Gamma Ray-Induced Synthesis of Silver Nanoparticles using Bacterial Cellulose as a MultiFunctional Agent and its Application

Antibacterial coatings based on bacterial cellulose (BC) have been widely used in many elds including food packaging and wound dressing. In this study, we aimed to synthesis of colloidal AgNPs and BC/ AgNP composite by using BC as a reducing and capping agent in one step reaction induced by gamma-ray. Bacterial strain Komagataeibacter rhaeticus N1 MW322708 was used for biosynthesis BC by inoculation on Hestrin and Schramm medium and incubated statically at 35 °C for 10 days. BC sheet was formed, harvested, puried, and dried, then used for the synthesis of AgNPs and BC/AgNP by soaked 0.05 g of dried BC in 10ml of 1mM aqueous AgNO 3 solution for 2h and then irradiated by gamma-ray under different doses. Color change from yellow to deep brown indicated the synthesis of AgNPs and BC/AgNP. The optical spectra of synthesized AgNPs revealed that the surface plasmon resonance was localized around 420 nm. DLS analysis showed that the mean diameter of AgNPs was 49.5 nm with a -19.36-mV value of zeta potential. TEM images revealed the spherical shape of synthesized AgNPs. The results of FESEM, FTIR, and XRD conrmed the formation of BC/AgNO 3 composite. The highly crystalline nature of the BC membrane and BC/AgNP composite was observed in XRD measurements with a crystal size of 5.416 and 5.409 nm, respectively. The antibacterial activity of BC and BC/AgNP against pathogenic bacterial isolated from Pastirma food samples revealed that BC does not show antibacterial activity, while BC/AgNP composite showed antibacterial potency against Staphylococcus aureus, Enterococcus faecalis, Listeria monocytogenes, Proteus mirabilis, and Escherichia coli, with an inhibition zone of (mm) 9±1, 9±0.57, 10±1.15, 8±0.5 and 7±0.28, respectively. We concluded that this novel method presented in this paper offers a promising route for both AgNPs and BC/AgNP composites synthesis using a green, renewable biopolymer as a multifunctional agent and potential to be applied in the future development of food packing, biomedical instruments, and therapeutics.


Introduction
Bacterial cellulose (BC) is a versatile biopolymer produced from the metabolic activity of cellulose-  Petrova et al., 2020). Cellulose synthesized by bacteria has a chemical composition similar to plant cellulose; however, thanks to its structural arrangements but, BC has remarkable physical properties. A study by Lin and the group showed that BC possesses high tensile strength, high crystallinity, high hydrophilicity, and excellent biocompatibility (Lin et al., 2013). BC has very high purity so that making other puri cation processes (such as deligni cation), which is very energy-consuming for plant cellulose, is unnecessary (Santoso et al., 2020). BC production gained more attention over the last several decades and its potential uses cover very varied elds including food (Padrao et (Xue et al., 2019). Nanoparticles and nanostructured materials have found their way into different elds such as food (Enescu et al., 2019), medicine (Jiji, 2020), environment (Umar et al., 2016), and so on. It is well known that silver nanoparticle materials have been proven to be the most useful because they showed great antimicrobial performance against a wide range of microorganisms including various bacteria and fungi (Siddiqi et al., 2018 andWang et al., 2018). AgNPs are extensively used in industrial applications than any other nanomaterial (Vance et al., 2015). However, the aggregation tendency of AgNPs may frustrate their unique properties at the nanoscale. We can prevent aggregation by the incorporation of AgNPs into a nanoporous polymer (Cai, 2009 (Barud et al., 2011). Some of these methods are cost, ecologically unfriendly, and unsafe that need to use toxic chemicals. Thus, the development of green approaches for the synthesis of colloidal AgNO 3 and BC /AgNP composite is necessary. Recently gamma radiation gains more attention in AgNPs synthesis. Gamma radiation was used to induces the reduction of Ag + into metallic Ag in different aqueous solutions; acetic water solution containing chitosan (Chen et al., 2007), aqueous silk broin (SF) solution (Madhukumar et al., 2017), and poly (N-vinylpyrrolidone) solution (Dhayagude et al., 2018). The main objective of this work is to synthesis of colloidal AgNPs and BC /AgNP composite by green, eco-friendly, free of toxic approach, characterization, and evolution of the antibacterial activity against some pathogenic bacteria.

