Hemicelluloses have been extracted from Okoumé sapwood by SE according to different pathways given in Fig. 1. A bleaching step performed before or after SE was performed with an oxidative agent (chlorite), SE and B/SE hemicelluloses were recovered in the liquid effluent of the SE process, performed at 170°C during 10 min and BAlc and SE/BAlc by an alkali extraction from the beached pulp. B/SE and SE/BAlc are the result of combined processes according to Fig. 1. All the hemicelluloses were isolated after ethanolic precipitation and dialysis.
3.1 Microscopy
Morphological analysis of the cell wall of untreated Okoumé sapwood, Pulp NaOH and Pulp H2O was investigated by astra blue-Safranine double staining of the cell wall. Representative pictures are shown on Fig. 2. A pink staining of the wood tissues indicated a low accessibility of the astra blue dye to the cellulose due to the lignin occurrence in the cell wall. A cell shape alteration with a reduction in sphericity was observed after SE as previously described by (Besserer et al., 2022) in Okoumé wood. Moreover, cell wall of the pretreated sample differed strongly in their staining. The blue color of NaOH-SE reveals a delignification of the S3 and S2 layers with a pink/red staining only occurring in the cell corner and in the S1/ middle lamella layers.
To investigate further the steam explosion effects on pulps, fluorescence microscopy was used to analyze the relative distribution of sugars and lignin within the cell wall of untreated okoumé sapwood and NaOH-SE (Fig. 3). Safranin is a metachromatic dye which bind to holocellulose and emits red fluorescence when cell wall is lignified and green signal in low lignin containing cell wall (Bond et al., 2008a). In agreement with the literature (Bond et al., 2008b; Lancha et al., 2021), Fig. 3A shows that the cell wall layers S1-S3 of untreated wood are composed of a mixture of sugars and lignin, which appears in a yellow stain, the middle lamella being rich in lignin (Fig. 3B). In contrast, in NaOH-SE large changes are observed in the distribution of sugars and lignin within the cell wall. Indeed, Fig. 3C-D shows a high holocellulose content in the S1-S3 sublayers of the cell wall. Comparison of the line profiles (Fig. 3B and Fig. 3D) suggests a migration of lignin at the lumen surface of the fibers. This is also well described in the literature (Besserer et al., 2022). These observations are due to lignin dissolution in basic conditions and are in accordance with the observation by optical microscopy after staining (Fig. 2). We can see by comparing Fig. 3B and Fig. 3D a visible increase in the accessibility of the wall polysaccharides after SE and thus a decrease in recalcitrance.
3.2 Characterization
Hemicelluloses fractions extracted according to the different pathways given in Fig. 1 have been quantified are characterized in terms of mass distribution (Tableau 1) and sugars and acetic acid (Tableau 2). B/Alc was isolated in high yield (18.7%) with a relatively high molecular weight. As expected for hardwood, xylose was the predominant sugars accounting for 80% w/w of the hemicellulose. The yield of hemicelluloses recovered from the liquid effluent of steam explosion (H2O-SE) was lower, exhibited lower molecular weight because of the acid-catalyzed hydrolysis of glyosidic linkages during the steaming step of steam explosion (Tableau 2).
Tableau 1. Molecular weight distribution of recovered hemicelluloses
| Treatments | Yield (%) | Mn (KDa) | Mw (KDa) | Mw/Mn |
| H2O-SE | 2.2 ± 0.3 | 6.6 | 9.1 | 1.2 |
| NaOH-SE | 4.4 ± 0.4 | 83.1 | 97.7 | 1.4 |
| B/SE | 10.2 ± 1.4 | 21.4 | 22.8 | 1.1 |
| SE/B/Alc | 11.3 ± 0.6 | 22.8 | 31.5 | 1.4 |
| B/Alc | 18.7 ± 0 .6 | 49.6 | 63.7 | 1.3 |
As shown in Tableau 2, relatively high amounts of mannose and glucose (11.3% and 9.3% respectively) were detected in H2O-SE and assigned to glucomannans (GM). This observation is in line with our previous study using a mild DMSO extraction. GM usually account for 3–4% w/w in hardwood but it has been shown by that Okoumé sapwood contained relatively higher amounts of GM (≈ 6–7%) with a ratio Glc/Man ≈ 1/1.
The xylose/arabinose (Xyl/Ara) ratio is a direct measure for the degree of substitution and an important indicator for structural feature of arabinoxylans (AX) molecules. The Xyl/Ara ratios given in Table 2 are in the following order: B/Alc < NaOH-SE < SE/B/Alc < H2O-SE ≈ B/SE. The relatively high value observed for H2O-SE and B/SE suggested a very low degree of branching which can be justified by an extensive hydrolysis of the arabinan chains during the steam explosion treatment. A comparison of the composition of B/Alc and SE/B/Alc can also provide insights into the effect of steam explosion pretreatment on the parietal hemicelluloses. Indeed, it was observed that SE/B/Alc had a lower Mw and a higher Xyl/Ara ratio than B/Alc (31.5 KDa and 40 versus 37.2 KDa and 18 respectively) suggesting a depolymerization of arabinan hemicellulose chains, in the wood cell wall during the steam explosion.
