3.1 Scanning Electron Microscopy (SEM/EDX)
From the SEM micrographs shown in figure 1, cellulose fibers can be observed with different surface morphologies among the samples. The unmodified sample (BC) appears to have a compacted fiber network with uniformly interconnected pores similar to what [41] reported. The modified samples, on the other hand, displayed different fiber networks depending on the CA concentration. At lower concentrations (MBC0.03, MBC0.07 and MBC0.15), porous fiber networks can be observed that could allow for more water absorption. The higher concentration (MBC0.30 and MBC0.70), in contrast, showed a bit compacted fiber similar to the untreated sample. This could be due to the high crosslinking density that occurs between the fibers, thus affecting the porosity and preventing the passage of water molecules leading to a low swelling rate, as explained in Section 3.5 and Figure 7.
The EDX spectra in Figure 2 represents the elemental composition (Carbon and Oxygen) typical of organic fiber, while the elemental and atomic weight percentages were presented in Table 1 for the pure and modified BC.
The slight shift of the elemental and atomic weight percentage (Table 1) of both carbon and oxygen in the pure sample (BC) in comparison is logically indicating the presence of CA in all modified samples. CA having the chemical formula (C6H8O7), have added to/compensated the percentage of oxygen atoms in the pure BC having the chemical formula (C6H10O5)n, thus reducing the proportion of carbon and increasing that of oxygen, similar to the trend reported for ascorbic acid modification on cellulose by [42]. The EDX data showing the carbon (C) and oxygen (O) peaks only (Figure 2) is also suggesting the notion that nata-de-coco-based BC is highly (~99%) pure [43].
3.2 Fourier Transformed Infrared (FTIR)
The FTIR spectra of the pristine and modified samples were shown in Figure 3. The signature peaks attributed to the dominant functional group of BCs' (OH-stretching) vibration were at 3346 cm-1. Peaks obtained at 2865 cm-1 and 1420 cm-1 were due to C-H stretching and 1450 cm-1 due to CH2 absorptions. Peaks obtained at 1719 cm-1 related to carbonyl/carboxyl (C=O) stretching [44] appears only on the crosslinked samples thus, confirming the presence of CA within the modified BC samples [30, 45, 46]. Peaks between 1055 cm-1, 1020 cm-1 were due to C-O-C interactions. The reduced intensity of the OH peaks on the crosslinked samples can also result from the chemical interaction with CA [47]. Overall, the low intense OH peaks at 3346 cm-1 and 1711 cm-1 on treated samples are indications that crosslinking modification on the BC was successful. The proposed mechanism of CA crosslinking on BC was presented in a schematic diagram in Figure 4.
3.3 X-ray Diffraction (XRD)
XRD patterns shown in Figure 5 represents the spectra obtained for the pure, modified samples. All samples showed peaks typical of cellulose I allomorph at lattice planes of 110, 1-10, and 200 corresponding to 2θ values of 14.6 º, 16.6 º, and 22.6 º, respectively as previously reported [32, 34, 48, 49]. Distinctive peaks with different intensities obtained at diffraction planes of 130, 042 and 040 corresponding to 2θ values of 19.4 º, 26.1 º and 34.3 º appear only on the modified samples, thus attributed to the CA crosslinking of the BC [50].
The peaks associated with BCs' crystallinity appear with similar intensities for all the samples, indicating that the CA modification has less effect on the crystalline structure and morphology of the BC [51, 52]. Even though [15] reported a decrease in crystalline peaks on sodium carboxymethylcellulose (NaCMC) crosslinked with CA, such may likely be due to one of the cellulose derivatives. De Lima et al. suggested that the decreased crystallinity they observed is ascribed to the viscosity increase of NaCMC or its interaction with cellulose nanofibers during crystallisation [53].
The crystallinity index and crystallite size values calculated from the XRD data were between 92% to 95% and 51Å to 56Å. This essentially shows that the CA crosslinking has less effect on the crystallinity and crystallite size of the MBC. Furthermore, cellulose I allomorph calculated using the Z-discriminant function also showed that all the samples have the same cellulose Iα rich (triclinic) form, the typical of bacterial cellulose [54, 55]. All calculated agreed with previously reported data [30] and indicate that the BC still maintained its crystalline nature after the crosslinking modification.
