3.1 In situ modified BC production
The yield for BC, agar-BC, Chitosan-BC was found to be 49%, 61% and 32%, respectively. Agar enhances the yield by increase in viscosity, free cells and regulating the oxygen transfer and also gets incorporated in the pellicle (Bae et al. 2004). On the other hand, the chitosan affects the pH and interferes with the assembly of cellulose nanofibers reducing the yield.
3.2. Chemical composition determination by FTIR.
The FTIR spectra of BC, Agar-BC, and Chitosan-BC are shown in Figure 1. The common peaks corresponding to cellulose are observed in the spectra corresponsing to BC - a broad characteristic peak at 3353 cm−1 for -OH stretching vibration, a peak at 2900 cm−1 attributed to aliphatic C-H stretching vibration, and a peak at 1436 cm−1 ascribed for C-H bending vibrations (Numata et al. 2019; Ullah et al. 2016b). The peaks observed at wavenumbers 1650, 1436, 1365, 1157 and 1064 cm-1 correspond to glucose carbonyl of cellulose, –CH2 bending vibrations, -CH bending vibration, C–O–C asymmetric stretching of glycoside bond and C–O stretching, respectively (Kačuráková et al. 2002). Therefore, The FTIR spectra confirm that the production of pure cellulose.
The FTIR absorption spectra of Agar-BC is similar to pure BC spectra, except that the intensity of peaks for -OH, CH2, C-H, C-O-C increased as agar comprises of galactose monomer units, which has same functional groups as cellulose. The peaks at 1157, 1114, and 1064 cm–1 indicate the presence of C2O2, C3O3, and C6O6 respectively (Shah et al. 2010). Thus, the FTIR spectra confirm that Agar is incorporated into the BC.
The Chitosan-BC spectrum was also similar to that of pure BC because most functional groups are common to both cellulose and chitosan (Munim et al. 2020; Urbina et al. 2018). The -OH stretching peak at 3358 cm‑1, CH stretching at 2896 cm‑1 and bending vibration peak at 1450 cm‑1 for α-CH2 bending are observed and an extra peak for C=O stretching is seen at 1737 cm‑1, which is mainly a characteristic of acetyl groups (Wong and Chan 2018). There is a peak shift to lower wave numbers for amide bond, which can be ascribed to its involvement in hydrogen bonding. (Munim et al. 2020).
3.3 Morphology and microstructure determination by SEM
The microstructures of BC, Agar-BC and Chitosan-BC were illustrated in Figure 2 (a, b, and c), which reveals a typical interwoven nanofibrous morphology. The average fibril diameter of BC was found to be 58 nm ± 15 nm, which is similar to the earlier reports (Khandelwal et al. 2016; Xiang and Acevedo 2017). The fibers of Agar-BC were more aggregated and produced a denser network than BC. The average fiber diameter of Agar-BC was 80 nm ± 25 nm which is higher and more widely distributed than that of BC. On the other hand, the average fibril diameter of Chitosan-BC was found to be lower than BC with a value of 49 nm ± 19 nm. Another interesting observation from the SEM images is that the Agar-BC nanofibers appear to be more bundled and coiled-up as compared to the fibres present in Chitosan-BC, where the fibers are straighter and more distinct. This has important implication on pore size and shape which determines the swellability and rheology.
3.4 Structural analysis by XRD
The X-ray diffraction profiles of BC, Agar-BC and Chitosan-BC are shown in Fig. 3 and the crystallinity values are tabulated in Table 1. The diffraction peaks of pure BC was obtained at 2 theta angle of 14.6°, 16.7° and 22.7° which correspond to the primary diffraction peak from crystal planes of (100), (010), and (110) in Iα cellulose. (Fang and Catchmark 2015; Kim et al. 2010; Mohite and Patil 2014). The crystallinity of pure BC was found to be 77%. Agar-BC shows similar crystalline diffraction pattern as BC with distinct peaks at 2θ = 14.6°, 16.7°, 22.7° and 34.1° and a higher crystallinity of 83%. It may be envisaged that the addition of agar to media increases the viscosity of the media which can reduce the disorder in the assembly of cellulose chains and thus can improve the crystallinity of BC. The diffraction pattern for Chitosan-BC was also similar to that of BC where the 3 major peaks at 2 theta angles of 14.6°, 16.7° and 22.7° are seen and the crystallinity was found to be 75%. The decrease in crystallinity can be attributed to the interference of chitosan molecules in the cellulose chain assembly process. (Urbina et al. 2018; Zhijiang et al. 2011).
