Morad Sawdust

Chadni

Nabil Grimi

Olivier Bals

Isabelle Ziegler-Devin

Nicolas Brosse

Morad Chadni
email: morad.chadni@utc.fr

STEAM EXPLOSION PROCESS FOR THE SELECTIVE EXTRACTION OF HEMICELLULOSES POLYMERS FROM SPRUCE

Introduction

The current industrial utilizations of non-cellulosic and non-starch polysaccharides focus on high molar mass guar gum, arabic gum, carrageenan, and xanthan gum, the molecular mass being one of the key features governing their beneficial properties [START_REF] Rana | Modified gums: Approaches and applications in drug delivery[END_REF][START_REF] Willför | Spruce-derived mannans -A potential raw material for hydrocolloids and novel advanced natural materials[END_REF]. In this context, the substitution of these polysaccarides by hemicelluloses extracted from wood could be of great interest because of their abundance and low cost. In recent years, the extraction of polymeric hemicelluloses with high molecular weight and minimal degradation of their native structure has gained growing attention and could stimulate the development of novel industrial applications such as hydrogels, food, health, papermaking, textile and cosmetic industries [START_REF] Ebringerová | Quaternized xylans: synthesis and structural characterization[END_REF]Mikkonen et al. 2012;Mikkonen and Tenkanen 2012) .

Spruce sawdust is a low cost industrial waste material available in large quantities [START_REF] El-Saied | Removal of Lead and Copper Ions from Polluted Aqueous Solutions using Nano-Sawdust Particles[END_REF][START_REF] Kovacova | Removal of Copper from Water Solutions by Adsorption on Spruce Sawdust[END_REF]. In native softwood, the dominant hemicelluloses are acetyl-galactoglucomannan (Ac-GGM) amounting to 14 -20% of wood [START_REF] Willför | Spruce-derived mannans -A potential raw material for hydrocolloids and novel advanced natural materials[END_REF][START_REF] Xu | Acetylation and characterization of spruce (Picea abies) galactoglucomannans[END_REF] and arabinoglucuronoxylans (AGX) for approximatively 10%. Ac-GGM has a backbone of β-D-(1→4)glucopyranosyl and β-D-(1→4)-mannopyranosyl units in a linear or slightly branched chain. Single α-D-galactopyranose units are attached to the main chain by (1→6)-bonds. The mannopyranosyl units are acetylated at C-2 or C-3 with a degree of acetylation of about 0.5 [START_REF] Capek | Galactoglucomannan from the secondary cell wall of Picea abies L. Karst[END_REF]. An approximate ratio of galactose-to-glucose-to-mannose is around 0.5-1.1:1:3.5-4.5 for water-soluble Ac-GGM [START_REF] Timell | Recent progress in the chemistry of wood hemicelluloses[END_REF]. The average weight molar mass Mw of native Ac-GGM has been reported to be between 20 and 50 kDa and the degree of polymerization varies from 100 to 400 [START_REF] Timell | Recent progress in the chemistry of wood hemicelluloses[END_REF][START_REF] Willför | Spruce-derived mannans -A potential raw material for hydrocolloids and novel advanced natural materials[END_REF].

The potential availability of softwood hemicelluloses is huge. These polysaccharides can be recovered from thermomechanical pulping [START_REF] Persson | Isolation of hemicelluloses by ultrafiltration of thermomechanical pulp mill process water-Influence of operating conditions[END_REF] or with methods such as hydrolysis, alkaline extraction, microwaves treatments. [START_REF] Alvarez-Vasco | Alkaline hydrogen peroxide pretreatment of softwood: Hemicellulose degradation pathways[END_REF][START_REF] Gulbrandsen | Extracting hemicelluloses from softwood and bagasse as oligosaccharides using pure water and microwave heating[END_REF][START_REF] Palm | Extraction of Hemicellulosic Oligosaccharides from Spruce Using Microwave Oven or Steam Treatment[END_REF]. However, the extraction of acetylated and high molecular weight hemicelluloses from the wood tissue with a minimum of degradation and/or depolymerization in an economically and environmentally feasible way is still challenging. The extraction methods described in the literature generally rely on recovering partially-degraded and generally de-acetylated GGM in mixture with other hemicelluloses (e.g. xylans) without any selectivity and with a low molecular weight.

However, the presence of acetyl groups in mannose units of high molar mass GGM is described to be an important structural feature for specific applications, the degree of acetylation being for critical the solubility in water [START_REF] Timell | Recent progress in the chemistry of wood hemicelluloses[END_REF].

