Modulation of morphology, water uptake/retention, and rheological properties by in-situ modification of bacterial cellulose with the addition of biopolymers

In situ modification of bacterial cellulose (BC) allows structural and morphological tuning which determines the crucial properties such as water absorption/retention and rheological behaviour. This work reports the effect of in situ modification carried out by adding two biopolymers Agar and Chitosan to the standard culture media for bacterial cellulose synthesis. The agar modified BC (Agar-BC) form a BC network with reduced pore volume, and a much denser network, leading to lesser water absorption and further lower retention time than BC. Agar-BC also demonstrates a higher storage modulus, while the yield point is observed at a lower shear strain. This indicates a behaviour similar to that of a crosslinked polymer with a low strain onset of plasticity. On the other hand, chitosan-modified BC (Chitosan-BC) also exhibits a lower pore volume with a lower densely packed structure and with lower swellability and water retention reduced to 1 h (7 h for BC). Chitosan-BC presents a lower modulus with a yield strain similar to that of unmodified BC. The water absorption-retention behaviour is discussed in detail considering the relative pore shape-size distribution, fibre dimension and surface area. The mechanism of viscoelastic deformation for each of the cases is explained.


Introduction
Cellulose is abundant in nature owing to its various sources such as plants, trees, microbes, and algae, which can be processed chemically and enzymatically to obtain cellulose (Lynd et al. 2002). Plant cell walls constitute the major source of cellulose, which also consists of several impurities such as lignin, pectin, hemicelluloses, and xylosans. Thus, intensive and extensive chemical processing is needed to extract pure cellulose for its various applications (Shoda and Sugano 2005;Szymańska-Chargot et al. 2017b). On the other hand, bacterial cellulose (BC) is produced as a 99.99% pure hydrogel with an ultra-fine nanofibrous network. BC possesses good mechanical properties and high water-absorbing and holding capacity (Islam et al. 2017). BC can be easily produced by the fermentation of culture media containing sugars using cellulose producing bacteria (such as acetobacter) in various morphologies such as sheets, spheres under agitated or static conditions (Singhsa et al. 2018). BC and its composites have been found suitable for many applications ranging from merely as an additive in food industries to wound dressings, skin treatments, and filtering membranes (Esa et al. 2014;Lin et al. 2013a). Further, due to its high purity, mechanical strength, crystalline nature, liquid-absorbing, and holding capabilities, biodegradability and most importantly its biocompatibility, BC is of particular interest in drug delivery, bioactive implants, artificial blood vessels, cosmetology and tissue scaffolds (Ullah et al. 2016a).
Due to the wide possibility of its applications, there is a huge interest in the modification of BC to further tune its properties by adding bio-derived polymers such as Chitosan, Agar, Pectin, Gelatin, Polylactic acid, Polyhydroxybutyrate, Sodium alginate and starch (Bae et al. 2004; Barud et al. 2011;Cacicedo et al. 2020;Kim et al. 2010;Lin et al. 2016Lin et al. , 2013bMartínez-Sanz et al. 2012;Ruka et al. 2013;Szymańska-Chargot et al. 2017a;Wan et al. 2009;Yin et al. 2020;Zhou et al. 2007). It has been observed that the addition of these polymers affects the structure, morphology, and composition which modulates its mechanical properties, liquid holding and retention capability, and yield. Such modifications can be carried out by two methods -a) in situmodifications introduced during cellulose synthesis (Khandelwal et al. 2016) and b) ex-situ modifications -modification introduced after production (Hu et al. 2014;Stumpf et al. 2018). The former is of particular interest as it allows uniform modification and changes at various levels of the hierarchical organization of cellulose. While various studies report in situ modifications and their effect on microstructure, the insights into their impact on crystallinity, pore size distribution, water retention and most importantly on rheological behavior remains poorly explored. Rheological behaviour is an important consideration in all the above-mentioned applications as they allow understanding of long-term and short-term viscoelastic behavior which helps in determining performance, processing, and shelf life.
Various approaches have been used for rheological modification of polymer gels (Mahendar et al. 2020;Nanda et al. 2013;Pandey et al. 2017;Quigley et al. 2020). Limited work is available on rheological modification of BC and in particular using the in situ methods. Amongst the various biopolymers used for modifying the properties of BC, Agar and Chitosan have been chosen for this study, given their high suitability in biomedical, pharmaceutical, and tissue engineering applications. Agar is commonly used in the form of nutrient media for the growth of various bacteria (Basu et al. 2015). Besides its role as a growth medium, it has a pronounced effect on the yield of BC (Bae et al. 2004;Chao et al. 2001;Ishida et al. 2003). Chitosan is a linear polysaccharide produced by the deacetylation of chitin, the second most abundant polysaccharide found on earth (Lim and Hudson 2003;Srinivasa and Tharanathan 2007). Chitosan modified BC finds many applications in food packaging, tissue engineering (Cai et al. 2009;Fernandes et al. 2009;Fu et al. 2009;Kim et al. 2011;Lin et al. 2013b), and in polymeric drug carrier (Cacicedo et al. 2020;Pavaloiu et al. 2014).
While there is abundant literature on ex-situ modifications of BC, the literature on in situ modification by chitosan and agar with a focus on understanding the effect on water absorptivity-retention and rheological properties is absent. This paper looks into the changes in structure, morphology, water absorption, and retention of BC produced by in situ modification with 0.1% Agar and chitosan, with emphasis on understanding the rheological characteristics.

