*3.1. Chitosan Nanofibers*

Figure 1 shows the photograph of ChNF suspensions treated with 10, 15 and 30 passes of the ACC treatment. After ACC treatment, the suspensions were changed to ivory color, and as the ACC pass increased the suspension color turned to a milky. The viscosity of ChNF suspensions was measured and Figure 2 shows the result. The viscosity of ChNF suspensions increased with the number of ACC pass. The higher is the number of passes at the ACC chamber, the higher is the viscosity of ChNF. Higher viscosity means a higher fibrillation of chitosan, thus reducing its size to nanofibers. Note that the low viscosity ChNF samples (10 and 15 ACC passes) easily flowed when the samples were placed upside down, while the 30 passes case did not flow when it was placed upside down. Interestingly, the viscosity values decreased with the time especially, for the 15 and 30 passes cases. This might be associated with the broken inner bonds in ChNF and the layer separation in the suspension. Figure 2b shows the viscosity change with the number of ACC pass when the time is 5 min. The viscosity linearly increased with the number of ACC pass.

The yield of ChNF was evaluated in each sample by centrifugation. The result shows that, after 10, 15 and 30 passes of the ACC treatment, the yields of ChNF were 13.2%, 15.5% and 27.7%, respectively. From the viscosity and yield data, it is clear that the higher is the number of ACC passes, the better is the isolation of ChNF.

The morphology changes of chitosan after the ACC treatment were observed by FE-SEM and AFM. Figure 3a–d shows the morphologies of the original chitosan and ChNFs with 10, 15 and 30 passes of the ACC treatment, respectively. The chitosan particles were around 50–100 μm. After the ACC treatment, it was changed to nanofibers, as shown in Figure 3b–d. After 30 passes, the ChNF size was reduced to 38 ± 16.5 nm in width and length of several microns. The width of ChNF was calculated in 100 measurements from the AFM images. This size of ChNF is larger and longer than that of CNF. Thus, in the preparation of ChNF–CNF composites, 30 passed ChNF was chosen.

**Figure 1.** Photograph of ChNF suspensions: (**a**) the original chitosan; (**b**) after 10 ACC passes; (**c**) after 15 ACC passes; and (**d**) after 30 ACC passes.

**Figure 2.** Effect of ACC passes on the viscosity of ChNF suspension: (**a**) viscosity change with time, and (**b**) viscosity change with ACC pass number.

**Figure 3.** Morphologies of chitosan and its nanofibers: (**a**) SEM image of the original chitosan particles; and AFM images of ChNFs with different ACC passes: (**b**) 10 passes; (**c**) 15 passes; and (**d**) 30 passes (AFM images are in 5 μm × 5 μm).