Tableau 2. Composition of sugars and acetic acid (% of dry mass of hemicelluloses molar mass of hemicelluloses) of hemicelluloses
| Treatments | Xy | Manc | Glcc | Arac | Galc | Rhac | GalAc | Xyl/Ara | Ac ac |
| H20-SE | 47.7 ± 3.1 | 11.3 ± 2.7 | 9.3 ± 1.2 | 0.5 ± 0.1 | 5.4 ± 0.3 | 4.6 ± 0.5 | 5.4 ± 0.3 | 95 | 8.4 |
| NaOH-SE | 30.7 ± 1.1 | - | 0.8 ± 0.0 | 1.4 ± 0.1 | 1.7 ± 0.1 | 1.4 ± 0.1 | 0.6 ± 0.0 | 38 | |
| B/SE | 45.5 ± 9.1 | 7.2 ± 2.4 | 18.2 ± 2.7 | 0.5 ± 0.1 | 4.5 ± 0.6 | 1.2 ± 0.1 | 5.7 ± 0.9 | 91 | 5.8 |
| SE/B/Alc | 60.1 ± 5.0 | 1.1 ± 0.1 | 1.5 ± 0.1 | 1.5 ± 0.1 | 1.5 ± 0.1 | 0.7 ± 0.0 | 0.9 ± 0.1 | 40 | |
| B/Alc | 80.7 ± 0.3 | - | 3.1 ± 0.1 | 4.4 ± 0.1 | 3.3 ± 0.1 | 2.1 ± 0.1 | 5.5 ± 0.1 | 18.3 | |
The acetylation degrees determined by HPLC after saponification of the hemicellulosic fractions are given in Table 2. As expected, NaOH-SE, B/Alc and SE/B/Alc were totally desacetylated because of the alkali conditions used for the hemicelluloses extraction. On the other hand, H2O/SE and B/SE were acetylated (8.4 and 5.8% respectively). The relatively high acetylation degree of Okoumé sapwood xylans was previously discussed ( Moukagni et al., 2021).
A comparison of H2O-SE and B/SE showed that the bleaching step prior to steam explosion greatly increased the hemicelluloses extractability. We observed an increase in yield of 5 to 10% respectively. The molecular masses of B/SE were also significantly higher than that of H2O-SE (23 KDa and 9 KDa respectively) with a lower acetylation degree (5.8 wt% for B/SE compared to 8.4 wt% for H2O-SE). These observations can be justified by the cleavage the lignocellulosic complex during bleaching previously observed by microscopy (Fig. 2 and Fig. 3) facilitating the extraction of high molecular weight hemicelluloses. This confirmed that the stability of the linkages between lignin and hemicelluloses may play a central role in hemicellulose recalcitrance during hydrothermal extraction. For B/SE a relatively higher amounts of Glc was recovered (≈ 18%). In order to rationalize this observation, an SEM study was performed and showed the presence of wood microparticles in the B/SE hemicelluloses sample. Micrographs are given in Supplementary material section SM1. These particles were not removed by the filtration technique used (Whatman paper) and were recovered after dialysis and lyophilization. This observation can be rationalized by the tearing and the ablation of wood small fragments by steam explosion, the delignified wood being more friable and fragile. We can deduct from these observations that B/SE promoted the extraction of relatively high molecular weight xylans, with a low degree of arabinan branching, low acetylation degree and associated to microsized wood fragments.
3.3 Spectroscopic characterization
The effects of the steam explosion on wood pulps (Pulp NaOH and Pulp H2O) and hemicelluloses extracts (NaOH-SE, SE/B/Alc and B/SE) were evaluated by near-infrared spectroscopy (NIRS) and compared to Okoumé wood. Result of the principal component analysis on second derivative of the spectra is plotted on Fig. 4. Sample clusters were correlated with the different sample type. Indeed, wood and pulps (cluster A and B) samples were clearly distinct from and hemicellulose extracts (clusters C-E). After loadings analysis and by comparison with the data previously published (Nisgoski et al., 2018; Sandak et al., 2010; Schwanninger et al., 2011) band influencing the clustering along PC1 and PC2 could be identified. On PC 1 axis, the clustering is mainly influenced by interaction of water with lignocellulosic matrix (4950 cm− 1, 5220 cm− 1), the 5944 cm− 1 (CH3 groups on hemicelluloses) and hemicelluloses characteristics bands (5236 cm− 1, 5804 cm− 1, 4598 cm− 1). Bands influencing the PC2 axis were assigned to O-H and C-H stretches in semi-crystalline or crystalline regions in cellulose (4802 cm− 1, 6300 cm− 1) and signals found in lignin Car–H str. and C = C str., 4700 cm− 1, first overtone strech C–H, 5890 cm− 1, first overtone C aromatic–H str 5938 cm− 1 and C–H stretch and C = O stretch, 4546 cm− 1.