Production and puri cation of bacterial cellulose
The bacterial strain Komagataeibacter rhaeticus N1 MW322708 strain isolated from Kombucha was used for BC synthesis. K. rhaeticus N1 MW322708 was inoculated into a sterile plastic box that contains 400 ml of Hestrin and Schramm (HS) medium (% w/v): 2% glucose, 0.5% peptone, 0.5% yeast extract, 0.27% Na 2 HPO 4, and 0.15% citric acid (Hestrin & Schramm, 1954) the medium was modi ed by adding 1.5% ethanol to nal concentration, the nal pH was adjusted to 7, then K. rhaeticus N1MW322708 statically incubated at 35°C. BC sheet formed in air-liquid phase after 10 days were harvested, treated with 0.1 M NaOH solution at 85 • C for 2h to remove the bacterial cells, then rinsed several times with deionized water to achieve a neutral pH; nally BC sheet was dried at 85 • C for 1h and then subjected to autoclave.

Synthesis of colloidal AgNPs and BC /AgNP composite under gamma radiation
Puri ed BC sheets (0.05 g, dry weight) were soaked in 10 ml of 1 mM aqueous AgNO 3 solution for 2h then irradiated by gamma-ray under different doses (0.2, 0.4, 0.8, 1, 5, 10, 20, 40, 80 and100 KGy). BC sheets were taken, rinsed several times with distilled water, and oven-dried at 40°C. Finally, the deep brown color solution and BC/AgNP sheet were stored in dark conditions at 4°C for future use.

Gamma radiation Source
Gamma irradiation was applied using a cobalt 60 irradiation source (Gamma cell 4000-A-India), located at the Egyptian Atomic Energy Authority (EAEA), Cairo, Egypt. The irradiation dose rate was 1.0 kGy/h at the time of the experiment and the irradiation process was carried out at ambient temperature.

Fourier transform infrared (FTIR)
Fourier transforms infrared (FTIR) spectrum was performed on ATI Mattson (Genesis series, Unicom, England). BC/AgNP sheet was scanned at the frequency range of 400 to 4000 cm − 1 .

X-ray diffraction analysis (XRD)
X-ray diffraction spectra of the dried BC and BC/AgNP composite were obtained using the XRD-6000 Shimdazu device (Japan). A standard Theta/2Theta diffractometer (using a copper X-ray source) was used. Scans were performed at 2 degrees per min from the diffraction angle ranged from 4 to 90°. The apparent crystal size (ACS) of BC and BC/AgNP was calculated using Scherrer's equation (Klug and Alexander, 1954), as follows: Where k is the unknown shape factor and usually considered as 0.9, λ is the X-ray wavelength, β is the full width at half maximum in radians and θ is the diffraction angle 2.5. Antibacterial activity of BC and BC/AgNP composites The antibacterial activity of prepared BC, BC/AgNP composites was determined using the disk diffusion method against Gram-positive (Staphylococcus aureus, Enterococcus faecalis, and Listeria monocytogenes) and Gram-negative (E. coli and Proteus mirabilis) bacteria species were isolated from Pastirma food samples collected from local markets in Cairo, Egypt, and identi ed by VITEK 2 compact automated system (Biomerieux Inc., Marcy I'Etoile, France). Tested bacteria were cultured into a tube containing 5 ml of Muller Hinton broth (CLSI, 2016) at 37°C for 24 h, then 0.1 ml of bacterial suspension with 0.5 McFarland turbidity was swapped on the surface of the Mueller Hinton agar medium, dried discs (6mm in diameter) of BC/AgNP composite and pure BC were placed on the agar medium along with amoxicillin/clavulanic acid (AMC) (20/10µg/ml), ceftazidime (CAZ) (30µg/ml) and streptomycin (S) (10µg/ml) against tested bacteria. The petri-dishes were incubated at 37°C for 24h. The antibacterial activities of the samples against tested organisms were monitored by observing the zone of inhibition

Results And Discussion
Bacterial isolates The bacterial strain used in this study was isolated from kombucha tea in Egypt and identi ed by morphological, and biochemical characteristics as well as by 16S rRNA gene sequence analysis and deposit in GenBank under accession number (MW322708) strain K. rhaeticus N1 MW322708. https://www.ncbi.nlm.nih.gov/nuccore/MW322708

Synthesis of bacterial cellulose
The BC produced by K. rhatecus in static culture is initially extruded from the pores on the cell surface as micro bres and results in the growth of a dense, white BC pellicle at the air-liquid interface of modi ed HS medium after 10 days of incubation. Figure (1) shows BC produced by K. rhaeticus N1 MW322708 strain after 10 days using static culture conditions (a), harvesting of BC (b), and BC after puri cation and drying (8 g/l) (c). Komagataeibacter rhaeticus is the most e cient BC producer, as it has the capacity to assimilate several different sugars and yields high levels of cellulose in a liquid culture medium (Rajwadeet al., 2015; Petrova et al., 2020).

