2.1 Raw material

Spruce sawdust with a particle size of 1–5 mm was produced from a spruce tree from the Lorraine region of France and stored at room temperature in sealed polyethylene bags in dark until use. The average moisture content of the raw material was 10% w/w. The hemicellulose content of the sawdust was approximately 30% w/w dry biomass. Carbohydrate composition of sawdust includes 72% glucan, 7.5% xylan, and 16.3% mannan and other sugars.

2.2 Impregnation

For each experiment, a mass of 20 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 h before treatment at room temperature.

2.3 Experimental methodology of extraction

2.3.3 Separation of extracted hemicelluloses

After each experiment, the extract was filtered on a 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% H₂SO₄. Ten milliliters was taken from the filtered extract for analyses. The extract solution was concentrated by vacuum evaporation (BUCHI, France) using a water bath at 50 °C. To precipitate high-molecular-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 redissolved in distilled water and dialyzed using a dialysis membrane with a cutoff of 3.4 kDa (Spectrum Laboratories, USA). Finally, dialyzed hemicellulose solutions were freeze-dried. Fig. 1 shows the experimental procedure of extraction and separation from spruce sawdust.

2.4 Measurement of monosaccharides and uronic acids using HPAEC-PAD (high-perforformance anion exchange chromatography – pulsed amperometry detection)

The monosaccharide content of the soluble fraction was analyzed according to the method of Hames et al [36]. The sugar concentrations were measured by HPAEC-PAD (high-perforformance anion exchange chromatography – pulsed amperometry detection) (ICS-3000 Dionex, Dionex Corp., Sunnyvale, USA) equipped with a Dionex CarboPac™ 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%/NaOCOCH₃(1 M)- NaOH (20 mM) 5%: 20–37 min; and pure water 40%/250 mM NaOH 20%/NaOCOCH₃ (1 M)- NaOH (20 mM) 40%: 37–41 min. Each elution was followed by a wash and subsequent equilibration step. 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.

2.5 Degradation products

Degradation products of hemicelluloses (furfural, hydroxymethylfurfural [HMF], formic acid, and levulinic acid) are analyzed according to the procedure followed by Sluiter et al [37]. All the products are quantified using 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.

2.6 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 UV detection at 210 nm instead of 280 nm.

2.7 Size-exclusion chromatography

A Shimadzu Prominence HPLC system is used to determine molecular-weight distribution of hemicelluloses by gel filtration chromatography. The stationary phase is a Phenomenex Polysep GFC-P guard column (7.8 × 35 mm), Phenomenex Polysep GFC-P3000 and GFC-P2000 analytical columns (7.8 × 300 mm). The mobile phase is 0.25 M aqueous NaNO₃. Samples are filtered using a 0.45-μm membrane, and 20 μL is injected. Samples are separated at 30 °C under a flow of 0.5 mL min⁻¹, and detection is done using 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 1000–80,000 were used to prepare the calibration curve.

2.8 Fourier-transform infrared

Fourier-transform infrared (FTIR) analysis for extracted hemicelluloses was performed by an FTIR spectrophotometer (Thermo Scientific iD3ZnSe ATR—Nicolet iS 5 FTIR Spectrometer unit). Hemicellulose powder was placed on a ZnSe crystal plate. Each infrared (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 Thermo Scientific OMNIC software.

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

Two-dimensional ¹H–¹³C heteronuclear single-quantum coherence (HSQC) spectra were recorded using a Bruker III 400 MHz spectrometer equipped with a 5-mm BBFO (Broad Band Fluorine Observe) probe. The experiment was conducted with 20 mg of sample dissolved in 1 ml of D₂O. The number of collected complex points was 1024 for the ¹H dimension, with a relaxation of 1.5 times and 256 times increments recorded in ¹³C dimension. HSQC spectra were taken at room temperature.

2.10 Soluble lignin

The acid-soluble lignin (ASL) content was determined from absorbance values at 205 nm according to the laboratory analytical procedure (LAP) provided by the National Renewable Energy Laboratory [36].

2.11 Wood solubilization

Wood solubilization (WS) was presented as the ratio of the wood dissolved to the weight of the wood used for extraction as expressed in Eq. 4:

3.1 Wood solubilization

Before microwave activation, spruce sawdust was impregnated in a solution of distilled water (neutral medium) or 1 M NaOH (basic medium) for one night. The aim of the impregnation step is to open the wood structure and to enhance the accessibility of hemicelluloses during the microwave extraction [38–40]. After microwave treatment, a diffusion step was carried out for 3 h at 70 °C. The effect of impregnation and diffusion steps on hemicelluloses yields +19% and 2.5%, respectively, in neutral medium and +16.4% and +30%, respectively, in basic medium. See Fig. S1 in Supplementary Material. The temperature profiles for conventional extraction and for MAE at different power are also given in SD section (Fig. S2).

