*2.3. Chemical Characterization of CC*

The total carbohydrates of CC were determined in triplicate by using a modified sulphuric acid method described previously [21]. Briefly, 300 mg of CC (untreated and pre-treated) was hydrolyzed with 72% (*v*/*v*) sulphuric acid at 30 ◦C for 1 h, diluted to 3% (*v*/*v*) sulphuric acid and autoclaved to solubilize the carbohydrate fraction. Following the hydrolysis, fractions were filtered to remove the insoluble lignin from the solution.

Determination of the alkali-extractable hydroxycinnamic acid content was carried out by treating 10 mg of biomass with 1 M NaOH solution for 24 h at room temperature and in the dark. The liquors obtained from alkaline treatments were separated from the solid fraction by centrifugation at 16,000× *g* for 5 min. The liquors were neutralized by 2 volumes of 1 M HCl and analyzed by HPLC as described in Section 2.4.

The morphological structure of untreated and pre-treated biomass was analyzed with a scanning electron microscope (SEM), JOEL JSM 840. CC samples were mounted on a metal stub with adhesive tape and coated with a thin layer of gold prior to SEM analysis.

In order to detect changes in functional groups, FTIR analysis of untreated and pre-treated CC was conducted by loading a few milligrams of pulverized samples in the universal ATR of a Spectrum 100 FT-IR spectrometer system (Perkin Elmer, Wellesley, MA). FT-IR spectra were recorded in quadruple at a range of 650–4000 cm−<sup>1</sup> with a resolution of 4 cm−<sup>1</sup> .
