Fr Ed Erique Peter-Valence

Carlos Llarena-Hernandez

Mich Ele Largeteau

Jean Michel Savoie

Florence Ruaudel

Fabio Ziarelli

Elis Ee Ferr

Anne Marie Farnet

Chemical Characterization of the Biomass of an Edible Medicinal Mushroom, Agaricus subrufescens, via Solid-State 13 C NMR

Keywords: Agaricus subrufescens, Agaricus bisporus, fruiting bodies, vegetative mycelium, β(1À3) polysaccharides

The biomass of 18 strains of Agaricus subrufescens and of 13 strains of Agaricus bisporus was chemically analyzed using solid-state 13 C NMR. The study focused on polysaccharides because they can play a major role as antitumor molecules. The study also examined whether biomass chemical properties varied between the vegetative mycelium and the fruiting bodies of A. subrufescens, and these data were compared with the mycelium of A. bisporus. Qualitative differences between vegetative mycelia and fruiting bodies were observed, whereas quantitative differences were measured between the two species with a higher percentage of polysaccharides in the biomass of A. subrufescens. This Agaricus species is thus an interesting potential source of polysaccharides with medicinal properties, both from vegetative mycelium obtained in liquid cultures and from fruiting bodies produced on composts.

' INTRODUCTION

The edible mushroom Agaricus subrufescens Peck., formerly known as Agaricus blazei or Agaricus braziliensis, is a Basidiomycete that has long been widely consumed in Brazil and Japan as a health food because of its nutritional properties. Furthermore, this mushroom is believed to prevent or to fight various diseases such as diabetes, hepatitis, hyperlipidemia, and cancer, although both the bioactive molecules it produces and their physiological roles are still not clear. An overall view of the question is available in reviews. [START_REF] Firenzuoli | The medicinal mushroom Agaricus blazei Murrill: review of literature and pharmaco-toxicological problems. Evidence-Based Complement[END_REF][START_REF] Oliveira Lima | Does the Agaricus blazei Murill mushroom have properties that affect the immune system? An integrative review[END_REF] Nevertheless, polysaccharides such as (1À6)-βglucan, (1À3)-β-glucan, (1À4)-R-glucan, or (1À2)-β-mannopyranosyl residues [START_REF] Firenzuoli | The medicinal mushroom Agaricus blazei Murrill: review of literature and pharmaco-toxicological problems. Evidence-Based Complement[END_REF][START_REF] Oliveira Lima | Does the Agaricus blazei Murill mushroom have properties that affect the immune system? An integrative review[END_REF] from this fungal species have been demonstrated to inhibit the growth of cancer cells and increase the immune activity in patients. [START_REF] Camelini | Structural characterization of β-glucans of Agaricus brasiliensis in different stages of fruiting body maturity and their use in nutraceutical products[END_REF][START_REF] Mizuno | Polysaccharides from Agaricus blazei stimulate lymphocyte T-cell subsets in mice[END_REF] The major carbohydrate structure in the water-soluble part of the fruiting bodies of A. subrufescens is (1À6)-β-glucan, and the yield and structural diversity of glucans are supposed to increase as the fruiting bodies mature. [START_REF] Camelini | Structural characterization of β-glucans of Agaricus brasiliensis in different stages of fruiting body maturity and their use in nutraceutical products[END_REF] Apart from the use of fruiting bodies, attempts have also been made for exopolysaccharide production in submerged cultures of mycelium [START_REF] Fana | Effect of nutritional and environmental conditions on the production of exopolysaccharide of Agaricus brasiliensis by submerged fermentation and its antitumor activity[END_REF] or polysaccharide production in solid cultures of mycelium. [START_REF] Chung | Isolation and characterization of a water-soluble polysaccharide from the mycelium of solid cultured Agaricus blazei Murill[END_REF] It thus seems of great importance to identify chemical markers that would reveal the presence of these polysaccharides in fungal biomass, that is, vegetative mycelium or fruiting body. These indicators would be useful to determine the effects of (i) the type of fungal material considered (vegetative mycelium or fruiting bodies), (ii) the age of the fruiting bodies, (iii) the culture conditions, and (iv) the genetic diversity on the concentrations of such polymers of medicinal interest in the fungal biomass.

Our study consequently focused on the chemical characterization of the fungal biomass, using solid-state 13 C NMR. This method has been widely used to study polymer structures because no preliminary extractions are required and this holistic approach thus allows for analysis of samples in their native state. This technology is efficient for various fields such as environmental research, to monitor organic matter transformation, [START_REF] Dignac | Effect of N content and soil texture on the decomposition of organic matter in forest soils as revealed by solid-state CPMAS NMR spectroscopy[END_REF] or the food industry, for instance, to characterize proteins and free amino acids in cheese. [START_REF] Bresca | Characterization of the geographical origin of buffalo milk and mozzarella cheese by means of analytical and spectroscopic determination[END_REF] Here, the fungal biomass of A. subrufescens is chemically described using both vegetative mycelium and fruiting bodies from a large number of strains from various geographical origins (Europe, North, South, and Central America, Asia). Vegetative mycelium from different strains of the button mushroom Agaricus bisporus is also analyzed to compare their specificities with those of A. subrufescens.

Production of the Vegetative Fungal Biomass. The vegetative fungal biomass was produced using 500 mL flasks containing 100 mL of liquid medium (Cristomalt 20 g/L, CuSO 4 5 mg/L) inoculated with a 1 cm wide plug of solid cultures (Cristomalt 20 g/L, agar 15 g/L). The flasks were incubated at 25 °C with continuous shaking (200 rpm) for 15 days. Then the cultures were transferred in 1 L flasks containing 200 mL of liquid Cristomalt medium under the same conditions for 15 days. The mycelium was recovered by filtration on gauze and frozen before being lyophilized for solid-state NMR analyses.

Production of Fruiting Bodies. The compost used for the cultures of 13 strains of A. subrufescens was provided by a company producing button mushrooms (Renaud SA, Pons, France). Eight kilograms of compost was inoculated with 2% of spawn (mycelium developed on rye grains), placed in trays, and incubated at 25 °C and 85% relative humidity during 20 days to allow the invasion of the compost by the mycelium. Then a casing layer (one-third ground limestone, onethird peat, one-third thin sand) was added. The ambient air temperature was maintained between 22 and 25 °C with 95% relative humidity and low concentrations of CO 2 . Two and 4 weeks after the addition of the casing layer, the trays were placed at about 18 °C for 8 h. The fruiting bodies were harvested young, tough, and medium size. A composite sample of 10 fruiting bodies for each strain was frozen immediately after harvest and lyophilized before the NMR analyses.

Cross-Polarization Magic Angle Spinning 13 C Nuclear Magnetic Resonance ( 13 C CP/MAS NMR) Procedure. The solid-state 13 C NMR spectra were obtained on a Bruker Avance-400 MHz NMR spectrometer operating at 13 C and 1 H resonance frequencies of 101.6 and 400.3 MHz, respectively, using a commercial Bruker double-bearing probe. About 60 mg of samples was placed in zirconium dioxide rotors of 4 mm o.d. and spun at a magic angle spinning rate of 10 kHz. The CP technique was applied with a ramped 1 H pulse starting at 100% power and decreasing until 50% during the contact time (2 ms) to circumvent HartmannÀHahn mismatches. To improve the resolution, a dipolar decoupling GT8 pulse sequence 9 was applied during the acquisition time. To obtain a good signal-to-noise ratio in 13 C CPMAS experiment 6000 scans were accumulated using a delay of 3 s (enough to ensure the complete 1 H relaxation). The 13 C chemical shifts were referenced to tetramethylsilane and calibrated with glycine carbonyl signal, set at 176.03 ppm. The 13 C NMR spectra were divided into seven chemical shift regions according to Dignac et al. [START_REF] Dignac | Effect of N content and soil texture on the decomposition of organic matter in forest soils as revealed by solid-state CPMAS NMR spectroscopy[END_REF] Dmfit 2003 software was used to determine each chemical shift region intensity. [START_REF] Massiot | Modelling one-and two-dimensional solid-state NMR spectra[END_REF] Statistical Analysis. The nonparametric MannÀWhitney U-test [START_REF] Mann | On a test of whether one of two random variables is stochastically larger than the other[END_REF] was used to separate significantly different means (P < 0.05) for each NMR signal (COOHÀC, O-alkyl-C, aromatic + phenolic-C, and alkyl-C). To compare vegetative mycelia and fruiting bodies, only the data from the 13 common strains were used. To compare the mycelium of A. bisporus and A. subrufescens, the data from all of the strains were available.

