*2.2. Pretreatment of Wheat Straw*

The severity factor (SF; Equation (1)) of different pretreatment conditions was determined as the logarithm of the reaction ordinate, as proposed by Overend and Chornet [25] (Equation (2)).

$$SF = L\text{og }R\text{o} \tag{1}$$

$$Ro = t \times \exp\left(\frac{Tr - 100}{14.75}\right) \tag{2}$$

where *t* is the holding time of pretreatment in minutes, and *Tr* is the pretreatment temperature in ◦C.

For each pretreatment batch, 37.5 g of wheat straw (on dry weight (DW) basis) was mixed with tap water (for A-HTP) or aqueous sulfuric acid (for SA-HTP) at a 7:1 liquidto-solid ratio in a 1-L reactor (Parr 4520, Moline, IL, USA). The loading of sulfuric acid (96%) was 0.5 g per 100 g wheat straw (DW). The reaction time was 15 min. For A-HTP, the temperature settings were 160, 175, 190, and 205 ◦C, which correspond to SF values of 2.9, 3.4, 3.8, and 4.3, respectively. For SA-HTP, the temperature settings were 160 and 190 ◦C, which correspond to SF values of 2.9 and 3.8, respectively. The pretreatment conditions were chosen within the ranges typically reported for wheat straw [8–10]. At the cooling stage of pretreatment, when the temperature was approximately 90 ◦C, a sample was carefully taken from the gas phase on the top of the reaction suspension. The sample was received under a water column in a 15-mL Falcon tube, and it was analyzed immediately (Section 2.6). After pretreatment, the solid and liquid phases were separated by vacuum filtration, and the solid phase was washed with 1 L water. A portion of the filter cake was stored frozen for analytical enzymatic saccharification assay, and the rest was air-dried for around one week; the yield of pretreated solids was determined gravimetrically based on DW. The weight of the portion used for enzymatic saccharification was included in the calculation of the DW of pretreated solids (DW of PSs).

The yield of pretreated solids (*PSs*) was calculated as follows:

$$\text{Yield of PSs (\%)} = \frac{\text{DW of PSs (g)}}{\text{Initial DW of biomass (g)}} \times 100\tag{3}$$
