*3.1. OxiOrganosolv Fractionation of Wheat Straw with ACO and EtOH as Organic Solvents*

Initially, wheat straw biomass was organosolv-pretreated by following the OxiOrganosolv process [6] with an H2O/ACO 50:50 or an H2O/EtOH 50:50 co-solvent system. The pretreatment time was kept constant at 120 min in all cases upon addition of 16 bar O<sup>2</sup> as a catalyst. The only parameter shifting was the temperature that was regulated at 150, 160 and 175 ◦C for both solvent mixtures, resulting in six different experimental runs. Pretreatment conditions as well as compositional analysis, cellulose/hemicellulose recovery and lignin removal of solid fraction are shown in Table 1. The results showed that, with a constant residence time of 120 min, increasing the pretreatment temperature results in a corresponding increase in the % biomass solubilization in the case of both ACO and EtOH. This was attributed to the removal of the hemicellulose and lignin fractions from the wheat straw substrate. Delignification peaked at 175 ◦C of pretreatment both in the case of ACO and EtOH, reaching 87.5 ± 0.6% and 86.9 ± 0.1% for sample S3 and S6, correspondingly. Notably, cellulose recovery in the solid fraction was almost 100% in almost all cases in both solvents, indicating only very limited hydrolysis of cellulose in the pre-treatment conditions that were studied. On the other hand, hemicellulose recovery in the solid fraction reached the highest values of 82.6 ± 0.8% in the case of ACO (sample S1) and 84.6 ± 0.1% in the case of EtOH (sample S4) in the lowest temperature of 150 ◦C, while increasing temperature resulted in increased hemicellulose removal and also combined with increasing lignin removal. As such, both solvents achieved very good delignification of the wheat straw, while ACO was also more effective for the removal of hemicellulose, resulting in the most cellulose-rich pulps.

**Table 1.** Pretreatment conditions, compositional analysis of solid pulps, lignin removal and % recovery of cellulose and hemicellulose. Standard error is ≤2.5% in all measurements.


<sup>1</sup> Unless stated, hemicellulose fraction consists only of xylose-containing structures. <sup>2</sup> Contains 28.1 wt% xylose and 0.3 wt% galactose. <sup>3</sup> Contains 21.8 wt% xylose, 0.5 wt% mannose and 0.8 wt% galactose. <sup>4</sup> Includes also extractives 15.5 wt% and inorganic content (ash) 3.1 wt%.

> The aqueous liquid fraction resulting from the organosolv pretreatment was examined for hemicellulose recovery after organic solvent evaporation and lignin removal by vacuum filtration. The results, presented in Figure 1 and in Table 2, demonstrate that the highest % of hemicellulose recovery in the form of monosaccharides and polysaccharides was achieved when the temperature was the highest for both organic solvents, reaching up to 32.7 ± 1.2% for ACO, 175 ◦C, 120 min (sample L3) and 34.4 ± 0.9% for EtOH, 175 ◦C, 120 min (sample L6). These values accounted for 119 and 88.3 mg/g of untreated wheat straw biomass after ACO or EtOH pretreatment, respectively. The aforementioned results are in agreement with the % hemicellulose recovery values in solid pulps, as depicted in Table 1, where the samples that underwent pretreatment at higher temperatures stood out for their high cellulose and low hemicellulose content. As such, the results in Table 2 demonstrated a dwindling trend in the oligosaccharides/monosaccharides ratio in the aqueous phase when the pretreatment conditions became more severe in the case of ACO. More specifically, the ratio was 10.5 for 150 ◦C, 120 min (sample L1) noting a remarkable decline to 2.2 for 175 ◦C, 120 min (sample L3), which was linked to the degradation of

oligosaccharides in severe conditions. Contrariwise, the corresponding data in the case of EtOH did not follow the same trend, instead exhibiting a similar ratio regardless of the pretreatment temperature, and more specifically, 10.8 for 150 ◦C, 120 min (sample L4) and 10.5 for 175 ◦C, 120 min (sample L6). EtOH did not follow the same trend, instead exhibiting a similar ratio regardless of the pretreatment temperature, and more specifically, 10.8 for 150 °C, 120 min (sample L4) and 10.5 for 175 °C, 120 min (sample L6). **Table 2.** Compositional analysis of monosaccharides/oligosaccharides in the liquid fraction. Stand-

are in agreement with the % hemicellulose recovery values in solid pulps, as depicted in Table 1, where the samples that underwent pretreatment at higher temperatures stood out for their high cellulose and low hemicellulose content. As such, the results in Table 2 demonstrated a dwindling trend in the oligosaccharides/monosaccharides ratio in the aqueous phase when the pretreatment conditions became more severe in the case of ACO. More specifically, the ratio was 10.5 for 150 °C, 120 min (sample L1) noting a remarkable decline to 2.2 for 175 °C, 120 min (sample L3), which was linked to the degradation of oligosaccharides in severe conditions. Contrariwise, the corresponding data in the case of

**Table 2.** Compositional analysis of monosaccharides/oligosaccharides in the liquid fraction. Standard error is ≤2.5% in all measurements. ard error is ≤ 2.5% in all measurements. **mg/g of Untreated % Hemicellulose Recovery in Liquid** 


*Fermentation* **2021**, *7*, 219 7 of 16

**Figure 1.** Compositional analysis and hemicellulose sugar profile of liquid fractions. The concentration of sugars in the form of (**a**) monosaccharides and (**b**) oligosaccharides is presented. Standard error is ≤ 2.5% in all measurements. **Figure 1.** Compositional analysis and hemicellulose sugar profile of liquid fractions. The concentration of sugars in the form of (**a**) monosaccharides and (**b**) oligosaccharides is presented. Standard error is ≤2.5% in all measurements.

The optimal temperature for efficient % hemicellulose recovery in the liquid fraction was 160 °C (sample L2) in the case of ACO, resulting in 25.4 ± 2.1% hemicellulose recovery in the form of oligosaccharides and only 2.8 ± 0.2% in the form of monosaccharides. In the case of EtOH, the optimal temperature for high hemicellulose recovery was 175 °C (sample L6), achieving 31.4 ± 1.4% hemicellulose recovery in the form of oligosaccharides and only 3.0% in the form of monosaccharides. Furthermore, no presence of sugar degradation products was detected, while phenolic compounds were found only in low quantities, as identified by the Folin–Ciocâlteu method (Supplementary Table S1). The optimal temperature for efficient % hemicellulose recovery in the liquid fraction was 160 ◦C (sample L2) in the case of ACO, resulting in 25.4 ± 2.1% hemicellulose recovery in the form of oligosaccharides and only 2.8 ± 0.2% in the form of monosaccharides. In the case of EtOH, the optimal temperature for high hemicellulose recovery was 175 ◦C (sample L6), achieving 31.4 ± 1.4% hemicellulose recovery in the form of oligosaccharides and only 3.0% in the form of monosaccharides. Furthermore, no presence of sugar degradation products was detected, while phenolic compounds were found only in low quantities, as identified by the Folin–Ciocâlteu method (Supplementary Table S1).
