*2.4. Enzymatic Hydrolysis*

Solids resulting from consecutive stages of autohydrolysis (AS) and delignification under selected conditions were subjected to hydrolysis using the enzymatic complex Cellic CTec2 (Novozymes, Denmark). The enzymatic activity of Cellic CTec2 was 137 FPU (Filter Paper Units)/g of enzyme [30]. Hydrolysis assays were performed in Erlenmeyer flasks kept at 50 ◦C in an orbital incubator (150 rpm). The pH of media was adjusted to 4.8 by adding 50 mM citrate buffer. Experiments were performed for 0–96 h at LSR = 15 g/g using an enzyme to solid ratio (ESR) of 15 FPU/g substrate. All assays

were performed by triplicate. Aliquots of samples were withdrawn at selected times, centrifuged, filtered, diluted and assayed for glucose by HPLC as described in Section 2.5. Cellulose conversion into glucose, and xylan conversion into xylose were calculated as follows:

$$\% \text{cellulos conversion into glucose} = 100 \times \frac{[Glu]}{[Glu\_{Pot}]} \tag{5}$$

where [*Glu*] is the glucose concentration and [*GluPot*] is the potential glucose concentration (calculated assuming total conversion of the cellulose contained in the substrate);

$$\% \text{ xylan conversion to xylose } = 100 \times \frac{[\text{Xyl}]}{[\text{Xyl}\_{Pot}]} \tag{6}$$

where [*Xyl*] is the xylose concentration and [*XylPot*] is the potential xylose concentration (calculated assuming total conversion of the xylan contained in the substrate).