The quality and composition of extracted hemicelluloses is also important for further applications. The sugar composition is extremely dependent on wood species and on the extraction conditions. Mannansrich hemicelluloses are mainly extracted from softwood [START_REF] Deloule | Production of hemicellulose oligomers from softwood chips using autohydrolysis followed by an enzymatic post-hydrolysis[END_REF][START_REF] Song | Separation of polymeric galactoglucomannans from hotwater extract of spruce wood[END_REF] while xylan-rich hemicelluloses are usually extracted from hardwood [START_REF] Tunc | Hemicellulose Extraction of Mixed Southern Hardwoods[END_REF] or agricultural residues.

However, a few publications reported that the sugar composition of hemicellulose fraction could be adjusted by the solvent of extraction. Adjusting the pH at values above 8 of suspensions of Norway spruce thermomechanical pulp (TMP), dissolved deacetylated GGM and at pH values above 10 AGX began to dissolve from the TMP [START_REF] Thornton | Effects of Alkaline Treatment on Dissolved Carbohydrates in Suspensions of Norway Spruce Thermomechanical Pulp[END_REF]. However, studies dealing with the effect of pH of impregnation and/or extraction on the composition of extracted hemicelluloses from wood are scarce [START_REF] Perez | Impact of the extraction conditions and wood species on the chemical composition of hemicelluloses[END_REF].

Steam explosion (SE) is one of the most common pretreatments used in the fractionation of lignocellulosic biomass [START_REF] Alvira | Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: A review[END_REF]. This hydrothermal pretreatment involves two main steps:

(1) A steam cracking which consists in causing the steam to penetrate under high pressure within the structure of the wood. Thus, the vapor condenses and the material becomes moistened. The condensed water coupled with high temperature induce the hydrolysis of the acetyl and methylglucuronic groups contained in xylans and galactomannan fractions. The acetic and uronic acid released decrease the pH of the medium and catalyze the depolymerization of hemicellulose fraction [START_REF] Blecker | La steam explosion: application en tant que prétraitement de la matière lignocellulosique[END_REF]. More drastic conditions (high temperature, long time of treatment), lead to the formation of monosaccharides and to the hydrolysis of a part of the cellulose.

(2) An explosive decompression which is caused by a sudden drop in pressure followed by a revaporization of a part of the condensation water present in the material provoke a mechanical breakup of the structure of the material [START_REF] Blecker | La steam explosion: application en tant que prétraitement de la matière lignocellulosique[END_REF]. SE is traditionally used as pre-treatment for chemical and biochemical conversion of biomass such as pulping, fermentation and bio-refinery processes [START_REF] Carvalheiro | Hemicellulose biorefineries: a review on biomass pretreatments[END_REF][START_REF] Galbe | A review of the production of ethanol from softwood[END_REF].

Several publications described the extraction of hemicelluloses in oligomeric and/or polymeric forms from agricultural residues [START_REF] Krawczyk | Isolation of hemicelluloses from barley husks[END_REF][START_REF] Schultz | Steam explosion of mixed hardwood chips, rice hulls, corn stalks, and sugar cane bagasse[END_REF][START_REF] Sun | Structure and thermal property of alkaline hemicelluloses from steam exploded Phyllostachys pubescens[END_REF] or wood [START_REF] Palm | Extraction of Hemicellulosic Oligosaccharides from Spruce Using Microwave Oven or Steam Treatment[END_REF][START_REF] Simangunsong | Steam Explosion of Beech Wood: Effect of the Particle Size on the Xylans Recovery[END_REF][START_REF] Wojtasz-Mucha | Hydrothermal pretreatment of wood by mild steam explosion and hot water extraction[END_REF] in the liquid effluents of a steam explosion pretreatment. In fact, this process has been shown to be effective in opening up the lignocellulose structure, increasing the accessibility of polysacharides to hydrolysis. Most of the published studies report a severe hydrolytic depolymerization of hemicelluloses during the steam process with a depolymerization of the polysaccharide chains into oligomers and monomers and their subsequent degradation (hydroxymethylfurfural and furfural) [START_REF] Li | Hot Water Extraction of Hemicelluloses from Aspen Wood Chips of Different Sizes[END_REF][START_REF] Wojtasz-Mucha | Hydrothermal pretreatment of wood by mild steam explosion and hot water extraction[END_REF]. However, few papers described the recovery of high molecular mass hemicelluloses from agricultural residues or wood by a steam explosion pretreatment performed after a basic impregnation [START_REF] Sabiha-Hanim | Fractionation of oil palm frond hemicelluloses by water or alkaline impregnation and steam explosion[END_REF][START_REF] Sun | Structure and thermal property of alkaline hemicelluloses from steam exploded Phyllostachys pubescens[END_REF][START_REF] Xing | Combination of Low-pressure Steam Explosion and Alkaline Peroxide Pretreatment for Separation of Hemicellulose[END_REF]. Taking into account the current industrial development of the steam explosion process for cellulosic bioethanol production, a full recovery of the feedstock through optimum utilization of all lignocellulosic components, including hemicellulosic components as marketable products is a major goal of optimizing a biomass-to-biofuel process. Therefore, there is a need to better understand the impact of SE conditions for the extraction of high molecular mass hemicellulose fractions.