Solubilization of wood was calculated using Eq. 3. As expected, the solubilization of wood is significantly higher in basic medium (Fig. 3). The MAE increased notably the effect of sodium hydroxide (+81% for 573 WW). The advantage of MAE over conventional extraction could be explained by the rapid and the selective heating of wood that lead to destabilization and structural damage of wood matrix, thus improving biomolecule extraction and also the heat and mass transfer which act in the same direction. Furthermore, the effect of MAE is related not only to the fast increase of temperature but also to the power transmitted to the wood matrix which destabilizes and weakens structural linkages between lignin and carbohydrates. In conventional extraction, heat transfer and mass transfer had opposite directions [41].

Wood solubilization steadily increased with the increase of microwave power. The solubilization of sawdust is mainly affected by microwave power which is the main parameter affecting the irradiation strength and the rate of temperature increase (Fig. 2). For basic impregnation, alkaline solutions increase the water absorption capacity, swell up cellulosic fibers and decrease their crystallinity, and thus increase the specific surface area [42]. Based on previous reports, the mechanism of action is due to the decrease of intermolecular hydrogen bonds by solvation [43], as well as lignin-hemicellulose bond scissions.

3.2 Hemicellulose extraction

Polymeric hemicelluloses extracted in the liquid fraction were quantified by subtracting the amount of monomeric sugars (determined by ionic chromatography) from the total sugar content in the liquid fraction obtained after extraction. As expected, Figs. 3(A and B) show that the extraction of hemicelluloses highly depends on the pH of the impregnation step, as it affects the hydrolysis of the hemicellulose-lignin linkages. In fact, at maximum power (573 W) when sodium hydroxide was used as solvent, the effect of microwave on the extraction yield was higher than that of the conventional extraction (32.3 and 22.5 mg/g of dry wood, respectively). On the other hand, lower extraction yields were observed using water as solvent. To explain the effect of MAE on wood, Panthapulakkal et al. [44] have suggested that the disruption of wood structure and the rupture of wood fibers are due to the selective interaction of the microwaves with the inhomogeneous lignocellulosic materials. Hu et al., and Panthapulakkal et al., have found that microwave treatment improves surface disruption and breaking of lignin structures in alkaline-treated switch grass, while in conventional heating, the fibers remain intact [34,44].

The composition of the liquid fraction in terms of monomeric sugars (after acid hydrolysis) at different conditions of microwave extraction is also given in Figs. 3(A and B). Polysaccharides extracted in the neutral medium are mainly composed of mannose (33%), glucose (18%), and galactose (20%). As a result, we can assume that the hemicellulosic extract is mainly composed of galactoglucomannans [9]. On the other hand, in the basic medium, the high contents of xylose (57%) and arabinose (10%) suggest the dominant extraction of arabinoxylans. This selectivity of extraction as a function of the pH of the medium is in accordance with previous reports. It has been described that in neutral or mild acid medium (pH ≈ 5), hemicelluloses extracted from softwood are rich in mannose and acetylated glucomannans, exhibiting a relatively high water solubility [9,31]. In basic medium, the high xylose content can be rationalized by the dominant extraction of arabinoglucuronoxylans. In fact, arabinoglucuronoxylans are substituted with methyl-glucuronic acid and arabinose moiety which affect their solubility [45]. Thornton et al. [46] have found from the Norway spruce wood that at pH values greater than 10, the concentration of extracted arabinose and xylose units increases, suggesting the release of arabino-(4-O-methylglucurono)xylans.

The oligomeric and polymeric fractions of the extracted hemicelluloses were calculated by subtracting the amount of monomeric sugars from the total sugar content in the extracts. The monomer proportion is very low (< 2%) whatever the extraction conditions, the mild conditions used in this study (T < 100 °C, pH > 5) limiting the hydrolytic cleavage of hemicelluloses. Based on literature data, the hydrolysis of hemicelluloses into monomeric sugars is promoted by extraction at higher temperatures for longer times and lower pH level [31].

Solubilization of lignin during the extraction of hemicelluloses is expected especially in the basic medium. As shown in Fig. 4B, the amount of soluble lignin increased after microwave sawdust irradiation. A significant difference was observed for MAE at high power input (4.8% and 0.49% at 573 W for 1 h compared with 3.3% and 0.3% at 100 °C for 1 h in basic and neutral medium, respectively). Acetic, uronic, formic and levulinic acids have also been detected in the liquid effluents. The carboxylic acid contents were relatively important in basic medium (Fig. 4A). Acetic and formic acids are the major carboxylic acids formed (6 and 1.7 g/l, respectively). Levulinic acid presents the lowest concentration, accounting for 0.3 g/l. The peeling reactions occurring during alkaline treatments lead to endwise degradation of polysaccharides with formation of formic acid and different dihydroxy and dicarboxylic acids [47]. Acetic acid is released from the hemicellulosic backbone by saponification of acetate groups.

