*3.4. Length-Frequency and Diameter-Frequency Histograms*

Length-frequency and diameter-frequency histograms (Figure 3) were prepared using the TEM data. Aggregation and overlapping nanofibers make it difficult to accurately measure dimensions; thus, only individual nanofibers with clearly identifiable ends were measured. The AH-NC exhibited a wide distribution of lengths whereas the diameter distribution had a narrower range of 2 to 8 nm with the maximum at 4.5 nm. The length distributions of HH-NC and US-NC were 25 to 275 nm and 75 to 350 nm, respectively. The diameter distributions of HH-NC and US-NC were 3 to 15 nm and 4 to 10 nm, respectively. The length and diameter distributions of HH-NC and US-NC were wider than those of AH-NC. Therefore, AH-NC had the smallest particles of the three preparation methods (Table 1).

**Figure 3.** Length-frequency and diameter-frequency histograms of (**a**) AH-NC, (**b**) HH-NC, and (**c**) US-NC.

These results are comparable to those of other studies [2,34,35]. The average aspect ratios (L/D) of HH-NC and US-NC were 21.20 and 23.20, respectively, which were lower than that of AH-NC (36.73). According to a previous report [36], the reinforcing effect of NC is expected to improve as the aspect ratio increases, which may further improve the mechanical properties of biocomposites. The length and diameter of NC prepared from various sources has been reported: 100–300 nm length and 3–5 nm

diameter from wood [37], 171.6 nm length and 14.6 nm diameter from cotton [38], 1160 nm length and 16 nm diameter from tunicate [39], and 100–1000 nm length and 10–50 nm diameter from bacterial cellulose [40].

The morphology and dimensions of NC depended on the preparation method and each method resulted in distinct features. Acid hydrolysis, a well-known method for preparing NC, results in a larger aspect ratio than the homogenization and ultrasonication methods. These mechanical process can break hydrogen bonds and disintegrate microfibers into nanofibrils, which form needle-like cellulose crystallites and consequently reduce the aspect ratio of NC.
