action on lignin. Selectively delignification of SCB by P. sanguineus could be an efficient way of pretreatment, in order to enhance enzymatic hydrolysis in second-generation bioethanol production. This mainly because can diminish 32.67 % of lignin, with less than 2 % of carbohydrate losses.

Crystallinity index (CI) is another important issue to account in biomass pretreatments. Here, we observed that solid-state fermentation with P. sanguineus increases 27.24 % the CI after 16 days, mainly when carminic acid was absence. The CI value indicates the relation between the amount of crystalline cellulose and amorphous cellulose [START_REF] Park | Cellulose crystallinity index: measurement techniques and their impact on interpreting cellulase performance[END_REF]; when the aim is to hydrolyze cellulose with cellulases, it is desired to have low CI values because amorphous cellulose is more easily degraded by cellulases [START_REF] Yu | Effect of lignin chemistry on the enzymatic hydrolysis of woody biomass[END_REF][START_REF] Yoshida | Effects of cellulose crystallinity, hemicellulose, and lignin on the enzymatic hydrolysis of Miscanthus sinensis to monosaccharides[END_REF]. When fungi degrade lignocellulose, generally the CI value increases [START_REF] Howell | Temporal changes in wood crystalline cellulose during degradation by brown rot fungi[END_REF]), as we observed with P. sanguineus; however, when carminic acid was added, the increment of CI was lower than the controls. According to our results, the addition of carminic acid at low concentrations (0.003 mM) in combination with P. sanguineus offers several advantages for SCB pretreatment, i.e., it induces the production of laccase and diminishes by 30.65 % of aromatic compounds; the increment of CI was lower in this treatments (18.79 %) than the control (27.24 %) and partially avoids the biomass production.

In the basis of the results obtained, we can recommended the use of P. sanguineus for SCB selective delignification, even though it increased its CI value; also, carminic acid is a good inducer of laccase both in liquid cultures and in SSF. However, its use to enhance delignification could be limited because it affects biomass production. Further experiments could be design to avoid this limiting.

This study showed that the use of carminic acid and alizarin yellow, two important textile and industrial dyes, enhance the production of laccase better in liquid cultures of P. sanguineus than in SSF (carminic acid). Also, the use of P. sanguineus for SCB delignification could bring many advantages, as a rapid and efficient lignin decay with very few polysaccharides losses, even in the absence of inducers. When carminic acid was added to sugarcane bagasse, cellulase activity decreased, preserving the carbohydrate fraction and increasing the cellulose crystallinity index. Finally, we found that higher laccase amount does not necessary entails to higher delignification yields, biomass and redox mediators could have important roles in this process.

Fig. 1

 1 Fig. 1 Biomass production in liquid media enriched with phenolic dyes at different concentrations. Bars indicate the mean and whiskers the standard error

Fig. 2

 2 Fig. 2 Laccase activity (U L -1 ) kinetics of liquid cultures with different dyes addition, in concentration from 0 to 0.01 mM. Middle points indicate the mean and whiskers the standard error. a A red glucosidal hydroxyanthrapurin dye. b an azo-dye. c a trisulfonate dye. d treatment without dye

Fig. 3

 3 Fig. 3 Enzymatic activities observed in SSF phase. a Endoglucanase activity, highest activity was registered in treatments without carminic acid at day 12. b Laccase activity was only registered after 4 and 8 days. Middle points indicate the mean and whiskers the standard error

Table 1

 1 Phenolic dyes used for laccase induction or for decoloration test in vivo with fungi

	Dye	Type	Fungi	Reference
	Reactive black 5	Azo dye	Leptosphaerulina sp.	Copete et al. (2015)
	Amaranth, reactive black 5, cibacron brilliant yellow	Azo dyes	Trametes versicolor	Champagne and Ramsay (2005)
	Amido black	Azo dye	Pleurotus pulmonarius	Zilly et al. (2002)
	Congo red	Azo dye	Pleurotus pulmonarius	Zilly et al. (2002)
	Bromophenol blue	Triphenylmethane dye	Pycnoporus sanguineus	Pointing and Vrijmoed (2000)
	Malachite green	Triphenylmethane dye	Pycnoporus sanguineus	Pointing and Vrijmoed (2000)
	Violet crystal	Triphenylmethane dye	Trametes maxima	Hernández-Luna et al. (2008)
	Amaranth	Azo dye	Trametes versicolor	Swamy and Ramsay (1999)
	Textile wastewater	Mixture of colorants	Pycnoporus sp. SYBC-L3	Lui et al. (2012)
	Remazol brilliant blue reactive	Anthraquinonic dye	Trametes villosa	Hernández-Luna et al. (2008)

Table 2

 2 Models utilized for phenolic dyes degradation

	Model equation	R 2
	[Aniline blue, peak 595 nm] = (γ 595 nm + 0.46)/1.93	0.82
	[Aniline blue, peak 610 nm] = (γ 610 nm + 0.643)/2.22	0.90
	[Violet crystal, peak 588 nm] = (γ 588 nm -2.813)/1.75	0.85

[Violet crystal, peak

592 nm] = (γ 592 nm -2.19)/1.98 0.90 [Alizarin yellow, peak 358 nm] = (γ 358 nm -0.913)/1.24 0.93 [Alizarin yellow, peak 363 nm] = (γ 358 nm -0.94)/1.105 0.92 [Carminic acid, peak 494 nm] = (γ 494 nm -0.176)/0.81 0.99

Table 5

 5

	Fungal biomass indicators (COOH, alkyl-C) in		COOH	Alkyl-C	U gDM -1	Biomass (ΣCOO, alkyl-C)	U biomass -1
	SSF after 8 days of culture. Their
	relationship with laccase produc-	Control	7.92	32.14	0.75206154	40.06	0.019
	tion (U ΣCOOH alkyl-C -1 )	Carminic acid 0.003	8.54	34.06	1.15373077	42.60	0.027
		Carminic acid 0.006	8.91	35.98	1.04263077	44.89	0.023
		Carminic acid 0.01	9.04	35.40	1.09390769	44.44	0.025

Table 6

 6 Chemical shifts in SCB after 16 days of culturing. Data are given in percentage of increment or decrement

	Treatment	Cellulose	COOH (organic	Alkyl C	O-alkyl C	Aromatic	Aromatic	Aromatic
		crystallinity ratio	acids)	(lipids)	(polysaccharides)	C	quaternary C	tertiary C
	Control	+27.24 %	+572.79 %	+2035.98 % NC	-32.67 % -25.89 %	-7.05 %
	Carminic acid	+18.74 %	+400.89 %	+1489.33 % -1.94 %	-30.65 % -34.82 %	-7.05 %
	0.003 mM
	Carminic acid	+15.94 %	+470.64 %	+1696.49 % -0.94 %	-16.33 % -27.00 %	-5.18 %
	0.006 mM
	Carminic acid	+15.94 %	+214.04 %	+936.99 % -1.35 %	-1.95 % -17.60 %	-5.57 %
	0.01 mM
	NC no changes

Acknowledgments The authors would like to thank to Mexican Consejo Nacional de Ciencia y Tecnología (CONACyT) for the Ph.D. scholarship of Hernández C, to ANUIES-CONACyT-ECOS-NORD (M13A02), Mexican and French government for financial, to Universidad Veracruzana (INBIOTECA), to Aix Marseille Université (IMBE, IRD and Spectropole) for technical support, and to the anonymous reviewers of this manuscript.

Compliance with ethical standards

Funding This study was funded by the Mexican Consejo Nacional de Ciencia y Tecnología (CONACyT) and the ANUIES-CONACyT-ECOS-NORD Program (M13A02).