' RESULTS AND DISCUSSION

This study reports for the first time the chemical comparison of fungal biomass from vegetative mycelium and fruiting bodies of a fungal species, A. subrufescens, using solid-state 13 C NMR. Interspecific chemical differences of vegetative mycelium biomass have been also investigated between two Agaricus species, A. subrufescens and A. bisporus. A large sample of both cultivated and wild strains from various origins has been studied, and thus the data can be considered as representative of each species.

The 13 C CP/MAS NMR data have been analyzed using each area of the spectrum that can be assigned to different categories of biomolecules: from 0 to 45 ppm, alkyl-C (mainly lipids from membranes); from 45 to 110 ppm, O-alkyl-C (polysaccharide moiety); from 110 to 160 ppm, aromatic-C (mainly aromatic amino acids and melanin); from 160 to 200 ppm, COOHÀC/ CONHÀC (mainly fatty acids and peptide bonds). [START_REF] Dignac | Effect of N content and soil texture on the decomposition of organic matter in forest soils as revealed by solid-state CPMAS NMR spectroscopy[END_REF] Furthermore, whatever the spectra considered (either from the vegetative mycelium or from the fruiting body), certain peaks may be more specifically assigned to certain functions from biopolymers. With regard to proteins, several peaks can be linked to carbons from side chains of amino acids: CR at 54 ppm; Cβ at 40 ppm (from Phe, Ile, Tyr, Asn, Asp) or 30 ppm (from Arg, Gln, Glu, His, Lys, Trp); Cζ at 155 ppm from Arg and Tyr. 13 Polysaccharide resonances can be found in the O-alkyl region (45À92 ppm) and di-O-alkyl region (90À110 ppm): the main peak at 72 ppm can be assigned to C2, C3, and C5 of carbohydrates, and the region from 99 to 105 ppm is linked to anomeric carbons with either R or β configuration. [START_REF] Saitô | A high-resolution solid-state 13 C NMR study of (1,3)-β-D-glucans from various sources. Conformational characterization as viewed from the conformationdependent 13 C chemical shifts and its consequences to gelation property[END_REF][START_REF] Ohno | Carbon-13 nuclear magnetic resonance spectroscopy of some edible mushroom[END_REF] Chemical Differences in the Fungal Biomass of Vegetative Mycelia and Fruiting Bodies. The 13 C CP/MAS NMR spectra of 13 strains of A. subrufescens have been compared between the vegetative mycelium biomass obtained in liquid cultures and the fruiting bodies. Figure 1 shows an example of two NMR spectra obtained from the vegetative mycelium and the fruiting bodies of strain CA570. This first result reveals significant differences for the functional groups COOH/CONHÀC, O-alkyl-C, and alkyl-C (Table 1). The fruiting bodies contain a higher percentage in Qualitative differences between peaks are mainly observed in the functional region assigned to polysaccharides. In the 13 C CP/ MAS NMR spectra from the vegetative mycelium, three peaks at 96.97, 92.54, and 60.85 ppm have been recorded that were not in the spectra of the fruiting bodies. On the other hand, three other peaks at 69.14, 67.63, and 63.48 ppm have only been obtained in the 13 C CP/MAS NMR spectra of the fruiting bodies (Figure 1).

To compare with more accuracy the results obtained from both the vegetative mycelia and the fruiting bodies, only certain peaks have been selected in the regions assigned to polysaccharides and proteins in the solid-state 13 C NMR spectra. A protein moiety can be detected in the spectrum by signals at 175À 173 ppm (CONH), at 20À50 ppm (CÀN, CÀH from peptide bonds), and at 120À165 ppm (aromatic side chains of amino acids). A polysaccharide moiety can also be described more precisely: 60À90 ppm signals can indeed be assigned to carbons C2, C3, C4, C5, and C6 from the glycosidic ring, [START_REF] Saitô | A high-resolution solid-state 13 C NMR study of (1,3)-β-D-glucans from various sources. Conformational characterization as viewed from the conformationdependent 13 C chemical shifts and its consequences to gelation property[END_REF] whereas anomeric carbon can be linked to a signal from 99 to 105 ppm. [START_REF] Ohno | Carbon-13 nuclear magnetic resonance spectroscopy of some edible mushroom[END_REF] Piazzoferato et al. [START_REF] Pizzoferrato | Solid-state 13 C CP MAS NMR spectroscopy of mushrooms gives directly the ratio between proteins and polysaccharides[END_REF] compared 13 C NMR of different mushroom species and focused on certain peaks for the proteins (170 ppm for CdO and 20 ppm for CH 3 groups) and for the polysaccharides (105 ppm for anomeric CH and 90 ppm for other CH groups). To estimate the relative percentage of proteins and polysaccharides in the fungal biomass, we have thus used peaks at 173 and 19 ppm for proteins and at 102 ppm for polysaccharides. Because the latter peak at 92 ppm is present only in the mycelium but not in the fruiting bodies, we did not calculate the ratios 19 ppm/92 ppm and 173 ppm/92 ppm. The results are presented in Table 2. For both ratios, it is more than twice as high in the fruiting bodies as in the vegetative mycelium. From the data of Piazzoferato et al., [START_REF] Pizzoferrato | Solid-state 13 C CP MAS NMR spectroscopy of mushrooms gives directly the ratio between proteins and polysaccharides[END_REF] the lowest ratio 19 ppm/ 102 ppm was obtained for the fruiting bodies of Lentinula edodes (0.69) and the highest for that of Volvariela volvacea (8.98). However, these results were based on data from only one strain per species. On the other hand, the same authors used five different strains of Pleurotus ostreatus in their study. Comparison of the average peak intensities at 173, 102, and 19 ppm between these 5 P. ostreatus and the 13 A. subrufescens strains we studied shows that only the difference in the peaks corresponding to the CH group of the protein moiety (19 ppm) was significant, with a 10 times lower value for A. subrufescens compared with P. ostreatus (Table 3). These data stress the relative low concentration in proteins of A. subrufescens biomass by comparison with other edible mushrooms. Furthermore, the observed differences between the vegetative mycelia and the fruiting bodies may be taken into consideration for the choice of the biomass to be used, depending on nutritional or medicinal objectives, to favor either high protein or high polysaccharide concentrations, respectively.

