*2.3. Compositional Analysis of the Raw Material and Pretreated Solids*

The content of extractives in raw wheat straw and pretreated solids was determined by extracting aliquots of each material with 200 mL ethanol in a Soxhlet system (Extraction System B-811, Büchi, Flawil, Switzerland). Structural carbohydrates and lignin were determined using TSSA. TSSA was performed essentially according to an NREL protocol [26] but using HPAEC for quantification of monosaccharides. HPAEC was performed using a Dionex ICS-5000 (Sunnyvale, CA, USA) instrument with pulsed amperometric detection (PAD), a separation column (4 × 250 mm), and a guard column (4 × 50 mm) (both CarboPac PA1, Dionex, Sunnyvale, CA, USA). Prior to the analysis, the samples were diluted with ultra-pure water and filtered through 0.2 µm Millex-GN nylon membranes (Merck Millipore Ltd., Cork, Ireland). Acid-insoluble (Klason) lignin was determined gravimetrically and acid-soluble lignin (ASL) was determined spectrophotometrically at λ 240 nm. Ash content was determined using an NREL protocol [27]. All analyses were performed in triplicates. Values are stated as mass fractions of initial masses of samples (DW).

The recoveries of glucan and xylan were calculated using the following equations:

$$\text{Glucan recovery} \left(\% \right) = \frac{\text{G}\_{\text{PS}} \times \text{Yield of PS}}{\text{G}\_{\text{RM}}} \times 100 \tag{4}$$

$$\text{Xylan recovery} \left( \% \right) = \frac{\text{X}\_{\text{PS}} \times \text{Yield} \, of \, \text{PS}}{\text{X}\_{\text{RM}}} \times 100 \tag{5}$$

Here, *GPS* and *XPS* are the mass fractions of glucan and xylan, respectively, in pretreated solids (PS), and *GRM* and *XRM* are their mass fractions in the raw material.

Total lignin recovery was calculated using the following equation:

$$\text{Lignin recovery} \left( \% \right) = \frac{(\text{KL} + \text{ASL})\_{\text{PS}} \times \text{Yield of PS}}{(\text{KL} + \text{ASL})\_{\text{RM}}} \times 100\tag{6}$$

Here, *KL* is the mass fraction of Klason lignin, and *ASL* is the mass fraction of acidsoluble lignin in pretreated solids (*PS*s) and raw material (*RM*).

### *2.4. Pyrolysis-Gas Chromatography/Mass Spectrometry (Py-GC/MS)*

Py-GC/MS analysis was performed at the Biopolymer Analytical Facility of the KBC Chemical–Biological Center (Umeå, Sweden). Prior to Py-GC/MS analysis, the pretreated and raw samples were freeze-dried and ball-milled (mixer mill MM400, Retsch, Haan, Germany). Then, 50 µg biomass powder was applied to a pyrolyzer equipped with an autosampler (PY-2020iD and AS-1020E, Frontier Lab, Fukushima, Japan) connected to a GC/MS machine (7890A/5975C, Agilent Technologies AB, Kista, Sweden). The pyrolysate was separated and analyzed, as described by Gerber et al. [28]. Areas were determined for peaks assigned as carbohydrates (C), guaiacyl (G), syringyl (S), *p*-hydroxyphenyl (H), generic phenolic constituents (P), known spectra unknown identification (U), and unknown spectra (0). Values are stated as percentages of total peak areas (C + G + S + H + P + U + 0). The fraction of lignin (L) corresponds to the sum of the fractions of G, S, H, and P.