The aim of this work is to study the effect of different parameters of extraction in the liquid stream of spruce hemicelluloses by steam explosion (severity factor and soda or water pre-impregnation) on the molar mass and chemical properties of extracted hemicelluloses from spruce sawdust. The impact of the experimental parameters of the steam explosion process on the hemicelluloses yields and composition, molecular mass and on the chemical selectivity has been examined.

Materiel and method

Raw material

Spruce sawdust with a particle size of 1 to 5 mm was produced from spruce wood (Picea abies) from Lorraine forest in France and stored at room temperature in sealed polyethylene bags in the dark until extraction. The average moisture content of the raw material was 10.01 % w/w. The hemicellulose content of the sawdust (~30 % w/w dry biomass) was calculated by analyzing no-cellulosic monosaccharides of spruce wood according to NREL/TP-510-42618 method. Carbohydrate composition of sawdust includes 72 % glucan, 7.5 % xylan, and 16.3 % of mannan and other sugars.

Soaking

50 g of spruce sawdust was soaked with a solution of 1 M sodium hydroxide (ratio solid/liquid = 1:7)

or water (ratio solid/liquid = 1:7). The sawdust was left for 15 hours before treatment at room temperature. The impregnated sawdust was treated directly without any filtration or pressing.

Steam explosion

Soaked sawdust was placed into a 2-liter reactor with heat jacket and automatic control for steam pressure and sampler retention time. The biomass was treated at different temperatures and retention times by injecting a saturated steam into a pressure resistance chamber. After a given reaction time, the steam saturated biomass is released into a big discharge tank "explosion chamber" as well the biomass.

The selected experimental conditions are based on literature data for hardwood [START_REF] Schultz | Steam explosion of mixed hardwood chips, rice hulls, corn stalks, and sugar cane bagasse[END_REF] and other lignocellulosic biomasses [START_REF] Vivekanand | Effect of different steam explosion conditions on methane potential and enzymatic saccharification of birch[END_REF][START_REF] Zhuang | Hemicelluloses solubilization from Populus tremuloides via steam explosion and characterization of the water-soluble fraction. II. Alkali-catalytic process[END_REF].

The treatment severity for each extraction is determined by a correlation between temperature process and retention time as described by the following equation (Chornet E and Overend R.P, 1988):

where: S = Severity Factor, T(t) = Process temperature (°C), t = Retention time (min), 14.75 = Activation energy value in the conditions where process kinetics are of first order and obey to Arrhenius law.

Hemicellulose recovery

After steam explosion, the condensed vapor and the biomass were recovered and filtered on qualitative filter paper with retention particle size of 5-13 µm (VWR International, Fontenay-sous-Bois, France).

The volume and end-pH of the extract solutions were measured at room temperature shortly after the extraction. The liquid fraction from 1 M NaOH impregnated sawdust was neutralized to a pH of 5.5-6 by a solution of 72 % H 2 SO 4 . 10 mL were taken from the filtered extract for analyses. The extract solution was concentrated by vacuum evaporation using a water bath at 50 °C. To precipitate highmolecular weight hemicelluloses, four volumes of 95 % ethanol were added to the liquid extracts. The liquid phase was separated from the precipitated hemicelluloses by settling at room temperature and decantation. Hemicelluloses were further separated from the remaining liquid by centrifugation at 4000 rpm for 10 min at room temperature. The precipitated hemicelluloses were re-dissolved in distilled water and dialyzed with a dialysis membrane with a cut-off of 3.4 kDa (Spectrum Laboratories, Inc., Rancho Dominguez, CA, USA) in order to remove salts and low molecular mass organic molecules. Finally, dialyzed hemicelluloses solutions were freeze-dried.

Measurement of monosaccharides and uronic acids using HAPE-PAD

The External sugar and uronic acid standards were used for calibration (7 points per curve). The reagents and standards are provided by Sigma-Aldrich and Fisher Scientific.