In this study, we also aimed to identify certain peaks that may be assigned to the polysaccharides of medical interest. This information is indeed of importance because it can be used as a chemical indicator of the presence of β glucans in fungal biomass. The availability of such a chemical marker is essential to determine which type of culture (solid-state fermentation or liquid culture) is the most relevant to obtain the best production of these molecules of medical interest. Furthermore, this would indeed be helpful to find the best culture conditions (either in liquid or solid medium) to obtain significant concentrations of these polysaccharides of interest in the fungal biomass of A. subrufescens. As described above, the anomeric carbon signal from glycosides ranges from 99 to 105 ppm. It should be noted that various data about the assignation of NMR signals to chemical functions are available and greatly depend on the material studied. For instance, Synytsya et al., [START_REF] Synytsya | Glucans from fruit bodies of cultivated mushrooms Pleurotus ostreatus and Pleurotus eryngii: structure and potential prebiotic activity[END_REF] assigned the peak at 103.3À103.6 ppm to anomeric-C with β configuration, whereas Chenghua et al., [START_REF] Chenghua | A β-D-glucan from the sclerotia of Pleurotus tuber-regium (Fr.) Sing[END_REF] assigned this function to the peak at 102.6 ppm. In our study, whatever the NMR spectrum considered, we found a signal at 102.7 ppm: this signal from the vegetative mycelia (average percentage of relative intensity = 7.0) was significantly different (P < 0.05) from that in the fruiting bodies (average percentage of relative intensity = 5.3), confirming the higher content in polysaccharides in the vegetative mycelium than in the fruiting body as found for total polysaccharides (Table 1). However, to accurately identify chemical indicators of polysaccharides of medical interest (with β configuration) from A. subrufescens fungal biomass, further studies should focus on extracting these polysaccharides to obtain pure polymers, which would be further analyzed via solid-state NMR. Journal of Agricultural and Food Chemistry ARTICLE No quantitative differences were observed for the aromatic-C signals where differences, partially due to various degrees of melanization, may have been found. However, other regions of the spectrum can be used as markers of melanization: Fuji et al. [START_REF] Fujii | Fungal melanin inhibitor and related compounds from Penicillium decumbens[END_REF] have found that different signals from 128.9 to 134.6 ppm can be assigned to olefinic carbons (CH 2 dCH 2 -CH 2 dCH 2 ) from melanin. In our study, a peak at 129 ppm was found only in the fruiting body biomass. This result can be explained by the fact that the fungal cap of A. subrufescens is particularly colored, clearly indicating melanization. However, Knicker et al. [START_REF] Knicker | 13 C and 15 N NMR analysis of some fungal melanins in comparison with soil organic matter[END_REF] have found that the solid-state 13 C NMR spectra of melanin from various fungal species showed large and significant interspecies differences. Thus, extracting melanin from the fungal biomass of A. subrufescens would be useful to clearly define the peaks that can be assigned to this polymer.

Chemical Differences in the Vegetative Mycelium of A. subrufescens and A. bisporus. The 13 C CP/MAS NMR spectra from the vegetative mycelium biomass of the 18 strains of A. subrufescens have been compared to those of the 16 strains of A. bisporus to test the hypothesis of interspecific chemical differences in the biomass of Agaricus spp. The spectra from the vegetative mycelium of both species do not show qualitative differences between the two species studied (Figure 2), whereas significant differences in the relative intensity are observed except for the signals assigned to aromatic-C. NMR signals in this region of the spectrum are the weakest: in fungal biomass they may be assigned to the aromatic amino acids or to melanin, which is not observed in the vegetative mycelium of Agaricus species. The results indeed show a significantly higher intensity for the O-alkyl-C region with A. subrufescens, whereas higher intensities for both the COOH/CONHÀC and the alkyl-C regions are found for A. bisporus (Table 4). These differences suggest that A. subrufescens biomass contains more polysaccharides, whereas that of A. bisporus contains a higher proportion of lipids. The polysaccharide moiety of the fungal biomass is an important chemical characteristic because this fraction potentially contains polymers of medical interest as previously mentioned. Many fungi such as L. edodes, 20 P. ostreatus, 21 or A. subrufescens 22 possess antitumor activities related to the presence of (1À3)-β-glucan [START_REF] Yoshioka | Antitumor polysaccharides from P. ostreatus (Fr.) qu el.: isolation and structure of a β-glucan[END_REF] and/or a β-glucanÀprotein complex. [START_REF] Kawagishi | Formolysis of a potent antitumor (1À6)-β-D-glucanÀprotein complex from Agaricus blazei fruiting bodies and antitumor activity of the resulting products[END_REF] Thus, finding that the relative intensity of the polysaccharide fraction from A. subrufescens species is higher than that from A. bisporus is noteworthy. However, A. bisporus mushrooms have been shown to have a potential breast cancer chemopreventive agent, as they suppress aromatase activity and estrogen biosynthesis. This is mainly due to unsaturated fatty acids, [START_REF] Chen | Anti-aromatase activity of phytochemicals in white button mushrooms (Agaricus bisporus)[END_REF] and vegetative mycelia may also be a source of such fatty acids.

The ratios from Piazzoferato et al. [START_REF] Pizzoferrato | Solid-state 13 C CP MAS NMR spectroscopy of mushrooms gives directly the ratio between proteins and polysaccharides[END_REF] have also been used to compare the fungal biomass of the two species of Agaricus. With both ratios 173 ppm/102 ppm and 19 ppm/102 ppm, no significant differences in protein and polysaccharide are found between both species (Table 2). In their study, Piazzoferato et al. [START_REF] Pizzoferrato | Solid-state 13 C CP MAS NMR spectroscopy of mushrooms gives directly the ratio between proteins and polysaccharides[END_REF] calculated protein/polysaccharide ratios from the different fungal species, and their results indicated that the chemical characteristic of fungal biomass can strongly vary depending on the species. However, more data are needed to draw conclusions because a small number of strains of P. ostreatus (five strains) were analyzed in their study.

Edible mushrooms can be considered as a source of active molecules, and thus investigating whether certain factors (such as intra-and interspecies diversity or culture conditions) can enhance this potential is of importance. This study reveals that a high proportion of polysaccharides is found in A. subrufescens biomass. This warrants further investigations to purify certain polysaccharides of therapeutic interest from this fungus to clearly identify solid-state NMR chemical markers that would be helpful to test the effects of culture conditions on such polysaccharide concentrations.

' AUTHOR INFORMATION

Corresponding Author *Phone/fax: +33 4 91 28 81 90. E-mail: a-m.farnet@ univ-cezanne.fr.

  Protein/ polysaccharide ratios (COOHÀC CONHÀC/O-alkyl-C) were calculated as described by Pizzoferrato et al.: 11 170 ppm/105 ppm, and 20 ppm/105 ppm.

Figure 1 .

 1 Figure 1. 13 C NMR spectra of vegetative mycelium (a) or fruiting body (b) biomass from strain CA570 of Agaricus subrufescens. The numbers (ppm) indicate the peaks from the O-alkyl region, which differ in the 13 C NMR spectrum of the vegetative mycelium or the fruiting body.

  Funding SourcesC.L.-H. thanks CONACYT, Mexico, for a Ph.D. fellowship. This work was supported by the French ANR, Project ANR-09-BLAN-0391.

Figure 2 .

 2 Figure 2. 13 C NMR spectra of strain CA565 of Agaricus subrufescens (a) and of strain Bs514 of Agaricus bisporus (b).

Table 1 .

 1 Means of Relative Intensities of Chemical Groups from13 C NMR Data from the Vegetative Mycelium or the Fruiting Bodies of 13 Strains of Agaricus subrufescens a

		type of mycelium
	chemical functions, ppm	vegetative mycelium fruiting bodies
	COOH/CONHÀC, 160À200 ppm	8.70 ( 1.54*	15.13 ( 0.85*
	aromatic-C, 110À160 ppm	2.54 ( 0.87	5.60 ( 0.76
	O-alkyl-C, 45À110 ppm	74.84 ( 4.86*	55.91 ( 2.72*
	alkyl-C, 0À45 ppm	13.92 ( 2.54 *	23.36 ( 1.34*
	a The asterisks (*) indicate significant differences between means (p <
	0.05) using the non-parametric MannÀWhitney U-test.

