**4. Discussion**

#### *4.1. Effect of Ultrasonic Irradiation on The Depolymerization Cellulose*

The term "ultrasonic" describes sound waves with a frequency greater than 20 kHz. Many studies have reported the exposure to this wave is responsible for a number of physical and chemical changes. The ultrasonication was the adopted method here to carry out partial depolymerization of native BC into microfibers. The utilization of ultrasonic waves offers a simple and versatile tool for synthesizing micro or nanostructured materials that are often unavailable by conventional methods. In this work, native BC was irradiated into a mixture of water/ethanol (50% *w*/*w*) at constant power of 100 W and frequency of 20 kHz by varying irradiation times. The influence of the length of ultrasonic irradiation period on the CrI is presented in Table 3.


**Table 3.** Influence of the length of ultrasonic irradiation period on Crystallinity Index, CrI.

> 1 Microcrystal Cellulose, MCC (commercial).

From the table, it could be concluded that the increase of CrI is dependent on the irradiation time. Ultrasonic irradiation in water/ethanol induced partial depolymerization of BC with a CrI increase of 8.4% during the first 30 min. The maximum CrI was observed to be 71.4% at 120 min. The results indicated that the irradiation leads to the rupture of amorphous cellulose chains. The disintegration of amorphous regions may be explained by acoustic cavitation. As native BC in a liquid medium was exposed to ultrasonic irradiation, the acoustic waves induce alternating high and low pressure; this creates bubbles (i.e., cavities) and makes them oscillate. A bubble can grow while absorbing the ultrasonic energy at each cycle, until it becomes unstable and finally collapses violently, releasing the energy stored within it, subsequently producing shock waves in the medium [58]. A shear deformation during the collapse of the bubbles is considered to be responsible for the chemical effects which induce disintegration of the amorphous regions of cellulose.

Cavitation occurs over a very wide range of frequencies, from 10 Hz to 10 MHz. Above that frequency regime, the intrinsic viscosity of liquids prevents cavitation from occurring. According to Suslick and coworkers [59], most high intensity ultrasonic horns operate within the range of 20 to 40 kHz. Several factors can affect acoustic cavitation, such as reaction temperature, hydrostatic pressure, frequency, acoustic power, and the type of the solvent medium used. In our study, the amorphous regions degradation increases slightly after 30 min irradiation, and the CrI of cellulose for all sample experiments is still lower compared with that of commercial MCC (75.0%). In our experiments, optimum crystallinity is observed with 30 min irradiation; then, when reaction time is increased, degradation might occurred in both the amorphous and crystalline regions, consecutively reducing the product crystallinity. Concerning the solvent medium, when more volatile solvent is used such as ethanol in water, the mixture is expected to produce more cavitation bubbles, which can significantly promote the reduction rate of amorphous regions. We therefore compare our results (using water/ethanol 50% *w*/*w*) to reactions done in pure water. From XRD spectra, it was calculated that the increase in CrI was less pronounced when the reaction was run in sole water (increase from 60.7 to 67.0%) than in water/ethanol mixture (increase from 60.7 to 69.1%). This result is in accordance with DSC observations, as the endothermic peak of sample that was irradiated with ethanol (the red line in Figure 5B) was detected broader than the one with pure water (blue line in Figure 5B). Both samples have the same melting temperature, i.e., around T m = 344 ◦C. The endothermic peak for sample as detected in the DSC curves becomes larger when the crystallinity of the sample increases. According to Ciolacu et al., 2011 [57], the broadening of endothermic peaks detected in the DSC curves of celluloses is in a linear relationship with the percentage value of the amorphous material from their crystalline structure.

**Figure 5.** The evolutions of crystalline regions formations for treatment of bacterial cellulose after 30 min irradiation time in pure water (blue curve) or a mixture of ethanol / water 50% ( *w*/*w*) (red line) observed by XRD ( **A**) and by DSC (**B** in insert) (untreated BC, dark line).

