*2.4. Detoxification and Enzymatic Hydrolysis of Liquid Fractions*

Detoxification and enzymatic hydrolysis of liquid fractions were required before the evaluation of pretreatment aqueous liquors as carbon source for *C. cohnii*. Even after the initial removal of the OxiOrganosolv organic solvent, as described in Section 2.1, some residual organic solvent with potential to alter the microalga metabolism was still present (as detected by HPLC analysis), so an additional evaporation step was necessary. After the removal of the organic solvent, the liquid fraction was filled with water up to its original volume in order to maintain the same sugar concentration as the corresponding sample prior to evaporation. Moreover, the OxiOrganosolv-derived liquid fractions contained phenolic compounds that are toxic to the microalgae used. Therefore, 5% (*w*/*v*) active carbon was used for the removal of phenol compounds, whose levels were determined with the Folin−Ciocalteu method [26].

The liquid fractions are richer in hemicelluloses in comparison with the solid fractions. However, most of the compounds are found in the form of oligosaccharides, which cannot be metabolized by *C. cohnii*. As a result, hydrolysis with Cellic® HTec2 enzyme cocktail (Novozymes A/S, Bagsværd, Denmark) was carried out in order to produce fermentable monosaccharides. The protein content was 75 mg/mL, as calculated by the Bradford method [23]. Hydrolysis experiments were performed in 500 mL glass flasks at 45 ◦C under agitation (100 rpm) with an enzyme loading of 10 mg/g of oligosaccharides at pH 5.5

(self pH, no addition of buffer). The total reaction volume to shake flask volume was 1/10. Samples were analyzed with HPLC by isocratic ion-exchange chromatography using an Aminex HPX-87P column with a micro-guard column at 85 ◦C (Bio-Rad Laboratories, Hercules, CA, USA) and using water as a mobile phase at a flow rate of 0.6 mL/min. With this HPLC analysis, the levels of glucose, xylose, mannose, galactose and arabinose were determined. The hydrolysis yield was calculated based on the increase of monosaccharides after enzymatic reaction.