Materials
Hestrin Schramm (HS) media was used for the production of BC. The media components include glucose, peptone, yeast extract, citric acid, and disodium hydrogen phosphate of microbiological grade, which were purchased from Himedia, India. Low molecular weight extrapure Chitosan (90% DA), granulated Agar (bactograde), and Sodium hydroxide (analytical grade) were procured from SRL chemicals, India. Cellulose producing bacteria Komagataeibacter hansenii (ATCC 23,769) was procured from LGC Promochem, India.

Production of BC and in situ modification
Hestrin Schramm (HS) media comprising of glucose 20 g/L, citric acid 1.15 g/L, disodium hydrogen phosphate 3.4 g/L, peptone 5 g/L, and yeast extract 5 g/L was prepared in DI water and adjusted to pH 4.5 for the production of BC (Adepu and Khandelwal 2018;Illa et al. 2019). A 100 mL of autoclaved media was poured in UV treated petriplates and inoculated with 3 ml of Komagataeibacter hansenii ATCC 23,769. The petriplates were wrapped with a parafilm sheet and incubated at 26C for 14 days. After 14 days, the produced BC pellicles were treated with 0.5 M NaOH to kill the bacteria and then washed with DI water to remove cell debris and neutralize pH. The purified BC was freeze-dried at -80°C for 24 h. For in situ modification, HS media was modified by adding 0.1 wt% of Agar and 0.1 wt% Chitosan to the media for obtaining Agar-BC and Chitosan-BC, respectively. In order to maintain optimum free cell volume, the concentration of modifier was fixed as 0.1wt% and as the agar modified media solidifies at higher concentrations at room temperature in static culture and in order to ensure consistency, the same was also used for chitosan. The freeze-dried BC pellicles were used for all characterization studies, except for the rheological measurements where never dried wet pellicles were used. The yield of BC and modified BC was calculated from the ratio of the weight of dried BC (purified and freeze-dried) to the weight of carbon source (Dglucose) used in HS media (as %) as well as the biomass yield was calculated from the ratio of the weight of dried BC (purified and freeze-dried) to the volume of the media (as g/L).