Conflict of interest

The authors declare that they have no competing interests.

Human and animal rights and informed consent This article does not contain any studies with human participants or animals performed by any of the authors.


Christian A Hernández

Norma Sandoval

Julieta Mallerman

José Antonio García-Pérez

Anne-Marie Farnet

Isabelle Perraud-Gaime

Enrique Alarcón
email: enalarcon@uv.mx

Ethanol induction of laccase depends on nitrogen conditions of Pycnoporus sanguineus

Keywords:

Background: Ethanol has been pointed out as a laccase inducer. However, there are controversial reports about its efficiency with some fungi. In this study, we hypothesized that ethanol laccase induced in Pycnoporus sanguineus depends on nitrogen nutriment conditions. To prove this, we assessed laccase production in submerged cultures of P. sanguineus, with different nitrogen concentrations and with, or without ethanol added in a factorial designed experiment.

Results: In order to analyze the effects of factors on the response variables, a factorial ANOVA, and response-surface models were performed. It was found that the nitrogen source was the main factor that affected laccase production in P. sanguineus. The treatments with yeast extract (2 g/L) and ethanol (3 g/L) induced the highest laccase activity (31.01 ± 4.9 U/L), while the treatments with urea reached the lowest activity (less than 1.6 U/L). Ethanol had positive and synergic effects on laccase production, in accordance with the surface response model, as long as simple nitrogen sources (urea) were not available. Conclusions: We suggest that laccase in P. sanguineus is regulated by a catabolic nitrogen repression mechanism; laccase activity is strongly inhibited by urea used as nitrogen source and it decreases when the amount of urea increases; contrarily, a synergic positive effect was observed between yeast extract and ethanol on laccase production.

Introduction

It is well known that nitrogen is one of the main nutrients for microbial metabolism because it is an essential component of proteins and nucleic acids, as well as carbon. Its concentration in the medium and type of source (e.g. mineral or organic) can affect enzyme production in submerged fungal cultures [START_REF] Boominadhan | Optimization of protease enzyme production using Bacillus sp. Isolated from different wastes[END_REF][START_REF] Shivam | Culture conditions for the production of α-galactosidase by Aspergillus parasiticus MTCC-2796: A novel source[END_REF][START_REF] Mikiashvili | Effects of carbon and nitrogen sources on Pleurotus ostreatus ligninolytic enzyme activity[END_REF]. In fungi, the nitrogen catabolite repression, homologous to the economic theory of microbial metabolism for carbon sets that, simple sources of nitrogen (e.g. ammonium) are preferred and consumed before complex sources (e.g. organic complex sources, such as yeast extract), with the objective of saving resources. For some enzymes linked to secondary metabolism, e.g. proteases, L-asparaginase and laccase, it is well known that this mechanism drives their extracellular production [START_REF] Parente | A secreted serine protease of Paracoccidioides brasiliensis and its interactions with fungal proteins[END_REF][START_REF] Poussereau | Regulation of acp1, encoding a non-aspartyl acid protease expressed during pathogenesis of Sclerotinia sclerotiorum[END_REF][START_REF] Moura | Production of L-asparaginase by filamentous fungi[END_REF][START_REF] Sotero-Martins | Asparaginase II-GFP fusion as a tool for studying the secretion of the enzyme under nitrogen starvation[END_REF]; thus, for fungal laccases, for example, some studies pointed that this enzyme is regulated by the nitrogen catabolite repression system [START_REF] Jiang | Negative roles of a novel nitrogen metabolite repression-related gene, TAR 1, in laccase production and nitrate utilization by the basidiomycete Criptococcus neoformans[END_REF] and besides, their production, it responds differentially to diverse nitrogen sources in the culture medium. Thus, it is assumed that fungal laccases are activated when carbon and nitrogen are limited [START_REF] Janusz | Fungal laccase, manganese peroxidase and lignin peroxidase: Gene expression and regulation[END_REF][START_REF] D'agostini | Low carbon/nitrogen ratio increases laccase production from basidiomycetes in solid substrate cultivation[END_REF]. Thereby, it has been observed that laccase production in submerged cultures is performed in the presence of nitrogen as a simple mineral source (such as ammonium nitrate and ammonium sulfate [START_REF] Eggert | The ligninolytic system of a white rot fungus Pycnoporus cinnabarinus: Purification and characterization of the laccase[END_REF]), or complex organic supplies (e.g. a mixture of amino acids, peptone, and yeast extract, [START_REF] Thongkred | Oxidation of polycyclic aromatic hydrocarbons by a tropical isolate of Pycnoporus coccineus and its laccase[END_REF][START_REF] Khanam | Laccases production by improved strain of Pycnoporus cinnabarinus in media with improvident[END_REF][START_REF] Sarnthima | Laccase production by Pycnoporus sanguineus grown under liquid state culture and its potential in Remazol Brilliant Blue R decolorization[END_REF]). In this sense, high laccase yields have been reported, when complex organic nitrogen was added to the medium, in contrast with a simple mineral nitrogen source, for Agaricus bisporius [START_REF] Kalisz | Regulation of extracellular laccase production of Agaricus bisporius by nitrogen sources in the medium[END_REF], Pleurotus ostreatus [START_REF] Mikiashvili | Effects of carbon and nitrogen sources on Pleurotus ostreatus ligninolytic enzyme activity[END_REF], Trametes hirsuta [START_REF] Bakkiyaraj | Enhance laccase production by Trametes hirsuta using wheat bran under submerged fermentation[END_REF] and Trametes pubescens [START_REF] Strong | Improved laccase production by Trametes pubescens MB89 in distillery wastewaters[END_REF]. Besides, the effect of the nitrogen source could be species and strain specific and varies with culture conditions (e.g. carbon source, C/N ratio, micro-nutriments content, presence of inducers). Hence, not only the source, but also the concentration of nitrogen in the medium, influences laccase production [START_REF] D'agostini | Low carbon/nitrogen ratio increases laccase production from basidiomycetes in solid substrate cultivation[END_REF][START_REF] Kachlishvili | Effect of nitrogen source on lignocellulolytic enzyme production by white-rot basidiomycetes under solid-state cultivation[END_REF].