3.5 Surface area, and pore size distribution by N2 adsorption
The specific surface area of BC, Agar-BC and Chitosan-BC was measured by N2 adsorption isotherms shown in Figure 4 (a) and the estimated pore size distribution is depicted in Figure 4 (b). The isotherms of all the samples are type IV isotherm with a hysteresis loop. Hysteresis at mid to high relative pressures is attributed to capillary condensation in mesopores. Particularly here, a H3 type of hysteresis is seen which suggests slit-like pores. In case of Chitosan-BC, low pressure hysteresis is also seen which can be attributed to the swelling of non-rigid slit like pores with infinite pore length, which may originate from lower interactions between the chitosan modified cellulose fibers and low density.
The BET specific surface area (Table 1) of BC was found to be 74.38 m2/g while that for Agar-BC and Chitosan-BC was 53.37 m2/g and 25.98 m2/g respectively. The pore volume of BC, Agar-BC and Chitosan-BC were found to be 0.245 cm³/g, 0.176 cm³/g, and 0.174 cm³/g respectively. Overall porosity of BC, Agar-BC and Chitosan-BC was found to be 88.2%, 86.1% and 87.8%. The calculated relative mesoporosity with respective to microporosity of BC, Agar-BC and Chitosan-BC was found to be 74%, 93% and 82%. The relative microporosity of modified BC was reduced or in other words the relative mesoporosity was increased using modification, the reason for which needs further investigation. However, it may be attributed to filling up of the micropores in case of Agar, while in case of chitosan, it is difficult to comment as it comprises of slit like pores. In case of Agar-BC, it can be clearly seen that the fiber diameter has increased which can contribute to lesser surface area while in the case of Chitosan-BC, relatively larger number of larger pores can cause an overall reduction in surface area. However, it must be remembered that in case of hysteresis at low pressure, BET surface area may not be accurate.
3.6 Water absorptivity and retention kinetics of BC Modifiers
The water absorption behavior of BC modifiers and pure BC is presented in Figure.5 (a) and also summarized in Table 1. The Water absorptive kinetics showed that after 6 hrs, the samples were almost saturated when immersed in deionized water. All the curves show three phases – a) a steep slope in the first 1 hour reaching up to about 95%, 75% and 65%, for BC, Agar-BC and Chitosan-BC, respectively followed by b) slowing up of uptake and c) a saturation plateau at 150% (achieved in 6 hours), 130% ( achieved in 5 hours) and 110% (achieved in 5 hours), for BC, Agar-BC and Chitosan-BC, respectively. The slowing down followed by increasing uptake may be because of further ingress of water into the interiors. The water absorptivity rate and extent of absorption for Agar-BC is lesser than BC due to a) denser network evident from SEM images, b) lower surface area and pore volume revealed by Nitrogen adsorption studies and c) lesser tendency to rearrange, given the higher crystallinity of nanofibers. The water absorptivity of Chitosan-BC was also lesser than BC due to lesser surface area and less dense network.
The water retention behavior for the samples is shown in Figure 5 (b), where distinct regions with a fast removal of unbound water, followed by sustained weight-loss, and a very slow evaporation rate in the final step, can be observed. BC showed a higher water retention with more retention time of 7 hours due to higher water content and a deep ultra-fine network with comparatively higher microporosity. The water retention by Agar-BC and Chitosan-BC was seen up to 6 hours and 1 hour respectively, owing to lower water content and lesser microporosity. The structural and morphological variations in BC greatly influences the water uptake-retention kinetics.
