Bacterial isolates
The bacterial strain used in this study was isolated from kombucha tea in Egypt and identified 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 microfibres and results in the growth of a dense, white BC pellicle at the air-liquid interface of modified 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 purification and drying (8 g/l) (c). Komagataeibacter rhaeticus is the most efficient 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 Ago 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 modification of BC, just pure BC without any surface modification soaked in AgNO3 solution under gamma-ray which promote the reduction process and were used for creating a hydrated electron and primary radicals in many studies (Chen et al., 2007; Park et al., 2012; Van Phu et al., 2014; Madhukumar et al., 2017; Dhayagude et al., 2018). It was found that no apparent color change of the BC membrane and solution at (0.2, 0.4, 0.8, 1, and 5 KGy). At 10 kGy visual observations showed that as the reaction started, the color shifted from pale yellow to deep brown for higher doses (20–100 kGy), signifying the reduction of Ag+ to Ago and formation of colloidal AgNPs and BC/AgNP composite. Recently, using of BC as a template in BC /AgNP composite synthesis gaining a lot of attention among researchers. The composite synthesis process and the mechanism of reaction in which the metal is reduced and binds onto BC surface have not been studied in much detail in many reports (Kaushik and Moores, 2016). Many researchers showed that it’s necessary to make the surface modification of BC by some compounds i.e. TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical), that oxidize BC and introducing surface-active carboxyl groups for BC /AgNP composite synthesis (Lal and Mhaske 2018; Elayaraja et al., 2020). Recently, Musino et al., (2021) showed that the hydroxyl groups on the surface of BC act as nucleation points for AgNPs through ion-dipole interaction, and OH groups represent the effective nucleation point for AgNPs synthesis on a BC solid surface. As far as we know it’s the first study for the synthesis of colloidal AgNPs and BC/AgNP composite using only BC as a reducing and stabilizing agent under gamma radiation.
UV–visible spectrum
Figure (2) showed UV–visible spectrum of AgNPs synthesized by BC under different doses of gamma-ray. The reduction of Ag+ to Ago by OH groups on BC surface under gamma-ray was preliminarily identified 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 brown- at 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 confirmed 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 flocculation 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 examination
FE-SEM images of BC and BC /AgNP composite shows three-dimensional structures of BC nanofibers. After the BC sheet was soaked in AgNO3 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 fibers (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− 1 region are assigned to the OH stretching vibration of the hydroxyl groups present in the BC nanofiber (Zhu et al., 2014; Cacicedo et al., 2020). Peaks at 3000 − 2800 cm− 1 were assigned to the stretching vibrations of the CH2 and CH2-OH groups (Liet al., 2011; Cacicedo et al., 2020). The band at 1640 cm− 1 can be assigned to C = O (Wang et al., 2017). The band at 1426.38 cm− 1 is assigned to the stretching vibrations of CH2 or OH in-plane bending (Barud et al., 2011). The peak at 1321.94 to 1369.67 can be assigned to O-H in-plane bending (Osorioet al., 2019).The band at 1022.36 cm− 1 for the C-O-C and C-O-H stretching vibrations of the sugar ring (Huang et al., 2015). The absorption at 1158.31 cm− 1 is coming from the C-O-C stretching vibration of the pure cellulose. A group of absorption peaks at the wavenumber region of 1200 − 900 cm− 1 arise due to the C-O and C-C stretching vibrations of the cellulose network (Cui et al., 2014). The FTIR spectrum of BC/AgNP composite (b) contained all the characteristic peaks of BC along with an additional new band for AgNPs at 1545.5 cm− 1 in the BC/AgNP composite resulting from the hydrogen bonding interaction between BC and AgNPs (Wang et al., 2017; Wan et al., 2020).
XRD analysis
XRD patterns provide information about the crystalline structure of BC and BC/AgNP composite. Figure (8) showed three diffraction peaks at about 14.32°, 16.82°, 22.57°, and 14.66°, 16.56°, 22.86° for BC and BC/AgNP composite respectively, the peaks corresponded to (110), (110), and (200) crystal planes of cellulose (French, 2014and Volova et al., 2018). In many studies, the XRD of pure BC membrane showed three characteristic peaks at 14.60°, 16.82°, and 22.78° (Yan et al., 2008; Ul-Islam et al., 2013; Wu et al., 2014). The XRD graph of BC/AgNP composite exhibited newly three peaks 27.4°, 32.4°, and 46.5° attributed to the diffractions from the planes of Ag. Mageswariet al ., (2015) reported diffraction peaks of 46°, 54°, and 68°, that attributed to 2 1 1, 2 2 0, and 2 2 2 lattice planes of the face-centered cubic crystal structure of silver, while diffraction peak at 26° and 32° was indexed to 1 1 0 and 1 1 1, planes of silver oxide. In many studies, the face-centered cubic crystal structure of crystalline Ag showed diffraction peaks at around 38 °, 46°, and 64° that was attributed to the planes of 1 1 1, 2 0 0 and 2 2 0, respectively (Prakash et al., 2013; Jyoti et al., 2016and Anjum and Abbasi, 2016). The peak at about 27° and 32° was also reported for the diffractions from the planes of silver (Kumar et al., 2012and Rose et al., 2019).
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.
Table (4) show the antibacterial activity of BC/AgNP composite, purified BC, AMC, CAZ, and S against Gram-positive (Staphylococcus aureus, Enterococcus faecalis, and Listeria monocytogenes) and Gram-negative (proteus mirabilis and E. coli) bacteria. Purified BC and amoxicillin/clavulanic acid did not show antibacterial activity against any tested bacteria. Listeria monocytogenes, Proteus mirabilis, and E. coli were resistant to ceftazidime. BC/AgNP composite and streptomycin showed antibacterial potency against both Gram-positive and Gram-negative bacteria. It was observed that Enterococcus faecalis, Staphylococcus aureus, and Listeria monocytogenes were more sensitive and gave higher inhibition zone for BC/ AgNP composite while Gram-negative E. coli and Proteus mirabilis were more resistant. Many studies reported that AgNPs have shown higher antimicrobial potency in Gram-positive than Gram-negative bacteria (Mandal et al., 2016; El-Sherbinyet al., 2020, Jiji et al., 2020). the antibacterial activity of AgNPs depends on many factors including size, charge, the concentration of AgNPs (Van Phu et al., 2014, Jiji et al., 2020), and also the stabilizer used (Phu et al., 2014). Various mechanisms have been proposed for the antibacterial action of AgNPs, the most common one can be that free silver ions uptake may preventing DNA replication or may cause direct damage to the bacterial cell wall by forming pits in the cell wall that lead to an increase in permeability of cell wall and final cell death (Jones and Hoek, 2010; Bapat et al., 2018).