Degradation products

Degradation products of hemicelluloses (furfural, HMF, formic acid, acetic acid and levulinic acid) are analyzed according to NREL/TP-510-42623 method. The products are measured with an Ultimate 3000 HPLC system. The components are separated in 45 min on an Agilent Hi-Plex H column at 55° C with an isocratic flow rate of 0.4 mL • min -1 of sulfuric acid (5. 10 -3 M). Acids were detected by UV at 210 nm, while HMF and furfural are detected by UV at 280 nm. An external calibration is performed with six calibration points per standard. Reagents and standards are provided by Sigma-Aldrich.

Acetyl content

Acetyl content of extracted hemicelluloses was calculated by measuring the acetic acid concentration of hemicelluloses after a saponification reaction. Acid acetic was measured following the same procedure as degradation products with an UV detection at 210 nm instead of 280 nm.

Size-exclusion chromatography

A Shimadzu Prominence HPLC system is used to determine molecular weight distribution of hemicelluloses by gel filtration chromatography (GFC). The stationary phase is composed of two columns linked in series: Phenomenex Polysep GFC-P as a guard column (7.8 x 35 mm), Phenomenex

Polysep GFC-P3000 and GFC-P2000 analytical columns (7.8 x 300 mm). Sodium nitrate (0.25 M) is used as an eluent with a 0.5 mL.min -1 flow rate. Samples are filtered with a 0.45 µm membrane and 20 µL are injected. Samples are separated at 30 °C and detection is done with a Refractive Index Detector (Shimadzu RID-20A). Each analysis runs for 70 min. aqueous solutions (1 mg/mL) of dextran standards with molecular weights of 1000; 5000; 12000; 24000; 50000 and 80,000 Da were used to prepare the calibration curve.

Two-dimensional (2D) 1H-13C heteronuclear single-quantum coherence (HSQC) analysis

1 H-13 C correlation 2D NMR (HSQC) spectra were recorded on a Bruker III 400 MHz spectrometer equipped with a 5-mm BBFO probe. Experiment was conducted with 20 mg sample dissolved in 1 ml D 2 O. The number of collected complex points was 1024 for the 1 H-dimension with a relaxation of 1.5and 256-time increments were recorded in 13 C-dimension. HSQC spectra were taken at room temperature.

Fourier Transform Infrared Spectroscopy

Fourier Transform Infrared (FTIR) analysis for extracted hemicelluloses were performed by a FTIR spectrophotometer (Thermo Scientific iD3ZnSe ATR-Nicolet iS 5 FTIR Spectrometer unit).

Hemicelluloses powder was placed on a ZnSe crystal plate. Each IR spectrum was obtained over the wave number range of 400-4000 cm -1 with eight scans at resolution of 4 cm -1 . The spectra were analyzed using ThermoScientific OMNIC software.

Statistical analysis

All experiments were conducted at least in duplicates. The means and error bars were calculated and were represented in all figures.

Results and discussions

The hemicelluloses were recovered after SE of spruce sawdust from the liquid stream by precipitation in ethanol and dialysis. The influence the severity of treatment and pH of the pre-impregnation step (Water Pre-soaked Sawdust, WPS, Sodium hydroxide Pre-soaked Sawdust, SPS) on the extraction of high molar mass hemicelluloses was examined. A visual comparison of the sawdust samples after SE is given in supplementary data section (S2) as well as Scanning Electron Microscopy (SEM) of untreated sawdust, sawdust after steam explosion (S3).

Yield of extracted hemicelluloses

Fig. 1 gives the variations of the hemicelluloses yields (recovered after precipitation and dialysis) related to the duration of SE for the different temperatures. For WPS, during the cooking step of the SE process acetyl groups present in the raw material are cleaved to produce acetic acid. Fig. 1 gives the pH profiles of the SE liquid streams. As a result, a decrease of the pH is observed with the increase of the severity of the SE. For SPS, the pH of the liquid after SE remind unchanged at 13 due to the neutralization of acetic acids and hydronium ions by OH -ions. Hemicellulosic fractions isolated from the WPS were obtained in high yields compared to those obtained from SPS. In fact, the maximum yield achieved was 57.78 mg. g -1 DW at severity factor (SF) of 3.65 for WPS and 43.52 mg.

g -1 DW at SF of 3.94 for SPS. For both presoaking conditions (SPS and WPS, Fig. 1), the extraction yields increased with the increase of SF until a SF of 3.94 which is in line with yields obtained after treatment of wood chips of Norway spruce (17, 19 and 43 mg. g -1 at SF of 2.7, 3 and 3.4 respectively) [START_REF] Wojtasz-Mucha | Hydrothermal pretreatment of wood by mild steam explosion and hot water extraction[END_REF]. A further increase of the SF of treatment led to a strong decrease in the hemicellulose's recovery.