As expected and in accordance with the microscopic study (see Figure 2), these results showed only weak effects of H2O-SE conditions on chemical composition of wood cell wall (Fig. 4, cluster A). Regarding NaOH-SE pulp, a shift in PC1 is justified by a significant reduction in the lignin content of the wood particles and an increased acess to carbohydrates. In turn, the carbohydrates composition as well as water interaction were slighly affected. Mean spectra on the different sample cluster were plot on Figure 5. For clarity reason, only hemicelluloses fractions are represented with native wood as reference. The most contributive bands identified by PCA analysis are indicated. Details are in supplemental data SM2. The hemicellulose fractions were characterized by a strong lignin removal and a decrease of crystalline cellulose. The B/SE fraction seems to be more acetylated with more fragmented hemicelluloses and interact less with water than the SE/B. Because SE/B has stronger interaction with water, similar hemicellulose abundance and opposite vibration signal at 5944 cm-1, it can be inferred that this sample is less acetylated than SE-NaOH and B/SE.
The hemicelluloses fractions B/SE and SE/B were analyzed by FTIR. The FTIR spectra presented in Fig. 6, show typical profiles of hemicelluloses observed by several authors in other lignocellulosic materials such as Eucalyptus and Epicea (Chadni et al., 2019; Morais de Carvalho et al., 2017). The high acetylation degree of B/SE observed by HPLC analysis (Table 2) was confirmed by a strong band at 1735 cm− 1 characteristic of carbonyl (C = O) stretching of ester function. The presence of acetate group could also be confirmed by the presence of a strong band at 1240 cm− 1 attributed to the C-0 δ (C-O) deformation bonds of the ester functions of xylans (Rowley et al., 2013). Regardless of extraction mode, all hemicellulose chains contained the band at 1614 cm− 1 attributed to adsorbed water and/or ν(COO-) uronic acids in the hemicellulose chains (Gil-Ramirez et al., 2018). This band was the most intense for B/SE. The band at 1508 cm− 1 was absent in all extracts confirming the relatively low residual lignin concentration in the hemicellulose fractions. The high xylan content in SE/B/Alc shown in Table 2 was confirmed by intense bands at 895 cm− 1 and 1040 cm− 1, assigned to C1-H bonds of the ß-(1–4)-D-xylopyranose and C-O bonds of xylans respectively (Sun et al., 2014) (Marques et al., 2010)
B/SE fraction was characterized by 1H and HSQC NMR (Fig. 7 and Fig. 8). The 1H NMR signals were assigned based on literature data (Morais de Carvalho et al., 2017; Sun et al., 2012; Teleman et al., 2003) and were reported in Table 3. The internal (1 → 4)-linked -ß-D-xylopyranosyl units of the glucurunoxylan backbone were clearly identified. The acetylation of B/SE was clearly observed by the signals at δH 2.0-2.4 ppm (CH3-CO-), δH 4.55 ppm (H3 in O-3 acetylated Xylp units), δH 4.57 ppm (H2 in O-2 acetylated Xylp units), at δH 5.12 ppm and δH 4.07 ppm (2,3- di-O-acetylated H-3 and H-4 respectively). In addition, the presence of 4-O-methylglucuronic acid was characterized by δH 5.3 ppm, 4.3 ppm and 3.5 ppm signals corresponding to the H-1, H-5, and methoxy protons, respectively, as well as a signal at 4.6 ppm assigned to the anomeric proton of MeGlcA substituted D-Xyl units. The δH peak 5.02 ppm is attributed to the H-3 of the 3-O-acetylated units substituted at O-2 by MeGlcA residues of the Xylp chain.
The distribution pattern of B/SE hemicelluloses were evaluated using HSQC NMR spectroscopy (Fig. 8). The main signals were assigned based on the literature data (Campestrini et al., 2013; Giummarella and Lawoko, 2017b; Sun et al., 2014; Yue et al., 2018). The 13C/1H chemical shifts of 102.21/4.40, 73.36/3.18, 74.00/3.4, 75.00/3.62, 63.05/4.00 (H-5eq), and 63.05/3.32 (H-5ax) ppm correspond to C-1/H-1, C-2/H-2, C- 3/H-3, C-4/H-4, and C-5/H-5 of the (1→4)-β-D-xylan skeleton, respectively. The C2/H2 cross-peak assigned to the mannose unit is detected at 3.91/68.56 ppm. The cross-peaks at 97.90/5.23, 82.42/3.13, 70.00/4.06, 60.54/3.75, and 60.00/3.40 ppm are attributed to the C-1/H-1, C- 4/H-4, C-5/H-5, and -OCH3 units of 4-O-methylglucuronic acid in the O-2 position, respectively. The presence of the acetylated O-2, O-3, and O-2,3 units detected at in the xylan backbone is confirmed by correlation at 73.28/4.62 ppm (C2/H2), 75.17/4.92 (C3/H3), and 71.32/4.75 ppm. The signal attributed to the acetylated O-3 unit corresponding to the correlation at 4.87/99.0 ppm (H1/C1) was detected. A comparison of this spectrum with that of H2O-SE showed that these two fractions exhibit great similarities in NMR. This result is in agreement with the composition of simple sugars and acetate given in Table 2.