**1. Introduction**

The most advantageous characteristics of the bio-based edible film are their edibility and inherent biodegradability [1]. Various biopolymers have been explored to reduce the use of non-degradable petroleum-based materials such as cellulose, chitosan, starch, collagen, pectin, etc. [2]. However, problems of strong hydrophilic character, high degradation, and inadequate mechanical properties in moist environments still limit the applications of biopolymers [3,4]. To become more applicable in practice, biopolymers have to be modified in terms of properties and functionalities [5]. In food packaging applications, for instance, the incorporation of reinforcement fillers [6,7] into the biopolymers matrix has shown to be an efficient strategy to overcome some critical issues [8] such as

low mechanical resistance [9], hydrophilicity [10], and poor barrier to water vapor [11,12] compared to those of pure polymer or conventional (microscale) composites. More importantly, the process is less expensive compared to the development of new synthetic polymeric materials [13].

Nanocomposites represent an alternative to conventional technologies for improving biopolymer properties, by adding nanoparticles for which at least one dimension is in the nanometer range [14]. Most composite materials consist of one or more discontinuous phases distributed in one continuous phase. Discontinuous phase materials are usually harder and possess superior mechanical properties compared to continuous phase materials. The continuous phase is called the matrix, and the discontinuous one is called reinforcement [15]. The entity of the interactions is strongly affected by the nature of the discontinue phase; this can be maximized by passing from iso-dimensional particles to nanotubes [4]. Preparation of hybrid polymeric materials filled with natural particles also allows the fabrication of films with smart functions, such as antibacterial [16–18] and antioxidant capacities [19–21]. Numerous studies have been done on potential applications of biopolymers. Cellulose is an appropriate candidate used as a reinforcing material. Cellulose is a fibrous, tough, water-insoluble biomaterial that can play a substantial role in blending with different biopolymers to produce various bio-based nanocomposites [22]. Cellulose is the most abundant renewable biopolymer produced in the biosphere, and is obtained mainly from vegetables (plants and some algae species) and microbes (bacteria) [23].

Bacterial Cellulose, BC is constituted of fermented fibers, and is commonly synthesized by bacteria that are members of the Gluconacetobacter genus. Compared to cellulose plant fibers, BC displays higher crystallinity, and possesses improved properties such as high purity (with the absence of lignin and hemicellulose), ultrafine fibrous structure, low density, high water-retention capacity, and biocompatibility [24]. All these features make BC a promising biomaterial for industrial applications [25,26]. BC and plant fibers are both biopolymers that have similar molecular units but present a different structural organization. Depending on the source, plant fibers are mainly composed of three major components: cellulose, hemicellulose, and lignin. In contrast, the fibers made by bacteria are of pure cellulose; therefore, they present different physical properties [27]. Cellulose is a linear polysaccharide which consists of D-anhydro glucopyranose units linked by β-1,4-glycosidic bonds. The cellulose microfibrils have two types of structural regions: (i) the ordered region (crystalline) and (ii) the disordered region (amorphous). The crystalline regions give important mechanical properties to the cellulose fibers. Cellulose crystallinity, the degree of organization of the cellulose lattice, is a parameter describing the relative amount of crystalline content in the cellulose [28]. Crystallinity is a major factor affecting the activity of most celluloses; its values vary depending on the source and the mode of chemical treatment of the fibers [29].

Nanocrystals Cellulose (NCC) can be obtained by removing the amorphous regions while keeping the crystalline regions through partial depolymerization and purification from fiber sources. A comparison of the preparation of NCC from different natural materials and synthesis routes are presented in [30]. The most commonly-employed method to produce NCC is via acid hydrolysis conducted by strong mineral acids such as sulfuric acid, H2SO4, or hydrochloric acid, HCl [31]. The reaction involves the preferential hydrolysis of amorphous regions, promoting cleavage of glycosidic bonds. This procedure leads to the removal of the individual crystallites, which are regularly distributed along the microfibers, and drives to the formation of rod-like nanocrystals. The type of acid used determines the characteristics of the obtained NCC. H2SO4 will promote sulfonation of the crystallites surface [32] that produces a stable colloidal suspension due to electrostatic repulsion [33]. However, the presence of sulfate groups induces some crystallites to degrade, and reduces the thermostability of NCC [34–36]. It is generally known that low thermal stability may limit the use of nanocellulose and the manufacturing of its nanocomposites at high temperatures [37]. Although residual sulfate can be removed by dialysis, it is a time-consuming process, and particle aggregation is very difficult to avoid [38–40]. On the other hand, HCl produces hydroxyl groups on the surface of crystallites [41]. It generates a low-density surface charge with limited NCC dispersibility, which tends

to promote flocculation in aqueous suspensions [42]. HCl is less corrosive than H2SO4, and though the yield is lower [41], it permits a significant increase in thermal stability of NCC [43]. To reach high yield value, a highly-concentrated aqueous solution of HCl is needed under hydrothermal conditions at 110 ◦C for a long period of the reaction [35].

As described in the previous paragraph, high-yield production of NCC is obtained using an excessive amount of mineral acids. Pollution to the environment, corrosion to the equipment, and the difficulty of controlling the reaction are the major limitations to synthesis using acid hydrolysis [44]. To overcome these issues, the aim of this study is to develop a fast, highly-efficient, and eco-friendly preparation method for the extraction of cellulose nanocrystals from Bacterial Cellulose, BC. A two-step process is considered, yielding Bacterial Cellulose NanoCrystals, BC-NC, namely: (1) partial depolymerization of BC under ultrasonic irradiation, (2) extraction of crystalline regions using microwave assisted by MnCl2-catalyzed hydrolysis. The effect of irradiation time on the partial depolymerization process and impact of MnCl2 concentration during the hydrolysis treatment is evaluated on the chemical structure, crystallinity index, thermal properties, and surface morphology of irradiated Depolymerized Bacterial Cellulose DP-BC and extracted BC-NC

#### **2. Materials and Methods**