This decrease in yield can be explained by degradation reactions and depolymerization of hemicelluloses chains (alkali-catalyzed for SPS or acid catalyzed for WPS) under harsher conditions. In fact, the increase of the SF led to the formation of furans and carboxylic acids in WPS and SPS media respectively. The high concentrations of theses degradation products were obtained at SF of 4.13 (Figs.

3A and 3B). Furthermore, the amount of monomers (not precipitated) extracted after SE treatments of WPS increased with increasing SF and reached a maximum at values of SF = 4.72 with a yield of 5.3 mg. g -1 DW (See Table S1 in supplementary data section). The increase of the amount of monomers at higher SF values could be due to the depolymerization of hemicelluloses chains under high temperatures and low pH. The amount of monomers after treatment of SPS was very low (less than 0.02 mg. g -1 DW).

SPS

WPS pH WPS

The average molecular masses of the extracted hemicelluloses were assessed by water-based sized exclusion chromatography. Table 1 shows the molecular weight as a function of SE parameters for WPS and SPS fractions. As expected, the data showed that the molecular weight of the SPS hemicelluloses fractions are significantly higher than the WPS hemicelluloses. It has been described that high pH and low temperature are known to reduce the kinetic of degradation of polysaccharides [START_REF] Rissanen | Obtaining spruce hemicelluloses of desired molar mass by using pressurized hot water extraction[END_REF]. On the other hand, low pH, high temperature and long treatment duration increase the rate of acid-catalyzed hydrolysis of glycosidic linkages in hemicellulose chains. As a result, the decrease of pH previously mentioned for WPS for the highest severities (Fig. 1) justifies the lower molar mass obtained. Hemicelluloses extracted from SPS had relatively higher molecular weight than those extracted from WPS.

Whatever the presoaking conditions, a significant molecular weight decrease was observed as the severity of the treatment increase's owing to hydrolysis and depolymerization during the cooking step of the process. The higher molecular weight hemicelluloses have been recovered SF of 2.36 and 2.76 for WPS (>25 kDa) and at SF of 2.76, 2.95 and 3.35 for SPS (> 68 kDa). Previous works dealing with the extraction of neutral polysaccharides from Norway spruce reported higher GGM molecular mass (20-60 kDa) [START_REF] Pranovich | Separation and preparative isolation of galactoglucomannan from TMP-water[END_REF][START_REF] Sjöström | Detrimental substances in pulp and paper production: approaches to chemical analysis of deposits and dissolved organic matter[END_REF]. The present results show that during the SE process performed after a water presoaking, a depolymerization of GGM occurs to some extent.

