Muhammad Wajid Ullaha,
Mazhar Ul-Islamab,
Shaukat Khana,
Yeji Kima,
Jae Hyun Janga and
Joong Kon Park*a
aDepartment of Chemical Engineering, Kyungpook National University, Daegu 702-701, Korea. E-mail: parkjk@knu.ac.kr; Fax: +82 539506615; Tel: +82 539505621
bDepartment of Chemical Engineering, College of Engineering, Dhofar University, Salalah, 211, Oman
First published on 22nd February 2016
In the current study, nanocomposites of bio-cellulose with titanium dioxide nanoparticles (TiO2-NPs) were synthesized by an in situ strategy using a cell-free system. The system was developed from Gluconacetobacter hansenii PJK through bead beating. A suspension of TiO2-NPs was prepared in 1% sodium dodecyl sulfate and added to the cell-free extract of G. hansenii PJK. The bio-cellulose/TiO2 nanocomposite was synthesized at 30 °C, pH 5.0 for 5 days (bio-cellulose/TiO2-I), 10 days (bio-cellulose/TiO2-II), and 15 days (bio-cellulose/TiO2-III) using 10 g L−1 glucose. Field-emission scanning electron microscopy (FE-SEM) confirmed the structural features and impregnation of TiO2-NPs into the bio-cellulose matrix. Fourier transform-infrared (FT-IR) spectroscopy confirmed the presence of Ti–O groups in the chemical structure of the nanocomposite. X-ray diffraction (XRD) analysis indicated the presence of specific peaks for bio-cellulose and TiO2-NPs in the nanocomposite. The TiO2-NP uptake by bio-cellulose was greatly increased with time and 40 ± 1.6% of the initially added nanoparticles were successfully impregnated into the nanocomposite after 15 days of incubation. NP release analysis revealed minute detachment though a prolonged treatment time of 10 days. The synthesized nanocomposite showed better thermal and mechanical properties compared to pure bio-cellulose. The antibacterial test revealed impressive results where the inhibition zones produced against E. coli by bio-cellulose, bio-cellulose/TiO2-I, bio-cellulose/TiO2-II, and bio-cellulose/TiO2-III were zero, 2.1 cm, 2.5 cm, and 3.7 cm, respectively. The current strategy can be effectively employed for the development of composite materials of biopolymers with several kinds of bactericidal elements.
Microbial cellulose, a biopolymer produced by several microbial species, has received immense consideration owing to its purity, improved physico-mechanical, and biological properties.7,8 It serves as a carrier in drug delivery systems, enzyme immobilization, and scaffold for tissue engineering, which further highlights its importance in several fields.9–11 Furthermore, it possesses a high potential to form composites with most biocompatible and bactericidal elements.9,12,13 Microbial cellulose is composed of a fibrous structure where thin fibrils are interconnected through inter- and intra-molecular hydrogen bonding that stabilize its reticulate structure.14 The fibrils are loosely arranged with empty spaces between them that result in expanded surface area and a highly porous matrix.15–17 Further, the strong and stable fibrils offer better resistance to applied force and resist any variation in its structure. Similarly, the empty spaces between the fibrils can accommodate liquids and media components as well as small particles, thus supporting the formation of composites with several nano- and biocompatible polymeric materials.
Several methods have been reported for the synthesis of composites of microbial cellulose with other materials such as in situ, ex situ, and solvent dissolution and regeneration methods.1,2 However, these methods have several limitations such as the in situ method encounters limitation due to the cytotoxic effects of bactericidal elements against microbial cells; the ex situ method is confined to only nano-sized materials owing to the difficulty of penetrating the well-arranged fibril network; and the solvent dissolution and regeneration method alters the reticulate structure of microbial cellulose, and consequently, its physico-mechanical and biological properties.1,2,18 Thus, the need was extensively felt to develop an alternative approach for producing cellulose and preparing its composites with a wide range of materials for multifarious applications. Recently, we have developed a cell-free system for production of bio-cellulose that showed improved yield.19 Moreover, the produced bio-cellulose exhibited improved physico-mechanical properties.20 The system was entirely comprised of enzymes and not the microbial cells, and hence, can be utilized for the in situ preparation of composites of bio-cellulose with a wide range of nanomaterials of any type and size.