Other factors also affect laccase production, like the type of inducer: metal ions (e.g. copper and manganese) [START_REF] Kenkebashvili | Effect of carbon, nitrogen sources, and cooper concentration on the ligninolytic enzyme production of Coriolopsis gallica[END_REF][START_REF] Preussler | Copper laccase induction in white rot fungi Trametes villosa (Sw.:Fr.) kreisel[END_REF][START_REF] Stajic | Effect of different carbon and nitrogen sources on laccase and peroxidases production by selected Pleurotus species[END_REF]; phenolic compounds; some organic molecules, like tannic acids [START_REF] Carbajo | Tannic acid induces transcription of laccase gene cglcc1 in the white-rot fungus Coriolopsis gallica[END_REF], and ethanol [START_REF] Eugenio | Synergic effect of inductors on laccase production by Pycnoporus sanguineus[END_REF][START_REF] Barreto | Trametes versicolor growth and laccase induction with by-products of pulp and paper industry[END_REF][START_REF] Alves | Highly efficient production of laccase by the basidiomycete Pycnoporus cinnabarinus[END_REF] in white-rot fungi. It has been reported that these inducers increase the production rate of fungal laccases, both in solid state fermentation and submerged fermentation. Ethanol has been pointed out as cheap and less toxic inducer for laccase production [START_REF] Valeriano | Production of laccase by Pycnoporus sanguineus using 2,5 xylidine and ethanol[END_REF]; nevertheless, little is known about its induction mode. Some have speculated that ethanol could cause oxidative stress via membrane disruption, and/or by the segregation of intracellular Ca 2 + , which acts as a secondary messenger and induces the laccase genes [START_REF] Lomascolo | Overproduction of laccase by a monokaryotic strain of Pycnoporus cinnabarinus using ethanol as inducer[END_REF]. Some authors had recommended the use of ethanol to increase laccase production, meanwhile other studies have not reported positive effects of using this alcohol as an inducer; even negative effects were observed in some cases [START_REF] Eugenio | Synergic effect of inductors on laccase production by Pycnoporus sanguineus[END_REF][START_REF] Barreto | Trametes versicolor growth and laccase induction with by-products of pulp and paper industry[END_REF]. The effectiveness of ethanol as an inducer of laccase could be related to other relevant culture conditions, e.g. the concentration and source of nitrogen. However, little is known in this field about P. sanguineus. It has been reported that 2,5-xylidine and cooper sulfate had a synergic and positive effect on laccase production when combined with organic nitrogen sources [START_REF] Bakkiyaraj | Enhance laccase production by Trametes hirsuta using wheat bran under submerged fermentation[END_REF][START_REF] Strong | Improved laccase production by Trametes pubescens MB89 in distillery wastewaters[END_REF], but there are no results with ethanol. With this in mind, we hypothesized that the different results reported so far about the inductive effect of ethanol on fungal laccase could be due to the differences in nitrogen availability. Thus, this study was conducted to evaluate the effectiveness of ethanol as a laccase inducer, in the tropical white-rot fungus Pycnoporus sanguineus, using different and contrasting nitrogen nutriment conditions (various sources and concentrations).

Materials and methods

Strain and inoculum

The strain of P. sanguineus used was isolated from a wild mushroom found in the sugar cane fields of Jalcomulco, Veracruz, Mexico (19°20′ 00″N, 96°46′00″W) and preserved in a potato-dextrose-agar medium (PDA) at 4°C. The collected fungus was identified by Ramírez-Guillén F., a classical taxonomy expert, and deposited at XAL Herbarium (INECOL A.C., Xalapa, Mexico) as F. Ramírez-Guillén 932. The fungus strain was also identified by sequencing the ITS 1 and ITS 4 regions, compared with sequences in GenBank and the DNA sequence obtained was deposited in GenBank under the accession number KR013138.

The fungus was reactivated in Petri dishes with PDA at 30°C for seven d prior to the experiment. As inoculum during the experimental phase, agar squares of 0.25 cm 2 in size with the reactivated mycelium of P. sanguineus were used.

Experimental design

To determine the response of laccase production in P. sanguineus under different culture conditions (Table 1) a factorial design was used, where the independent variables were: i) source of nitrogen with two levels, yeast extract and urea; ii) concentration of nitrogen in the source with three levels, 1, 2 and 3 g/L; and iii) ethanol as laccase inducer with two levels, 0 and 3 g/L.

The dependent variables measured were: i) laccase activity (U/L), ii) extracellular protein (mg/mL), and iii) simple sugars released from the substrate (g/L). Each treatment was replicated four times. Samples for the determination of laccase activity, protein production and simple sugars released were taken each 4 d for a 20 d period.

Culture conditions

The experimental units were glass containers with 150 mL of basal medium [START_REF] Eggert | The ligninolytic system of a white rot fungus Pycnoporus cinnabarinus: Purification and characterization of the laccase[END_REF] with the following composition per liter: 1 g of KH 2 PO 4 ; 0.26 g of NaH 2 PO 4 ; 0.317 g of (NH 4 ) 2 SO 4 ; 0.5 g of MgSO 4 × 7H 2 O; 0.5 mg of CuSO 4 ; 74 mg of CaCl 2 ; 6 mg of ZnSO 4 ; 5 mg of FeSO 4 ; 5 mg of MnSO 4 ; and 1 mg of CoCl 2 supplemented with sugar cane bagasse as a carbon source (2%). The experimental units were incubated in darkness at 30°C in an environmental chamber (Binder, GmbH), without agitation.

Statistical analysis

To identify if the independent variables have significant effects on laccase production, a factorial ANOVA was carried out (α = 0.05). The combined effect of nitrogen concentration in the source and the ethanol concentration in the medium was analyzed by response surface methodology [START_REF] Box | On the experimental attainment of optimum conditions[END_REF]. The analysis was carried out using Statistica 7 (Statsoft, Inc.) and GraphPad PRISM (GraphPad Software, Inc.) software.

Analytical methods

Lacasse activity was estimated using syringaldazine as a substrate, according to Leonowicz and Grzywnowicz [START_REF] Leonowicz | Quantitative estimation of laccase forms in some white rot-fungi using syringaldazine as a substrate[END_REF]. 10 μL of a solution of syringaldazine at 5 mM in 0.1 M sodium acetate buffer, pH 4.5, were added to 990 μL of culture sample, according to Criquet [START_REF] Criquet | Laccase activity of forest litter[END_REF]. The oxidation kinetics from syringaldazine to quinone was followed at

0 Y 1 E- Yeast extract 2 0 Y 2 E- Yeast extract 3 0 Y 3 E- Yeast extract 1 3 Y 1 E+ Yeast extract 2 3 Y 2 E+ Yeast extract 3 3 Y 3 E+ Urea 1 0 U 1 E- Urea 2 0 U 2 E- Urea 3 0 U 3 E- Urea 1 3 U 1 E+ Urea 2 3 U 2 E+ Urea 3 3 U 3 E+
525 nm (ε = 65,000 M -1 cm -1 ) for 90 sec. The activity was expressed as μmol of quinone formed from syringaldazine per min (U) per one liter of culture (U/L). The releasing of simple sugars was determined by the spectrophotometric method of 3,5-dinitrosalicylic acid (DNS), according to Miller [START_REF] Miller | Use of dinitrosalicylic acid reagent for determination of reducing sugar[END_REF]. 500 μL of the sample was mixed with 500 μL of DNS reagent and boiled for 5 min; later the reaction mixture was cooled for 10 min in icy water. Subsequently, the reaction mixture was diluted with 5 mL of dH 2 O and quantified at 540 nm. The absorbance was transformed into glucose (g/L) through calibration according to a standard curve. The total of extracellular protein was determined according to the Bradford method [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF]. 500 μL of the sample were mixed with 500 μL of Bradford reagent (Sigma, USA), the mixture was left standing for 5 min and quantified spectrophotometrically at 595 nm. The absorbance was transformed into protein (mg/mL) using a standard curve of bovine serum albumin (BSA).

Results

Laccase production was highly dependent on nitrogen supply conditions (P b 0.05, Table 2). In cultures with yeast extract as nitrogen supply (Fig. 1a), the highest activity was observed (31.01 ± 4.9 U/L) after 12 d in a culture treated with 2 g/L of yeast extract as a nitrogen source and 3 g/L of ethanol (Y 2 E +). Meanwhile, in all treatments with urea as nitrogen supply (Fig. 1b) laccase activity was lower than 1.6 U/L.

