## 3 Results and Discussion
### Chemical Characterization of Feedstock and Processed Solids
#### 3.2.2 Xylo-Oligosaccharide Production and Byproduct Release  
In addition to monosaccharides, xylo-oligosaccharides with a low degree of polymerization (DP \(<\) 6) (xylotriose, X3, and xylotetraose, X4) were also detected in the hydrolysate after enzymatic and alkaline treatment of the residues. We observed that during the hydrothermal pretreatment, xylo-oligosaccharides were not detected, except a very low amount of xylobiose (0.01 g/L) in the hydrolysate at 180 \({}^{\circ}\)C, which might be caused by the autohydrolysis conditions, for instance, the long residence time (30-40 min) and pressure conditions (12 bar) in the reactor until it reached the desired temperatures. [PERSON] et al. applied an acidosis treatment with acetic acid to wheat straw and reported that the yield of released XOS was also dependent on the structural composition of the raw material, specifically the wax content and inert structure, which inhibit the production of XOS [42]. However, after the enzymatic and alkaline treatment of the residues, statistically significant differences were noticed. During the enzymatic treatment, xylanase was the most efficient in the hydrolysates obtained at 160 \({}^{\circ}\)C, with an amount of 1.23 \(\pm\) 0.12 g/L XOS released, compared to the enzymatic mix, which showed an increasing trend of liberated XOS from 0.59 \(\pm\) 0.03 g/L in the hydrolysates at 140 \({}^{\circ}\)C and at 160 \({}^{\circ}\)C to a maximum amount of 0.78 \(\pm\) 0.03 g/L in the liquids at 180 \({}^{\circ}\)C. When combining enzymatic with alkaline treatment, which was shown to improve enzymatic digestibility, higher yields of XOS were obtained, showing statistically significant differences when compared to the enzymatic treatment. Xylotriose and xylotetraose were detected in the hydrolysates at 140 \({}^{\circ}\)C, with a content of 1.48 \(\pm\) 0.2 g/L, and 1.33 \(\pm\) 0.13 g/L in the filtrate at 180 \({}^{\circ}\)C, after 48 h of enzymatic hydrolysis with xylanase at pH 4.6. [PERSON] et al. reported concentrations of 1.94 \(\pm\) 0.045-2.07 \(\pm\) 0.068 mg XOS/mL (X2-X5) at pH 7 and 8, respectively, after enzymatic hydrolysis at 60 \({}^{\circ}\)C of wheat straw using an endoxylanase from _B. halodurans_[58]. Production of XOS from wheat straw was also studied by [PERSON] et al., who reported 0.079 g XOS/g xylan after enzymatic hydrolysis with endoxylanase from _Aspergillus niger_[59]. Slightly lower yields were obtained when using the enzymatic mix Cellic CTec2, with an increasing trend of XOS being observed in the hydrolysates, from 1.11 \(\pm\) 0.05 g/L in the hydrolysate at log R\({}_{0}\) = 2.35 to a maximum of 1.26 \(\pm\) 0.06 g/L in the hydrolysate at log R\({}_{0}\) = 3.53. [PERSON] et al. obtained higher yields of xylotryose and xylotetraose after enzymatic hydrolysis of pretreated wheat straw at 180 \({}^{\circ}\)C for 20 min (X3 + X4: xylotriose: 3.1 \(\pm\) 0.7 g/L). The literature shows that XOS produced via alkaline enzymatic treatment have low aqueous solubility, since the acetyl and uronic groups are completely degraded [2,21]. During autohydrolysis, polysaccharides and monosaccharides could further decompose in degradation products like acetic acid, furfural and hydroxymethylfurfural (HMF). Figure 2a,b show the results of these degradation products. It was noticed that the yield of acetic acid increased with the severity of the hydrothermal treatment, from 0.15 \(\pm\) 0.02 g/L to 1.32 \(\pm\) 0.10 g/L in the hydrolysate at 180 \({}^{\circ}\)C. The highest content of acetic acid was detected after enzymatic treatment with xylanase and the combined alkali enzymatic with the same enzyme in the hydrolysates at 180 \({}^{\circ}\)C, with yields of 1.71 \(\pm\) 0.12 g/L and 1.76 \(\pm\) 0.13 g/L, showing statistically significant differences when compared to the other enzymatic treatment applied. Acetic acid originated from the hydrolysis of acetyl groups on the hemicellulose backbone, with the increase in the concentrations of byproducts leading to a decrease in the pH of the filtrate (from 5.2 to 3.9 after HTP).