#### *4.2. Effect of MnCl2 Concentration on The Extraction of Crystalline Regions*

The microwave treatment assisted by HCl-MnCl2 catalyzed hydrolysis was evaluated to hydrolyze cellulose. In this part, the use of a microwave reactor was performed to ge<sup>t</sup> a higher conversion and a shorter reaction time for catalyzed hydrolysis of depolymerized cellulose. In comparison, a

conventional heating microwave is a high-frequency radiation that possesses both electrical and magnetic properties [60]. Regarding the addition of catalysts, MnCl2, was utilized to improve the extraction rate of crystalline regions during the hydrolysis. It was already shown that metal chlorides, due to their Lewis acid property, exhibit higher catalytic activity than inorganic acids [61]. The concentration of HCl used in this work was significantly reduced to 0.1 M, instead of 6 M, as used in many reported works.

Table 4 shows the effect of the metal chlorides catalyzed hydrolysis reaction on the crystallinity index. In the absence of a catalyst (0%) in the 0.1M HCl medium, thermal hydrolysis could not occur effectively, and the CrI obtained was only 0.5% higher than for the starting material (DP-BC) from 69.1% to 69.6% for 30 min of reaction. Conversely, with the addition of MnCl2, the CrI was increased to 71.3%, 72.7%, and 79.4% for 1%, 2.5%, and 5% ( *w*/*w*), respectively. A similar result was obtained for the use of FeCl3·6H2O, 5% ( *w*/*w*) as catalyst with a CrI increased from 69.1% to 77.8%. It was also found that the CrI of all experiments showed a lower value compared with that of the commercial NCC (85.0%). Nevertheless, it appears that the presence of a catalyst plays an important role in the extraction of crystalline regions. In our hypothesis, during partial depolymerization, the ultrasonic treatment leads to the distortion of the amorphous parts and eases the accessibility of chemical reagen<sup>t</sup> to loosen them. Thus, the protons could more easily penetrate into the disordered regions during catalyzed hydrolysis, and as a result, greatly promote the hydrolytic cleavage of glycosidic bonds even in the diluted HCl medium. For this step, the hydrolysis reaction at 0.1M of HCl and 5% *w*/*w* of both metal chlorides (MnCl2 and FeCl3·6H2O) for 30 min reaction can enhance the removal of the amorphous regions, even though the CrI obtained is still less than 80%.


**Table 4.** Influence of concentration of catalyst MnCl2 and FeCl3·6H2O on Crystallinity Index, CrI.

> 2 NanoCrystal Cellulose, NCC (commercial).

The rapid degradation of amorphous regions during catalyzed hydrolysis can be explained by the Lewis acid character. According to Stein et al., 2010 [62], some metal chlorides such as FeCl3, AlCl3, CuCl2, and MnCl2 could form hydrated complexes in aqueous solution and coordinate the glycosidic oxygen of cellulose. This helps to scissor the glycosidic linkages and to facilitate the hydrolysis process, while the chloride anions attack the hydroxyl atoms [61,63,64]. Introducing metal chloride salts into acid solution can further improve catalytic performance at which the intra- and inter-molecular hydrogen bonds can be broken, and the degradation of the amorphous regions can be accelerated by permeating the internal structure of irradiated cellulose (DP-BC) to acid. Moreover, under microwave and hydrothermal conditions, the easy diffusion of metal cations and chloride anions into the hydrogen bond network, as well as the strong ability of chloride anions to disrupt the hydrogen bond, can be achieved; thus, the hydrolysis rate is greatly enhanced.

Considering the thermal behavior analysis, the summary of DSC results is presented in Table 5 as follows:


**Table 5.** Characteristic thermal behavior of sample celluloses.

It was found that compared with the DP-BC, the degradation temperature of the BC-NC decreased by approximately 62.7 ◦C. Similar results were obtained for commercial MCC and NCC sample references. The nano-sized NCC exhibited lower degradation temperature than the micro-sized MCC, i.e., by 53.7 ◦C. The reason is that the thermal stability of nanocrystals is related to several factors including their dimension, crystallinity, and composition, which in turn depend on extraction conditions [65,66]. So, the NCC with the highest crystallinity would exhibit the highest thermal stability, but smaller dimensions should also cause a decrease of the degradation temperature. The FT-IR analysis demonstrated that the chemical structures of BC-NC remained unchanged after MnCl2-catalyzed hydrolysis process.