Nitrogen concentration and ethanol had significant effects on laccase production only at certain moments. Nitrogen concentration had significant effects after 8 d (F = 5.54; P = 0.007) and 12 d (F = 72.03; P b 0.001) of culture (when laccase expressed high activity), and ethanol concentration had significant effects only at d 12 (F = 42.82; P b 0.001). Combined effects of ethanol and nitrogen supply/nitrogen concentration were found at d 8 and 12, and as well as between nitrogen supply and nitrogen concentration (Table 2). The surface response analysis showed combined effects of ethanol and nitrogen concentration on laccase production when yeast extract was supplied (Fig. 2); ethanol showed positive effects after 8 (m = 0.73; Fig. 2b), 12 (m = 3.07 Fig. 2c) and 16 d (m = 1.13). At d 4, non-synergic effects were observed between ethanol and nitrogen concentrations (nitrogen concentration did not show effect, Fig. 2a). However, at d 8 and 12 the highest laccase activities were observed in treatments with 3 g/L of ethanol and low nitrogen concentration (Fig. 2b, Fig. 2c).

In cultures with urea, non-effects were observed when ethanol was added (Fig. 3), and urea concentration only showed an effect after 16 d of culture (Fig. 3d). When urea was used, the highest laccase activities were observed at d 16 on treatments with 1 g/L (Fig. 3d).

Extracellular protein production was slightly higher in treatments with yeast extract than those with urea (Fig. 4a and Fig. 4b). However, no effects of ethanol addition or nitrogen concentration were observed. The sugar content in cultures with urea was slightly higher than those with yeast extract (Fig. 5), but no significant effect was observed.

Discussion

In accordance with previous reports for other fungi [START_REF] Mikiashvili | Effects of carbon and nitrogen sources on Pleurotus ostreatus ligninolytic enzyme activity[END_REF] such as P. ostreatus, our ANOVA results indicate that the source of nitrogen is the most significant source of variation in laccase activity, and that the yeast extract (an organic complex source) promotes more laccase production in a submerged culture than urea (a mineral simple source).

Some studies have reported better laccase production with mineral sources of nitrogen than with organic ones [START_REF] Stajic | Effect of different carbon and nitrogen sources on laccase and peroxidases production by selected Pleurotus species[END_REF], for several species of Pleurotus. However, it is accepted that inorganic sources lead to lower laccase yields than organic sources, although both of them promote good fungal (biomass) development [START_REF] Piscitelli | Induction and transcriptional regulation of laccase[END_REF]. However, the source of nitrogen added to the medium and its consequent effect on laccase activity is species and strain specific, as determined by Mikiashvili et al. [START_REF] Mikiashvili | Effects of carbon and nitrogen sources on Pleurotus ostreatus ligninolytic enzyme activity[END_REF]. In this study, we observed that P. sanguineus preferred a medium with an organic nitrogen source for the production of laccase, as reported previously. Eugenio et al. [START_REF] Eugenio | Synergic effect of inductors on laccase production by Pycnoporus sanguineus[END_REF] stated that asparagine is the best nitrogen source (even better than yeast extract) for laccase production using P. sanguineus.

According to our results, the nitrogen source concentration used showed significant effects. When yeast extract (at d 8 and 12) and urea (at d 16) concentrations were significant, the highest laccase activities were registered. It suggests that nitrogen concentration has a strong effect on laccase production during the late stages of development of fungi, when secondary metabolism is active [START_REF] Vining | Functions of secondary metabolites[END_REF]. Negative correlations between concentrations of an inorganic nitrogen source (ammonium tartrate) and laccase production were reported for P. sanguineus by Pointing et al. [START_REF] Pointing | Optimization of laccase production by Pycnoporus sanguineus in submerged liquid culture[END_REF]. That study also reported that the highest laccase production was reached when low carbon (glucose) and low nitrogen (ammonium tartrate) sources were added to the medium. Laccase repression occurred at high nutriment (carbon/inorganic nitrogen) conditions. Our surface response results tend to the same conclusion of Pointing et al. [START_REF] Pointing | Optimization of laccase production by Pycnoporus sanguineus in submerged liquid culture[END_REF]: the amount of inorganic nitrogen sourceurea, in our caseaffects the production of laccase negatively in P. sanguineus.

In our study, the highest laccase activity was observed in the treatments with the lowest urea concentrations (1 g/L). The preference of an organic nitrogen source and the negative correlation between urea concentration and laccase activity may suggest that laccase production in P. sanguineus could be subjected to a nitrogen catabolic repression; a mechanism for saving resources that was reported for laccase production in other basidiomycetes, like Cryptococcus neoformans [START_REF] Jiang | Negative roles of a novel nitrogen metabolite repression-related gene, TAR 1, in laccase production and nitrate utilization by the basidiomycete Criptococcus neoformans[END_REF]. It has also been suggested that Phanerochaete chrysosporium (wild-type) has a regulatory system for laccase that inhibits its production under sufficient carbon and nitrogen conditions; this can be deregulated in mutant strains [START_REF] Qiu | Carbon-nitrogen regulation of a laccase-producing mutant of Phanerochaete chrysosporium resisting carbon and nitrogen nutritional repression[END_REF].

The catabolic nitrogen repression states that in the presence of simple nitrogen sources the expression of genes for more complex nitrogen sources (like genes of protease, γ-glutamyl transpeptidase, L-asparaginase, and permease) is repressed [START_REF] Lee | Nitrogen metabolite repression of metabolism and virulence in the human fungal pathogen Cryptococcus neoformans[END_REF]. In this study we suggest that this regulatory mechanism operates and regulates laccase production in P. sanguineus; however, more research must be done to prove that. Nevertheless, the effect of the nitrogen source added to medium on laccase activity is evident.

In this study, we evaluated submerged cultures of P. sanguineus, but other studies have reported that in solid-state fermentation, an increment of urea concentration accompanied an increment of laccase production for P. ostreatus, Lentinula edodes, and Agaricus blazei [START_REF] D'agostini | Low carbon/nitrogen ratio increases laccase production from basidiomycetes in solid substrate cultivation[END_REF]. This shows how the response of laccase to nitrogen supplies and culture conditions could change accordingly for fungi species.

According to our results, ethanol had a significant effect on laccase activity at d 12 and showed combined effects with nitrogen conditions at d 8 and 12. As we previously hypothesized, the effect of ethanol as an inducer of laccase is linked to other factors. We observed that ethanol induces laccase production when simple nitrogen sources (urea) are not available. In previous reports, Eugenio et al. [START_REF] Eugenio | Synergic effect of inductors on laccase production by Pycnoporus sanguineus[END_REF] and Barreto et al. [START_REF] Barreto | Trametes versicolor growth and laccase induction with by-products of pulp and paper industry[END_REF] have established that the effect of adding ethanol to the medium was null or negative on laccase production. Eugenio et al. [START_REF] Eugenio | Synergic effect of inductors on laccase production by Pycnoporus sanguineus[END_REF] used ammonium tartrate (Kirk's medium) to produce laccase with P. sanguineus. Barreto et al. [START_REF] Barreto | Trametes versicolor growth and laccase induction with by-products of pulp and paper industry[END_REF] used glutamine (Trametes defined medium), a simple organic source of nitrogen to produce laccase with T. versicolor. In both cases, the nitrogen source of the medium corresponded to simple sources, one inorganic and one organic. When more complex mediums were used, ethanol addition had a positive effect as a laccase inductor. Valeriano et al. [START_REF] Valeriano | Production of laccase by Pycnoporus sanguineus using 2,5 xylidine and ethanol[END_REF] used malt extract medium for the production of laccase, Alves et al. [START_REF] Alves | Highly efficient production of laccase by the basidiomycete Pycnoporus cinnabarinus[END_REF] used a mixture of yeast extract and ammonium sulfate as nitrogen sources, and Lomascolo et al. [START_REF] Lomascolo | Overproduction of laccase by a monokaryotic strain of Pycnoporus cinnabarinus using ethanol as inducer[END_REF] used yeast extract; all those studies suggested that the ethanol is a good laccase-inducer. Following this line of knowledge, and supported by our results, we conclude that ethanol can act as a weak inducer of laccase only in the presence of complex nitrogen sources. Moreover, we hypothesize that this works only when nitrogen catabolite repression is not operating.