Table 2 .

 2 Means of Ratios of Peaks Assigned to Proteins or Polysaccharides (Proteins/Polysaccharides) As Described by Piazzoferrato et al. 11a These ratios were calculated from13 C NMR data of 18 strains of Agaricus subrufescens (for the vegetative mycelium and the fruiting body) and 16 strains of A. bisporus (for the vegetative mycelium).

		A. subrufescens	A. bisporus
		fruiting	vegetative	vegetative
	ratio	body	mycelium	mycelium
	173 ppm/102 ppm	2.93 ( 0.49	1.27 ( 0.35	2.14 ( 0.38
	19 ppm/102 ppm	0.31 ( 0.06	0.17 ( 0.07	0.23 (0.07

a

Table 3 .

 3 Comparison of Relative Intensities of Different Regions from the 13 C NMR Spectra of Fruiting Bodies of Pleurotus ostreatus and Agaricus subrufescens

	fungal species

a Data from Piazzoferrato et al. 11 dx.doi.org/10.1021/jf2017622 |J. Agric. Food Chem. 2011, 59, 8939-8943

Table 4 .

 4 Means of Relative Intensities of Chemical Groups from 13 C NMR Data in Vegetative Mycelium from 18 Strains of Agaricus subrufescens and 16 Strains of Agaricus bisporus a The asterisks (*) indicate significant differences between means (p < 0.05) using the nonparametric MannÀWhitney U-test.

	fungal species

a

dx.doi.org/10.1021/jf2017622 |J. Agric. Food Chem. 2011, 8939-8943 Journal of Agricultural and Food Chemistry ARTICLE ' ACKNOWLEDGMENT

We thank Diego Cunha Zied for purchasing Brazilian A. subrufescens strains and ARP for some A. bisporus strains. We are grateful to Christiane Coldefy and Ludovic Devaux for mushroom production.


Anne Marie Farnet Da Silva

L Qasemian

G Gil

Elisée Ferré

The importance of

teaching and research institutions in France or abroad, or from public or private research centers.

The importance of water availability in the reaction equilibrium of hydrolases in forest litters from a Mediterranean area: a study on lipases

Introduction

Mediterranean ecosystems are subjected to severe climatic conditions, mainly characterized by long summer droughts. Moisture contents in soils or litters can thus vary strongly according to spatio-temporal patterns [START_REF] Fioretto | Decomposition of Cistus incanus leaf litter in a Mediterranean maquis ecosystem: mass loss, microbial enzyme activities and nutrient changes[END_REF]. Water influences the biological processes of organic matter transformation at different scales: microbial growth, enzyme production by microorganisms, protein conformation [START_REF] Wehtje | Enantioselectivity of lipases: effects of water activity[END_REF] and/or the equilibrium of certain enzymatic reactions such as hydrolases, for which water is a substrate of the reaction. For instance, lipases (EC 3.1.1.3) can catalyse either the hydrolysis or the synthesis of esters depending on the amount of water available. Summer drought may favour the synthesis pathway of lipase activity by limiting water availability, leading to carbon storage in litters through the esters produced. On the other hand, when amounts of water are greater (in spring or autumn under the Mediterranean climate), the hydrolytic activity of lipases may be enhanced and favour carbon assimilation [START_REF] Smyth | Including the effects of water stress on decomposition in the carbon budget model of the Canadian Forest Sector CBM-CFS3[END_REF]. While studies on enzyme activities in litters or soils have focused mostly on hydrolysis to assess carbon assimilation by microorganisms [START_REF] Fioretto | Enzyme dynamics on decomposing leaf litter of Cistus incanus and Myrtus communis in a Mediterranean ecosystem[END_REF][START_REF] Qasemian | Effects of anthracene on microbial activities and organic matter decomposition in a Pinus halepensis litter from a Mediterranean coastal area[END_REF], considering the balance between synthesis and hydrolysis and its dependence on available water can be of major importance because it gives valuable information about the carbon cycle (carbon storage or assimilation). Moreover, water in environmental samples is, most of the time, characterized only as humidity, though mass loss after drying can result from volatile compounds and very tightly-bound water molecules can still be sorbed on soil particles. Taking into account the quantity of water actually available is possible using thermodynamic water activity, a w , which is used widely in the food industry [START_REF] Mathlouthi | Water vapour sorption isotherms and the caking of food powders[END_REF]. Thermodynamic water activity can be defined as the ratio of water vapour pressure in the system over pure water pressure at constant pressure and temperature.

We suggest that a w may be a useful concept to apply to soils and the aim of this study was to investigate how a w may influence the reaction equilibrium of hydrolases in litter, using lipases as model enzymes. To do so, a reaction medium with organic solvent was used; this was of crucial importance because, under these conditions, the amount of water was controlled and thus, the influence of the water actually available in the litter on hydrolysis or transesterification can be described. Furthermore, by describing the relationship between humidity and a w , we examined whether the chemical composition of litters of different plant species, Quercus pubescens L., Q. ilex L. and Pinus halepensis L., may affect water availability.

Materials and methods

Litter sampling: litters of Quercus pubescens (QP), Q. ilex (QI) and Pinus halepensis (PH) were collected as three composite samples for each species at Petit Sambuc (Var, France, 43 • 37 17.868 N, 5 • 39'1.835 E) in February 2012. For each litter, horizon L was sieved (2 mm diameter). All the experiments described below were performed for each type of litter.

Water activities were measured in an incubator at 25 • C with the Hygropalm probe (Rotronic, Fisher Scientific, Illkirch, France) using 2 g litter (fresh mass). In order to obtain different a w values, litter was gently dried at room temperature (around 22 • C) with a fan. Lipase activities (both hydrolysis and transesterification) were then measured from litters with the different a w values found. The litter moisture was measured after 72 hours of incubation at 80 • C using 1 g fresh litter and was expressed as the percentage of water in fresh litter. The experiment was performed three times for each sample.

Lipase hydrolysis activities were measured as described by [START_REF] Farnet | A modified method based on p-nitrophenol assay to quantify hydrolysis activities of lipases in litters[END_REF]; this assay was adapted for litters from the method of [START_REF] Pencreac'h | Hydrolysis of p-nitrophenyl palmitate in n-heptane by the Pseudomonas cepacia lipase: a simple test for the determination of lipase activity in organic media[END_REF] and using MTBE (methyltertiobutylether) as the organic solvent. The reaction mixture was 10 mm p-nitrophenyl laurate dissolved in 2 ml MTBE and 1 g litter. The reaction mixture was incubated at 30 • C under magnetic stirring at 500 rpm for 2 hours. Then, 500 ml of the organic phase was added to 4 ml 0.1 m NaOH and p-nitrophenol was measured at 412 nm with a spectrophotometer (Biomate; Fisher Scientific).

Lipase transesterification activities were based on an assay adapted for litters by [START_REF] Goujard | A method to quantify transesterification activities of lipases in litters[END_REF] from the method described by [START_REF] Teng | A modified p-nitrophenyl palmitate assay for lipase synthetic activity determination in organic solvent[END_REF] and using also MTBE as the organic solvent. The reaction mixture was 10 mm of p-nitrophenyl laurate solubilized in MTBE, 400 μl pentanol, 1 g of litter in 3.6 ml MTBE. The protocol was the same as described earlier. Three replicates were performed for each experiment. Activities were expressed as μmoles of p-nitrophenol released per hour per g dry mass. A calibration curve of p-nitrophenol dissolved in MTBE was made with and without litter under the same experimental conditions.