3.4 Water Contact Angle (WCA.)
The wetting behaviour of a materials' surface is closely related to the molecular terminal groups present, and contact angle studies give information on the wettability properties of a material [56]. In theory, a surface is considered hydrophilic or super hydrophilic when its WCA is smaller than 90º or 10º, respectively [35]. Figure 6 represents the mean contact angle measured for the pure and modified BC samples. All, including the pure BC, fall between 0º and 33.90º, signifying that all samples were either hydrophilic or super hydrophilic depending on their contact angles. However, it noteworthy that the modified samples have shown a decreasing WCA up to 0º (MBC0.30 and MBC0.60) where the water droplet is no longer capturable (disperses as soon as dropped). BC's hydrophilicity could be attributed to the additional carboxyl groups [44, 57] that can form hydrogen bonds with water molecules [58]. Even though a native BC is inherently hydrophilic, the angle of contact with water tends to decrease with increasing the CA concentration. Essentially here, the CA modification on BC has improved its surface chemistry to attract more water further.
3.5 Swelling rate (SR.)
Generally, polymeric materials' water absorption and swelling behaviour are through capillary action and diffusion and electrostatic repulsion between the ions on the polymer chains that forces it to expand and swell [57]. The swelling rate (SR) for the pure and modified BC samples was presented in Figure 7. Modified samples have shown an increased SR, mostly at lower and decrease higher concentrations of CA to a rate even below that of the pure BC. The decrease in SR with the increase in CA concentration could be due to the numerous crosslinker points formed within the BCs' fiber networks, thus reducing the spaces for water to enter [57]. It is evident for samples (MBC0.30 and MBC0.60) having the lowest absorption rates, a packed fiber geometry on the SEM micrographs in Figure 1, and the sample images in figure 7, which could result from the high concentration of CA Water absorption/swelling is especially advantageous for BCs' medical applications, such as wound dressings [28]. Interestingly, all samples showed similar SR in both SBF and DI water. The SR results reported here agreed with the previous report that BC water holding capacity is between 60 to 700 times its dry weight [59].
3.6 Thermal Gravimetric Analysis (TGA)
An important property of BC is its thermal stability, especially for applications in biomedicine where higher temperatures are applied for sterilisation processes. Figure 8 shows the thermal behavior of the pristine and modified BC evaluated in this study. An initial weight loss observed for all the samples at a temperature between 45 to 120 ºC was due to absorbed moisture evaporation. Except for samples with the highest CA concentration (MBC0.30 and MBC0.60) that displayed a partial decomposition between 120 to 300 ºC, all other modified samples were not different from the pristine BC. They all showed a maximum weight loss at a temperature between 300 to 392 ºC due to dehydration, decomposition, and dissociation of the glycosidic linkages [60-62]. The partial, total and residual mass loss observed at maximum temperatures 300 ºC, 392 ºC, and 620.93 ºC were 25.928%, 88.149%, and 7.875%, respectively. The partial decomposition observed may also be due to the high concentration of CA that attracts more moisture than the lower concentrations. Our result implies that the CA modification has less effect on the thermal properties of the BC [63].
3.7 Tensile Testing
Table 2 represents the tensile test results for all the samples with the modified showing improved mechanical strength compared to the unmodified, except for the lowest CA concentration (MBC0.03), which exhibits a very low tensile strength value. The decrease in the mechanical strength displayed could result from lesser crosslinking degrees within the fiber networks due to the low amount of the crosslinker, which can also be seen from the SEM micrographs Figure 1. The sample thickness and spongy appearance after freeze-drying could also lead to the loosening of the fibers. It can also be observed that, despite having a lower modulus value, the elongation at break is within the same range as other modified samples, implying that the elasticity of the fibers is close to other modified samples after reaching the maximum yield limit.Like the modulus, the tensile strength also follows the trend as increasing with the CA concentration if not for the lowest concentration. However, the elongation at break showed a different pattern where it increases from the lowest and decreases at the highest concentration, especially in the lowest concentration. Therefore, it can be hypothesised that high concentration CA treatment on BC may have a negative effect on the stretching ability of the BC fiber.