Our study contributes to a better understanding of the use of ethanol as an inducer of laccase in P. sanguineus, and our results showed that its functioning as an inducer is dependent on nitrogen nutriment conditions.

Conclusion

In accordance with our results, we conclude that: i) laccase production in P. sanguineus is strongly dependent on the type of nitrogen source added to medium in submerged cultures; urea inhibits its production (simple mineral source) and yeast extract (organic complex source) promotes it. We hypothesize that these results suggest the presence of a mechanism of nitrogen catabolite repression for P. sanguineus; however, genomic studies are required to assert it. ii) Ethanol only functions as a laccase inducer when simple nitrogen sources are not available in the culture medium; in the presence of complex nitrogen sources, such as yeast extract, ethanol showed positive synergic effects on laccase production.

Fig. 2 .

 2 Fig. 2. Influence of yeast extract and ethanol concentrations on laccase produced by P. sanguineus after (a) 4 d; (b) 8 d; (c) 12 d, and (d) 16 d. Changes in color patterns indicate changes on the observed laccase activity. The response surface graph follows a linear fit of the data; the proposed model is showed.

Fig. 1 .

 1 Fig. 1. Laccase activity observed during 16 d of culture with (a) yeast extract, and (b) urea, as nitrogen source; and with 3 g/L (+) or 0 g/L (-) of ethanol. Bars indicate the mean of the data, and whiskers the standard error, n = 3. The subscripts numbers indicate concentration in g/L. Note the differences on the ordinate scale (Y axis).

Fig. 3 .

 3 Fig. 3. Influence of urea and ethanol concentrations on laccase produced by P. sanguineus (a) after 4 d; (b) 8 d; (c) 12 d, and (d) 16 d. Changes in color patterns indicate changes on the observed laccase activity. The response surface graph follows a linear fit of the data; the proposed model is showed.

Fig. 5 .

 5 Fig. 5. Sugar content kinetics in the culture medium during 16 d of culture with (a) yeast extract, and (b) urea, as nitrogen source; and with 3 g/L (+) or 0 g/L (-) of ethanol. Bars indicate the mean of the data, and whiskers the standard error, n = 3. The subscript numbers indicate concentration in g/L.

Fig. 4 .

 4 Fig. 4. Extracellular protein kinetics produced during 16 d of culture with (a) yeast extract, and (b) urea, as nitrogen source; and with 3 g/L (+) or 0 g/L (-) of ethanol. Bars indicate the mean of the data, and whiskers the standard error, n = 3. The subscripts numbers indicate concentration in g/L.

Table 2

 2 ANOVA effect of all measured parameters on laccase activity, i.e. N source (yeast extract/ urea), N concentration (1, 2, 3 g/L), ethanol concentration (0, 3 g/L) and their interactions. Significant effects are showed in bold.

	Day Effect	Df MS	F value P
	4	N source	1	2.04	7.28	0.01
		N concentration	2	0.22	0.80	0.24
		Ethanol concentration	1	0.69	2.46	0.12
		Ethanol concentration * N concentration 2	0.02	0.08	0.91
		Ethanol * N source	1	0.29	1.04	0.31
		N concentration * N source	2	0.33	1.20	0.31
		Ethanol * N concentration * N source	2	0.10	0.35	0.70
	8	N source	1	601.98	59.08 b0.001
		N concentration	2	56.53	5.54	0.007
		Ethanol concentration	1	14.63	1.43	0.23
		Ethanol concentration * N concentration 2	52.95	5.19	0.01
		Ethanol * N source	1	14.90	1.46	0.23
		N concentration * N source	2	57.26	5.61	0.007
		Ethanol * N concentration * N source	2	53.18	5.22	0.01
	12	N source	1	1584.39 282.12 b0.001
		N concentration	2	404.55	72.03 b0.001
		Ethanol concentration	1	240.51	42.82 b0.001
		Ethanol concentration * N concentration 2	369.76	65.84 b0.001
		Ethanol * N source	1	240.41	42.80 b0.001
		N concentration * N source	2	404.59	72.04 b0.001
		Ethanol * N concentration * N source	2	369.80	65.84 b0.001
	16	N source	1	222.70	11.15	0.001
		N concentration	2	44.51	2.22	0.12
		Ethanol concentration	1	34.25	1.71	0.19
		Ethanol concentration * N concentration 2	65.02	3.25	0.05
		Ethanol * N source	1	35.19	1.76	0.19
		N concentration * N source	2	45.49	2.27	0.11
		Ethanol * N concentration * N source	2	64.58	3.23	0.05

Table 1

 1 Levels of factors evaluated.

	Nitrogen supply	N concentration	Ethanol concentration	Code
		(g/L)	(g/L)
	Yeast extract	1

C.A. Hernández et al. / Electronic Journal of Biotechnology 18 (2015) 327-332

Acknowledgments

The authors would like to thank the anonymous reviewers of this paper, as well as the Universidad Veracruzana for its support and the academic group Structure and Functioning of Forestall Ecosystems (CAUVER-324), and also to the biologist F. Ramírez-Guillén, from INECOL A.C., Xalapa, Mexico, for the taxonomical identification of the fungus.

Conflict of interest statement

The authors declare that there are no conflict of interest.

Financial support

We thank to CONACYT (Mexico), for C. Hernández Ph.D. scholarship, and to ECOS-ANUIES-CONACYT Program for the ECOS Project M13A02.


Corinne Coquet

Elisée Ferré

Dominique Peyronel

Claude Dal

Anne Marie Farnet Da Silva

Anne Marie Farnet
email: m.farnet@univ-cezanne.fr

Philippe Morat

Identification of new molecules extracted from Quercus

Keywords: 3-friedelanol, 2,6-heptanediol, Quercus suber L., Suberin, Terpens 3-friedelanol, 2,6-heptanediol, Quercus suber L., Subérine

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Suberin is a natural aliphatic-aromatic cross-linked polymer located in plant tissues where it favours protection from environmental damage. Suberin is, indeed, located in various parts of plants: roots, leaves, periderms from tubers, and in the outer bark of many higher plants. Although several models have been proposed [START_REF] Kollatukudy | Lipids polymer and associated phenols, their chemistry, biosynthesis and role in pathogenicity[END_REF][START_REF] Bernards | Demystifying suberin[END_REF], suberin structure remains unknown since in situ polymer analysis is hard to perform and available techniques such as 13 C solid NMR provide only holistic data about the structure [START_REF] Gil | A 13 C solid state nuclear magnetic resonance spectroscopic study of cork cell wall structure: the effect of suberin remova[END_REF]. Furthermore the association of suberin with other plant cell-wall polymers such as lignin or cellulose has not yet been clearly established. However, suberin depolymerisation has allowed one to identify several aliphatic and aromatic monomers. Thus, many studies [START_REF] Holloway | Some variation in the composition of suberin from the cork layers of higher plants[END_REF][START_REF] Pereira | Chemical composition and variability of cork from Quercus suber L[END_REF][START_REF] Graça | Feruloyl ester of ω-hydroxyacids in cork suberin[END_REF][START_REF] Cordeiro | Cork suberin as a new source of chemicals. 1. Isolation and chemical characterization of its composition[END_REF] based on various extraction protocols have described some of these aliphatic units: fatty alcohols, fatty acids, long chain ω-hydroxyacids, α, ωdiacids. These monomers may also have unsaturations as well as epoxyde or diol groups. The aromatic compounds isolated from suberin are close to those observed in lignin (coniferyl, synapilic and coumarylic alcohols) but acidic forms are mainly found in suberin [START_REF] Bernards | Demystifying suberin[END_REF][START_REF] Bernards | The polyphenolic domain of potato suberin: a non-lignin cell wall biopolymer[END_REF].