Chemical characterization of litter was carried out by solidstate NMR of 13 C; CP-MAS 13C NMR spectra were obtained on a Bruker DSX 400 MHz (Bruker, Milano, Italy) spectrometer operating at 100.7 MHz. Samples (600 mg) were spun at 10 KHz at the magic angle. Contact times of 2 ms were applied with a pulse width of 2.8 μs and a recycle delay of 3 s. Chemical shift values were referenced to glycine signal (carbonyl C at 176.03 ppm). The 13 C NMR spectra were divided into seven chemical-shift regions according to [START_REF] Ziarelli | Applications et développements des méthodes de résonance magnétique nucléaire haute résolution à l'état solide pour les matériaux organiques et inorganiques[END_REF]. Dmfit 2003 software was used to determine the intensity of each chemical-shift region [START_REF] Massiot | Modelling one-and two-dimensional solid-state NMR spectra[END_REF]. An aromaticity ratio was also calculated to estimate the proportion of aromatic compounds in each litter [START_REF] Baldock | Assessing the extent of decomposition of natural organic materials using solid-state C-13 NMR spectroscopy[END_REF].

One-way anova was performed to compare variations in hydrolysis and transesterification activities taking into account the type of litter (Quercus pubescens, Q. ilex and Pinus halepensis). Data were transformed as necessary to respect the normality and homogeneity of variance. Then pairwise comparison of means using Student's test was made. The peaks assigned to each chemical region of the NMR spectrum (COOH-C, aromatic-C, O-alkyl-C and alkyl-C) were compared taking into account the type of litter by using this procedure. Statistica Vs 6 (StatSoft, Maison-Alfort, France) was used for statistical analysis and P values < 0.05 were considered as being significant.

Results

Chemical characterization of litters from three Mediterranean species

Relative percentages of COOH-C, aromatic-C, O-alkyl-C and alkyl-C were calculated from the solid-state NMR data obtained from the three types of litter under study (Table 1). One-way anova showed that the type of litter strongly influenced the amount of apolar molecules assigned to aromatic compounds (phenols) and lipids. More precisely, aromatic-C and alkyl-C signals were significantly larger in the Pinus halepensis and Quercus ilex litters, respectively. On the other hand, the O-alkyl signal, assigned to polysaccharides, was similar in the three types of litter.

Sorption isotherms in litters

The relationship between humidity and a w can be described using sorption isotherms. Figure 1 shows the isotherms found for the three litters and demonstrates that a similar pattern was obtained whatever the litter studied. These isotherms are characteristic of type-2 sigmoidal isotherms as described by [START_REF] Brunauer | On a theory of Van derWalls adsorption of gases[END_REF]. This type of isotherm is characterized by the presence of multilayers of water molecules at the surface of a material. Here, and according to [START_REF] Brunauer | On a theory of Van derWalls adsorption of gases[END_REF], the sorption isotherms found can be described as follows: for a w ranging from 0 to 0.2, water is strongly bound to the material, from 0.2 to 0.6, molecules of water are organized in multi-layers, and from 0.6 to 1, molecules of water are free or loosely bound to the external surface of the multilayer.

Variations in lipase activities dependent on a w and moisture content

It is noteworthy that both hydrolysis and transesterification activities were detected at the same time for a w ranging from 0.2 to 1 and that the type of litter did not strongly influence the variations observed for both hydrolysis and transesterification activities (Figure 2a,b). These activities followed the same pattern, increasing with a w , more strongly at values greater than 0.6 when the moisture content became more than 15% (Figure 2a,b).

The results of comparisons of means using anova on both hydrolysis and transesterification activities showed that litter significantly affected both enzyme activities (Table 2). Moreover, pairwise comparison of means showed that both activities differed significantly between Quercus pubescens litters on the one hand and both Q. ilex and Pinus halepensis litters on the other (with P < 0.01 in each case). The lipase activity patterns (both hydrolysis and transesterification) obtained from Q. pubescens litter were different from those observed in the P. halepensis or Q. ilex litters. These activities seemed to be less influenced by the increase in free water molecules from a w = 0.6. For all the litters studied, a decrease in hydrolysis and transesterification activities was observed for water activities near, or equal to, 1 (Figure 3a,b). Here, quantitative differences in lipase activities between the different litters cannot be taken into account because variations in microbial communities resulting from the type of the litter may influence the enzymatic activity level.

Discussion

Water availability in litters from different vegetal species

We first investigated how the amount of available water, characterized by a w , varied with moisture content depending on the type of litter considered (from Quercus pubescens, Q. ilex or Pinus halepensis). We hypothesized that various percentages of polar molecules (such as polysaccharides and proteins) and apolar molecules (mainly aromatics and lipids) could be found in the structure of three different species typical of Mediterranean areas and thus could influence water sorption on the vegetal polymers. Here, a greater quantity of alkyl-C and aromatic-C was obtained for P. halepensis and Q. ilex litters respectively, which has been reported before, although not demonstrated using solidstate NMR of 13 C [START_REF] Coulis | The fate of condensed tannins during litter consumption by soil animals[END_REF]. However, these differences in chemical structure did not influence water sorption because a similar isotherm was obtained. Type-2 isotherms [START_REF] Brunauer | On a theory of Van derWalls adsorption of gases[END_REF] are frequently found in food products but, to our knowledge, have never been described before for litters. This demonstrates that free water molecules are available in the three litters studied from a w values greater than 0.6, because 0.6 is a point of inflexion of the curve, from which the shape of the sorption isotherm becomes concave [START_REF] Mathlouthi | Water vapour sorption isotherms and the caking of food powders[END_REF]. This can be linked with the results found considering variations in lipase activities, which increased from a w of 0.6. Free water is thus available for microbial activities from 15% of water content (a w 0.6) in litters from three typical Mediterranean species.

Influence of a w on the balance between hydrolysis and transesterification activities of lipases

We hypothesized that the dynamics of hydrolysis and transesterification activities depend on the amount of water available and that a succession of activities (hydrolysis for larger a w values and transesterification for smaller a w ) may occur in litters. However, we found that both activities were taking place at the same time. This trend had also been observed by [START_REF] Chowdary | The influence of water activity on the lipase catalyzed synthesis of butyl butyrate by transesterification[END_REF] and [START_REF] Ma | Water activity dependance of lipases catalysis in organic media explains successful transesterification reactions[END_REF] with purified lipases (purchased from either Sigma Chemicals, St Louis, MI, USA, or Amano Enzyme Co. Ltd., Nagoya, Japan) from Candida rugosa and Rhizopus oryzae.

We also found that both activities increased with a w and more precisely from a w = 0.6; as noted earlier, sorption isotherms showed that free molecules of water were available from a w = 0.6, which may favour both enzyme conformation and interaction with substrate. However, lipase activities in Q. pubescens litter were less influenced by the increase in free water molecules from a w = 0.6; the chemical composition of this litter, with a smaller amount of apolar molecules than Q. ilex and Pinus halepensis litters, may have modified interactions between water, enzymes and the substrate. Physico-chemical characteristics of the material, such as porosity and hydrophobic molecule contents, strongly influence the effective moisture diffusivity [START_REF] Roca | Moisture migration in a cereal composite food at high water activity: effects of initial porosity and fat content[END_REF]. Thus here water mobility probably differs between the litter of Q. pubescens and that of Q. ilex and Pinus halepensis leading to a different pattern in lipase activities. [START_REF] Chowdary | The influence of water activity on the lipase catalyzed synthesis of butyl butyrate by transesterification[END_REF] noted that the optimum a w for synthetic activities of lipases is strongly and positively correlated with the quantity of substrate. Thus, substrate concentration has a large effect on the optimum water activity controlled by any limitation of water's mass transfer. Here, a large concentration of p-nitrophenyl laurate was used (to reach concentrations for enzyme saturation), which explains a large optimum a w for the synthetic activities of lipases. In fact, the activities were measured here with a large amount of p-nitrophenyl laurate; under natural conditions, and for the same quantity of water, variations in lipase activities (both hydrolysis and transesterification) greatly depend on the relative proportion of available substrates, such as alcohols, acids or esters. From our results, we can conclude that both types of lipase activities potentially occur even for very small water activities (ranging from 0.2 to 0.4). Thus, carbon assimilation can still be realized via hydrolytic activities (though to a lesser extent) under drastic drought conditions (with moisture contents ranging from 10 to 20%).