Sugar composition of hemicelluloses

The composition of the hemicellulosic fractions isolated in terms of monomeric sugars has been established, after hydrolysis, by ionic chromatography. The average compositions corresponding to the 12 extraction conditions from WPS and SPS are given in Table 2. All the data are given in the SM section. Whatever the experimental parameters, the data strongly suggest that after a neutral (WPS) and a basic (SPS) presoaking, the hemicellulosic fractions are mainly composed of galactoglucomannans and arabinoxylans respectively. In fact for WPS, the mannose is the dominant sugar (52-64%), followed by glucose (3-16%), galactose (10-13%), the other sugars like xylose (6-13%), glucuronic (0.17%) and galacturonic (1.06 %) acids being observed as minor constituents. As a result, we can assume that the mannonse, glucose and almost all the galactose can be assigned to GGM. On the other hand, hemicelluloses extracted from a basic impregnation (SPS) appear to be mainly composed of arabinoxylans (AX), xylose and arabinose are the most abundant (68-81 % and 7-13% respectively), and mannose is present at low level (0.2-4%). From these results, it is remarkable to note that the pH of the The obtained results were in agreement with those reported by [START_REF] Thornton | Effects of Alkaline Treatment on Dissolved Carbohydrates in Suspensions of Norway Spruce Thermomechanical Pulp[END_REF] who found that for pH values above 10, arabino-(4-0-methyIglucurono)xylans began to dissolve from Norway spruce thermomechanical pulp, and at pH values bellow 8, the solubilization of galactoglucomannans is promoted. In this paper, a considerable decrease of mannose units was observed when the suspension was prepared at pH 9 and an increase in concentration of arabinose and xylose units was observed for pH > 10 suggesting the release of arabino-(4-O-methylglucurono)xylans. [START_REF] Perez | Impact of the extraction conditions and wood species on the chemical composition of hemicelluloses[END_REF] have also found that spruce hemicelluloses extracted in acidic conditions are primarily composed of galactoglucomannans with a high proportion of mannans (at least 45 %, up to 57 %), then glucans (16 to 25%) and galactans (13 to 19%). Some arabinoxylans are also present (up to 15 %, depending on the extractions conditions), while, the fractions obtained in alkaline conditions are a mixture of arabinoxylans and galactoglucomannans with a heavily contamination with lignin (up to 18 %). According to [START_REF] Svärd | Rapeseed straw polymeric hemicelluloses obtained by extraction methods based on severity factor[END_REF], the nature of the polysaccharides extracted from lignocellulosic biomass is function of the extraction pH because their solubility is affected by the substituents on the polymer chain [START_REF] Ebringerová | Hemicellulose[END_REF]. As a result, neutral and acidic media could promote the solubilization of water soluble acetylated galactoglucomannans [START_REF] Lundqvist | Isolation and characterization of galactoglucomannan from spruce (Picea abies)[END_REF]. On the other hand, alkaline medium yielded a xylose-rich extract because of the methyl glucuronic acid and arabinose substituents of AGX [START_REF] Andrewartha | Solution properties of wheat-flour arabinoxylans and enzymically modified arabinoxylans[END_REF][START_REF] Croon | The 4-O-methyl-D-glucuronic acid groups of birch xylan during sulphate pulping[END_REF][START_REF] Kabel | Effect of pretreatment severity on xylan solubility and enzymatic breakdown of the remaining cellulose from wheat straw[END_REF]. These results could explain the slight increase in yields for WPS seen in Fig. 1. In fact, the amount of GGM in spruce wood (20% of wood) is very high compared to AGX (10 %) [START_REF] Timell | Recent progress in the chemistry of wood hemicelluloses[END_REF]. Therefore, Since GGM are water-soluble at high temperatures and AGX are alkali-soluble, the yield obtained in WPS is slightly high (at short time, the effect of autohydrolysis is more operative than the chemical effect of sodium hydroxide).

Acetylation degree of extracted hemicelluloses

It is well known that the hydroxyl groups of the backbone units of softwood GGM are partly substituted by O-acetyl groups [START_REF] Timell | Recent progress in the chemistry of wood hemicelluloses[END_REF] and that this acetyl substitution is critical for further utilization of the hemicelluloses [START_REF] Hannuksela | Sorption of spruce O-acetylated galactoglucomannans onto different pulp fibres[END_REF][START_REF] Xu | Acetylation and characterization of spruce (Picea abies) galactoglucomannans[END_REF]. The degree of acetylation of GGM (WPS extracts) determined by HPLC after saponification, are given in Fig. 2. The molar Ac/Man ratios were used as a measure for the acetylation degree (AD) of GGM. Regarding SPS extracts, the acetylation degree is much lower (AD < 0.06) since (1) most of the extracted hemicelluloses are arabinoglucoronoxylans, native softwood xylans being considered to be non-acetylated, [START_REF] Fengel | Wood: chemistry, ultrastructure, reactions[END_REF][START_REF] Sjöström | [END_REF] and (2) because of a further deacetylation by saponification reaction under basic conditions. Interestingly, for the SE performed at a SF > 2.95, the AD observed (0.27-0.35) are very closed to the values reported for native AcGGM, determined by NMR and titration methods [START_REF] Hannuksela | NMR structural determination of dissolved O-acetylated galactoglucomannan isolated from spruce thermomechanical pulp[END_REF][START_REF] Xu | Acetylation and characterization of spruce (Picea abies) galactoglucomannans[END_REF]. These acetylation values are higher compared to those reported in previous studies for AcGGM isolated from thermomechanical pulping or from spruce wood [START_REF] Kishani | Solubility of Softwood Hemicelluloses[END_REF][START_REF] Song | Effects of pH control with phthalate buffers on hot-water extraction of hemicelluloses from spruce wood[END_REF]. From this result one can deduce that SE allow to extract representative and slightly modified AcGGM samples from softwood. At low SF treatments, the extraction of lower substituted GGM was observed. The optimum SF for the extraction of highly acetylated GGM is 3.65 after a water presoaking of the spruce sawdust.