The current study was aimed to develop nanocomposites of bio-cellulose with TiO2 nanoparticles through an in situ strategy using a cell-free system. The synthesis mechanism of nanocomposite by a cell-free system was described, and its structural features and antibacterial activity against bacterial cells were investigated. This developed approach for the synthesis of nanocomposite can be effectively extended to the synthesis of other composite materials of diverse nature and a wide range of applications.
The TiO2 nanoparticles suspension was prepared by sonication after suspending the nanoparticles in distilled water with different concentrations of SDS solutions (0.5, 1, 2, and 5%). A suspension of TiO2 nanoparticles prepared in distilled water was used as a reference. All suspensions were sonicated at 25 min intervals until all the nanoparticles were in suspended form. Thereafter, the suspensions were regularly observed for 15 days, after which their absorbance was determined at 315 nm.
The crystallite size of both samples was calculated using the WHFM values through the Scherrer equation given as follow:
(1) |
(2) |
Similarly, the FT-IR spectra of freeze-dried samples of bio-cellulose and the bio-cellulose/TiO2 nanocomposite were recorded by using a Perkin Elmer FTIR spectrophotometer [Spectrum GX & Autoimage, USA, spectral range: 4000–400 cm−1; beam splitter: Ge-coated on KBr; detector: DTGS; resolution: 0.25 cm−1 (step selectable)]. For analysis, the samples were mixed with KBr (IR grade, Merck, Germany) pellets and processed further to obtain IR data that was transferred to a PC to acquire the spectra as reported previously.15
Similarly, the tensile properties of bio-cellulose and the bio-cellulose/TiO2 nanocomposite were measured using an Instron Universal Testing Machine (Model 4465, USA) according to the procedure described by the American Society for Testing and Materials (ASTM D 882). Briefly, two metal clamps were placed at either end of each 100 mm × 10 mm rectangular strip of freeze-dried samples and then mounted on an Instron 4465 that measured both elongation and maximum tensile load before fracture. The experiment was repeated several times, and the average values were taken for each sample.
A naked-eye observation of TiO2 nanoparticles suspensions in both distilled water and various concentrations of SDS solutions after sonication showed that the nanoparticles started settling down with the passage of time (Fig. 1A). Nearly all TiO2 nanoparticles suspended in distilled water settled after 24 h. However, the settling rate was much slower for the nanoparticles suspended in different concentrations of SDS solutions. It was observed that nearly all of the nanoparticles suspended in the SDS solutions remained suspended for 15 days, which is in agreement with previous observations.29,30 These results show that the resuspension ability of TiO2 nanoparticles was extended by the addition of detergent during sonication. This improved feature could be very useful during the in situ synthesis of a nanocomposite of bio-cellulose with TiO2 by a cell-free system.
Fig. 2 shows the SEM micrographs of surface and cross section of bio-cellulose, bio-cellulose/TiO2-I, bio-cellulose/TiO2-II, and bio-cellulose/TiO2-III nanocomposites synthesized after 5, 10, and 15 days, respectively by the cell-free system. It shows that the TiO2 nanoparticles were impregnated into the bio-cellulose matrix, confirming the successful synthesis of nanocomposite through the in situ strategy. During nanocomposite synthesis, the suspended TiO2 nanoparticles in the culture medium interact with the developing subfibrils from β-1,4-glucan chains that form the micro and macro fibrils, bundles, and ribbons.16,17 The nanoparticles are encaged within the bio-cellulose matrix through hydrogen bonding.15 The synthesis of bio-cellulose/TiO2-I showed that the TiO2 nanoparticles were attached to the fibrils only and were not impregnated into the matrix as shown in Fig. 2a. These observations were in agreement with the cross sectional analysis of the bio-cellulose/TiO2-I nanocomposite that displayed the presence of TiO2 nanoparticles towards the outer surface only and not in the interior of the bio-cellulose matrix (Fig. 2b). Such arrangement of TiO2 nanoparticles in the bio-cellulose matrix could be attributed to the early phase of synthesis by the cell-free system that possessed a loosely arranged matrix. The compactness of the bio-cellulose matrix increases with time as more fibrils and pellicles are produced with the time and added to the pre-existing ones.19,32,33 This resulted in an increased density of TiO2 nanoparticles within the matrix of bio-cellulose/TiO2-II as shown in Fig. 2c. These observations were in agreement with the cross-sectional analysis of bio-cellulose/TiO2-II nanocomposite that displayed the presence of TiO2 nanoparticles in the interior of bio-cellulose matrix (Fig. 2d). The density of TiO2 nanoparticles kept on increasing in the bio-cellulose matrix with time, and a nanocomposite with uniform and deeply impregnated nanoparticles was synthesized as shown by the surface (Fig. 2e) and cross-sectional analyses of the bio-cellulose/TiO2-III nanocomposite (Fig. 2f).