Under our experimental conditions, both lipase activities decrease for large water activity values. In organic media, the hydration level of enzymes strongly influences both hydrolysis and transesterification activities, which is probably linked to the effect of mass transfer as previously explained. Moreover, [START_REF] Yamane | Intramolecular esterification by lipase powder in microaqueous benzene: effect of water content[END_REF] have suggested that aggregation of lipases may occur for large water activities, leading to a decrease in enzyme reaction yields. In our previous study [START_REF] Farnet | A modified method based on p-nitrophenol assay to quantify hydrolysis activities of lipases in litters[END_REF], 2 ml of water were added for hydrolysis quantification (a w more than 1), which obviously optimized the hydrolysis reaction yields, the huge quantity of water favouring both enzyme conformation and reaction. Here, the reaction mixture with organic solvent was not complemented with water to measure hydrolysis activity; this allowed us to quantify the level of lipase hydrolysis activity obtained with water present only in the litter. Thus these experimental conditions may be more representative of enzyme activity levels potentially expressed in the field.

Conclusion

Thermodynamic water activity, a w , should be taken into account to understand enzyme functioning under hydric stress better, as it implies experimental conditions that are more representative of in natura conditions by using an organic phase instead of the usual buffered aqueous-phase conditions. This study also demonstrated that certain enzymes can still be active at small water contents in litters. This has major relevance for understanding carbon dynamics in soils or litters and defining which conditions favour either carbon storage or assimilation. This balance in enzyme activities could be a useful indicator for soil management because it would determine how certain landscape uses (for both agrosystems and forests) could mitigate the effects of the climatic changes predicted through carbon storage in soils. This type of study should be extended to other enzymes such as glycosidases, which can catalyse both transglycosylation and hydrolysis.

  A . -M . F a r n e t a , L . Q a s e m i a n a , G . G i l b & E . F e r r éa a Institut Méditerranéen de Biodiversité et d'Ecologie marine et continentale (I.M.B.E.), Aix Marseille Université, UMR CNRS IRD

  Correspondence: A. M. Farnet. E-mail: anne-marie.farnet@imbe.fr Received 29 August 2012; revised version accepted 25 February 2013

Figure 1

 1 Figure1Sorption isotherms for litters of Pinus halepensis ( ), Quercus pubescens ( ) and Q. ilex ( ). Each point is a mean of five values; standard deviations were < 3%.

Figure 3

 3 Figure 3 Hydrolysis (a) and transesterification (b) activities in litters of Pinus halepensis ( ), Quercus pubescens ( ) and Q. ilex ( ) depending on moisture content. Each point is a mean of three values; standard deviations were < 3%.

Table 1

 1 Chemical characterization of litters of Pinus halepensis, Quercus pubescens and Q. ilex

	(a)	COOH-C Aromatic-C O-Alkyl-C Alkyl-C
	Pinus halepensis	4.53 ± 0.07 11.17 ± 0.21 58.92 ± 1.02 25.72 ± 0.85
	Quercus ilex	8.21 ± 0.32 14.15 ± 0.75 61.79 ± 2.01 15.85 ± 0.98
	Quercus pubescens 5.98 ± 0.58 9.2 ± 0.90 65.58 ± 1.87 19.26 ± 0.74
			Sum of	Mean
	(b)	d.f.	squares	square	F	P
	Between controls	2	0.954	0.460	3.56	< 0.05
	Within controls	30	10.618	0.253
	Total	32	11.538
	(a) Averages of relative percentages and standard deviations of each
	chemical regions were calculated from solid-state NMR data (10 peaks
	for each chemical regions, n = 3). (b) One-way anova on each chemical
	region as variables was performed.

Table 2

 2 (a) Means of hydrolysis and transestrification activities and standard deviations (n = 10) calculated for the three litters. (b) One-way anova on both hydrolysis and transesterification activities of lipases as variables measured in litters of Pinus halepensis, Quercus pubescens and Q. ilex

	(a)	Means of hydrolysis activities / g -1 DW	Means of transesterification activities / g -1 DW
	Quercus ilex	1.280 ± 0.293		1.364 ± 0.314
	Quercus pubescens	3.899 ± 1.200		3.927 ± 1.098
	Pinus halepensis	0.950 ± 0.240		0.906 ± 0.196
	(b)	d.f.	Sum of squares	Mean square	F	p
	Hydrolysis activities
	Between groups	2	45.122	22.561	49.599	< 0.0001
	Within groups	25	11.372	0.455	-	-
	Total	27	56.494	-	-	-
	Transesterification activities
	Between groups	2	45.572	22.786	58.934	< 0.0001
	Within groups	25	9.666	0.387	-	-
	Total	27	55.238	-	-	-

© 2013 The Authors Journal compilation © 2013 British Society of Soil Science, European Journal of Soil Science

Acknowledgements

We are very grateful to Mrs Marjorie Sweetko and Mr Patrick Fournier for their very helpful assistance with the English language for all these years and to Mr I.M. Da Silva for his technical support. We would like to thank Dr F. Ziarelli for solidstate NMR analysis. This work is dedicated to Oscar.


Christian A Hernández

Fabio Ziarelli

Isabelle Perraud Gaime

Anne Marie Farnet Da Silva

Gabriela García

José A García-Pérez

Beatriz Gutierrez-Rivera

Enrique Alarcón

Christian A Herna ´ndez

Annemarief Arnet

Da Silva

Gabriela Garcı

Chemical and Biological Pretreatments on Sugarcane Bagasse to Enhance its Enzymatic Hydrolysis

Keywords: alkaline pretreatment, bioethanol, cellulose crystallinity, Pycnoporus sanguineus

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Sugarcane bagasse (SCB) is ap romising source of raw material for bioethanol production [START_REF] Cardona | [END_REF] through enzymatic hydrolysis. The efficiency of this method depends on ap re-treatment aimed at reducing the crystallinity of the raw material, as well as its hemicellulose and lignin content, lignin being ac ellulase adsorbent. [2,3] Thus, for an efficient lignocellulose hydrolysis, a pretreatment is necessary in order to decrease lignin, the cellulose-crystallinity index, and the hemicellulose content.