Fig. 2. Acetylation degree of extracted hemicelluloses from WPS

Degradation products

As expected for WPS, the increase of acidity of the media previously observed lead to the formation at high temperature of degradation products, furfural and hydroxymethylfurfural (HMF) formed by dehydration of C5 and C6 sugars respectively [START_REF] Fengel | Wood: chemistry, ultrastructure, reactions[END_REF]. The Fig. 3B gives the furans derivatives content as a function of the SE conditions. At SF < 2.95, the furfural and HMF concentrations are very low, under the limit of detection for the short duration treatments. On the other hand, the increase of the severity of SE is followed by a steadily increase in furans content. For SF = 4.13, 15.8 mg/l of HMF and 5.7 mg of furfural were formed in the liquid effluent of SE, while 19,6 mg/l and 6.9 mg/l were formed respectively at SF = 4.72.

In basic conditions (SPS), the degradation of polysaccharides proceed though distinctly different mechanisms. Primarily by depolymerization reactions, leading to the formation of carboxylic acid (peeling reactions). Acetic acid is also produced primarily by saponification of acetate groups. Fig. 3A gives the concentrations of formic, acetic and levulinic acids according to the SE parameters.

Spectroscopic characterization 358

The composition the hemicellulosic Ac-GGM and AGX fractions was confirmed by a 2D NMR study 359 accordance with published data of spruce GGM. H1/C1 cross peaks assigned to galactose, glucose and mannose units are detected at 4.96/98.9 ppm, 4.58/104.4 ppm and 4.68/100.2 ppm respectively. Signals assigned to 2-O-acetylated--Manp units are clearly observed at 4.87/99.0 ppm (H1/C1), 5.44/71.6 ppm (H2/C2), and the presence of 3-O-acetylated--Manp units is confirmed by correlation at 5.03/73.2 ppm (H3/C3), 4.75/99.9 ppm (H1/C1). From the intensity of the cross peak, we can observe that a slightly larger amount of 2-O-acetylated mannose units was detected compared to the 3-O-acetylated counterpart. This is in accordance with a previous study performed on Ac-GGM isolated from spruce thermomechanical pulp [START_REF] Hannuksela | NMR structural determination of dissolved O-acetylated galactoglucomannan isolated from spruce thermomechanical pulp[END_REF][START_REF] Teleman | Isolation and characterization of O-acetylated glucomannans from aspen and birch wood[END_REF]). The low intensity signal of reducing end -mannopyranosyl unit was also detected through its characteristic cross-peak at 5.11/93.8 ppm.

Regarding the SPS fraction (Fig. 4B FTIR has been also used for hemicelluloses identification. The results of FTIR spectroscopy are presented in Fig. 5. The absorptions at 1460, 1424, 1392, 1320, 1247, 1166, 1122, 1119, 1043, 985, and 898 cm -1 are associated with hemicelluloses [START_REF] Peng | Comparative Study of Hemicelluloses Obtained by Graded Ethanol Precipitation from Sugarcane Bagasse[END_REF]. The intense pic at 1043 cm -1 for SPS spectrum, which is assigned to the C-O and C-C stretching vibration and the glycosidic linkage (C-O-C) contributions in hemicelluloses, indicates a typical absorbance for xylans in the isolated hemicelluloses [START_REF] Peng | Comparative Study of Hemicelluloses Obtained by Graded Ethanol Precipitation from Sugarcane Bagasse[END_REF], which is in agreement with the sugar composition of extracted hemicelluloses Table 2.

The acetyl and uronic ester groups of hemicellulose absorb at 1745 cm -1 . The intensity of this band is quite intense in the WPS spectrum, confirming that the hemicellulose fraction from WPS is acetylated while those extracted from SPS are totally deacetylated. The presence of a small band at 1514 cm -1 in the SPS spectra can be assigned to the presence of small amounts of associated lignin in the hemicelluloses. In fact, extraction of hemicelluloses in basic medium, promotes the co-extraction of lignin residues, due to the action of alkali ions on the ester and ether linkage between hemicelluloses and lignin [START_REF] Alvarez-Vasco | Alkaline hydrogen peroxide pretreatment of softwood: Hemicellulose degradation pathways[END_REF][START_REF] Perez | Impact of the extraction conditions and wood species on the chemical composition of hemicelluloses[END_REF][START_REF] Thornton | Effects of Alkaline Treatment on Dissolved Carbohydrates in Suspensions of Norway Spruce Thermomechanical Pulp[END_REF].