Here we used different methods to depolymerise suberin from cork cropped from Quercus suber L. cultivated in the Maures (Var, France). The cork industry in Mediterranean regions is mainly developed in Portugal, which produces 50% of the world production. In the South of France, the industrial transformation of cork is linked to the production of furniture, isolating materials, local craftwork products. Recently, molecules extracted from cork have aroused interest in the cosmetic industry: in this context we have produced cosmetic-active extracts which have a smoothing effect on skin [START_REF] Coquet | Quercus suber cork extract displays a tensor and smoothing effect on human skin: an in vivo study[END_REF].

Here we tested various methods of pre-extraction and extraction in order to select and to optimise a methodology of extraction and to contribute to the description of this polymer. Methanolysis as well as hydrolysis under various experimental conditions were performed and molecules from these different extracts have been identified using Gas-Chromatography-Mass-Spectrometry (GC-MS).

Material and methods

Plant material. Cork samples (reproduction cork collected at the second crop) were collected from Quercus suber L., provided by 'Melior Liège' (Fréjus, Var, France). They were grounded and sieved (0.5 mm diameter) before extraction.

Extraction of the wax fraction. 5 g of cork powder were mixed with 750 ml of the solvent tested (hexane, dichloromethane, chloroform, ethyl acetate, acetone and ethanol). The wax fraction was then saponified in 15 volumes of KOH 2 M dissolved in water, and refluxed for 4 h. Both saponified and unsaponified wax fractions were analysed by GC-MS as described in paragraph GC-MS analysis.

Cork depolymerisation. Pre-extraction of cork: cork was pre-extracted sequentially in chloroform, ethanol and water, air-dried and used as such for the following reactions. All the experiments of pre-extraction and extraction were performed twice.

Alcalin methanolysis: 5 g of pre-extracted cork were mixed with ten volumes of sodium methanoate, 3% in methanol, were added. The mixture was refluxed for 4 h then acidified to pH 5 with sulphuric acid 47.5% [START_REF] Marquez | On the determination of suberin and other structural components in cork from Quercus ilex L., Anais do instituto superior de agronomia[END_REF] and extracted with the same volume of chloroform for 1 h. The organic fraction was washed twice with 100 ml of distilled water, the solvent was evaporated and the extract was finally dried over anhydrous sodium sulphate.

Acid methanolysis: 5 g of pre-extracted cork were added to 750 ml of chloridric acid, 0.4 M in methanol and stirred for 4 h at 80 • C. The mixture was then extracted as described above.

Basic hydrolysis: 5 g of pre-extracted cork were added to 750 ml of KOH dissolved in water or ethanol and refluxed for 4 h. The following KOH concentrations were tested: 1, 2 or 5 M. The mixture was then extracted as described above.

Acidic hydrolysis: 5 g of pre-extracted cork were added to 750 ml of chlorydric acid in methanol or water and refluxed for 4 h. Different chlorydric acid concentrations were tested: 1, 2 or 5 M. The mixture was then extracted as described above. The same experiment was performed at room temperature or refluxed.

After each extraction, the volatile fraction was obtained from the polymeric fraction by distillation with temperature ranging from 100 to 150 • C.

GC-MS analysis. Ethylic esters were obtained by mixing 60 mg of the extract with 2 ml of BF 3 , 14% in methanol and 2 ml of anhydrous methanol. The mixture was incubated at 100 • C for 2 h and then extracted with 8 ml of chloroform/distilled water (50/50). The solvent is evaporated and the sample was sylilated at 50 • C for 2 h using N,O-bis-trimethylsilyltrifluoroacetamide (BSTFA) and dimethyl formamide (Shanchun et al., [START_REF] Shanchun | Origins and simulated thermal alteration of sterols and keto-alcohols in deepsea marine sediments of the Okinawa Trough[END_REF]). 1 µl was directly injected into the gas chromatography. GC-MS was performed on an Agilent (Agilent, DE, USA) Gas chromatograph 5973N interfaced to an Agilent MSD II detector. The chromatograph was equipped with a HP5 capillarity column (30 m × 0.25 I.D., 0.23 µm film thickness, J&W Scientific, CA, USA). The injection was performed at 300 • C in the split less mode and helium was used as carrier gas with a constant flow rate of 1 ml/min. A temperature gradient ranging from 150 • C to 325 • C was realized (4 • C/min). Electron impact mass spectra were measured at acceleration energy of 70 eV.