The potential application of a nanocomposite is highly dependent on the amount of impregnated nanoparticles. The conventionally reported strategies of nanocomposite synthesis such as in situ, ex situ, and solvent dissolution and regeneration approaches encounter the limitation of inefficient nanoparticles uptake. This study attempted to overcome this limitation through the development of an in situ strategy using a cell-free system. The nanoparticles uptake during the in situ synthesis of nanocomposite by a cell-free system was determined through two methods: dry-weight analysis and optical density method. The difference of initial nanoparticles concentration added to the mixture and after harvesting the nanocomposite after the respective time period gave the amount of nanoparticles impregnated into the bio-cellulose/TiO2 nanocomposites. The detailed results are described below:
The dry-weight analyses of pure bio-cellulose and bio-cellulose/TiO2 nanocomposite produced under the same experimental conditions by a cell-free system were done to determine the amount of TiO2 nanoparticles impregnated into the nanocomposite. Table 1 shows that 0.0271 ± 0.0038 g, 0.0578 ± 0.0105 g, and 0.0832 ± 0.0108 g bio-cellulose was produced after 5, 10, and 15 days, respectively by the cell-free system. On the other hand, the dry-weights of bio-cellulose/TiO2-I, bio-cellulose/TiO2-II, and bio-cellulose/TiO2-III nanocomposites were found to be 0.0281 ± 0.0042 g, 0.0604 ± 0.0123 g, and 0.0885 ± 0.0084 g, respectively. This indicates the impregnation of 0.0010 g, 0.0026 g, and 0.0054 g corresponding to 3.55%, 4.30%, and 5.99% of the total weights of bio-cellulose/TiO2-I, bio-cellulose/TiO2-II, and bio-cellulose/TiO2-III nanocomposites, respectively. These results show that the impregnation of TiO2 nanoparticles kept increasing with increased time, and 39.4% of the initially added TiO2 nanoparticles (i.e. 0.0137 ± 0.0021 g) were successfully impregnated into the nanocomposite after 15 days. These results are justified by the SEM micrographs that show a clear and increasing trend of nanoparticles impregnation into the bio-cellulose matrix with increasing time (Fig. 2).
Nanocomposite | Experiment I (dry-weight method) | Experiment II (optical density method) | |||
---|---|---|---|---|---|
Dry weight (g) | TiO2 up taken in bio-cellulose/TiO2 (g) | Amount of TiO2 in culture medium (g) | TiO2 up taken in bio-cellulose/TiO2 (g) | ||
Bio-cellulose | Bio-cellulose/TiO2 | ||||
Bio-cellulose/TiO2-I | 0.0271 ± 0.0038 | 0.0281 ± 0.0042 | 0.0010 | 0.0104 ± 0.0011 | 0.0008 |
Bio-cellulose/TiO2-II | 0.0578 ± 0.0105 | 0.0604 ± 0.0123 | 0.0026 | 0.0078 ± 0.0031 | 0.0034 |
Bio-cellulose/TiO2-III | 0.0832 ± 0.0108 | 0.0885 ± 0.0084 | 0.0054 | 0.0055 ± 0.0022 | 0.0057 |
The results of the optical density method indicated a similar trend to that of dry-weight analysis. Optical density analysis of sample medium after harvesting the bio-cellulose/TiO2-I, bio-cellulose/TiO2-II, and bio-cellulose/TiO2-III nanocomposites was carried out using a TiO2 standard curve. The initial amount of TiO2 nanoparticles in the culture medium was found to be 0.0137 ± 0.0021 g determined by the standard curve. The amounts of TiO2 nanoparticles impregnated into the nanocomposites, and non-impregnated nanoparticles in the culture medium are given in Table 1. The optical density analysis of the culture medium shows that 0.0104 ± 0.0011 g, 0.0078 ± 0.0031 g, and 0.0055 ± 0.0022 g of nanoparticles were still present in the culture medium after the synthesis of bio-cellulose/TiO2-I, bio-cellulose/TiO2-II, and bio-cellulose/TiO2-III nanocomposites, respectively. This indicates that 0.0008 g, 0.0034 g, and 0.0057 g of TiO2 nanoparticles, corresponding to 5.83%, 24.81%, and 41.60% of the initially added nanoparticles, were successfully taken up by the bio-cellulose/TiO2-I, bio-cellulose/TiO2-II, and bio-cellulose/TiO2-III nanocomposites, respectively. This is also justified by the SEM micrographs of bio-cellulose/TiO2-I, bio-cellulose/TiO2-II, and bio-cellulose/TiO2-III nanocomposites synthesized in situ by the cell-free system.