Conclusion

Steam explosion was assessed for the extraction of high molecular weight hemicelluloses from spruce sawdust. It has been shown that the conditions of the pre-impregnation step before SE strongly influence the selectivity of the extraction. After a basic impregnation, ~90% of extracted hemicelluloses are arabinoglucuronoxylans whereas after a water presoaking ~85% of acetylated galactoglucomannans were recovered. In these latter conditions, the GGM isolated are highly acetylated, with an acetylation degree very closed to the values reported for native Ac-GGM. This study show that SE can be an efficient and selective process for a mild extraction in the water stream of close-to-native and highly acetylated GGM or high molecular weight arabinoglucuronoxylans from softwood. This approach paves the way for the recovery of high value-added co-products (e.g. acetylated GGM) from the liquid stream of the steam explosion pretreatment.

  monosaccharide content of the soluble fraction was analyzed according to NREL/TP-510-42618 procedure. The sugar concentrations were measured by HAPE-PAD (ICS-3000 Dionex, Dionex Corp., Sunnyvale, USA) equipped with a Dionex CarboPac TM PA-20 (3 × 150 mm) analytical column. Filtered samples (20 μL) were eluted at 35°C and at 0.4 mL/min with the following composition: pure water 99.2 %/250 mM NaOH 0.8%: 0-20 min; pure water 75 %/250 mM NaOH 20%/ NaOCOCH 3 (1 M)-NaOH (20 mM) 5% 20-37 min; and pure water 40%/250 mM NaOH 20%/NaOCOCH 3 (1 M)-NaOH (20 mM) 40 % 37-41 min. Each elution was followed by a wash and subsequent equilibration step.

Fig. 1 .

 1 Fig. 1. Yield of extraction of hemicelluloses versus severity factor

  pre-soaking medium before SE strongly influences the selectivity of the extraction. In basic medium (SPS), ~90% of extracted hemicellulose are arabinoxylans (β-D-Xylp 10; α-L-Araf 1,5) and ~5 % are alkali-soluble galactoglucomannans. In acidic medium (WPS), an opposite trend is observed: ~85% of water-soluble galactoglucomannans (β-D-Manp 5; β-D-Glup 1; α-D-Galp 1) and ~15% arabinoglucuronoxylans are recovered.

Fig. 3 .

 3 Fig. 3. Degradation products in the bulk liquid versus severity factor: (A) SPS fraction; (B) WPS 356 fraction 357

  1 H-13 C HSQC (seeFigs. 4A and 4B respectively). The HSQC spectrum of AcGGM is in good 360

  , signals at 5.23/97.6 ppm (H1/C1), 3.70/72.4 ppm (H2/C2), 3.85/77.1 ppm (H3/C3), 3.15/82.4 ppm (H4/C4), 4.28/72 ppm (H5/C5) assigned to 4-Omethylglucuronic acid residues reveal the presence of this residue not detected by ionic chromatography. The presence of xylan and arabinan signals, reveals that AGX was the major hemicellulose extracted for a basic impregnation. The chemical shifts of Ara, Xyl and OMeGlcUresidues were in accordance with those described for glucuronoxylans from other material[START_REF] Moine | Structural characterization and cytotoxic properties of a 4-O-methylglucuronoxylan from Castanea sativa[END_REF].

Fig. 4 .

 4 Fig. 4. 2D HSQC NMR spectral analysis of extracted hemicelluloses from WPS (A) and SPS (B)

Fig. 5 .

 5 Fig. 5. FTIR spectrograms of extracted hemicelluloses from SPS and WPS

Table 1 :

 1 Molecular weight (M w ) and Polydispersity index (PI) of extracted hemicelluloses versus

			severity factor
		WPS		SPS
	Severity factor	M W	PI	M W	PI
	2.36	27.68	2.39	67.6	1.42
	2.76	25.12	2.38	69.9	1.29
	2.95	21.34	2.55	69.4	1.33
	3.35	14.14	2.66	69.4	1.39
	3.54	16.47	2.52	67.8	2.04
	3.65	14.05	2.50	64.7	2.10
	3.94	14.31	2.48	63.5	1.67
	4.13	9.98	2.54	35.2	1.30

Table 2 :

 2 contents of neutral sugars and uronic acids (mol %) of the extracted hemicelluloses from WPS and SPS fraction (for more details, see Fig.S2in supplementary data section)

	SPS	WPS

Acknowledgements

This work was financially supported by SOFREN Company and ANRT "Agence National de la Recherche et de la Technologie". The authors appreciate the financial support from SOFREN and ANRT. LERMAB is supported by the French National Research Agency through the Laboratory of Excellence ARBRE (ANR-12-LABXARBRE-01).

Abbreviations

AD