Ag ood pre-treatment includes alkaline agentsl ike NaOH, Ca(OH) 2 and KOH. [4] Alkali attacks lignin via saponification of ester-linkages between cellulose and hemicellulose with lignin, [5] although it may generate alkaline-celluloseb yi on exchange. [6] Also, as opposed to acids, alkaline agents for biomass delignification rarely generate compounds that are harmful to microorganisms. [7] Biological pre-treatmentsu sing live fungi, on the other hand, have been found to enhance enzymatic hydrolysis. [8] Fungi (mainly white-root fungi) can oxidize lignin via phenoloxidase enzymes (i. e., laccases, Mn-peroxidases, lignin peroxidases) andp roduce cellulases and hemicellulases (e. g. xylanases), decreasing the stock of polysaccharides and, consequently,t he production of bioethanol. And since fungi also transform some lignocellulosem ateriali nto fungal biomass, the outcome is ad ifferent raw materiala ltogether.T he structuralc haracterization of SCB fibres pre-treated with live fungi is key to the understanding of how fungal enzymes affect fibre composition, and how this structuralc omposition affects in turn the hydrolysis/fermentation processes downstream. Classicalm ethods for fibre analysisb ased in subsequent detergent hydrolysis (e. g. VanS oest and Wine [9] )a re not enough to explain the complex modifications of lignocellulose fibre by fungi or chemicalp re-treatments. Thus, for lignocellulose characterization, the use of other techniques, such as near infrared spectroscopy (FTIR) and 13 Cs olid-state nuclear magnetic resonance (NMR), are preferred. 13 CC PMAS NMR spectroscopy is ap owerful technique of analysis, successfully used on soil litter, [10] the lignification process, [11] and the extent of condensation in lignin characterisation, [12] and can also be used to evaluate changes in fungal biomass. [13] Solid-state NMRa llows for chemical analysis to be carried out in an atural state, and is an excellent choice for samples with restricted solubility,a sa re lignin residues, or when the physical structure of cellulosei st ob ee xamined. [14] Therefore, the aim of this study was to characterize SCB fibre by 13 CC PMAS NMR spectroscopya fter ab iological pre-treatment, using the fungus Pycnoporus sanguineus and two alkaline pre-treatments with NaOH and Ca(OH) 2 .For more details see SI. After that, pre-treated SCB was hydrolysed with Aspergillus niger cellulases and fermented with Saccharomyces cerevisiae, and the yields of both steps werer elatedt ot heir structural composition using multivariate statistical methods.

Results and Discussion

Chemical shifts in SCB submitted to different pre-treatments Both chemical and biological pre-treatments lead to changes in the chemicalc omposition of SCB. The O-alkyl Cs ignal (45-110ppm) corresponds to the carbohydrate (i. e., holocellulose) content of SCB, indicating that the original composition of SCB had 79.76 %h olocellulose, and that after an alkaline pretreatment the proportion increased to 88.86 %w ith NaOH, and to 84.13 %w ith Ca(OH) 2 (Figure1). Conversely,b iological pre-treatment decreased the amount of holocelluloset o5 0.65 %. The aromatic Cr egion,w hich correspondst ol ignin, had an original proportion of 12.87 %, and the pre-treatmentsd ecreasedt his value as follows:C a(OH) 2 to 10.26 %, NaOH to 7.03 %, and P. sanguineus to 6.53 %. All treatments led to a decreasei na romatic Cc ontent, but the highest lignin decay was achieved with P. sanguineus (Table 1).

The values of the regions Carboxyl Ca nd Alkyl Ca re associated to proteins andlipids, and their peaks correspond to the acetate groups in the hemicellulose that appearsi nt his region. Ta king them together, they were very low in the original composition of SCB (3.57 %a nd 2.45 %, respectively). Alkaline pre-treatments diminish the amount of Carboxyl Ct o1 .48 % (NaOH) and 1.60 %[ Ca(OH) 2 ], and in general they did not affect the Alkyl Cregion. On the other hand, the pre-treatment with P. sanguineus strongly increased both regions:C arboxyl Ci n-creasedto9.31 %, and Alkyl Cto33.49 %(Figure 1).

The signals corresponding to the Ca,C bandC dof amino acids increased with the treatments with P. sanguineus from 5.61 %t o1 8.38 %( as did the signals corresponding to chitin and the Cg of amino acids, from 3.47 %t o8 .49 %), whereas alkaline treatmentsd iminished or did not changet hose signals (Figure 1). The ergosterol signal was near to 0i nt he original composition of SCB, and remained the same after the alkaline pre-treatments. However,w hen P. sanguineus was used, ergosterol increased from 0.24 %t o4 .33 %. Thus, P. sanguineus transformed ap ortion of SCB into fungal biomass, as was hypothesized at the beginning.

The proportion of amorphous cellulose and crystalline cellulose, estimated by the signal positiono ft he C4 of pyranoglucose, [20] indicatest hat SCB has an originalc rystallinity index (CI) of 0.29. This value changeds lightly according to the pretreatments employed: SCB pre-treated with NaOH increased its CI to 0.32, decreased to 0.27 when Ca(OH) 2 was used, and

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remainedunchanged with the P. sanguineus pre-treatment.This indicates that NaOH promotes amorphous cellulose removal, which may affect the performance of hydrolysis. Finally,t he signals of aromatic carbonsd ecreasedi na ll treatments, P. sanguineus being the treatment that obtained the highest decreaseo faromatic carbons, followed closely by the SCB pretreated with NaOH,a nd then by the SCB pre-treated with Ca (OH) 2 .

Hydrolysis step

In all treatments,c ellulase activity decreased rapidly within the first 24 hours, while total sugar contenti ncreased within the first 24 hours, remaining almost unchanged afterwards (Figure 2).

The area under the curve of FPU activities showedt hat when SCB pre-treatedw ith P. sanguineus (99.21 FPU for 96 h) and NaOH (115 total FPU for 96 h) was hydrolysed, the enzymatic stability was higher. Conversely,less FPU activitywas registeredi nt reatmentsw ithS CB pre-treated with Ca(OH) 2 (56.91 total FPU in 96 h), and with untreated SCB (57.33 total FPU for 96 hours).C ellulase stability was positively correlated with O-alkyl C, Xylan C4 and Cellulose crystallinity,a nd negatively correlated with Alkyl C, CarboxylC ,c arbon amino acids, chitin and ergosterol (Table 2).

Like total reducing sugars release, glucosep roduction was faster within the first 24 ho fh ydrolysis. SCB pre-treated with P. sanguineus produced the highest amount of glucose( 29.76 AE 2.2 mg.ml À1 ), followedb yS CB pre-treated with Ca(OH) 2 (28.19 AE 0.45 mg.ml-1), NaOH (23.84 AE 2.0mg.ml À1 ), and the untreated SCB (9.95 AE 15.93 mg.ml À1 )( Figure 2). Saccharose contentd ecreased slightly during SCB hydrolysis in all treatments (Figure 2), reaching af inal concentration of 4.38 AE 0.36 mg.ml À1 when pre-treated with Ca(OH) 2 ,4 .26 AE 0.4 mg.ml À1 when pre-treated with P. sanguineus,3 .66 AE 0.3 mg.ml À1 when pre-treated with NaOH, and 1.48 AE 2.4 mg.ml-1 when untreated (Figure 2). The glucose released was positively correlatedw itha lkyl C, carboxyl C, amino acids content,c hitin and ergosterol. These variablesp ositivelya ffected the hydrolysis yield too (Table 2). Meanwhile, cellulose crystallinity,a romatic C, and tertiary, quaternary and p-hydroxyphenyl aromatic C, negatively affected glucose release and the hydrolysis yield. Saccharose was positively correlated with the variables associated to the presenceo ff ungal biomass,a nd negatively correlated with lignin compounds (aromatic carbons) (Table 2).

The treatment that promoted the highest glucosea nd hydrolysisyield was P. sanguineus.