Table 1 shows a slight difference between the initially added nanoparticles and the sum of impregnated and unattached nanoparticles in the culture medium. This difference could be attributed to the amount of loosely bound nanoparticles that are removed during the washing of nanocomposites. The amount of TiO2 nanoparticles impregnated into the bio-cellulose matrix determined through the above two different approaches were comparable. A significant difference in the dry-weights of bio-cellulose and bio-cellulose/TiO2 nanocomposites and optical density values of culture media before and after the harvesting the nanocomposites showed that TiO2 nanoparticles were successfully impregnated into the bio-cellulose matrix. Further, the content of impregnated nanoparticles kept on increasing with time and 40 ± 1.6% of the initially added TiO2 nanoparticles were successfully impregnated into the matrix after 15 days.
From the above results, it can be concluded that nanoparticles interact with the microfibrils of bio-cellulose during the early phase of synthesis by a cell-free system. More nanoparticles get impregnated with increasing time and are entrapped more towards the interior of bio-cellulose matrix due to the addition of fibrils and pellicles. Several pellicles containing the impregnated nanoparticles interact with each other and form the larger bio-cellulose sheet containing a large number of impregnated TiO2 nanoparticles (i.e. nanocomposite). The thickness of nanocomposite increases in all directions when more pellicles containing the impregnated nanoparticles are attached. This process continues until all of the substrate available in the medium is consumed, and thus, a nanocomposite with a large number of impregnated nanoparticles is formed. These observations suggest that the in situ synthesis approach using a cell-free system ensures the uniform distribution of nanoparticles within the bio-cellulose matrix.
The FT-IR spectra of bio-cellulose contained basic peaks for all chemical groups in cellulose and thereby confirmed the basic structure of pure cellulose. The spectra of bio-cellulose showed characteristic peaks for OH stretching at 3364 cm−1, which are in agreement with previous observations.14,18,20 A broader peak for bio-cellulose indicated stronger OH bonding.20 Similarly, peaks were obtained for a CH stretching vibration at 2924 cm−1 as reported previously.14,18,20 The presence of the CH group was further supported by the appearance of several peaks corresponding to CH bending vibrations at 1450–1200 cm−1.13,20 In addition, two characteristic peaks at 1453 cm−1 and 1396 cm−1 were observed.20 The peaks due to C–O–C stretching vibrations appeared at 1060 cm−1.13,20,34 The FT-IR spectrum of bio-cellulose/TiO2-III nanocomposite contained additional small peaks at 621 cm−1, 594 cm−1, 549 cm−1, and 412 cm−1, which are in agreement with previous observations.35 The presence of these characteristic Ti–O peaks of titania confirms the successful synthesis of bio-cellulose/TiO2-III nanocomposite by a cell-free system through an in situ strategy. The peaks at 1000–1300 cm−1 for bio-cellulose/TiO2-III nanocomposite due to C–OH stretching (1060 cm−1) and C–O–C bending vibrations (1163 cm−1), are weakened in comparison to the peaks in bio-cellulose because the TiO2 nanoparticles grow on the surface of bio-cellulose. These results are in agreement with previous observation.36