Characterization techniques
The FTIR spectroscopy was performed using an Alpha-P (Bruker Corporation, USA) machine in the wavenumber range of 4000 to 450 cm -1 in transmittance mode to determine the chemical composition. (Adepu and Khandelwal 2020) The surface morphology of all freeze-dried samples was observed by a JEOL JSM-7800 Field Emission scanning electron microscope (FE-SEM) operating at an accelerating voltage of 5 kV. Before analysis, samples were gently fixed on an aluminum stab with two-sided adhesive tape and gold coated with a sputter coater to deposit 10-15 nm layer of gold. The average fiber diameter was calculated from SEM images with the consideration of at least 100 fibers by using Image J software, across several images. The crystallinity of freeze-dried samples was determined from X-ray Diffraction (XRD) patterns obtained in a Bruker X-ray diffractometer using Cu Ka radiation with a step size of 0.02 AE and a dwell time of 3 s, from 10 to 50 AE (2h angle). The degree of crystallinity was calculated according to the Eq. 1.
Here, A Cryst is the sum of area under crystalline peaks; and A Total is the total area under the diffractogram. The peak deconvolution was done by Gaussian fitting using Origin 9.0 pro software as example of which is included in supplementary information. The area under the deconvoluted peaks was used for the calculation of crystallinity. (Adepu and Khandelwal 2020).
The specific surface area was estimated from Nitrogen adsorption isotherms using an Autosorb iQ, Quantachrome, USA, instrument, using BET analysis. Before analysis, the samples were subjected to degassing at 373 K for 2 h. Pore size distribution was obtained by BJH analysis from the isotherm. The porosity of the sample was determined using the following equation Eq. 2.
Here, V a is Apparent volume and V g is the actual volume of the sample.
Mesoporosity (%) was calculated from the ratio of pore volume for pore size 2-50 nm to the total pore volume for pore size upto 50 nm. (Adepu and Khandelwal 2020;Illa et al. 2019;Treesuppharat et al. 2017).
The water absorption was measured through swelling studies by the gravimetric method (Lin et al. 2013b). The initial weight of the pellicles for a sample size (2 cm by 1 cm) was noted after freezedrying (W 1 ). The samples were immersed in a beaker containing 30 ml of deionized water and the wet weight of the swollen membranes (W 2 ) was measured at various time points. The excess water was removed by gently blotting with a filter paper before weighing. The Water Absorptivity (%) or swelling was calculated by the following equations: The water retention of the samples was evaluated as per the reported method (Li et al. 2011). The weighed freeze-dried samples (W d ) were completely saturated with water. The saturated samples were wiped with a filter paper to remove excess water and weighed as W 0 . The swollen membranes were placed in a wideopen dish at room temperature. The samples were taken out and weighed (W t ) at several time points and then placed back into the dish. The Water Retention (WR) was calculated as follows.
These studies were carried out in triplets. The rheological properties of the samples were measured by using a rheometer (MCR 94 SN82806453, Antonpaar, USA) with parallel plates geometry. Wet pellicles were cut into discs (25 mm) for amplitude sweep and frequency sweep measurements. Amplitude sweep was used to identify the linear viscoelastic limit to determine the strain for frequency sweep measurement in the scanning range of 0.01-100 Hz at 26°C. (Gao et al. 2016;Numata et al. 2019).

Results and discussion
In situ modified BC production The digital photographs of pellicles produced with and without modification are shown in Fig. 1. The yield in terms of conversion of carbon substrate to cellulose for BC, agar-BC, Chitosan-BC was found to be 49%, 61% and 32%, respectively and biomass yield for BC, agar-BC, Chitosan-BC was found to be 9.8 g/L, 12.2 g/L and 6.4 g/L, respectively. The biopolymer 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 of the media and is also known to be an anti-microbial (Cacicedo et al. 2020;Lin et al. 2013b), which may reduce the yield.
Chemical composition determination by FTIR.
The FTIR spectra of BC, Agar-BC, and Chitosan-BC are shown in Fig. 2. The common peaks corresponding to cellulose are observed in the spectra corresponding 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, -CH 2 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 are similar to pure BC spectra, except that the intensity of peaks for -OH, CH 2 , C-H, C-O-C increased as agar comprises of galactose monomer units, which has the same functional groups as cellulose. The peaks at 1157, 1114, and 1064 cm -1 indicate the presence of C 2 O 2 , C 3 O 3 , and C 6 O 6 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 a-CH 2 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).

Morphology and microstructure determination by SEM
The microstructures of BC, Agar-BC and Chitosan-BC were illustrated in Fig. 3 (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.