The highest hydrolysisy ield wasa chieved when SCB was pre-treated with P. sanguineus,followed by pre-treatments with Ca(OH) 2 and NaOH. The lowest yield was obtainedw ith untreated SCB. The fungal biomass gain did not negatively affect enzymatic hydrolysis. In fact, the highest hydrolysis yield was obtained with SCB pre-treated with P. sanguineus. The effect of proteins or lipids on cellulase catalysis is not well documented, but it is known that some lipids can act as surfactants, and avoid the absorption of cellulases by cellulose, increasing the hydrolysis yield. [23,24] Some proteins,l ike bovine serum albumin, can performthe same role. [25] Thus, the enrichmentofproteinsand ergosterol by the pretreatment with P. sanguineus lead to ad ecrease in the absorption of cellulase by the SCB fibre, improving enzymatic hydrolysis. The decay in the aromatic Cand O-alkyl Cregions in this pre-treatmentare mainly due to the production of laccases and cellulases(as previously reported [27] ). Many authors suggest that this fungus is ag ood producer of laccase, [26,28] and therefore an excellent choice for delignification processes. [29] According to our PrincipalC omponent Analysis, the hydrolysis yield was mainly affected by the content of aromatic Ca nd xylan C4, which,asp reviously reported, [2] corresponds to the content of lignin and hemicellulose. The cellulose crystallinity index (CI) was negatively related to the hydrolysis yield, according to Mussato et al.. [3] In this study,t he pre-treatment with NaOH increased CI, which is related to lower hydrolysis, compared to the SCB pre-treated with P. sanguineus. Pretreatments with NaOH and Ca(OH) 2 (of less effect) increased cellulose crystallinity,b ut also increased the O-alkyl region and cellulase stability. Untreated SCB had the highest amounts of lignin compounds, andwas negatively associated to the release of glucoseand the hydrolysis yield (Figure 3).

Fermentation step

As with enzymatic hydrolysis, SCB pretreated with P. sanguineus had the highest fermentation yield and the second highest production of ethanol. Differences in the hydrolysis yield led to differences in the initial glucose content of the fermentation cultures (Figure 3). Glucose decreased near to 0mg.ml À1 in all treatments after 48 ho fc ulturing with S. cerevisiae,w hile saccharose metabolized more slowly (Figure3).

On the other hand, S. cerevisiae biomass (Figure 4) and ethanol increased after 96 h. S. cerevisiae cell growth (biomass) was positively correlated with the glucose consumed, but negativelyr elated with the fermentation yield and ethanol production (Table 3).

An egative effect of hydrolysates pretreatedw ith P. sanguineus on S. cerevisiae biomass was observed, which could be related to as econdary metabolite produced by fungus during the pretreatment. Cinnabarin and poliporin, produced by P. sanguineus,h ave antimicrobial properties; [30,31] however, non-antifungal metabolites have been reported for this fungus. In fact, hydrolysed SCB pre-treated with P. sanguineus does not completely inhibit the growth of S. cerevisiae,a lthough it does induce cell compilation. This interestingp henomenon needs furtherstudy.

SCB hydrolysatesp re-treated with NaOH (3.26 AE 0.3mgml À1 )and P. sanguineus (2.99 AE 0.2 mg.ml À1 )were the ones that produced more ethanol, followedb yu ntreatedS CB (2.11 AE 0.8 mg.ml À1 )a nd SCB pre-treated with Ca(OH) 2 (1.50 AE 0.3 mg.ml À1 )( Figure 3). The highest fermentation yield was attained from the hydrolysate of SCB pre-treated with P. sanguineus,f ollowed by untreatedS CB, SCB pre-treated with NaOH,and, finally,SCB pre-treated with Ca(OH) 2 .

No strong relations were observed between those variables measured with ethanol production and fermentation yields. Nevertheless, aP CA biplot indicatest hat the hydrolysed composition resulting from SCB pre-treated with P. sanguineus reachedbetter ethanol and fermentation yields (Figure 4), while the least efficient pre-treatment was that with Ca(OH) 2 .

Having examinedt he effects of alkaline and biological pretreatments on the downstream process, we suggest that when the aim is to produce ethanol, the use of P. sanguineus is preferable to the use of NaOH or Ca(OH) 2 ,s incei tl eads to higher hydrolysis and fermentation yields than alkali treatments.Further studies will focus on the effect of ergosterol and fungal proteins on cellulase catalysis, and the use of other fungus in the SCB pre-treatment.

Conclusions

In summary,b oth alkalinea nd biological pre-treatments were able to decrease the intensity of aromatic C( lignin) signals.

However,b iological pre-treatmentsc onverted ap ortiono ft he O-alkyl C( carbohydrates) into fungalb iomass (lipids and proteins). The ergosterol and protein gained by the SCB fibre by the pre-treatment with P. sanguineus might be ap ositive factor to enhancet he hydrolysis yield. These beneficiale ffects include better fermentation yields than the ones obtained by hydrolysates from SCB pre-treated with alkaline solutions.

Figure 1 .

 1 Figure 1. 13C CPMASNMR spectraofsugarcane bagassebeforeafter pretreatments (indicated with colours). Important regionsare shown.

Figure 2 .

 2 Figure 2. Kinetics of the different parameters measured during the hydrolysis phase. a) glucose released, b) cellulase activity (FPU), c) saccharose released, d) total reducingsugars released. Different colours indicate different pretreatments. Middle points represent the means, and bars the standard error.

Table 1 .

 1 Regions of13 CCPMAS NMR spectrautilizedfor assesschemical shifts in SCB

	Component	Position (ppm)	Reference
	Alquil-C region	0-45	[15]
	Aminoacidscarbon g	16.1-24.7	[15]
	Aminoacidscarbon b	28-37.6	[15]
	CH 3 of quitin	23.32	[16]
	Ergosteroland deri-
	vates C4, C24, C20,	37.05-42.85	[17,18,19]
	C13, C10
	O-alquilCRegion	45-110
	Cellulose crystallinity	Is calculated by dividing the total
	index	area of the crystalline peak (87 to
		93 ppm) by the total area assigned	[20]
		to the C 4 peak (80 -93ppm).
		53.5-61.3
	Aminoacids carbon a	47.5-49.2	[15]
	Aminoacids carbon d	81.2 -81.7	[15]
	Xylane C4		[21]
		110-160
	Aromatic CRegion	110-123
	Aromatic tertiaryC	123-160	[22]
	Aromatic quaternary C	157-162	[22]
	p-hydroxyphenyl		[22]
		160-190
	Carboxil-C Region	173.7
	QuitinC = O	170.9-174.5	[16]
	Carbon of COOHfrom		[15]
	aminoacids

  Table2. Pearson correlations (r) between chemicalcomponents of pretreated SCB and hydrolysis products/enzymaticactivity.

	Compounds	Cellulase	Glucose	Hydrolysis	Saccharose fi-
		stability	released	yield	nal content
	Alkyl C-0.592	0.746	0.923	0.435
	Carboxyl C-0.500	0.545	0.786	0.158
	Aromatic C0 .117	-0.653-0.668-0.739
	O-alkyl C0 .597	-0.634-0.847-0.254
	Aag + chitin	-0.478	0.489	0.742	0.087
	aaCbda	-0.675	0.797	0.950	0.483
	Ergosterol	-0.	0.762	0.932	0.459
		99
	Xyl	0.497	-0.593-0.823-0.232
	ne C4
	Cellulose CI	0.793	-0.311-0.268-0.036
	Te rtiary aromatic	0.257	-0.757-0.740-0.833
	C
	Quaternary aro-	0.057	-0.604-0.632-0.694
	matic C
	p-hydroxyphenyl	0.247	-0.736-0.633-0.908
	aromatic C

ChemistrySelect 2017, 2,4213 -4218 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemistrySelect 2017, 2,4213 -4218

Acknowledgements

The authors would like to thank the anonymous reviewers of this paper,a sw ell as the Universidad Veracruzana for its support, the academic group Structure andF unctioningo f Ecosystems (CAUVER-324), and also to the biologist F. Ramı ´rez-Guille ´n, from INECOL A.C., Xalapa, Mexico, for the taxonomical identification of the fungus. Finally,authors would like to thank to the ECOS-NORD-CONACYT-ANUIES project (M13 A02) for the financial support.

Supporting Information Summary

For experimental details, such as chemical and biological treatments, enzymatic hydrolysis and fermentation,a nd analytical methods ( 13 CC PMAS NMR), ands tatistics, please refer to the SupportingInformation (SI).

Conflict of Interest

The authors declare no conflict of interest.