*3.4. Crystallinity of Cellulose*

− − − The effect of organosolv pretreatment on the crystallinity of OPEFB was determined by calculating the total crystallinity index (TCI) value (absorbance ratio at wavenumbers 1248 and 897 cm−<sup>1</sup> ). Crystallinity index can be used to observe changes in cellulose digestibility after pretreatment. Glucan-rich fraction spectra showed a higher absorption band at 897 cm−<sup>1</sup> and a lower absorption band at 1428 cm−<sup>1</sup> (Figure 5). The result shows an increase in amorphous cellulose and a decrease in crystalline cellulose after pretreatment which indicates an increase in cellulose digestibility. The TCI value of the untreated OPEFB was 1.25 ± 0, whereas after pretreatment in the best conditions, i.e., 0.07% H2SO4, 210 ◦C, 90 min, and a solid-to-liquid ratio of 1:10, the TCI was 0.95 ± 0.03, representing a reduction of 24.48%.

− − **Figure 5.** FTIR spectra of **(1)** untreated oil palm empty fruit bunches (OPEFB) and **(2)** glucan-rich fraction from the best organosolv pretreatment condition (0.07% H2SO<sup>4</sup> , 210 ◦C, 90 min, and solid-to-liquid ratio of 1:10). Bar **(a)** shows the absorbance at 1428 cm−<sup>1</sup> whereas bar **(b)** shows the absorbance at 897 cm−<sup>1</sup> . − −

### *3.5. Enzymatic Hydrolysis*

Untreated OPEFB and a mixture of evaporated glucan-rich fraction and hemicellulosic compounds-rich fraction were digested using 10 FPU of Cellic®® Ctec3 enzyme per gram of glucan, for 24 h. The use of organosolv pretreatment led to an increase of 4.56-fold in glucan digestibility (Figure 6a). Enzymatic hydrolysis of evaporated hemicellulosic compounds-rich fraction resulted in 0.25 g/L glucose after 18 h. Since an enzyme loading of 10 FPU/g glucan could not achieve 100% glucan digestibility, higher enzyme loadings were used (Figure 6b). There was no difference on glucan digestibility between 18 h and 24 h of enzymatic hydrolysis. Therefore, enzymatic hydrolysis for 18 h resulted in glucan digestibility of 36.29 ± 2.84%, 55.45 ± 3.72%, 82.66 ± 3.09%, and 94.06 ± 4.71% at enzyme loading of 10, 15, 20, and 30 FPU/g glucan, respectively.

**Figure 6.** Glucan digestibility of (**a**) untreated OPEFB and of a mixture of evaporated glucan-rich and hemicellulosic compounds-rich fractions at enzyme loading of 10 FPU/g glucan and (**b**) a mixture of evaporated glucan-rich and hemicellulosic compounds-rich fractions using different enzyme loadings. Error bars represent ± 2× the standard deviation.