Structural analysis by XRD
The X-ray diffraction profiles of BC, Agar-BC and Chitosan-BC are shown in Fig. 4 and the crystallinity values are tabulated in Table 1. The diffraction peaks of pure BC were obtained at 2 theta angles of 14.6°, 16.7°and 22.7°which correspond to the primary diffraction peak from crystal planes of (100), (010), and (110) in Ia 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 2h = 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%. Previously a decrease in crystallinity has been attributed to the interference of chitosan molecules in the cellulose chain assembly process, however, in this work the change is not significant owing to low chitosan concentration (Urbina et al. 2018;Zhijiang et al. 2011). It has been observed that addition of modifiers can affect crystallisation, not only in terms of crystallinity but also the orientation. Depending on the interaction between cellulose and the modifier, the orientation may be affected. (Khandelwal et al. 2016). Agar as well as chitosan both interact with cellulose and depending on their molecule size and specific interaction can result in different crystallinity and orientation. Further investigation to understand the interactions and effect on crystallisation may be pursued in future work.
Surface area, and pore size distribution by N 2 adsorption The specific surface area of BC, Agar-BC and Chitosan-BC was measured by N 2 adsorption isotherms shown in Fig. 5 (a) and the estimated pore size distribution is depicted in Fig. 5 (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 slitlike 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 m 2 /g while that for Agar-BC and Chitosan-BC was 53.37 m 2 /g and 25.98 m 2 /g respectively. The pore volume of BC, Agar-BC and Chitosan-BC were found to be 0.245 cm 3 /g, 0.176 cm 3 /g, and 0.174 cm 3 /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 respect 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 seen that the fiber diameter has increased which can contribute to the lesser surface area while in the case of Chitosan-BC, the 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.

Water absorptivity and retention kinetics of BC Modifiers
The water absorption behavior of BC modifiers and pure BC is presented in Fig. 6 (a) and is also summarized in Table 1. The water absorptive kinetics showed that after 6 h, the samples were almost saturated when immersed in deionized water. All the curves show three phases -a) a steep slope in the first 1 h 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 h), 130% ( achieved in 5 h) and 110% (achieved in 5 h), for BC, Agar-BC and Chitosan-BC, respectively. The slowing down followed by increasing uptake maybe 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 of fibrils to bend and accommodate water 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 Fig. 6 (b), where distinct regions with 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 higher water retention with more retention time of 7 h 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 h and 1 h respectively, owing to lower water content and lesser microporosity. The structural and morphological variations in BC greatly influence the water uptake-retention kinetics.

Rheological behavior
The dynamic viscoelastic parameters, storage modulus (G 0 ) and loss modulus (G 00 ), of BC and modified BC are shown as a function of shear strain in Fig. 7. In the linear viscoelastic region (LVER), G 0 values are higher than that of G 00 , which is a signature of gel-like solid behavior. The G 0 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 are 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 0 = G 00 ) 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, 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 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 that 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. Further, an angular frequency sweep was carried out at strains within the LVER limits for each sample. The storage modulus (G 0 ), loss modulus (G 00 ) and complex viscosity (g*) of all samples are plotted as a function of angular frequency in Fig. 8 (a & b). The higher values of storage modulus (G 0 ) as compared to loss modulus (G 00 ) 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 all hydrogels. The complex viscosity of all samples decreases linearly with an increase in frequency indicating a shear-thinning behavior.
Thus, it is shown that modification of growth media can lead to change in morphology, water uptake and retention, and also rheological behaviour (yield point and flow point) of BC. This tool can be used to tune the properties of BC for suitable applications.

Conclusions
The possibility of modulating water uptake-retention and rheological behaviour by tuning the morphology and structure has been successfully demonstrated by using In situ modification of bacterial cellulose. The addition of agar and chitosan leads to a considerable change in fibre dimensions, pore size-shape and surface area. Agar-BC exhibited a lower water uptake owing to a denser and rigid network, while Chitosan-BC showed a huge reduction in water uptake as well as retention time, due to a lower densely packed network. The modifications also led to an overall reduction in micropores (as compared to mesopores). The  rheological properties reveal higher gel strength of agar-BC and lower gel strength of Chitosan, which is due to the interaction of biopolymers with pure cellulose. Agar acts as reinforcement at very low strains leading to an increase in strength, which eventually deforms and leads to flow at relatively lower strains. Chitosan is known to interfere with cellulose assembly during synthesis leading to lower yield and thus a weaker network. The rheological measurements also establish the long-term stability of materials within the elastic limit. This work successfully demonstrates the effect of in situ modification on crystallinity, fiber network, surface area, pore size distribution, water swelling-sustain kinetics, yield stress and flow stress. These morphological, structural, and rheological parameters are essential parameters for the application of BC in cosmetics, drug carriers and regenerative medicine.