*2.3. Enzymatic Hydrolysis of Solid Pulps*

Both the cellulose-rich solid fraction and the hemicellulose-rich liquid fraction of the OxiOrganosolv pretreatment were exploited for the production of DHA with *C. cohnii*. An SHF approach (separated hydrolysis and fermentation) was employed because the conditions of these operations are different.

The solid fraction was enzymatically hydrolyzed to fermentable sugars following a procedure previously reported [19]. The reaction took place by employing a commercial enzyme cocktail, namely Cellic® CTec2 (Novozymes A/S, Bagsværd, Denmark), with a protein content of 95.6 mg/mL, as determined by Bradford assay [23]. Hydrolysis was performed in 250 mL glass flasks, at 50 ◦C, under agitation (160 rpm) and at an initial dry matter (DM) of 9% (*w*/*v*), in order to obtain a hydrolysate with high sugar concentration, suitable for *C. cohnii* cultivation. An enzyme loading of 9 mg/g of biomass was used, and the pH levels were maintained constant at 5.5 upon addition of 80 mM MES (2-Nmorpholino-ethanesulfonic acid) buffer solution. The final ratio of the reaction volume to shake flask volume was 1/10.

In order to track down the glucose and total reducing sugars (TRS) production, the glucose oxidase/peroxidase (GOD/POD) [24] and 3,5-dinitrosalicylic acid (DNS) method [25] were used, respectively. Samples were taken at different time intervals (8, 24, 48 and 72 h), centrifuged before each analysis, and the supernatant was filtered with 0.22 µM pore size filters. As a complement, in order to accurately determine the monosaccharide (glucose, xylose) and acetic acid concentration, a chromatographic analysis was performed (HPLC) by isocratic ion-exchange chromatography using an Aminex HPX-87H column with a micro-guard column, at 50 ◦C (Bio-Rad Laboratories, Hercules, CA, USA), using 3 mM H2SO<sup>4</sup> as a mobile phase at a flow rate of 0.6 mL/min. After 72 h of hydrolysis and analysis of released products, all supernatants containing fermentable sugars were collected after vacuum filtration and kept in the freezer (−20 ◦C) for future use.
