2.8.3. Thin-Layer Chromatography

Thin-layer chromatography (TLC) was performed to qualitatively analyse the xylooligomeric composition of liquid samples. The samples (5 µL) and the standard mixture (5 µL) containing xylose and xylo-oligosaccharides (DP2-DP6) (Megazyme, Bray, Ireland) were dotted on a silica gel plate. The analysis was performed using butanol/acetic

acid/water (2:1:1) as running solvent and revealed by water/ethanol/sulfuric acid (20:70:3) solution with 1% *v/v* orcinol over flame [40].

## 2.8.4. Determination of Oxygen Transfer Rate

Oxygen transfer rate (OTR) in shake-flask fermentation systems was determined by multiplying the maximum level of dissolved oxygen concentration (C\*) achievable and the gas–liquid mass transfer coefficients (kLa). The C\* was determined by optical oxygen sensor (VisiFerm DO 120, HAMILTON Bonaduz AG, Switzerland) in shake flasks containing semidefined xylose medium or xylose-rich hydrolysates (WB1/S, GRS/S) at 30 ◦C. The kLa was determined by a non-fermentative gassing-out method [41]. Dissolved oxygen was removed from the fermentation medium by bubbling it with nitrogen gas. After that, the headspace of the flasks was washed out with air, and the shaking was restarted. Then the dissolved oxygen concentration was continuously increased until reaching a constant value (maximum level of dissolved oxygen concentration (C\*)). The expression of -ln(1-(C/C\*)) was plotted as a function of time, where C is the actual dissolved oxygen concentration at that given point in time. A linear curve was fitted to the plotted data. The slope of the fitted linear curve was equal to the value of kLa [42]. The OTR in 100-mL shake flasks filled with 35, 50, and 65 mL semi-defined xylose medium were 1.1 (0.1), 2.1 (0.1), and 3.1 (0.1) mmol O2/(L × h), respectively, with the standard deviations indicated in parenthesis. The OTR values of the wheat bran and rice straw hydrolysates were 1.6 (0.2) and 2.1 (0.2) mmol O2/(L × h), respectively. OTR values of 1.1, 2.1, and 3.1 mmol O2/(L × h) in semi-defined medium correspond to kLa values of 5, 10, and 15 (1/h), respectively. OTR values of 1.6 mmol O2/(L × h) in WB1/S and 2.1 mmol O2/(L × h) in GRS/S correspond to kLa values of 8 and 10 (1/h), respectively.
