*2.4. Analytical Methods*

The moisture content of untreated OPEFB and of the glucan-rich and lignin-rich fractions, to determine recovery yields, was quantified through sample drying in an oven at 70 ◦C until constant weight. The total solids of the evaporated mixture of glucan-rich and hemicellulosic compounds-rich fractions were determined according to Sluiter et al., (2008) [36]. The lignin-rich fraction, glucan-rich fraction, and untreated OPEFB were also analyzed for carbohydrates, lignin, and ash according to the methods described by Sluiter et al., (2008) [37], in order to determine the purity of the fractions. The percentage of lignin recovery was calculated using the following Equation (1)

$$(\%)\text{ Ligning recovery} = \frac{\text{Log} \times (\%)\text{ ligning purity}}{\text{Lf}} \times 100\% \tag{1}$$

Lop = weight of lignin-rich fraction obtained after organosolv pretreatment; Lf = weight of lignin on OPEFB feed

Derived sugars from the acid treatment of samples for compositional analysis and glucose released during enzymatic hydrolysis were measured using High Performance Liquid Chromatography (HPLC) (Waters, Milford, MA, USA). The system was equipped with a hydrogen-based column (Aminex HPX-87P, Bio-Rad, Milford, MA, USA) operating at 60 ◦C and using 0.6 mL/min of 5 mM H2SO<sup>4</sup> as the eluent. A refractive index (RI) detector (Waters 2414) was used to quantify the compounds.

The crystallinity of the untreated and pretreated OPEFB was analyzed using a Fourier Transform Infrared (FTIR) spectrometer using Nicolet OMNIC 4.1 software (Impact 410 iS10, Nicolet Instrument Corp., Madison, WI, USA). The spectral data were obtained with an average of 64 scans and resolution of 4 cm−<sup>1</sup> , in the range of 400–4000 cm−<sup>1</sup> . The total crystallinity index was calculated by the absorbance ratio of wavenumbers 1428 cm−<sup>1</sup> and 897 cm−<sup>1</sup> [38].

The nitrogen content of OPEFB was analyzed using the Kjeldahl method according to Mahboubi et al., (2017) [39] and the crude protein was obtained by using a nitrogen-to-protein conversion factor of 6.25.