3.3 Composition of the recovered hemicellulose powder

The sugar composition of polymeric hemicelluloses recovered after precipitation and dialysis is similar to the sugar composition of the liquid fraction (Fig. 5). Galactoglucomannans and arabinoxylans are the main polysaccharides extracted in neutral and basic medium, respectively. In neutral medium, MAE promotes the extraction of arabinose and galactose compared with conventional extraction. In fact, hemicelluloses obtained from neutral medium account for 24% and 19.5% of galactose and 10.5% and 5% of arabinose for mircowave (573 W) and conventional extraction, respectively. The advantage of MAE not only seems to be due to the temperature but also is owed to the physicochemical interactions induced by microwave heating of the wood matrix.

In native softwood, mannopyranosyl units are acetylated at C-2 or C-3 with a degree of acetylation of about 0.5 [9,17]. In our study, the precipitated hemicelluloses recovered from a neutral medium had a lower degree of acetylation, in the range of 0.10–0.15. In basic medium, the acetyl groups have been totally released from the hemicellulosic backbone by saponification.

These results have been confirmed by an FTIR study. In Fig. 6, two weak absorbances at 1735 and 1247 cm⁻¹ due to acetyl groups attached to the hemicellulose chains were observed for the fractions extracted in neutral medium but not for the basic medium extracts. Moreover, the absorption at 1515 cm⁻¹ observed in all the hemicellulosic fractions was assigned to lignin's aromatic skeletal vibrations [48]. The higher intensity of this signal for the fractions isolated from basic media confirms the higher lignin contamination for hemicelluloses obtained in basic medium than those obtained in neutral medium previously observed (Fig. 4B).

Two-dimensional ¹H–¹³C HSQC spectra clearly show the typical signal spots expected for the hemicellulosic moieties. In basic medium (Fig. 7B), β-d-xylopyranoside units are specified by these signals at δC/δH 60/3.4; 62/4; 72/4.3; 72.5/3.2; 74/3.5; 72.5/3.7. The signals at 72/3.5 are assigned to α-d-xylopyranoside of xylans reducing end, and those at 62/3.3 and 71/3.5, to β-d-xylopyranoside of xylans nonreducing end. In neutral medium (Fig. 7A), the signals at 3.4/74; 3.5/74; 3.8/76; 4.2/70 are attributed to β-d-mannopyranoside. The same results were found in the study by Giummarella and Lawoko [49]. The signal in neutral medium at 5.45/72 was assigned to 6-O-acetyl β-d-mannopyranoside. In fact, as confirmed by the FTIR analysis, the extraction in neutral medium preserved some of the acetyl groups of the backbone units of GGM. These results are in close agreement to those obtained by Thornton et al [46] who found that deacetylation of the O-acetyl-galactoglucomannans appeared to begin at pH values above 8. The presence of acetyl residues is critical for the water solubility of GGM. The acetyl moieties are generally cleaved during the extraction at high temperatures leading to the release of acetic acid. The signals at 98/5.25 and 82/3.1 are assigned to 4-O-methyl-α-D-glucuronic acids that are spotted only in basic medium which confirms the extraction of arabinoglucuronoxylans in these conditions.

Weight-average (M_w) and number-average (M_n) molecular weights, as well as the polydispersity index (PDI), were determined by gel-permeation chromatography. The results in Table 1 show that hemicellulose fractions isolated from microwave irradiation display higher molecular mass than the conventionally extracted fractions (for conventional extraction, M_w ∼38,000 and ∼56,000 g mol⁻¹ in neutral and basic medium, respectively, and ∼42,000 and ∼67,000 g mol⁻¹ for hemicelluloses extracted with microwave activation in neutral and basic medium, respectively). It can be also observed that the hemicelluloses extracted in neutral medium had much lower molecular weights than those isolated in basic medium. These results were in close agreement to those reported by Sun et al [50] who found that increasing the alkali concentration during extraction increased the M_w of extracted hemicelluloses by 46%. They suggested that the extraction of high M_w hemicelluloses was owed to the increase of the coextraction of lignin-carbohydrates complexes. Considering the sugar composition given in Fig. 5, it can be concluded that the neutral medium released GGM with relatively low molecular weight, while basic extraction allowed the recovery of high-molecular-mass xylans.