Fig. 3B shows the comparative XRD patterns of the extended linear scanning (10–70°) of bio-cellulose and bio-cellulose/TiO2-III nanocomposite. The XRD spectrum of bio-cellulose showed two broad peaks at 2θ 11.78° and 20.32° arising from the (11) and (110) crystallinic planes, respectively, which represents the cellulose II structure.20 The XRD pattern of bio-cellulose/TiO2-III nanocomposite showed the diffraction pattern of both bio-cellulose and TiO2 nanoparticles that exhibit all characteristic peaks at 2θ 11.78°, 20.32°, 25.23°, 36.95°, 37.71°, 38.56°, 48.06°, 53.80°, 55.07°, 62.75°, 68.71°, and 70.32° arising from the (101), (103), (004), (112), (200), (105), (211), (118), (116), and (220) planes of anatase TiO2 nanoparticles.2,28 The slight decrease in the peak intensity of bio-cellulose in the bio-cellulose/TiO2-III spectra could be due to the TiO2 content.2
The degree of crystallinity of bio-cellulose and bio-cellulose/TiO2-III nanocomposite was calculated from the relative integrated area of the crystalline and amorphous peaks (eqn (2)). The ratio of crystalline to amorphous regions varies between samples and is dependent on the cellulose type, microbial strain, medium constituents, and processing conditions.37 The relative crystallinity of bio-cellulose was 31.98%. This lower crystallinity of bio-cellulose was clearly demonstrated by the absence of sharp crystallinic peaks in its XRD spectrum (Fig. 3B), and can be attributed to the incomplete growth of crystallite during its synthesis by a cell-free system.20,38 The impregnation of TiO2 nanoparticles did not significantly affect the crystallinity of bio-cellulose, which was slightly reduced to 31.08%.2 The crystallite size of bio-cellulose was calculated through the Scherrer equation and is summarized in Table 2.
Sample | d-Spacing (Å) | Crystallinic planes | FWHM | Crystallite sizes (Å) | Crystallinity index (%) |
---|---|---|---|---|---|
Bio-cellulose | 6.1001 | (1−10) | 1.611 | 47 | 31.98 |
3.8932 | (110) | 1.428 | 555 |
In the present study, both bio-cellulose and the bio-cellulose/TiO2-III nanocomposite displayed two major weight loss zones (Fig. 4A). In the first step, about 2–3% weight loss occurred at a temperature range of 90–100 °C in bio-cellulose. This weight loss in bio-cellulose could be attributed to the loss of moisture content adsorbed on the surfaces and interlayer coordinated water molecules.41,42 The weight loss in the first step was lower for the bio-cellulose/TiO2 nanocomposite, indicating that the sample had a lower water content.1 Negligible weight loss was observed as the temperature was increased to 290 °C and 310 °C for bio-cellulose and the bio-cellulose/TiO2 nanocomposite, respectively. The second phase revealed a sharp weight loss due to the degradation of the main cellulose skeleton in both samples.15,20,41 The onset temperatures of bio-cellulose and the bio-cellulose/TiO2 nanocomposite were 298 °C and 333 °C, respectively. The improved thermal stability of nanocomposite could be attributed to the impregnated TiO2 nanoparticles. During this phase, the weight loss in bio-cellulose was 84%. In contrast, a lower weight loss was recorded in the bio-cellulose/TiO2 nanocomposite (68%). Similarly, the endset temperatures of bio-cellulose and bio-cellulose/TiO2 nanocomposite were 346 °C and 411 °C, respectively. The overall results indicate that the thermal stability of bio-cellulose/TiO2 nanocomposite was higher than bio-cellulose. Fig. 4A also indicates that there was no further decomposition of TiO2 nanoparticles after bio-cellulose degradation. Inorganic materials are thermally stable and most degrade above 600 °C.1 The nanoparticles impregnated into polymers, such as cellulose, offer a barrier for the main skeleton by absorbing heat, which ultimately results in shifting the degradation process towards higher temperature and reduced weight loss.