Table 1. Results obtained from XRD, BET, Water Absorptivity and Water Retention Kinetics
Sample name
|
Crystallinity (%)
|
Surface area (m2/g)
|
Pore volume
(cm³/g)
|
Maximum WAC
(%)
|
Maximum
WRT
(h)
|
BC
|
77
|
74.38
|
0.245
|
153
|
7.0
|
Agar-BC
|
83
|
53.37
|
0.176
|
130
|
6.0
|
Chitosan-BC
|
75
|
25.98
|
0.174
|
110
|
1.0
|
3.7 Rheological behavior
The dynamic viscoelastic parameters, storage modulus (G′) and loss modulus (G′′), of BC and modified BC are shown as a function of shear strain in Figure 6. In linear viscoelastic region (LVER), G′ values are higher than that of G′′, which is a signature of gel-like solid behavior. The G′ values are constant at low strains and later decreases with strain. The point of deviation from linearity is referred to as the yield point (limit of the LVER), where irreversible structural deformation such as junction disruptions start happening. The calculated yield stresses for all the samples is tabulated in Table 2. It can be seen that the yield stress values are highest for Agar-BC and lowest for Chitosan-BC.
Further, the flow point is defined as the point of intersection of storage and loss modulus curves (G′=G″) when the material shows a change in behavior from solid gel-like to a viscous liquid-like property. The strain and stress corresponding to the flow point are also tabulated in Table 2. It is interesting to observe that while the flow stress values are highest for Agar-BC, the flow strain values are highest for BC. Chitosan-BC demonstrates the lowest flow stress as well as strain.
Overall, a higher storage modulus values with smaller values of yield and flow strains are observed for Agar-BC, as compared to BC. This implies Agar-BC possesses a more gel strength or rigid network requiring a higher shear stress for deformation. However, the flow stress values of both, BC and Agar-BC, are comparable as in one case strain is higher while in the other case modulus is higher. This implies that BC is a more reliable hydrogel which retains its elastic nature for larger strain, and for low strain, Agar-BC offers a higher modulus, making it more suitable .
Further, the Chitosan-BC is lower in modulus and also flows at lower strain, but yields at strain higher than Agar-BC and similar to BC. In other words, Chitosan-BC allows perhaps higher elastic strain before permanent changes. However, owing to lesser interconnections evident in the SEM images, the flow point is observed at lower strain and stress. This makes Chitosan-BC a more pliable and processible hydrogel. Broadly it can be said that Chitosan-BC behaves more like a low modulus plastic material (low modulus, low strain transition), while Agar-BC behaved like a high modulus plastic material (high modulus, low strains transition) with Agar acting as a reinforcement polymer to BC.
Table 2: Viscoelastic parameters for BC, Agar-BC and Chitosan-BC
Sample
|
Yield stress (G') [kPa]
|
Yield point
γ (%)
|
Flow point modulus (G'= G″) [kPa]
|
Flow point
(G'= G″)
γ (%)
|
Flow stress [kPa]
|
BC
|
116
|
0.0485
|
4.8
|
23.32
|
1.564
|
Agar-BC
|
170
|
0.019
|
11.8
|
10.02
|
1.677
|
Chitosan-BC
|
16
|
0.0497
|
1.5
|
9.761
|
0.214
|
Further, an angular frequency sweep was carried out at strains within the LVER limits for each sample. The storage modulus (G′), loss modulus (G′′) and complex viscosity (η*) of all samples are plotted as a function of angular frequency in Figure 7 (a & b). The higher values of storage modulus (G′) as compared to loss modulus (G′′) in the entire frequency range confirms the retention of gel-like solid behavior for short as well as long relaxation times. This makes these materials stable and thus symbolizes a good shelf life. It is also seen that both moduli were independent of frequency in the all hydrogels. The complex viscosity of all samples decreases linearly with an increase in frequency indicating a shear-thinning behavior.
For further understanding, the deformation behavior of BC hydrogel can be understood using the morphological illustration for interaction of fibers network among the BC modifiers, as shown in Figure 8. This illustration can explain the deformation behavior of bacterial cellulose, Agar-BC and Chitosan-BC as a application of shear stress. The initial strains would lead to an elastic deformation followed by a combination of elastic and viscous deformation at higher strains. In case of Agar-BC, depicting the higher gel strength nature which shows plastic deformation at low strains. This would be a specific hygrogen bonding between BC fibers and agar. So that which strengthens the BC network. In case of Chitosan-BC, there might be a non specific bonding like eclectrostatic interactions between BC fibers and amine charge molecules of Chitosan. So that it might loosen the strength of BC network