Results and discussion

Extraction and partial characterisation of waxes from cork

Most of studies on cork have used pre-extractions with different solvents in order to eliminate molecules which are not covalently bound to the polymer. This fraction is mainly composed of waxes. Different solvent were used: hexane, dichloromethane, chloroform, ethyl acetate, acetone and ethanol. Extraction yields were calculated as g of extract (dry weight)/g of cork (dry weight). The best extraction yield was obtained with chloroform (6.5 ± 0.5%) while the weakest were obtained when polar solvents (acetone and ethanol) were used (respectively 2.5 ± 0.2% and 3.2 ± 0.3%). Furthermore, extraction with these solvents may lead to polar molecule extraction such as phenols. These results are similar to those obtained previously [START_REF] Pereira | Chemical composition and variability of cork from Quercus suber L[END_REF]. GC-MS analysis has allowed us to identify several molecules before and after saponification of the wax extract. We identified three different triterpens from the samples extracted with chloroform: friedelin, friedelanol and betulin, with friedelin as the major compound (Table 1). These molecules were identified by their base peak m/z as well as by retention time and fragment m/z [START_REF] Budzikiewicz | Mass spectometry in structural and stereochemical problems, Pentacyclic triterpenes[END_REF][START_REF] Corsino | Biosynthesis of friedelane and quinonemethide triterpenoids is compartmentalized in Maytenus aquifolium and Salacia campestris[END_REF][START_REF] Bauer | Composition of the surface waxes from bell pepper and eggplant[END_REF][START_REF] Manoharan | Cycloartane type triterpenoids from the rhizomes of Polygonum bistorta[END_REF][START_REF] Otto | Conifer and angiosperm biomarkers in clay sediments and fossil plants from the Miocene Clarkia Formation, Idaho, USA[END_REF][START_REF] Queiroga | Evaluation of the antiulcerogenic activity of friedelan-3β-ol and friedelin isolated from Maytenus ilicifolia (Celastraceae)[END_REF]: for friedelin, base peak m/z 426, fragment m/z (%) 341 [START_REF] Marquez | On the determination of suberin and other structural components in cork from Quercus ilex L., Anais do instituto superior de agronomia[END_REF], 302 (36), 205 (57), 123 (84), 109 (77), 95 (91), 81 (74), 69 (100), 55 (77) for friedelanol (sylilated) base peak m/z 500, fragment m/z (%) 485 (23), 410 [START_REF] Cordeiro | Cork suberin as a new source of chemicals. 1. Isolation and chemical characterization of its composition[END_REF], 395 [START_REF] Bernards | The polyphenolic domain of potato suberin: a non-lignin cell wall biopolymer[END_REF], 347 [START_REF] Shanchun | Origins and simulated thermal alteration of sterols and keto-alcohols in deepsea marine sediments of the Okinawa Trough[END_REF], 321 [START_REF] Manoharan | Cycloartane type triterpenoids from the rhizomes of Polygonum bistorta[END_REF], 305 [START_REF] Conde | Variability of suberin composition of reproducction cork from Quercus suber through out industrial processing[END_REF], 257 [START_REF] Queiroga | Evaluation of the antiulcerogenic activity of friedelan-3β-ol and friedelin isolated from Maytenus ilicifolia (Celastraceae)[END_REF], 237 (71), 231 (36), 197 (31), 147 (38), 121 (56), 95 (100), 62 (66), 50 (68) and for betulin, base peak 442 m/z and fragment m/z (%) 363 [START_REF] Marquez | On the determination of suberin and other structural components in cork from Quercus ilex L., Anais do instituto superior de agronomia[END_REF], 234 [START_REF] Graça | Glycerol and glyceryl-esters of ω-hydroxyacids in cutins[END_REF], 207 (48), 189 (100), 175 (45), 161 (32), 135 (74), 107 (58), 95 (79), 81 (68), 69 (50), 55 (61), 41(60). In this study, cerin has not been identified in the wax fraction. Previous works [START_REF] Conde | Variability of suberin composition of reproducction cork from Quercus suber through out industrial processing[END_REF] have shown that cerin was the less abundant terpen in waxes of cork from various sites in Spain and that the proportions of terpens (friedelin, betulin and cerin) varied depending on crop localisation. The results found here may be explained by the geographic origin of our samples: Conde et al. [START_REF] Conde | Variability of suberin composition of reproducction cork from Quercus suber through out industrial processing[END_REF] have worked on reproduction cork which has been collected from various sites in Spain while we studied cork collected in South Eastern France. Friedelin and betulin have been extracted from cork and from various other tissues of higher plants as reported by previous studies [START_REF] Corsino | Biosynthesis of friedelane and quinonemethide triterpenoids is compartmentalized in Maytenus aquifolium and Salacia campestris[END_REF][START_REF] Otto | Conifer and angiosperm biomarkers in clay sediments and fossil plants from the Miocene Clarkia Formation, Idaho, USA[END_REF][START_REF] Castola | Composition and chemical variability of the triterpene fraction of dichloromethane extracts of cork (Quercus suber L.)[END_REF][START_REF] Silva | Cork: properties, capabilities and applications[END_REF]. However, friedelanol has never been isolated from cork before. This molecule has been found in waxes from bell pepper [START_REF] Bauer | Composition of the surface waxes from bell pepper and eggplant[END_REF] or in leaves and root bark of Celastraceae and of Hippocrataceae respectively [START_REF] Corsino | Biosynthesis of friedelane and quinonemethide triterpenoids is compartmentalized in Maytenus aquifolium and Salacia campestris[END_REF] as well as in the rhizomes of Polygonaceae [START_REF] Manoharan | Cycloartane type triterpenoids from the rhizomes of Polygonum bistorta[END_REF]. This molecule, which biosynthesis requires oxidosqualene, is known to be a precursor of friedeline [START_REF] Corsino | Biosynthesis of friedelane and quinonemethide triterpenoids is compartmentalized in Maytenus aquifolium and Salacia campestris[END_REF].

Saponification of the wax extract led to the hydrolysis of esters and the acid or neutral fractions obtained were analysed using GC-MS. The acid and neutral fractions represent 12 and 88% of the wax extract respectively (Table 2). Furthermore, we showed that molecules extracted under acid conditions were monoacids (palmitic, thapsic, stearic, eicosanoic, docosanoic, tetracosanoic acids) and diacids (eicosanedioic, docosanedioic, tetracosanedioic acids) while extraction under neutral conditions led to the identification of long chain alcohols (eicosanol, docosanol, tetracosanol) as well as sterols and triterpens (β-sitosterol, stigmasterol, friedelanol, friedelin and betulin). These results seem to show that sterols such as β-sitosterol and stigmasterol may be esterified with fatty acids since these molecules have not been directly identified after extraction with chloroform but are observable after saponification.

The identification of certain diacids in the chloroform extract which has been saponified is in agreement with Lopes et al. [START_REF] Lopes | Composition of suberin extracted upon gradual alkaline methanolysis of Quercus suber L[END_REF]. The authors have shown that some of them (C16:0, C18:0, C20:1) were easily extracted from cork using alcalin methanolysis with a sodium methanoate concentration of 0.01 M. Thus the localisation of certain diacids in suberin structure remains unclear: they may be either covalently linked to suberin but easily extractable or free in the cork structure and extractable without hydrolysis.

Partial depolymerisation of cork suberin

In most of previous studies, alcalin methanolysis has been extensively used in order to depolymerise suberin. Here, cork suberin was partially depolymerised using various methodologies (methanolysis, alcalin or acidic hydrolysis under different experimental conditions) to test whether these techniques lead to different yields and/or different molecules extracted. Whatever the technique used, three fractions were obtained in various proportions (Fig. 1): a polymeric fraction, a depolymerised fraction which contains some suberin monomers and a volatile fraction. Both depolymerised and volatile fractions were analysed by GC-MS.

The quantification of suberin monomers was performed after calibration of GC-MS system as described by Lopes et al. [START_REF] Lopes | Composition of suberin extracted upon gradual alkaline methanolysis of Quercus suber L[END_REF]: only one monomer (docosanoic acid, docosanol, 16-hydroxyhexadecanoic acid and hexacosanedioic acid) of each main chemical group was used for calibration since all the monomers from suberin are not commercialised. The identification of the molecules was performed using both software NIST 05, Software Version 2.0 (Agilent Technologies, CA, USA) and literature [START_REF] Cordeiro | Cork suberin as a new source of chemicals. 1. Isolation and chemical characterization of its composition[END_REF][START_REF] Lopes | Composition of suberin extracted upon gradual alkaline methanolysis of Quercus suber L[END_REF][START_REF] Eglinton | Gas-chromatographic-mass spectrometric studies of long chain hydroxyl acids III[END_REF][START_REF] Graça | Glycerol and glyceryl-esters of ω-hydroxyacids in cutins[END_REF]. Depolymerisation yields were higher using basic or acidic hydrolysis in alcoholic medium. The optimum concentrations of KOH and HCl for depolymerisation were 2 M. 5 M for both KOH and HCl may be considered as drastic conditions which may provoke a major degradation of monomers. Hydrolysis and methanolysis led to the same monomers observed in previous studies [START_REF] Holloway | Some variation in the composition of suberin from the cork layers of higher plants[END_REF][START_REF] Cordeiro | Cork suberin as a new source of chemicals. 1. Isolation and chemical characterization of its composition[END_REF]23]: ω-hydroxylated acids, epoxy acids and diacids are the main components respectively about 40-80, 20-30 and 10-15% depending on the methodology used. Alcalin hydrolysis in alcoholic medium allowed the best extraction yields for monohydroxyacids (about 80%) with 22-hydroxydocosanoic acid as the main compound (Table 2).

Since monomer proportion was the highest using alcalin hydrolysis in aqueous medium (low extraction yields for polymerised and volatile fraction), we tested room temperature as another experimental condition in order to obtain non-degraded monomers. We found that alcalin hydrolysis using room temperature gave very interesting results since similar yields of depolymerisation were obtained compared to reflux conditions: 25 and 22%, respectively. Furthermore the extract composition varies depending on the methodology used: when extraction was performed at room temperature, higher yields of extraction were observed for dihydroxyacids or trihydroxyacids while ω-hydroxy fatty acids were found in a less extent (Table 3). These experimental conditions seem to be less drastic and favour the extraction of certain molecules such as polyhydroxylated acids. Furthermore, non-oxygenated diacid extraction was more favoured at room temperature than at reflux (32 and 22%, respectively), which can be correlated with our previous results where certain diacids were found to be directly extractible with chloroform. The polymeric fraction found here has been described before [START_REF] Lopes | Composition of suberin extracted upon gradual alkaline methanolysis of Quercus suber L[END_REF][START_REF] Bento | Fragmentation of suberin and composition of aliphatic monomers realised by methanolysis of cork from Quercus suber L. analysed by GC-MS, SEC and MALDI-MS[END_REF]. This fraction is probably due to partial depolymerisation of suberin.