Fig. 4B shows the mechanical properties of bio-cellulose and bio-cellulose/TiO2-III nanocomposite. The maximum tensile strength value at the breaking point for bio-cellulose was recorded to be 17.54 MPa. This high value could be attributed to the thick, compact, and well-arranged fibrils of bio-cellulose.20 Such compact and uniform arrangement of fibrils in bio-cellulose could give a uniform response to applied force, and thus, result in improved tensile strength.15,37 The tensile strength for bio-cellulose/TiO2-III nanocomposite was increased to 20.98 MPa. Similar increases in tensile properties of polymer–nanoclay and polymer–nanoparticles have previously been reported.1,43,44 The Young's modulus for the bio-cellulose/TiO2-III nanocomposite was significantly increased to 0.97 GPa comparing to 0.38 GPa of bio-cellulose. These results demonstrate that the impregnation of TiO2 nanoparticles exerts a positive effect on the mechanical properties of bio-cellulose. It has been reported that the binding potential of nanoparticles to the polymer surface is readily affected by the physical phenomenon caused by rough surfaces and chemical interactions by hydrogen bonding and van der Waals forces.1,45 The binding of TiO2 and ZnO nanoparticles with microbial cellulose with OH moieties have already been reported.1,2 The TiO2 nanoparticles attached to bio-cellulose improves its overall toughness and restricts its mobility, ultimately resulting in the improved mechanical strength of the nanocomposite.1,46 The average strain of bio-cellulose and the bio-cellulose/TiO2-III nanocomposite was recorded to be 3.29 and 2.74%, respectively. The low strain of bio-cellulose could be attributed to the closely packed fibrils that cause the chains to be almost immobile and results in a very low level of elasticity.20 The strain of the nanocomposite was significantly decreased upon the incorporation of TiO2 nanoparticles into bio-cellulose. Several studies have reported a decrease in elasticity of composite material that can be attributed to the incorporation of nanoparticles into the main cellulose skeleton.1,15,46 The binding interaction between the TiO2 nanoparticles causes rigidity and restricts the mobility of bio-cellulose microfibrils, thus, resulting in decreased strain.
The antibacterial activity against E. coli of bio-cellulose and the bio-cellulose/TiO2 nanocomposites developed by a cell-free system was investigated using the agar disc diffusion and optical density methods. The results of are shown in Fig. 5A and B. During the disc diffusion method, the impregnated nanoparticles immediately begin to diffuse outwards from the nanocomposite disc. The released nanoparticles create a gradient in the agar such that the highest concentration is found in the vicinity of the disc while decreasing the concentrations further away from the disc. For bio-cellulose, the disc did not produce any inhibition zone (Fig. 5A), indicating that it does not possess any bactericidal activity, which is in agreement with previous studies.1,14,50 On the other hand, clear inhibition zones or ‘areas of no growth’ were produced by the bio-cellulose/TiO2-I, bio-cellulose/TiO2-II, bio-cellulose/TiO2-III nanocomposites discs after an overnight incubation. Precisely, a maximum of 3.7 cm, 2.5 cm, and 2.1 cm zones of inhibition were produced by the bio-cellulose/TiO2-I, bio-cellulose/TiO2-II, bio-cellulose/TiO2-III nanocomposites, respectively. The results obtained with disc diffusion method were in agreement with those of the optical density method that showed similar trends of antibacterial activity. The curves for optical density values versus culture time for bio-cellulose and bio-cellulose/TiO2 nanocomposites are shown in Fig. 5B. The results indicate that bio-cellulose did not show any antibacterial activity and, in fact, the E. coli growth was higher than the control, which is in agreement with previous reports.2,51 In contrast, the nanocomposites showed considerable antibacterial activity against E. coli, which was higher for bio-cellulose/TiO2-III compared to the bio-cellulose/TiO2-II and bio-cellulose/TiO2-I nanocomposites. This indicates that the bactericidal effect of the bio-cellulose/TiO2 nanocomposite is dependent on TiO2 concentration and release rate.52,53 These observations are in agreement with the SEM micrographs of the nanocomposites (Fig. 2). This can be further explained by the fact that with increasing time the bio-cellulose fibrils become more compact and hold the nanoparticles more firmly, which allows for the slow release of nanoparticles that show bactericidal activity over a prolonged time. Consequently, this will improve the potential of application of bio-cellulose/TiO2 nanocomposites in the biomedical field.
The release behavior of ions or nanoparticles indicates the strength of their interaction with the polymer matrix and their toxicological level.1,2 Further, the constant and controlled release of nanoparticles is necessary for biomedical and other applications, which may otherwise cause hazardous effect if high concentrations are released or if a release occurs in an uncontrolled fashion. A nanocomposite of cellulose with TiO2 nanoparticles shows its antibacterial activity due to the release of Ti4+.2 Therefore, the Ti4+ release behavior from bio-cellulose/TiO2-III nanocomposite was determined.
