*3.2.2 Methods of nanocellulose isolation*

In the last decades, various methods to manufacture CNF from regenerated cellulose and natural raw materials have been developed. These methods include electrospinning [49, 50], biosynthesis [51, 52], and mechanical isolation. Some review papers have been published to compare these methods [53–55]. The conventional needle-based electrospinning techniques for producing CNF deteriorates from the use of toxic solvents, very low productivity (spinning rate: 10 ml/h), the use of high voltage [56] and poor crystallinity. Biosynthesis by bacteria yields nanofibers with narrow diameter distributions, high aspect ratios and high crystallinity. However, it has several constraints such as the requirement for strict and high-cost production, poor reproducibility between the bacteria of different generations, and the complex post-treatment purification procedures. According to Sharma [36], the extraction of nanocellulose may be performed via three different methods; acid hydrolysis, enzymatic hydrolysis or mechanical treatment processes.

#### *3.2.2.1 Acid hydrolysis*

Acid hydrolysis is a common method used to isolate NC. The amorphous region of the cellulose fibrils may be hydrolysed using strong acids such as sulfuric acid via the esterification of hydroxyl groups by the sulphate ions. It will subsequently make the crystalline region of cellulose fibers to produce a stable colloidal dispersion of nanocellulose materials in the remaining reaction mixture. Sulfuric acid is the most used acid for the hydrolysis process. Other researchers have also used several mild acids such as formic, acetic, phosphoric and chloric acids for hydrolysis as summarized in **Table 5**. This reaction depends on three primary reaction parameters; reaction time, temperature and acid concentration, which also influences the properties of the nanocellulose product. The washing procedure is normally conducted using cold water followed by centrifugation or using sodium hydroxide until neutral pH is obtained. For instance, Pelissari et al. [40] mixed 1% v/v H2SO4 solution with the insoluble residue at 80°C for 1 h to eliminate the mineral traces and hydrolyse amorphous cellulose, providing the nanofibers product. The insoluble residue was then neutralized with alkaline or acid solution (5% KOH or 10% acetic acid)

depends on the pH followed by centrifugation (10,000 rpm; 5°C; 20 min) until neutral pH was obtained. The final residue was diluted with deionized water and the suspension was stored at 4°C in a sealed bottle.