However the volatile fraction pointed out in this study has never been shown. The proportion of this fraction is greater when acidic hydrolysis in alcoholic medium was used. Its composition has been analysed by GC-MS after both acidic and basic hydrolysis: low molecular-weight molecules which are probably resulting from the transformation of the aliphatic or aromatic compounds of suberin, were identified (Table 4). For instance, the three hydroxyacids identified (glycolic, 4-hydroxybutanoic and 6-hydroxyhexanoic acid) are probably formed by β-oxidation of long chain hydroxyacids while benzyl alcohol, benzoic acid and anisole would come from the transformation of aromatic compounds from suberin or lignin. Interestingly, 2,6-heptanediol has been identified in this fraction as the major molecule (20-40%) and for each extraction tested. Because of its structure, this molecule can hardly be a result of chemical degradation (no carbonyl and/or carboxylic functions). 2,6-heptandiol may be a natural compound which may come from the biological transformation of various oxygenated acids from suberin. For instance, nonadioic acid has already been identified in suberin by Lopes et al. [START_REF] Lopes | Composition of suberin extracted upon gradual alkaline methanolysis of Quercus suber L[END_REF] and may be an oxidative product of this transformation. Beta-oxidation and decarboxylation of nonadioic acid may lead to heptanedione which may be reduced in 2,6-heptanediol. Thus, this molecule may be either a product of degradation from plant metabolism or a structural compound of suberin since high amount can be found and because of its diol structure.

Conclusions

The findings reported here have underlined the variability of chemical composition of cork which can depend, for instance, on the geographical localisation of the sample. Further investigations should compare the molecular composition of cork using chosen environmental parameters (climate, edaphic conditions ...). Among the triterpens already identified in suberin (friedelin and betulin), we have found out friedelanol which has never been described before in this polymer.

Furthermore, 2,6-heptanediol has been depicted in the volatile fraction obtained after depolymerisation. Extraction conditions which lead to incomplete depolymerisation and using enzymes such as cellulases, pectinases [START_REF] Rocha | Enzymatic isolation and structural characterisation of polymeric suberin of cork from Quercus suber L[END_REF] or lipases and phenoloxidases, should be considered. This technique may indeed provide information about the sequence of depolymerisation of these molecules and thus their potential localisation in the net structure of this polymer.

Fig. 1 .

 1 Fig. 1. Percentages of the different fractions obtained using different methodologies of extraction: monomeric (1), polymeric (1) and volatile (2). Concentrations in KOH (alcalin hydrolysis) or HCl (acidic hydrolysis) are specified.

Table 1

 1 Relative composition of waxes extracted with chloroform and saponified under neutral or acid conditions (neutral and acid fractions, respectively)

	Molecules extracted	Molecule/chloroformic
		extract (%, w/w)
	Neutral fraction
	Triterpenes	52
	Friedeline	30
	Friedelanol	12
	Betuline	10
	Fatty alcohols	10
	Sterols	8
	Non-identified	18
	Acid fraction
	Fatty acids	4
	Diacids	8
	1,22-docosanedioic acid	2

Table 2

 2 Relative composition of suberin monomers (%, w/w) using different conditions of extraction

	Suberin monomers	Basic	Acidic	Acidic			Basic hydrolysis		Basic hydrolysis
		methano	methano	hydrolysis		in ethanol		in water
		lysis	lysis	[HCl]			[KOH]			[KOH]
				1 M	2 M	5 M	1 M	2 M	5 M	1 M	2 M	5 M
	Fatty alcohols	2.4	-	1	0.7	3.9	2.6	2.4	4.8	1.9	2.5	2.8
	Fatty acids	0.4	-	0.5	0.5	1.8	1.2	0.3	0.2	0.9	1.5	0.5
	Fatty diacids	15.8	10.5	10	14.6	9	16.2	9.2	9.3	17.9	20	21.1
	1,18-octadecen-9-dioic acid	5.3	4 .5	5	4 .5	3.1	5.5	2.5	2.3	6.7	11	9.2
	1,22-docosanedioic acid	3	3.4	3 .5	3.5	3.4	6.5	3	2.6	4.8	5.1	5
	Monohydroxylated fatty acids	41.9	44.6	48	47.5	37.6	73.8	80.2	80.3	74.1	76	71.8
	18-hydroxyoctadecen-9-oic acid	19.9	1 4 .1	1 5	1 3 .5	8.3	29.9	26.9	30.8	24.3	26	28.5
	22-hydroxydocosanoic acid	11.9	1 8 .9	2 3 .2	16.9	20.2	43.9	41.4	45.9	34.6	42	32.1
	9,10,18-trihydroxyoctadecanoic acid	14.3	6.8	1.4	1	6.5	3.7	5.9	3	3.1	3.5	2.3
	9,10-dihydroxy-1,18-octadecanedioic acid	7.6	0.8	1.4	1	7.5	2.5	2	2.3	2.1	3.5	1.5
	9,10-epoxy-18-hydroxyoctadecanoic acid	11.9	18.8	18.6	17.3	14.8	-	-	-	-	-	-
	9,10-epoxy-1,18-octadecanedioic acid	6.8	18.3	19.5	19.5	18.9	-	-	-	-	-	-

Table 3

 3 Relative composition of suberin monomers using acidic hydrolysis at room temperature (RT) or at reflux

		Experimental conditions
	Suberin monomers	RT	Reflux
	Fatty acids	0	0
	Fatty alcohols	1.5 ± 0.1	2.5 ± 0.15
	dicarboxylic acids	32 ± 2	2 0± 2.5
	1,18-octadecen-9-dioic acid	9.5 ± 0.3	11± 1
	1,22-docosanedioic acid	3.9 ± 0.15 5.9 ± 0.2
	ω-hydroxylated fatty acids	53.5 ± 2.4 76± 3.8
	18-hydroxyoctadecen-9-oic acid	22 ± 1.4	26± 2.1
	22-hydroxydocosanoic acid	16.5 ± 1.3 42± 2.5
	9,10-dihydroxyoctadecane-1,18-dioic acid 6 ± 0.8	3.5 ± 0.3
	9,10,18-trihydroxyoctadecanoic acid	7 ± 1.2	3.5 ± 0.7
	Average and standard deviation were calculated for two experiments.

Table 4

 4 Relative composition of the volatile fraction extracted under different experimental conditions

	Molecules	Molecules/extract (% w/w)
	identified	Alcalin hydrolysis	Acidic hydrolysis
		Alcoholic	-	(hydro-alcoholic)
		conditions
	Glycolic acid	/	/	6
	Butanoïc acid	/	10	10
	3-ethoxy-1-propanol	/	/	9
	Butanol	/	/	10
	4-hydroxybutanoic acid	6	/	/
	6-hydroxyhexanoic acid	11	/	/
	2,6-heptanediol	37	40	20
	2-heptanone	/	10	15
	Cyclohexanone	7	/	4
	Benzylic alcohol	6	/	2
	Benzoïc acid	/	/	5
	Anisole	/	5	/
	Non identified	33	35	19
	Standard deviations are under 3%.