Synthesis of AgNPs and BC/AgNP composite under gamma-ray irradiation:
In this work, gamma-ray was attempted to induce the reduction of Ag + to Ag o by surface OH groups on BC surface for synthesis of colloidal AgNPs and BC/AgNP composite, with no chemicals involved in the chemical reaction or no surface modi cation of BC, just pure BC without any surface modi cation soaked in AgNO 3 solution under gamma-ray which promote the reduction process and were used for creating a The reduction of Ag + to Ag o by OH groups on BC surface under gamma-ray was preliminarily identi ed by observing the change in color of the reaction medium from clear to yellowish-brown. The change in color to the pale yellow of the reaction mixture started at a dose of 10 KGy and gradually turned to deep brownat higher doses (20, 40, 80, and 100KGy). It is well known that AgNPs show a yellowish-brown color in water or aqueous solution; these colors arise due to the excitation of surface plasmon vibrations in the metal nanoparticles (Shankar et al., 2004). This color change was associated with the development of a strong absorption band at 420 in the UV-vis. spectrum. This UV-Visible absorption peak at around 420 nm was attributed to the surface plasmon resonance (SPR) absorption peak of AgNPs, which con rmed the AgNPs formation with small size and narrow size distribution. Appeared peak around 420 nm was previously reported as a spherical or quasi-spherical Ag NPs SPR band (Sivasankar et al., 2018;Tabaiiet al., 2018).

DLS analysis
To accurately measure the mean diameter of synthesized AgNPs DLS analysis was used. The results of the DLS analysis (Fig. 2) showed that the mean diameter of AgNPs was 49.5 nm. The size distribution of colloidal AgNPs illustrated by details in a table (1) Zeta potential analysis Zeta potential measures the electric charge on the surface of nanoparticles. Zeta potential value delivers information about the stability of nanoparticles. When nanoparticles in suspension have a large negative zeta potential value, nanoparticles will tend to repel with each other, thus there will be no tendency of the nanoparticles to agglomerate together. In contrast, in the case of low zeta potential values, no force to prevent the nanoparticles from coming together, thus nanoparticles tend to agglomerate (Roy et al., 2013). The value of the zeta potential of colloidal AgNPs was − 19.36 mV (Fig. 4). Obtained results from zeta potential proved that synthesized AgNPs were poly-dispersed, due to the high negative zeta potential value. The electrostatic repulsive force between nanoparticles results in the prevention of occulation of nanoparticles and has an important role in nanoparticles' long-term stability in the solution (Kotakadi et al., 2016).
TEM examination TEM examination of synthesized AgNPs was used to obtain information about the morphology and size of metal nanostructures. The obtained result revealed that the shape of obtained AgNPs was spherical (Fig. 5).

FE-SEM images of BC and BC /AgNP composite shows three-dimensional structures of BC nano bers.
After the BC sheet was soaked in AgNO 3 for 2h and then irradiated, silver ions reduced by OH surface groups of BC to AgNPs (white dots) which appeared to adhere to the surface of BC bers (Fig. 6).
FTIR spectrum was performed to detect the interaction between BC and Ag-NPs. Figures (7) and table (2) show the FTIR spectra of BC and BC/Ag nanocomposites. For BC (a) characteristic bands of cellulose that appeared at the 3200-3400 cm −  The apparent crystal size (nm) of BC and BC/AgNP composite are reported in Table (3). According to the Scherrer equation, the peak that was used for calculating the crystalline size of BC and BC/AgNP was 22.57° and 22.86° respectively. The average size of the BC membrane is determined to be 5.416 nm and 5.4091nm for BC/AgNP composite.

Conclusion
In this work, a facile green method for the preparation of colloidal AgNPs and BC/ AgNP composite under the induction effect of gamma radiation. BC soaked in AgNO 3 was used as a reducing and capping agent. There is no need for using a chemical reducing agent or a supplementary catalyst in this way. The prepared composite exhibit antibacterial activity against Gram-positive and Gram-negative bacteria. Therefore, the BC/ AgNP composite has the great potential to be applied in the future development of biomedical instruments, food packaging, and therapeutics such as wound dressing.

Declarations
Con ict of interest The authors declare that they have no con ict of interest.