In the current study, the amount of Ti4+ released from the bio-cellulose/TiO2-III nanocomposite in water was determined using the ICP method. The nanocomposite showed a very low level of ion release throughout the entire observation period. Precisely, the ions release level reached to only 0.1123% of the initially impregnated nanoparticles in the nanocomposite after 10 days of incubation in water under static conditions at room temperature (Table 3). Conversely, the nanocomposite retained 99.98% of nanoparticles impregnated after 10 days of incubation. This slow release of ions indicates the strong interaction of Ti4+ with bio-cellulose fibers at both the surface and inner matrix as shown by the highly compact fibril arrangement in the nanocomposite (Fig. 2).
Days | Initial amount of NPs in nanocomposite (g) | Amount of NPs released from nanocomposite | Amount of NPs retained in nanocomposite | ||
---|---|---|---|---|---|
(g) | % | (g) | % | ||
0 | 0.0054 | 0 | 0.0000 | 0.005400 | 100 |
2 | 0.0054 | 9.53 × 10−7 | 0.0177 | 0.005399 | 99.982335 |
4 | 0.0054 | 3.15 × 10−6 | 0.0585 | 0.005397 | 99.941548 |
6 | 0.0054 | 4.55 × 10−6 | 0.0843 | 0.005395 | 99.915652 |
8 | 0.0054 | 5.84 × 10−6 | 0.1083 | 0.005394 | 99.891698 |
10 | 0.0054 | 6.063 × 10−6 | 0.1123 | 0.005394 | 99.887721 |
Bio-cellulose synthesized by a cell-free system possesses a more compact and well-distributed fibril arrangement20 that favored the effective uptake of TiO2 nanoparticles (Table 1). Further, the TiO2 nanoparticles were impregnated into the bio-cellulose matrix (Fig. 2) and remained firmly attached to the fibers as shown by the slow release of Ti4+ from the synthesized bio-cellulose/TiO2-III nanocomposite (Table 3). On the other hand, the TiO2 nanoparticles are mostly attached to the bacterial cellulose (BC) surface in the ex situ method and a major portion are released during the washing (e.g. with sodium carbonate solution) of the BC/TiO2 nanocomposite.52 Further, the in situ synthesis of nanocomposite by the cell-free system favored the uniform distribution of TiO2 nanoparticles due to the continuous synthesis of cellulose fibers and their interaction with TiO2 nanoparticles as shown by the FE-SEM micrographs (Fig. 2). In contrast, the formation of agglomerates on the surface of a composite is a common phenomenon during the ex situ synthesis of BC composites with TiO2 or other nanomaterials.52 The bio-cellulose/TiO2 nanocomposite synthesized by the cell-free system showed better thermal properties as shown by the thermogravimetric analysis (Fig. 4A). The degradation temperature of bio-cellulose/TiO2 nanocomposite synthesized by the cell-free system was found to be 414 °C compared to 280–300 °C for BC/TiO2 nanocomposites as reported in previous studies.36,54 On the other hand, regeneration method of composite synthesis alters the reticulate structure of microbial cellulose and ultimately its physico-mechanical properties.1,2 Furthermore, the bio-cellulose/TiO2 nanocomposite synthesized by the cell-free system displayed better antibacterial activity compared to RBC/TiO2 nanocomposite created by the regeneration method.2 This could be due to the fact that uniformly distributed TiO2 nanoparticles in the bio-cellulose/TiO2 nanocomposite synthesized by the cell-free system are slowly and uniformly released and showed bactericidal activity against E. coli for a prolonged time (Fig. 5B).
From the above discussion, it can be concluded that a cell-free system can offer several advantages compared to microbial cell system in composite syntheses, such as in situ synthesis of composites with a wide range of bactericidal elements, better uptake and uniform distribution of nanoparticles, and cost effectiveness due to a better yield of bio-cellulose. Similarly, the thicker, compact, and well-distributed fibers in bio-cellulose could favor the synthesis of a composite with better physico-mechanical, antibacterial, and biological properties.
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